Pathology of Solid Organ Transplantation

Pathology of Solid Organ Transplantation

Helen Liapis  •  Hanlin L. Wang (Editors)

Pathology of Solid Organ Transplantation

Editors Helen Liapis, MD Washington University School of Medicine Department of Pathology & Immunology 660 S. Euclid Avenue Campus Box 8118 Saint Louis, Missouri 63110-1093 USA [email protected]

Hanlin L. Wang, MD, PhD Cedars-Sinai Medical Center Department of Pathology and Laboratory Medicine 8700 Beverly Blvd. Los Angeles, CA 90048 USA [email protected]

ISBN  978-3-540-79342-7 e-ISBN  978-3-540-79343-4 DOI  10.1007/978-3-540-79343-4

Springer Heidelberg Dordrecht London New York

Library of Congress Control Number: 2010935946 © Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Product liability: The publishers cannot guarantee the accuracy of any information about dosage and application contained in this book. In every individual case the user must check such information by consulting the relevant literature. Cover design: eStudioCalamar, Figueres/Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

To my husband Athanasios and my daughters Anastasia and Katerina, for time taken from them. Helen Liapis To Michelle, Sean, and Jason for their tremendous support and encouragement. Hanlin L. Wang



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Preface 

Solid organ transplantation is now the treatment of choice for end-stage kidney, heart, liver, pancreas, and lung diseases and increasingly so for intestinal disease. Experimental transplantation in animals provided the impetus for transplantation in humans, first with the kidney [1–3]. A brief account of successful human “experiments” in the time frame of the last 50 years is given in the Table 1 below. These successes were preceded by multiple failures or short-lived grafts. It is through the combined efforts of many researchers, surgeons, and the development of successful immunosuppressive drugs that graft survival and patient outcomes improved [4, 5]. More than a dozen Nobel prices since 1901 were awarded to those who worked on the fundaments of transplantation [6]. Currently, the search for agents that perfect induction of tolerance is intensified and transplant services and organ sharing continue to improve [7]. In this remarkable journey of pioneer surgeons, transplant immunologists, and chemists, pathologists were instrumental in recognizing allograft rejection, and more recently, defining the criteria that distinguish acute from chronic rejection, rejection from drug toxicity, and recurrent from de novo disease. Pathology has also been in the forefront of the endeavor of new therapies participating in the evaluation of the effects of drugs on tissue, thus maintaining clear and ethical views in the search for better treatments. Pathologic interpretation of the transplanted organs in humans was first described for the kidney in the 1960s by Gustav Dammin at Harvard and Kendick Porter at St. Mary’s Hospital in London [8, 9]. It soon became the most reliable tool to distinguish rejection from other complications of transplantation such as drug toxicity, recurrent/de novo disease, and infection. Transplantation pathology is now an indispensable guide to prompt therapy. As the field of transplantation advances, so is transplant pathology. New criteria for donors and the effectiveness of alternative immunosuppression drugs are better understood by histopathologic study of the tissue immune response in the graft, short- and long-term. Innovative approaches of immune tolerance, such as mixed allogeneic chimerism, monoclonal antibodies, and fusion proteins and stem cells for immune modulation, may in the next decade become a reality, therefore changing the pathology of grafted organs [10–12]. Finally, new molecular mechanisms to explain early dysfunction or late graft loss may eventually become diagnostic tools. This book aims to present a thorough account of the pathology of solid organ transplantation in the down of the twenty-first century. The book is organized in a detailed practical diagnostic approach which we hope the reader will find didactic and clear. Molecular studies are discussed when relevant to diagnosis. Introductory chapters are written by our clinical colleagues who describe the immune response from their perspective on treatment and management issues. A chapter on xenotransplantation and

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viiiPrefa Table 1  Human transplantation: a brief account 1902

Alexis Carrel

Technique to join blood vessels

1943

Willem J. Kolf

First artificial kidney

1944

Peter Medawar

Rejection is an immune-related phenomenon

1954

Joseph Murray

First kidney transplant in identical twins

1958

Georges Mathé

First bone marrow transplantation

1960

Renè Kuss

First living related ABO compatible kidney transplant

1966

William D. Keyy Richard Lillehei

First kidney/pancreas transplant

1967

Christian Barnard

First heart transplant

1967

Thomas Starzl

First liver transplant

1983 1986

Joel Cooper The Southeast Organ Procurement foundation (SEOPF) is formed

First successful lung transplant Scientific organization for transplant professionals Predecessor of the “United Network for Organ Sharing “UNOS”

1981

Bruce Reitz Norman Shumway

First heart–lung transplant

1983

Santoz Ltd. (Basel)

Cyclosporine A was FDA approved

1984

NOTA

National Organ Transplant Act establishes national system of matching donors to recipients UNOS separates from SEOPF

1986

Alexander Patterson

First bilateral lung transplant

1987

Pittsburgh surgeons team

First successful intestinal transplant

1982

William C. DeVries

First artificial heart transplant

1989

Fujisawa

Tacrolimus was introduced

1991

Ray V. Rajotte

Islet transplantation with insulin independence

1997

Antonio Secchi

Islet transplantation with insulin independence

1998

Jean Michel Dubernard

First hand transplant

2005

Duvauchelle, Dubernard

First face transplantation

2007

Transplant Growth and Management Collaborative Group

Assessment of nationwide transplant center capacity

organogenesis is a forecast for possible solutions in organ transplantation and one that will, if successful, may change the field and patient care. We would like to thank with gratitude all our colleagues who contributed their invaluable time and experience. We hope that the book will be useful to our colleagues, also in countries around the world, where transplantation is becoming increasingly more available and frequently the only organ replacement modality within financial reach. St. Louis, MO, USA

Helen Liapis Hanlin L.Wang

Prefa

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References   1. Groth, C.G., Longmire Jr., W.P. (eds.): Historical Landmarks in Clinical Transplantation. Springer, New York (2000)   2. Cinqualbre, J., Kahan, B.D., Küss, R.: Fifty years of retroperitoneal placement of renal transplants. Transplant Proc. 34, 3019–3025 (2002)   3. Cooper, J.D.: The history of surgical procedures for emphysema. Ann Thorac Surg. 63, 312–319 (1997)   4. Murray, J.E., Merrill, J.P., Harrison, J.H., Wilson, R.E., Dammin, G.J.: Prolonged survival of human-kidney homografts by immunosuppressive drug therapy. N Engl J Med. 268, 1315–1323 (1963)   5. Stähelin, H.F.: The history of cyclosporin A (Sandimmune) revisited: another point of view. Experientia 52, 5–13 (1996)   6. Starzl, T.E.: Liver transplantation. Gastroenterology. 112, 288–291 (1997)   7. Sung, R.S., Galloway, J., Tuttle-Newhall, J.E., Mone, T., Laeng, R., Freise, C.E., Rao, P.S.: Organ donation and utilization in the United States, 1997–2006. Am J Transplant. 8(4 Pt 2), 922–934 (2008)   8. Glassock, R.J., Feldman, D., Reynolds, E.S., Dammin, G.J., Merrill, J.P.: Human renal isografts: a clinical and pathologic analysis. Medicine (Baltimore). 47, 411–454 (1968)   9. Kincaid-Smith, P.: Histological diagnosis of rejection of renal homografts in man. Lancet. 21(7521), 849–852 (1968) 10. Atala, A.: Advances in tissue and organ replacement. Curr Stem Res Ther. 3, 21–31 (2008) 11. Kawai, T., Cosimi, A.B., Spitzer, T.R., et al.: HLA-mismatched renal transplantation without maintenance immunosuppression. N Engl J Med. 358, 353–361 (2008) 12. Vincenti, F., Kirk, A.D.: What’s next in the pipeline. Am J Transplant. 8, 1972–1981 (2008)

Contents

Part I Immunology, Clinical, and Laboratory Aspects of Organ Transplantation   1 Immunology of Organ Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . Sevgi Gurkan, Bernd Schröppel, and Barbara Murphy

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  2 Current Concepts of Immunosuppression and Side Effects . . . . . . . . . Anand Khurana and Daniel C. Brennan

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  3 Clinical Aspects of Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rouba Ghoussoub and Daniel C. Brennan

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  4 Clinical Evaluation of Alloantibodies in Solid Organ Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gerald P. Morris and T. Mohanakumar   5 Frontiers in Organ Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marc R. Hammerman

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Part II  Transplant Pathology of Organ Systems   6 Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Helen Liapis, Matthew J. Koch, and Michael Mengel

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  7 Lung . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Anja C. Roden and Henry D. Tazelaar   8 Liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199 Hanlin L. Wang, Christopher D. Anderson, Sean Glasgow, William C. Chapman, Jeffrey S. Crippin, Mathew Augustine, Robert A. Anders, and Andres Roma



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  9 Heart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315 Dylan V. Miller, Hannah Krigman, and Charles Canter 10 Small Intestine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347 Frances V. White and Sarangarajan Ranganathan 11 Pancreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371 Raghava M. Munivenkatappa, John C. Papadimitriou, and Cinthia B. Drachenberg 12 Vascularized Composite Allotransplantation . . . . . . . . . . . . . . . . . . . . . 393 Linda C. Cendales, Jean Kanitakis, and Carolyn Burns Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 401

Contents

Part Immunology, Clinical, and Laboratory Aspects of Organ Transplantation

I

1

Immunology of Organ Transplantation Sevgi Gurkan, Bernd Schröppel, and Barbara Murphy

1.1 Introduction Transplantation has become the treatment of choice for end stage kidney, liver, heart, and lung failure and is being explored as therapy for failure of a variety of other organs. The major hurdle to successful transplantation is immune-mediated rejection, a process that has been partially prevented through the use of potent immunosuppressive medications. The goal of this chapter is to summarize the immune response induced to a transplanted organ so as to provide a foundation upon which to base rational clinical decision-making aimed at prolonging graft survival. Knowledge of the basis of transplant immunology is necessary for the clinician to diagnose and treat immunological complications after transplantation.

1.2 Basic Transplantation Immunology 1.2.1 Components of the Immune System The human immune system consists of two major components, the innate and the adaptive components that have evolved to be complementary as there are important interactions between the two systems [1]. The innate immune system, also called the native or

natural immunity, mediates the initial and rapid immune response directed against microbes or cells that have been damaged by microbes. The innate response is initiated following recognition of molecules whose expression is shared by groups of microbes, referred to as pathogen associated molecular patterns (PAMPs). The components of the innate immune system include natural barriers such as skin, mucosal epithelia, cells which express receptors which recognize PAMPs (e.g., macrophages, neutrophils, natural killer cells, and eosinophils), and a series of nonpolymorphic proteins (e.g., defensins, cytokines, toll-like receptors (TLR), and complement). The adaptive immune system, also referred to as antigen specific or acquired immunity, is triggered when T or B cells recognize molecules called antigens initiating cellular and antibody mediated immune mechanisms [1, 17]. Antigens may be proteins, lipids or polysaccharides, produced by infectious and noninfectious pathogens. The adaptive immune system is distinguished from the innate immune response by virtue of its antigen specificity, the ability to clonally expand and to mount a memory response [1]. CD4+ and CD8+ T cells originating from the thymus mediate the cellular immune response, while humoral immunity is comprised of bone marrow derived B cells and antibodies secreted by mature B cells, called plasma cells [1].

1.3 The Adaptive Alloimmune Response S. Gurkan, B. Schröppel, and B. Murphy (*) Department of Medicine, Division of Nephrology, Mount Sinai School of Medicine, BOX 1243, One Gustave L. Levy Place, New York, NY 10029, USA e-mail: [email protected]

The alloimmune response is a well orchestrated reaction that can be divided into four major phases: recognition, activation with clonal expansion, recruitment of other effector mechanisms, and ultimately resolution.

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_1, © Springer-Verlag Berlin Heidelberg 2011

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1.3.1 Recognition of Alloantigen

Signal 1 TCR

MHC

In humans, the recognition of self vs. non-self occurs as a result of the recognition of a repertoire of major histocompatibility antigens expressed on the cell surface, called human leukocyte antigens (HLA). There are two classes of MHC molecules and both of which are encoded by the major histocompatibility complex (MHC) gene located on the short arm of chromosome 6. Class I molecules are heterodimers composed of a single transmembrane polypeptide chain (a chain) and a b2 microglobulin, and class II molecules consist of homologous peptides (a and b chains) [19, 20]. These highly polymorphic proteins form peptide binding grooves and are capable of binding a different array of peptides derived from foreign proteins for presentation to T cells. Class I HLA molecules (HLA A, B, C) are expressed on all nucleated cells and present endogenous peptides derived from within the cell itself, identifying that cell as an infected or damaged cell that is subsequently killed by the CD8+ T cell [1, 17]. In contrast to class I, the expression of class II HLA molecules (HLA DR, DP, DQ) is restricted to specialized antigen presenting cells (APC) (e.g., macrophages, dendritic cells, and B cells) but can also be induced on other cell types (e.g., renal tubular cells) in the context of inflammation. Class II molecules bind peptides derived from proteins that have been taken up from the environment by the cell and present to CD4+ T cells thereby alerting the immune system to a pathogen in the vicinity, augmenting the inflammatory and immune response [1, 17].

1.3.2 Cellular Alloimmunity Following the recognition of MHC by the T cell receptor (signal 1), activation occurs in the presence of a costimulatory signal (signal 2), which is necessary to lower the activation threshold of the T cell [8]. In the absence of the costimulatory signal the cell may undergo apoptosis (deletion) or become unresponsive to future encounters with antigen (anergy) (Fig.  1.1). The best characterized costimulatory molecules are the membrane-bound molecules CD28 and CD154 and their APC-expressed ligands CD80/86(B7-1/B7-2) and CD40, respectively [7, 35]. If T cell receives both

APC

T cell CD80/86

ACTIVATION

CD28

Signal 2 Signal 1 MHC

ANERGY

TCR

APC

T cell CD80/86

CD28

Signal 2

APOPTOSIS

Fig. 1.1  Costimulation. Full T cell activation requires two signals, the initial interaction of the T cell receptor with the MHC molecule and its associated peptide (Signal 1), and a second costimulatory signal of which the best characterized is the interaction of CD28 on the T cells with CD80/86 on the APC (Signal 2)

signals, a number of intracellular activation steps ensue. An increase in intracellular calcium activates the calcium/calmodulin sensitive molecule, calcineurin. Activated calcineurin dephosphorylates the transcription activating factor NF-AT (nuclear factor of activated T cells) allowing its translocation into the nuclease where it binds to the IL-2 promoter resulting in the transcription and translation of interleukin (IL)-2. IL-2 then binds to its receptor on the cell surface, comprised of three subunits one of which is the a subunit (CD25) [17]. Signaling through CD25 initiates a kinase-dependent cascade mediated in part through a protein called mammalian target of rapamycin (mTOR) causing the progression of cell from the G1 to S phase of the cell cycle, resulting in proliferation and differentiation. Once the T cell has been activated, it stimulates other T cells and B cells to mount an immune response. The cellular arm of alloimmunity starts with the recognition of graft antigens in secondary lymphoid organs. Primed T cells migrate back to the allograft where they re-encounter antigen and mediate their effector functions [22]. Recognition of alloantigen by T cells occurs via two distinct pathways: the direct and the indirect pathways (Fig.  1.2) [15]. Recognition of intact MHC on donor APCs by the recipient T cells is referred to as the direct (unique to transplantation), while recognition of donor alloantigen presented in the form of peptides by recipient MHC on recipient APC is referred to the indirect pathway. This latter pathway is the way in which nominal antigens from bacteria or viruses are recognized [32]. Over time donor APCs

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a

b

DIRECT ALLORECOGNITION

INDIRECT ALLORECOGNITION

Recipient CD4 or CD8 T Cell

Target Cell Gz

CTL Gz Pf

Gz Pf

Gz

Recipient CD4 T Cell FasL

MHC

Fas

Secretory Perforin (Pf) + Granzyme B (GZ) Mediated

Non-Secretory Receptor Mediated

Allopeptide

Peptide TCR

Donor APC

Recipient APC

Fig. 1.2  Direct and indirect T cell allorecognition. Alloreactive T cells recognize transplant antigens through two distinct pathways. (a) Direct: Recipient CD4+ and CD8+ T cells recognize and respond to donor MHC: donor peptide complexes on graftderived cells. (b) Indirect: Recipient APCs can endocytose donor APCs and or donor proteins, and process and present these peptides on the cell surface in the context of recipient HLA to T cells. The majority of indirectly presented peptides derive from donor HLA polymorphisms

migrate out of the allograft and are replaced by infiltrating recipient APCs, thus indirect recognition emerges as the dominant effector pathway directed against the allograft [4, 34]. In addition to MHCantigens, graft-reactive T cells recognize minor histocompatibility (mH) antigens. Minor transplantation antigens are donor-derived peptide determinants that can serve as targets of rejection even when the MHC is identical between donor and recipient. Once the cellular arm of the alloimmune response is activated graft injury is mainly mediated by CD8+ or cytotoxic T lymphocytes (CTLs) and macrophages. Antigen specific CTLs kill their targets through direct cell–cell contact by either a secretory pathway causing cell lysis/apoptosis, or ligand induced apoptosis (programmed cell-death) mediated by the Fas ligand (FasL)/ Fas pathway (Fig. 1.3). Macrophages kill their targets in an antigen non-specific manner by phagocytosis, or by release of soluble factors such as reactive oxygen intermediates, proteases, and tumor necrosis factor.

1.3.3 Humoral Alloimmunity The humoral arm of the alloimmune response starts with the recognition of allogeneic-HLA molecules

Apoptosis

Fig. 1.3  Cytotoxic T cells (CTLs). Recognition of allo-MHC on the surface of donor APCs by CTLs results in the direct killing of the cell through a ligand or secretory pathway. The secretory pathway in which perforin and granzyme are release and induce apoptosis is the predominant mechanism

through surface bound IgM receptors of the alloreactive B cells. Costimulatory signals, in particular the CD40/CD154 pathway, are required for full activation and differentiation of alloreactive B cells into antibody secreting plasma cells [9, 10]. Donor-specific antiHLA antibodies (DSA) produced by the B cells participate in graft injury by activation of the complement cascade via the classical pathway. Some complement activation by-products (i.e., C3a, C5a) also act as chemoattractants for inflammatory cells. C4d, another complement split product has now become an important indicator of complement activation and hence a tool in the determination of antibody-mediated rejection on kidney allograft biopsy [1]. DSAs may also be present prior to a transplant due to sensitization (e.g., through a failed allograft, blood transfusion, or pregnancy), or may have developed secondary to crossreactivity between an HLA molecule and an environmental antigen with a similar structure [3, 14, 42]. The presence of DSAs prior to a transplant can predispose the patient to hyperacute rejection. Alloantibodies can also develop post-transplantation and these de novo alloantibodies have been associated with acute and chronic graft injury [43]. Anti-donor humoral immune responses also target blood group antigens. With the exception of infants, who have yet to develop natural antibodies, or patients that have undergone desensitization protocols, transplantation across ABO differences is generally precluded due to complement-mediated hyperacute rejection [47].

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1.3.4 Resolution of the Alloimmune Response Termination of the immune response is an essential protective mechanism that is mediated by activationinduced apoptosis of T cells and negative regulation by costimulatory molecules. An expansive list of costimulatory molecules has now been identified, some of which are stimulatory while others down-regulate the immune response (Table  1.1). The relative contribution of these molecules finely orchestrates the intensity of the immune response. A dominance of negative “costimulation” helps terminate the alloimmune response. These pathways are now being investigated as a means to manipulate the response to an allograft. Generation of memory T cells follows the effector response. Memory T cells can rapidly respond to a reencounter with the same antigen due to a lower/different costimulatory requirements and lower activation thresholds as compared to naïve cells [24, 33, 45]. Memory cells are beneficial for the prevention of reinfection; however, secondary to their resistance to the effects of immunosuppressive agents they form a formidable barrier in the setting of transplantation [31]. Memory T cells directed toward pathogens following previous exposure can also cross-react with alloantigen leading to graft damage, this is referred to as heterologous immunity [46]. Resolution of the effector response is achieved in part by a subgroup of CD4+ T cells, so called

Table 1.1  T cell costimulatory pathways Molecule Ligand

regulatory T cells (Tregs). Tregs recognize antigen in a similar fashion as the other T cells but after their initial interaction with the antigen, they produce inhibitory cytokines (i.e., TGFb, IL-10) or express surface molecules (e.g., CTLA-4) that are able to inhibit the effector response. This mechanism not only facilitates the termination of the immune response once it is no longer necessary but also controls tolerance to self [17].

1.3.5 Transplant Tolerance Tolerance to the graft can be defined as the absence of detrimental immune response directed at the transplanted organ and indefinite graft survival with normal graft function in an immunocompetent host in the absence of immunosuppression. The mechanisms mediating tolerance include central (thymic negative selection), and peripheral. There are several potential mechanisms by which peripheral tolerance can be mediate, including elimination of the donor-reactive immune cells (apoptosis), immunologic ignorance (failure of the immune system to see the transplant antigens), induction of anergy (non-responsiveness), and active inhibition by regulatory/suppressor T cells (Tregs) [2, 18, 21, 26, 40, 41]. Multiple studies are investigating the mechanisms which may successfully be used to prolong graft survival and potentially induce allograft tolerance [27].

Signal

Effect

CD28:B7 superfamily CD28

CD80 or CD86

+

Activation of naïve T cells

CTLA4

CD80 or CD86

−

Resolution of an active response

PD-1

PD-1L or PD-2L

−

Resolution of an active response

ICOS

ICOS-L

+

Induction of TH2 immunity and reactivation of memory cells

CD40

CD154 (CD40L)

+

CD134 (OX-40)

CD134L(OX-40L)

+

Activation of naïve T cells and induction of antibody isotype switching in B cells Reactivation of effector and or memory cells

TNF-TNF-Receptor superfamily

CD27 CD70 + Reactivation of effector and/or memory cells CTLA4 cytotoxic T-lymphocyte-associated protein; ICOS inducible costimulatory; L ligand; PD programmed death; Th T-helper; TNF tumor necrosis factor

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1.4 Organ Specific Effects and Clinical Applications of Transplant Immunology The alloimmune response differs according to the type of organ transplanted. For example, the greatest beneficial effect of HLA matching is seen in bone marrow and kidney transplantation, where efforts are made to match at the HLA-A, B and DR loci. The avoidance of HLA mismatches improves kidney graft survival and reduces the incidence of acute rejection, with mismatches to HLA-DR having the strongest impact followed by the B and then A locus [12, 13, 30, 42]. The influence of HLA in the allocation of donor hearts is overridden by other factors such as the desire to minimize cold ischemia time, clinical urgency, and the requirements for size and age matching bet­ ween the donor and the recipient. Similar to the kidney, HLA-DR has the greatest impact on acute cellular rejection and overall heart allograft outcome [29, 38, 39]. The influence of HLA match in lung transplantation is controversial. As with heart transplantation, donated lungs are allocated without consideration of HLA compatibility. Recent data suggest that HLA mismatches (in particular HLA-DR and B) influence acute rejection rates and increase the risk for the development of bronchiolitis obliterans syndrome, one of the most significant post-transplant obstacles to long-term graft function [6, 36, 37, 44]. Liver allografts are believed to be more tolerant to HLA mismatches and ABO blood group incompatibilities than kidney, heart and lung transplants. However, recent data suggest that HLA-mismatches are associated with an increased risk of acute rejection and poor allograft survival, whereas ABO incompatibilities pose a higher risk of chronic rejection [11]. Small bowel is extremely vulnerable to rejection and a source for graft versus host disease (GvHD) secondary to its rich lymphatic tissue. The factors that affect long-term survival are still not well known. Current practice only requires the recipient and donor to have ABO compatibility, since incompatibility is associated with both GvHD and acute rejection [16, 28]. Pancreas transplantation is most commonly performed simultaneously with a kidney transplant or after a successful kidney transplant. The pancreas is considered a highly immunogenic organ and similar to the kidney, poor HLA matching is associated with an increased risk of

acute rejections and thus may impact long-term graft survival [25]. Sera from prospective transplant recipients (in the case of kidney) are routinely screened for the presence of HLA antibodies against a panel cells, representing most antigens encountered in the general population. Using a form of complement-dependent-cytotoxicity assay (CDC) the results are reported as PRA (the percent of panel cells that are killed by reacting with the HLA antibodies in patient’s serum). In order to specifically determine anti-HLA antibodies, solid-phase assays, enzyme-linked immunosorbent assays (ELISA) or flow cytometry bead based assays (Flow Specific Beads™ and FlowPRA™, which are purified HLA antigens coupled to microparticles) have been developed [5]. A positive T cell CDC cross-match is an absolute contraindication for kidney transplantation. The more sensitive but less specific flow cytometry cross-match (FCXM) detects DSAs independent of complement fixation. This test does not discriminate between cytotoxic and non-cytotoxic antibodies and to confirm the specificity of positive FCXM, specific solid phase assays need to be done. The clinical significance of CDC B cell or flow cytometry T or B cell positive cross-matches was previously considered controversial; however, there are now data to support their impact of short and long-term graft survival [14]. A positive B cell CDC or B cell FCXM may indicate anti-class II, weak anti-class I, or anti-immunoglobulin antibodies, which are abundant on B cells [23].

References   1. Abbas, A.K., Lichtman, A.H., Pillai, S.: Introduction to the immune system. William R Schmitt (ed) In: Cellular and Molecular Immunology, 6th ed. Saunders/Elsevier, Philadelphia (2007)   2. Ansari, M.J., Sayegh, M.H.: Clinical transplantation tolerance: the promise and challenges. Kidney Int. 65, 1560–1563 (2004)   3. Baid, S., Saidman, S.L., Tolkoff-Rubin, N., et al.: Managing the highly sensitized transplant recipient and B cell tolerance. Curr. Opin. Immunol. 13, 577–581 (2001)   4. Banasik, M., Klinger, M.: Chronic allograft nephropathy– immunologic and nonimmunologic factors. Ann. Transplant. 11, 7–10 (2006)   5. Bray, R.A., Nickerson, P.W., Kerman, R.H., et al.: Evolution of HLA antibody detection: technology emulating biology. Immunol. Res. 29, 41–54 (2004)   6. Chalermskulrat, W., Neuringer, I.P., Schmitz, J.L., et  al.: Human leukocyte antigen mismatches predispose to the

8 severity of bronchiolitis obliterans syndrome after lung transplantation. Chest 123(6), 1825–1831 (2003)   7. Clarkson, M.R., Sayegh, M.H.: T-cell costimulatory pathways in allograft rejection and tolerance. Transplantation 15, 555–563 (2005)   8. Croft, M., Dubey, C.: Accessory molecule and costimulation requirements for CD4 T cell response. Crit. Rev. Immunol. 17, 89–118 (1997)   9. Delves, P.J., Roitt, I.M.: The immune system. first of two parts. N Engl J. Med. 6, 37–49 (2000) 10. Delves, P.J., Roitt, I.M.: The immune system. second of two parts. N Engl J. Med. 13, 108–117 (2000) 11. Doran, T.J., Geczy, A.F., Painter, D., et al.: A large, single center investigation of the immunogenetic factors affecting liver transplantation. Transplantation 15, 1491–1498 (2000) 12. Dyer, P.A., Johnson, R.W., Martin, S., et al.: Evidence that matching for HLA antigens significantly increases transplant survival in 1001 renal transplants performed in the northwest region of England. Transplantation 48, 131–135 (1989) 13. Festenstein, H., Doyle, P., Holmes, J.: Long-term follow-up in london transplant group recipients of cadaver renal allo­ grafts. the influence of HLA matching on transplant outcome. N Engl J. Med. 2, 7–14 (1986) 14. Gebel, H.M., Bray, R.A., Nickerson, P.: Pre-transplant assessment of donor-reactive, HLA-specific antibodies in renal transplantation: contraindication vs. risk. Am. J. Transplant. 3, 1488–1500 (2003) 15. Gould, D.S., Auchincloss, H.: Direct and indirect recognition: the role of MHC antigens in graft rejection. Immunol. Today 20, 77–82 (1999) 16. Gundlach, M., Schmidt, P., Hell, K., et al.: The influence of major histocompatibility complex subloci differences on graft rejection in small-bowel transplantation. Transplant. Proc. 22, 2474–2475 (1990) 17. Hale, D.A.: Basic transplantation immunology. Surg. Clin. N. Am. 86, 1103–1125 (2006). v 18. Izcue, A., Coombes, J.L., Powrie, F.: Regulatory T cells suppress systemic and mucosal immune activation to control intestinal inflammation. Immunol. Rev. 212, 256–271 (2006) 19. Klein, J., Sato, A.: The HLA system. first of two parts. N Engl J. Med. 7, 702–709 (2000) 20. Klein, J., Sato, A.: The HLA system. second of two parts. N Engl J. Med. 14, 782–786 (2000) 21. Lakkis, F.G.: Transplantation tolerance: a journey from ignorance to memory. Nephrol. Dial. Transplant. 18, 1979–1982 (2003) 22. Lakkis, F.G., Arakelov, A., Konieczny, B.T., et  al.: Immunologic ‘ignorance’ of vascularized organ transplants in the absence of secondary lymphoid tissue. Nat. Med. 6, 686–688 (2000) 23. Le Bas-Bernardet, S., Hourmant, M., Valentin, N., et  al.: Identification of the antibodies involved in B-cell crossmatch positivity in renal transplantation. Transplantation 27, 477–482 (2003) 24. London, C.A., Lodge, M.P., Abbas, A.K.: Functional responses and costimulator dependence of memory CD4+ T cells. J. Immunol. 1, 265–272 (2000)

S. Gurkan et al. 25. Malaise, J., Berney, T., Morel, P., et al.; EUROSPK Study Group: Effect of HLA matching in simultaneous pancreaskidney transplantation. Transplant. Proc. 37, 2846–2847 (2005) 26. Mezrich, J.D., Benjamin, L.C., Sachs, J.A., et al.: Role of the thymus and kidney graft in the maintenance of tolerance to heart grafts in miniature swine. Transplantation 27, 1663–1673 (2005) 27. Newell, K.A., Larsen, C.P.: Toward transplantation tolerance: a large step on a long road. Am. J. Transplant. 6, 1989–1990 (2006) 28. Niv, Y., Mor, E., Tzakis, A.G.: Small bowel transplantation–a clinical review. Am. J. Gastroenterol. 94, 3126–3130 (1999) 29. Opelz, G., Wujciak, T.: The influence of HLA compatibility on graft survival after heart transplantation. the collaborative transplant study. N Engl J. Med. 24, 816–819 (1994) 30. Opelz, G., Wujciak, T., Dohler, B., et al.: HLA compatibility and organ transplant survival. Collaborative transplant study. Rev. Immunogenet. 1, 334–342 (1999) 31. Pearl, J.P., Parris, J., Hale, D.A., et al.: Immunocompetent T-cells with a memory-like phenotype are the dominant cell type following antibody-mediated T-cell depletion. Am. J. Transplant. 5, 465–474 (2005) 32. Pietra, B.A., Wiseman, A., Bolwerk, A., et al.: CD4 T cellmediated cardiac allograft rejection requires donor but not host MHC class II. J. Clin. Invest. 106, 1003–1010 (2000) 33. Pihlgren, M., Dubois, P.M., Tomkowiak, M., et al.: Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro. J. Exp. Med. 1, 2141–2151 (1996) 34. Saiki, T., Ezaki, T., Ogawa, M., et al.: In vivo roles of donor and host dendritic cells in allogeneic immune response: cluster formation with host proliferating T cells. J. Leukoc. Biol. 69, 705–712 (2001) 35. Sayegh, M.H., Turka, L.A.: The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J. Med. 18, 1813–1821 (1998) 36. Schulman, L.L., Weinberg, A.D., McGregor, C., et  al.: Mismatches at the HLA-DR and HLA-B loci are risk factors for acute rejection after lung transplantation. Am. J. Respir. Crit. Care Med. 157, 1833–1837 (1998) 37. Schulman, L.L., Weinberg, A.D., McGregor, C.C., et  al.: Influence of donor and recipient HLA locus mismatching on development of obliterative bronchiolitis after lung transplantation. Am. J. Respir. Crit. Care Med. 163, 437–442 (2001) 38. Sheldon, S., Yonan, N.A., Aziz, T.N., et al.: The influence of histocompatibility on graft rejection and graft survival within a single center population of heart transplant recipients. Transplantation 27, 515–519 (1999) 39. Smith, J.D., Rose, M.L., Pomerance, A., et al.: Reduction of cellular rejection and increase in longer-term survival after heart transplantation after HLA-DR matching. Lancet 18, 1318–1322 (1995) 40. Starzl, T.E., Murase, N., Abu-Elmagd, K., et al.: Tolerogenic immunosuppression for organ transplantation. Lancet 3, 1502–1510 (2003) 41. Starzl, T.E., Zinkernagel, R.M.: Transplantation tolerance from a historical perspective. Nat. Rev. Immunol. 1, 233–239 (2001)

1  Immunology of Organ Transplantation 42. Sumitran-Holgersson, S.: HLA-specific alloantibodies and renal graft outcome. Nephrol. Dial. Transplant. 16, 897–904 (2001) 43. Terasaki, P.I., Ozawa, M.: Predicting kidney graft failure by HLA antibodies: a prospective trial. Am. J. Transplant. 4, 438–443 (2004) 44. van den Berg, J.W., Hepkema, B.G., Geertsma, A., et  al.: Long-term outcome of lung transplantation is predicted by the number of HLA-DR mismatches. Transplantation 15, 368–373 (2001)

9 45. Viola, A., Lanzavecchia, A.: T cell activation determined by T cell receptor number and tunable thresholds. Science 5, 104–106 (1996) 46. Welsh, R.M., Selin, L.K.: No one is naive: the significance of heterologous T-cell immunity. Nat. Rev. Immunol. 2, 417–426 (2002) 47. West, L.J., Pollock-Barziv, S.M., Dipchand, A.I., et  al.: ABO-incompatible heart transplantation in infants. N Engl J. Med. 15, 793–800 (2001)

2

Current Concepts of Immunosuppression and Side Effects Anand Khurana and Daniel C. Brennan

2.1 Introduction The first successful human solid organ transplant was a renal transplant between two identical twin siblings, on 23 Dec 1954 [103]. Given the monozygosity, essentially no immunosuppression was used. The recipient never had a rejection episode but died 8 years later from recurrent glomerulonephritis. The introduction of immunosuppression with prednisone, azathioprine, and occasionally antilymphocyte globulin (ALG) in the 1960s allowed successful nonidentical living donor and deceased donor transplants. Through the 1970s and early 1980s, 1-year survival rates and acute rejection rates were around 60%. In the mid1980s, cyclosporine was introduced and rejection rates decreased to 40–50% and 1-year survival rates increased to 75–85%. In the last 2 decades with the introduction of newer immunosuppressive induction agents such as basiliximab, daclizumab, and thymoglobulin and maintenance agents including tacrolimus, mycophenolate, and sirolimus, transplant patients are able to achieve 1 year graft survival rates in excess of 90% and acute rejection rates of 5–20%. Over the last several years, the focus of even newer immunosuppressive drugs regimens has included immunosuppression targeting the co-stimulatory pathways and avoiding toxicities associated with steroids and the calcineurin inhibitors cyclosporine and tacrolimus.

A. Khurana and D.C. Brennan (*) Washington University in St. Louis, 4104 Queeny Tower, One Barnes-Jewish Hospital Plaza, St. Louis, MO 63110, USA e-mail: [email protected]

Long-term allograft survival depends on controlling the allo-immune response and preventing toxicity. The allo-immune response is most intense after the placement of the allograft and initially requires broad and high levels of immunosuppression targeting multiple pathways to minimize the risk of rejection. These pathways have been reviewed previously (Chap. 1). In general solid organ transplant immunosuppression is divided into an induction phase and a maintenance phase of immunosuppression. For the purpose of this review “induction agents” will refer to those drugs used only during the initial few days or weeks after transplantation and usually refers to the use of lymphocyte depleting or lymphocyte targeted therapy. Maintenance immunosuppressive medications are often similar to those that are used during the induction phase but at lower doses when the recipient requires less immunosuppression to prevent rejection. Both induction and maintenance agents may be associated with side effects and allograft pathology.

2.2 Induction Drugs Induction agents were used in less than 10% of renal transplants during most of the 1980s and mid 1990s and typically used for those recipients perceived to be at increased risk for rejection. Agents used during this period in the US were equine Minnesota antilymphocyte globulin (MALG), equine antithymocyte globulin (ATGAM), or monomuromab (OKT3), a mouse antihuman monoclonal agent that targets the CD3-complex. The use of induction agents has increased over the last decade [89]. As of 2003, approximately 70% of

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_2, © Springer-Verlag Berlin Heidelberg 2011

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patients with renal transplant received induction immunosuppression therapy [68]. Of these, approximately 35% received rabbit ATG (thymoglobulin); 20% received basiliximab 15% received daclizumab; 4% received alemtuzumab (Campath), and OKT3 or ATGAM were used in <1%. A recent multivariate analysis showed that use of induction therapy over the last several years was associated with a reduced risk of rejection of 26% in deceased donors and 13% in living donor transplantation [20].

2.3 OKT 3 OKT 3 was the first agent approved by the FDA for induction therapy. It was approved in 1986 after decreased acute rejection rates were noted in comparison to no induction therapy.

A. Khurana and D.C. Brennan

because of it various side effects that have been des­ cribed below. Its main role in transplantation is now restricted to the treatment of steroid resistant allograft rejections. Dosage OKT3 is given as a dose of 5 mg intravenously, daily and peripherally for 7–14 days. Its efficacy on re-use is diminished because of the formation of antimurine antibodies which neutralize its effect. About 45–50% patients exposed to OKT3 develop anti OKT3 antibodies [56, 154]. These may be anti-isotypic or anti-idiotypic. Both of these antibodies differ in their capacity to neutralize the therapeutic effect of OKT3. Anti-idiotypic antibodies, which are directed toward the variable portion of OKT3, are more likely to limit efficacy than anti-isotypic antibodies, which are directed toward the murine component of the antibody [29]. In patients that do not develop anti-idiotypic antibodies, OKT3 can be used for retreatment.

2.3.3 Side Effects 2.3.1 Mechanism of Action OKT 3 is a murine derived monoclonal antibody that targets the epsilon subunit of the CD3 complex. It causes an initial period of activation that is followed by subsequent inactivation of the T lymphocyte [68]. This initial period of activation causes a massive cytokine release which is responsible for the first dose effect associated with OKT3. T cells subsequently become ineffectual and are eventually opsonized and removed from the circulation. T cells usually appear in the circulation in about 3–5 days but lack CD3 and are immunologically incompetent [22]. The subsequent T cell paralysis helps prevent and treat acute rejection episodes.

The initial cytokine release is responsible for a major and sometimes life threatening “first dose effect” of this medication manifests as a flu-like syndrome with fever, tachycardia, diarrhea, nausea, myalgia and pulmonary edema and hypotension [68]. The package insert recommends that patients should be euvolemic prior to OKT3 to avoid serious pulmonary edema; however, this predisposes to acute tubular necrosis (ATN) (Fig. 2.1). Other

2.3.2 Efficacy OKT3 was initially used as an induction agent in renal transplantation. Its efficacy as an induction agent was highlighted by fewer rejection episodes and a longer time to initial rejection in comparison to placebo, along with maintenance regimen of prednisone, azathioprine and cyclosporine [3, 109]. The use of OKT3 as an induction agent has declined in the past few years

Fig. 2.1  Acute tubular necrosis. Blue Arrows show epithelial cell detachment from the tubular basement membrane

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2  Current Concepts of Immunosuppression and Side Effects

2.4.1 Mechanism IL-2 receptor antibodies bind to the alpha subunit of the IL-2R (CD25) which activates the intracellular kinases that promote T cell proliferation. The alpha subunit is expressed only on activated T cells, which are rare at the time of transplantation. Resting T cells do not express CD25 and are unaffected by IL-2R antagonists [110]. Basiliximab also impairs IL15 signaling through down regulation of the IL2g/ IL15 receptor b chain [10, 27].

Fig.  2.2  Cyclosporine induced thrombotic microangiopathy. Black Arrow shows an arteriole occluded by fibrin deposition which is also apparent in the intraglomerular capillaries

side effects include nephrotoxicity which manifests as allograft thrombosis not only within the arteries and veins but also the glomerular capillaries with histology similar to thrombotic microangiopathy [4, 122] (Fig. 2.2). OKT3 can also be associated with neurotoxicity which manifests as aseptic meningitis, seizures and rarely akinetic mutism [117]. There is also an increased incidence of cytomegalovirus (CMV) infections after OKT3 therapy (see Chap. 3) and B cell lymphoma or post-transplant lymphoproliferative disease (PTLD). This is especially true among patients who have received multiple courses of OKT3 [21, 111]. Among all the induction agents currently in use, OKT3 carries the highest relative risk of PTLD [30]. In patients with liver transplantation, the use of OKT3 has been associated with early and severe recurrence of Hepatitis C [128].

2.4 IL-2 Receptor Antagonists (Anti CD25 Antibodies) There are 2 IL-2 receptor antagonists available for use in organ transplantation- basiliximab and daclizumab. They are both chimeric, murine antibodies which have been humanized to decrease immunogenicity. Basi­ liximab is 75% human and daclizumab is 90% human [146].

2.4.2 Dosage Basiliximab is the more commonly used agent of the two drugs. It is given as a dose of 20 mg at days 0 and 4 and impairs the IL-2 receptor for a mean of 4–6 weeks [27, 110, 158]. The concomitant use of antimetabolites – azathioprine and mycophenolate mofetil (MMF) prolongs the duration of IL-2R alpha saturation [27]. The humanized content results in lower immunogenicity and longer half life of basiliximab in comparison to OKT3 [110]. A study of 339 patients using basiliximab as the induction agent found the incidence of anti-idiotype antibody formation to be 1%, which is much lower than OKT3 [27]. Daclizumab is the less commonly used IL-2 receptor antibody. It has not been studied as rigorously as basiliximab and has a more prolonged regimen for induction extending up to 8 weeks post transplant. Conventionally, it is given as 1  mg/kg on the day of the transplant and 4 doses subsequently 14 days apart. This regimen causes CD25 saturation for a mean period of 59 days [158]. An abbreviated regimen of 2 doses of 2 mg/kg daclizumab has also been compared to the conventional 5 dose regimen in patients with simultaneous kidney pancreas transplantation with favorable results [145].

2.4.3 Efficacy Basiliximab has been compared to placebo in patients on maintenance therapy with cyclosporine, steroids

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with/without azathioprine. It showed a lower rate of acute rejection with no difference in terms of overall graft and patient survival [62, 107, 108, 120, 159]. It has also been compared to placebo in combination with cyclosporine, mycophenolate and prednisone based triple immunotherapy regimens [77]. There was a 42.5% reduction in the rejection rate in comparison to placebo. Although it was not statistically significant, the study was not powered to detect a statistical difference between the two groups. In more recent studies, it has been used with prednisone, tacrolimus and mycophenolate based regimens [99]. The addition of basiliximab to the triple immunotherapy regimen was associated with a lower incidence of acute rejection in comparison to placebo and allowed for a lower dose of tacrolimus, thereby decreasing the risk of nephrotoxicity. Follow-up analyses showed improved acute rejection, graft loss and death in patients in the basiliximab group at 3 years that was not sustained at 5 years [62, 108]. Similar results have been obtained in another 5 year randomized controlled trial that demonstrated no significant long term benefit with basiliximab in comparison to placebo [134]. The data on daclizumab are not as extensive as basiliximab. In regimens using prednisone and cyclosporine, daclizumab has reduced the risk of early acute rejection and improved patient survival in comparison to placebo [107]. It has been compared with ALG in one small study where it showed better graft survival [121]. The two IL-2R antagonists have been compared head to head in limited trials and have shown mixed results [84, 114]. A modified two dose regimen of dac­ lizumab was compared with the standard two dose regimen of basiliximab and found to be inferior in terms of preventing acute rejection episodes in one study [84]. However, another study found that modified two dose daclizumab was as effective as basiliximab, resulted in better renal function and was more cost effective [114]. A meta analysis of randomized trials using IL2 antagonists found that adding basiliximab to a double-drug or triple-drug therapy regimen had the same benefit as adding daclizumab in preventing acute rejection (at 6 months: basiliximab RR 0.67; CI 0.59–0.77 vs. daclizumab RR 0.66; CI 0.53–0.82) [171]. Both agents are felt to be equally efficacious despite the lack of randomized trials directly comparing the two agents.

A. Khurana and D.C. Brennan

Use of IL-2R antagonists to allow for steroid free and early steroid withdrawal has been studied in the FREEDOM and CARMEN trials. In the FREEDOM trial, the incidence of acute rejection at 3 months was 20.6% in the steroid avoidance group, 15.6% in early steroid withdrawal group and 5.9% in the steroid group [158, 165]. It was felt that 65–90% patients could be maintained on steroid free regimens using IL-2 receptor antagonists and early withdrawal of steroids is probably better than complete avoidance [165]. An initial pilot study using daclizumab induction followed by cyclosporine and mycophenolate was successful in avoiding steroids in about 65% of patients, especially with low immunologic risk [32]. In the CARMEN study group, the regimen of daclizumab/tacrolimus /mycophenolate was compared with prednisone/tacrolimus and mycophenolate. Eighty eight percent of patients in the daclizumab group were able to avoid steroids at the end of 6 months with similar rates of rejection, patient, and graft survival [129]. Basiliximab induction therapy along with tacrolimus and sirolimus therapy has enabled early withdrawal of steroid therapy (4 days post transplantation) in renal transplant patients with 79% patients staying off steroids and with 100% graft survival at the end of 1 year [173]. This study, however, excluded African American patients who generally suffer an unacceptably high rejection rate with steroid avoidance regimens [6]. Studies that have tried IL2 antagonists to enable complete avoidance of calcineurin inhibitors have been associated with unacceptable high rejection rates and this practice is not currently recommended.

2.4.4 Side Effects The IL-2R antagonists are well tolerated and the incidence of side effects in studies has been reported to be similar to the placebo. The cytokine release syndrome does not occur although hypersensitivity reactions have been reported with both initial and re-exposure to both basiliximab and daclizumab [85]. The most frequent side effect is gastrointestinal upset [27]. The incidence of bacterial and viral infections and malignancies including PTLD are similar to placebo [27, 85, 111]. There do not seem to be any directly associated histopathologic side-effects of IL-2R antagonists.

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2.5 Polyclonal Antibodies (Thymoglobulin and ATGAM) Two preparations of polyclonal antibodies are currently available in the US – thymoglobulin and ATGAM. ATGAM is manufactured from immunization of human thymocytes in horses. Thymoglobulin is derived from rabbits [85].

2.5.1 Mechanism of Action Both target multiple T-cell markers such as CD2, CD3, CD4, CD8, CD11a, CD18, CD25, CD44 and CD45, co-stimulatory and adhesion molecules on antigen presenting cells, T and B cells as well as MHC molecules and natural killer (NK) cells. The primary mechanism of action is felt to be lymphocyte-depletion [85, 136]. The duration of lymphopenia can be up to 1 year with the use of thymoglobulin while with the Equine ATG, the lymphopenia is much shorter, about 14 days [51]. Long term specific depletion of the CD4+ lymphocyte subset and the preferential generation of a CD8+, CD57+ immunomodulatory subset of T cells has been postulated to explain the long-term success of polyclonal immunosuppression [136]. Unlike the IL2 receptor antagonists, thymoglobulin leads to the generation of T-regulatory cells in  vitro [86]. Even low concentrations of ATGs can induce a near complete disappearance of lymphocyte functioning antigen (LFA-1) on monocytes, granulocytes and lymphocytes and inhibit endothelial inflammatory and adhesion molecules [67]. Thus, they may be useful in ischemia reperfusion injury. Antibody affinity to CD 45 may also be important in controlling rejection and inducing tolerance [40, 85]. Binding to CD45RB alters the CD45 isoform expression on the T cells, which is associated with upregulation of CTLA 4 expression and induction of peripheral tolerance [40].

2.5.2 Dosage The half life of thymoglobulin is 2–3 days and the usual dose is 1.5 mg/kg/day for 4–7 days. It is usually given by large central vein infusion because of its

propensity to cause phlebitis although it has also been administered peripherally with heparin and hydrocortisone without significant thrombosis [93]. Data on bolus dose thymoglobulin are accumulating [5, 59, 105, 133, 176]. Three day bolus dose thymoglobulin (3  mg/kg intraoperatively followed by 1.5  mg/kg for 2 more days) has been shown to be as effective as the standard 7 day regimen in terms of preventing acute rejection, overall graft and patient survival [5]. Pretreatment with bolus dose anti lymphocyte therapy with thymoglobulin (5  mg/kg) can facilitate alloengraftment so that minimal immunosuppression is required for maintenance therapy in most patients [133].

2.5.3 Clinical Efficacy Thymoglobulin has been shown to be more efficacious compared to ATGAM [17, 45, 85, 151]. Thymoglobulin causes a more profound depletion of lymphocytes, leads to less severe biopsy proven rejection, better event-free survival, less cytomegalovirus disease, and fewer serious adverse events [17, 45, 85, 151]. The short term effects of thymoglobulin vs. basiliximab induction therapy have been compared. In patients with high risk for delayed graft function or rejection, thymoglobulin was found to reduce the frequency of acute rejection in comparison to basiliximab [16]. There was no difference in the incidence of delayed graft function, graft loss and death between the two agents [16]. A higher incidence of bacterial and viral infections but a lower incidence of CMV disease was noted with thymoglobulin therapy [16]. In patients with low immunologic risk, thymoglobulin and basiliximab have shown comparable efficacy in terms of rates of rejection, allograft and patient survival [78, 102, 142].

2.5.4 Side Effects Because of their xenogenic origin and significant antibody dose, polyclonal antibodies may cause allergic reactions or serum sickness. The most common adverse effects are urticaria and fever, chills, and rash especially after the first dose [85]. The cytokine release syndrome, more common with OKT3, can still occur

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with the polyclonal antibodies, especially with bolus regimens. Polyclonal antibodies may cause phlebitis when administered peripherally and thus are most commonly given through a central vein to minimize the risk. Hypertension, diarrhea, and headache can be seen with therapy. Hypotension, leucopenia or thrombocytopenia may require slowing of the infusion rate or either a reduction in dose or termination of therapy. There is an increased incidence of CMV infections with polyclonal antibodies after induction or treatment for rejection with intravenous anti-lymphocyte therapy. Use of polyclonal antibodies has also been implicated in the increased incidence of PTLD [21, 111]. The use of thymoglobulin increases the risk of PTLD by about fourfold. This risk increases further if anti rejection treatment is required [111].

2.6 Alemtuzumab 2.6.1 Mechanism of Action Alemtuzumab (Campath 1H) is a monoclonal antibody directed against CD52 and approved for treatment of chronic lymphocytic leukemia (CLL) [168]. The CD 52 antigen is highly expressed on B and T cells and monocytes, macrophages, dendritic cells and NK cells and therefore alemtuzumab causes profound lymphocyte depletion [8, 124, 168]. Alemtuzumab complexes with CD52 and causes cell death through complement mediated killing or antibody dependent cellular toxicity [8]. The CD52 molecule may be involved in cell to cell adhesion and signal transduction [8]. Recent studies have shown that alemtuzumab causes activation of CD4+ regulatory T cells. These cells may suppress the polyclonal responses of both CD4+ and CD8+ T cells with polyclonal or allogeneic stimulation [8].

2.6.2 Dosage After initial success in the treatment of CLL, Alemtuzumab is now being used as an induction agent for organ transplantation. The optimal dose for renal transplantation is unknown. Although the dose for treatment of CLL is 30  mg dose three times weekly,

A. Khurana and D.C. Brennan

typical regimens for renal transplantation are 20  mg after surgery and repeated on day 1 [8, 170]. This reflects lower numbers of lymphocytes in a transplant patient compared to ten times higher numbers in patients with CLL [8].

2.6.3 Clinical Efficacy A randomized control trial compared alemtuzumab to placebo for induction therapy [170]. It found no significant difference between the two groups in terms of delayed graft function, or patient and graft survival. The acute rejection rates were not different in the two groups although it occurred later in patients treated with alemtuzumab [170]. In this trial, patients that were treated with alemtuzumab were given lower doses of cyclosporine and steroids. In a non-randomized, retrospective study with variable follow up, there was a significant reduction in the incidence of acute rejection episodes compared historically, with other induction therapies including thymoglobulin, basiliximab, daclizumab and OKT 3 [70]. There was no increase in the incidence of infections or malignancy in the alemtuzumab cohort. However, the duration of follow-up for the alemtuzumab group was about 1 year on average and 5–6 years for the other cohorts in the study. A 3 year prospective pilot study of Campath induction therapy followed by maintenance therapy with sirolimus as a single agent reported high incidence of early acute rejection, with a distinct predominance of humoral antibody mediated rejection [11].

2.6.4 Side Effects Alemtuzumab for the treatment of CLL is associated with fever, chills and rigors and flu- like syndrome which require premedication and resolves with continued use [42]. Because of prolonged lymphopenia, patients can develop opportunistic infections like CMV, herpes zoster and herpes simplex infections among others [42]. When used as an induction agent for transplant, however, there was no reported increase in incidence of infectious complications in comparison to the control group. There was no increase in the incidence of PTLD with alemtuzumab [170]. However,

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serious hematological toxicity has been reported with alemtuzumab. In 2005, the FDA issued a warning because of three cases of severe immune thrombocytopenic purpura (ITP) noted in a study on effectiveness of alemtuzumab with multiple sclerosis. Use of alemtuzumab has also been associated with development of autoimmune thyroiditis in multiple sclerosis patients and transplant recipients [32, 69].

effects besides their effect on lymphocytes. They cause stabilization of the lysosomal membranes, suppression of prostaglandin synthesis, reduction of histamine and bradykinin release and impairment of monocyte/macrophage function.

2.7 Maintenance Drugs 2.7.1 Prednisone Corticosteroids have been an important part of maintenance immunosuppression since the earliest days of transplantation. The many side effects associated with their use has led investigators to explore ways to eliminate or minimize their use recently.

2.7.1.1 Mechanism of Action Corticosteroids are available in two formulations: prednisolone and prednisone. Prednisolone is primarily used in Europe and prednisone is used in North and South America. Prednisone is metabolized in the liver to prednisolone which is the active compound [149]. The bioavailability of prednisone is 80% of that of prednisolone. The efficacy of the two drugs is similar with a similar mechanism of action. Corticosteroids bind to glucocorticoid receptors in the cytoplasm. This complex then translocates into the nucleus and attaches to the glucocorticoid response elements (GREs) on the promoter sequence for various genes. Corticosteroids enhance the promoter enhanced transcription of I-kappa-B (IkB), interleukin (IL)-1 receptor-II (IL-1RII), lipocortin-1 (annexin I), IL-10, alpha-2-macroglobulin, and secretory leukocyte protease inhibitor, which are anti-inflammatory mediators and block the function of the transcription factors nuclear factor kappa B (NF-kB) and activator protein-1 (AP-1) that are required for transcription of proinflammatory mediators [149]. Corticosteroids also diminish the stability of mRNA encoding IL-1, IL-2, IL-6, IL-8, tumor necrosis factor (TNF), and granulocyte-macrophage-colony stimu­lating factor (GM-CSF). Corticosteroids exert general immunosuppressive side

2.7.1.2 Dosage Steroids are usually administered as a “pulse” intraoperative dose of 5–10  mg/kg of methylprednisolone, which is followed by 1  mg/kg/day of prednisone. Steroids are currently tapered to approximately 0.1 mg/ kg/day of prednisone by the end of 1 month to 6-months.

2.7.1.3 Clinical Efficacy Corticosteroids were formerly used in very high doses previously until it was shown that when combined with azathioprine 2 mg/kg/day, lower doses were as effective as higher doses with less morbidity [36, 101]. However, the prolonged use of corticosteroids, even in low dose has been associated with significant side effects. The focus in the past few years has been to use immunosuppressive regimens that minimize or avoid the use of steroids. These regimens have varied from very low maintenance dose, early withdrawal to complete avoidance of steroids. The usual trend in most transplant centers has been to taper the steroids dose quickly to 5 mg/ day and maintain it at this level unless acute rejection occurs. A meta-analysis of studies using early withdrawal of steroids found that this strategy was associated with an unacceptably high rejection rate [53]. A randomized, double-blind study comparing corticosteroid withdrawal to low dose prednisone showed that at 5-years there was no difference in the primary composite endpoint (death, graft loss, or moderate/severe rejection) or in any of the individual components of the primary endpoint. Renal function, assessed by serum creatinine or estimated glomerular filtration rate (eGFR), did not differ at any time-point out to 5-years. There were, however, higher rates of for cause biopsyproven acute rejection (BPAR) in the withdrawal group and chronic allograft nephropathy (CAN) was more than twice as high in the withdrawal group [172]. Late withdrawal of steroids has yielded conflicting results [64, 112]. In a large prospective study, 1,110

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cadaveric kidney recipients underwent slow glucocorticoid withdrawal after at least 6 months post transplantation. Seven year follow up noted improved graft, and patient survival in comparison to matched controls [112]. A meta-analysis of twenty glucocorticoid withdrawal studies, however, reported a higher relative risk of graft failure and increased risk of acute rejection with steroid withdrawal [64]. Various immunosuppressive combinations have been tried to enable steroid withdrawal without increasing the risk of rejection. Induction therapy with basiliximab, with maintenance regimen of sirolimus, mycophenolate and low dose tacrolimus was able successful in steroid withdrawal at 3 months without increasing the incidence of acute rejection [163, 164]. Similarly, induction therapy with thymoglobulin and maintenance therapy with mycophenolate and sirolimus has allowed for protocols with calcineurin minimization and early steroid withdrawal [55].

2.7.1.4 Side Effects Corticosteroids have multiple adverse effects, including cushingoid habitus, susceptibility to infection, impaired wound healing, growth suppression in children, osteoporosis, aseptic necrosis of bone, cataracts, glucose intolerance, fluid retention, hypertension, emotional liability, insomnia, manic and depressive psychosis, gastric ulcers, hyperlipidemia, polyphagia, obesity, and acne [139, 149].

2.7.2 Calcineurin Inhibitors The calcineurin inhibitors, first with the introduction of cyclosporine in the mid-1980s and then with and tacrolimus in the mid-1990s, have been the mainstay of immunosuppressive regimens for the past 25 years [16].

2.7.2.1 Mechanism of Action Cyclosporine binds to cyclophilin which helps concentrate the cyclosporine in the cytoplasm. Tacrolimus binds to FK binding protein 12 (FKBP 12). Although they bind to different cytosolic proteins, both drugs exert similar effects downstream [16]. The drug – cytosolic protein complex binds to calcineurin, a calcium/

A. Khurana and D.C. Brennan

calmodulin-activated protein phosphatase. Calcineurin dephosphorylates nuclear factor of activated T cells (NFAT) so that it can enter the nucleus and activate cytokine transcription genes. Binding of calcineurin by cyclosporine and tacrolimus causes failure of transcription of factors IL 2, IL3, IL 4, IL 5, CD 40 ligand, GM CSF, IFNg and TNFa, which are activated by NFAT. An additional effect of tacrolimus is blockade of cytokine receptor expression and cytokine effects on target cells [130]. This may explain why tacrolimus is equally effective at preventing rejection despite less calcineurin inhibition compared to cyclosporine at clinically used doses and levels achieved [72].

2.7.2.2 Dosage Historically doses of cyclosporine in transplantation were up to 17  mg/kg [119]. These high doses were associated with frequent side effects and poor tolerance. With further experience, it was realized that lower maintenance doses were as efficacious and less toxic. Tacrolimus achieves similar immunosuppression with 20–50 fold lower doses than cyclosporine [72]. Cyclosporine and tacrolimus doses are adjusted on blood levels. The dose of cyclosporine is adjusted to maintain 12 h trough levels of 200–300 ng/mL for the first 3 months post-transplant; after this period, trough levels of 50–150 ng/mL are generally adequate. For tacrolimus, doses are adjusted to attain target whole-blood trough concentrations of 8–10 ng/mL for the first 3 months, and 3–8 ng/mL after this period.

2.7.2.3 Clinical Efficacy Cyclosporine alone or in combination with azathioprine and corticosteroids led to a dramatic lowering in acute rejection rates and marked improvement in 1-year graft survival compared to the use azathioprine and corticosteroids. Cyclosporine, however, is nephrotoxic and the reduced rate of acute rejection has not translated into improved long term graft survival [91, 119]. Over the last decade, tacrolimus has replaced cyclosporine as the calcineurin inhibitor of choice at most centers. Use of tacrolimus has been associated with less rejection, lower serum creatinines and fewer side-effects in comparison to cyclosporine in some studies. The combination of tacrolimus with azathioprine showed reduced rate of acute rejection in comparison to cyclosporine

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and azathioprine combination [94, 116]. Further benefit of switching from cyclo­sporine to tacrolimus after the first episode of acute rejection was shown to reduce subsequent rejection episodes [18]. With the use of current induction regimens, use of mycophenolate as the antimetabolite, and conversion to modified cyclosporine, the graft and patient survival rates are not significantly different between tacrolimus and cyclosporine basedregimens [7, 58, 92]. The choice of calcineurin inhibitors at present is generally by center preference.

can also cause arteriolopathy with nodular protein deposits in the arterial wall and mucinoid thickening of the intima (Fig.  2.4). This is only partially reversible with reduction in dose. Long term cyclosporine use has been implicated in the development of CAN manifested as interstitial infiltrates, striped fibrosis, and arteriolar hyalinosis (Fig.  2.5). This CAN is irreversible. Similar nephrotoxicity has been noted with tacrolimus [98, 140]. Immunologic factors cause the initial tubulo-interstitial damage but calcineurin inhibitors are responsible for the major histological damage noted in CAN [106]. Calcineurin inhibitors may also cause hemolytic uremic syndrome/thrombotic thrombocytopenic purpura (HUS/TTP) or thrombotic microangiopathy (Fig. 2.2) [43]. This may be dose-related or idiosyncratic. Lower

2.7.2.4 Side Effects Both cyclosporine and tacrolimus have been associated with frequent side effects. Urinary tract infections are the most common infections and CMV infection is also seen frequently. The incidence of infections does not seem to differ among cyclosporine and tacrolimus [58, 92]. Nephrotoxicity, both acute and chronic, has been the major concern with calcineurin inhibitors. Cyclosporine and tacrolimus can cause acute nephrotoxicity because of intense arterial vasoconstriction that they produce. The vasoconstriction is associated with increased levels of endothelin1, decreased nitric oxide and increased TGF b [23, 157]. Acute CNI toxicity is usually a dose dependent phenomenon and resolves as the dose is decreased [119]. A variety of histopathological changes are noted with the use of cyclosporine. Isometric tubular vacuolization is a characteristic histopathologic change of acute calcineurin toxicity (Fig.  2.3). Cyclo­sporine

Fig.  2.4  Black arrows show arteriolar beaded hylanosis of chronic cyclosporine toxicity

Fig. 2.3  Black arrowheads indicate cyclosporine induced isometric vacuolization

Fig.  2.5  Black arrows show striped interstitial fibrosis of chronic cyclosporine toxicity

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doses of CNIs are currently used and the incidence appears to be decreasing from 3–14% in older literature to 0.8% in the current literature [127, 175]. Hypertension is also a known side effect of cyclosporine and tacrolimus therapy. It develops within a few weeks to months after therapy with calcineurin inhibitors. It seems to be independent of nephrotoxicity. In contrast to nephrotoxicity of the two drugs, which is comparable, the effects on blood pressure are more pronounced with the use of cyclosporine [24, 83]. The effect seems to more dependent on vasoconstriction than salt retention as demonstrated by hypertension present in an anuric transplant on cyclosporine [167]. Hyperlipidemia is a complication of the cyclosporine therapy. Patients treated with cyclosporine have higher LDL levels in comparison to tacrolimus based regimens, although triglyceride levels are similar [31, 57]. This may be related to binding of the LDL receptor or decreased lipoprotein lipase activity but the exact cause is not clear. Post transplant diabetes mellitus is a significant side-effect of current immunosuppressive regimens. Steroids induce insulin resistance but the calcineurin inhibitors, especially tacrolimus, have detrimental effects on beta cell function. Tacrolimus causes beta cell damage and reduced insulin secretion [132]. The incidence of hyperglycemia has been previously reported to be as high as 24% at 36 months with the use of tacrolimus [65]. The risk of de novo diabetes mellitus has now decreased because of lower doses of steroid therapy and lower doses of tacrolimus that are currently used [148, 155]. Risk factors for the development of diabetes are the dose of tacrolimus, concomitant use of steroids and African American race. Cyclosporine can decrease beta cell volume and increase the risk of post transplant diabetes. The risk of diabetes is lower with cyclosporine than tacrolimus. Both drugs are associated with neurological side effects: tremor, headache, neuralgia and peripheral neuropathy, with tremors more common with tacrolimus [116]. However, serious neurological complications can occur including seizures, encephalopathy, visual and auditory hallucinations, cerebellar ataxia, motor weakness and encephalopathy [12]. Other side effects include hirsutism, gingivitis and gum hyperplasia, which are more common with use of cyclosporine [116]. There is a 1–1.5% risk of

A. Khurana and D.C. Brennan

malignancy with the use of the calcineurin inhibitors [94]. This risk may be reflective of the overall degree of immunosuppression rather than any specific drugs. Skin cancers and lymphomas are the most common cancers. Post transplant lymphoproliferative disorder is equally prevalent among adult patients with the use of cyclosporine and tacrolimus. The pediatric literature, however, suggests an increased incidence of PTLD with the use of tacrolimus [37]. Dose-dependent hyperkalemia due to tubular aldosterone resistance has been noted with cyclosporine [46, 147]. Cyclosporine also causes hyperuricemia and gout and hypomagnesemia because of its effects on tubular handling of uric acid and magnesium respectively. Tacrolimus causes these disorders as well but is reported less often.

2.7.3 Mycophenolate Mycophenolate is an antimetabolite that is available in two formulations: mycophenolate mofetil (MMF, CellCept) and enteric coated mycophenolic acid (MPA, MyFortic). Both inhibit inosine monophosphate dehydrogenase (IMPDH).

2.7.3.1 Mechanism of Action Lymphocytes require IMPDH for replicating genetic information during cell division. Other cells in the body may use alternate enzyme pathways when the IMPDHdependent pathway is inhibited. MMF, introduced in 1995, is the morpholinoethyl ester of MPA, a selective antimetabolite that blocks de novo purine synthesis in lymphocytes. Because it is a stronger inhibitor of type II isoform of IMPDH, which is expressed preferentially in activated lymphocytes, it is useful for transplantation and treatment of autoimmune diseases. In addition, MPA inhibits recruitment of monocytes and macrophages and decreases TNF a and IL1 production which are essential in recruitment of fibroblasts [9]. This contrasts with the calcineurin inhibitors which increase TGF b expression in the grafted kidneys and promote fibrogenesis. Mycophenolate also strongly interferes with the adhesion of lymphocytes to the vascular endothelium [9].

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2  Current Concepts of Immunosuppression and Side Effects

2.7.3.2 Dosage The dose of mycophenolate for renal transplantation is 1,000 mg twice daily when used with cyclosporine but lower doses are used with tacrolimus because tacrolimus, unlike cyclosporine, does not block P-glycoprotein and thus does not block enterohepatic circulation leading to higher drug exposure compared to concomitant use of cyclosporine [35]. Doses up to 3 g/day may be required in African American patients for adequate immunosuppression. MyFortic which is the enteric coated formulation is given as 720  mg twice daily dosage.

2.7.3.3 Clinical Efficacy Compared to placebo or low dose azathioprine with a non-modified preparation of cyclosporine (San­ dimmune), mycophenolate was shown to reduce acute rejection by about 50% [1, 104, 118, 141]. This prompted the increased use of mycophenolate in transplant centers and the gradual phasing out of azathioprine as the anti metabolite of choice. In long term studies, the reduced rejection rates have not translated into better allograft survival. A recent randomized-prospective study showed that when used with a modified cyclosporine preparation and standard azathioprine doses that rejection rates were similar at 1, 2, and 5 years as was graft survival even among those who had steroid withdrawal [125, 126].

occult-gastrointestinal CMV in the absence of viremia among patients with GI side-effects on mycophenolate [63]. Invasive CMV in the kidney may also be seen with very low or absent CMV viremia [82]. Myco­ phenolate is not associated with any increase in incidence of post-transplant malignancies and has a satisfactory safety profile for long term immunosuppression [169].

2.7.4 Rapamycin (Sirolimus) Sirolimus is an inhibitor of mTOR (mammalian target of Rapamycin).

2.7.4.1 Mechanism of Action Sirolimus binds to the FK binding proteins, similar to tacrolimus but this complex binds to the mTOR complex. It blocks the effect of mTOR and blocks the activation of IL-2 and inhibits the progression of the T cell from the G phase to the S phase. Besides its effects on IL-2 and IL4, sirolimus also affects IL7, IL12 and IL15 [39]. Sirolimus, but not cyclosporine has been shown to prevent the CD28 mediated down regulation of IkB. This causes persistent inhibition of NF-kB and prevents transcription of IL2 and other cytokines [73].

2.7.4.2 Dosage 2.7.3.4 Side Effects Mycophenolate, unlike calcineurin inhibitors, has no direct cardiovascular, hemodynamic or renal side effects. It is also free of metabolic side effects. This along with its effects on reducing antidonor antibodies and TNF a and IL1 have been speculated to be the reason for decreasing CAN in transplant recipients [97, 153]. Its main side effects include gastrointestinal side effects including abdominal pain, nausea, diarrhea and hematological side effects including anemia, and leucopenia [169]. Patients with gastrointestinal complaints can be switched from MMF to Myfortic without loss of efficacy [19]. Mycophenolate may predispose to a slightly higher risk of CMV infections with higher doses (3 g/day) [25, 169]. There is a high incidence of

The loading dose of sirolimus can vary from 6 to 15 mg. The maintenance dose is 2–5 mg and is adjusted based on sirolimus whole blood trough concentrations targeted to 5–15 ng/mL.

2.7.4.3 Clinical Efficacy Studies have shown that in comparison with azathioprine, sirolimus is associated with a significant reduction of both the incidence and severity of biopsy proven rejection episodes [60, 61, 88]. When sirolimus was substituted for high-dose cyclosporine, there was an improvement in the graft function with no significant increase in the rejection rates [50, 52].

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Sirolimus has antiproliferative effects, causes inhibition of vascular smooth muscle cells and intimal proliferation and thus has been utilized for drug coated coronary stents [48]. It has been used with promising results in the treatment of variety of tumors including small cell lung cancer, pancreatic cancer, leukemia, lymphoma, rhabdomyosarcoma, neuroblastoma and breast cancer [54].

2.7.4.4 Side Effects Sirolimus is associated with increased incidence of delayed graft function [82, 95, 137, 143]. In a study of 144 patients with first cadaveric or living donor kidney allograft recipients, the incidence of delayed graft function was 25% with the use of sirolimus in comparison to 9% in those without [143]. A retrospective analysis of the cadaveric renal transplant patients in the USRDS system found that sirolimus was associated with a twofold increase in the incidence of delayed graft function although the graft and patient survival rate was unaffected [137]. Other side effects include increased incidence of lymphoceles, hernia, synergistic nephrotoxicity in combination with calcineurin inhibitors, hyperlipidemia, edema, anemia, proteinuria, thrombotic micro­ angiopathy, thrombosis, and pneumonitis [2, 28, 47, 50, 100]. There has been a small case series of three patients who developed lymphedema after exposure to sirolimus [2]. Sirolimus has been associated with synergistic nephrotoxicity with calcineurin inhibitors. This may be in part related to the increased drug levels of cyclosporine and/or increased TGF b levels with the combination therapy [135]. Like the calcineurin inhibitors, it has metabolic side effects including hyperlipidemia. Hyperlipidemia usually starts at 1-month post transplant and peaks at 3-months [28, 61, 100]. This invariably requires lowered cholesterol intake and the use of statins. Other metabolic side effects include impaired glucose tolerance. Sirolimus is associated with insulin resistance with hyperglycemia and hyperinsulinemia [74]. In a study of peripheral blood mononuclear cells of 30 transplant patients on chronic sirolimus therapy, a marked decrease of basal and insulin-stimulated AKT phosphorylation was noted

A. Khurana and D.C. Brennan

[38]. The combination of sirolimus with tacrolimus synergistically decreases islet cell size and increases islet cell apoptosis [74]. Sirolimus increases the de  novo development of proteinuria. Non nephrotic and nephrotic range proteinuria have been noted when calcineurin inhibitors were withdrawn and sirolimus therapy was initiated. This may have been a consequence of calcineurin inhibitor withdrawal and subsequent hyper-filtration in the setting of impaired glomerular permeability and CAN [123]. However, convincing reports of focal segmental glomerulosclerosis related to sirolimus have now emerged [81]. Immunohistochemistry has shown diminished expression of the podocyte-specific epi­ topes synaptopodin and p57, reflecting dedifferentiation and podocyte dysregulation. Moreover, a decrease in vascular endothelial growth factor (VEGF) expression has been observed. A variety of pulmonary effects have been attributed to the use of sirolimus. These have varied from lymphocytic alveolitis, lymphocytic interstitial pneumonitis, bronchoalveolar obliterans organizing pneumonia, focal fibrosis, pulmonary alveolar hemorrhage, or a combination thereof [115]. Sirolimus discontinuation or dose reduction resulted in clinical and radiologic improvement in all 15 patients in this series within 3 weeks. Anemia is another side effect which has been seen with the use of sirolimus [100]. This side effect was notable when higher trough levels of sirolimus were targeted but with current trough levels of 5–12 ng/mL, the incidence of this side effect has decreased [41]. Microcytosis has been noted in some studies [44, 66, 100]. It may be related to decrease in levels of hepcidin, the key regulator of iron metabolism, although the exact mechanism is not clear [90]. Sirolimus has been associated with an increased incidence of herpes virus infection and pneumonia but not CMV infections [28]. The incidence of skin cancers may be less with use of sirolimus. Use of sirolimus in place of an antimetabolite has been associated with reduction in appearance of new lesions [152]. Other beneficial effects have included a decrease in BK viremia in one small study. However, it is unclear if both effects were related to conversion to sirolimus or reduction in the level of immunosuppression related to stopping the antimetabolite [80].

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2  Current Concepts of Immunosuppression and Side Effects

2.8 Newer Immunosuppressive Medications 2.8.1 Janus Kinase (JAK) 3 Inhibitors JAK3 associates specifically with the common gamma chain of the interleukin-2 (IL-2) receptor and is found primarily on hematopoietic cells [174]. The genetic mutation of JAK3 causes abnormal lymphoid cell development and severe combined immunodeficiency [87, 174]. The association of JAK3 with the TcR/CD3 machinery as well as the IL-2R suggests a crucial role of this kinase in the regulation of both early T-cell activation and cytokine-driven cell growth [144, 150]. JAK 3 may also have a role in expression of eosinophilic airway inflammation [156]. JAK 3 inhibitors have had significant success in murine models of cardiac transplantation with prolongation of survival [71]. The most commonly tested JAK 3 inhibitor has been CP 690550. It produces a 20–100-fold more potent inhibition of JAK 3 in comparison to JAK 1 and 2 [26]. This compound is thus more immunosuppressive than other JAK inhibitors and carries a lower risk of hematological toxicity such as anemia and leucopenia. In in  vitro studies, CP-­ 690,550 caused a significant reduction of IL-2enhanced IFN-gamma production by T-cells, T-cell surface expression of CD25 and T-cell proliferative capacity [113]. Similar results have been replicated in animals. In addition, transplanted animals displayed significant reduction of NK cell and CD8+ T cell numbers in a dose- and time-dependent manner [33]. Though CD4+ T cells were unaffected, their number increased significantly within 2 weeks of the last dose of CP-690550. CP-690550 also inhibited IL-15induced CD69 expression in NK cells [33]. CP-690550 has been shown to reduce allograft rejection in nonhuman primates in combination with mycophenolate [15]. CP-690550 has also been shown to prevent allograft vasculopathy in a rodent model of aortic transplantation [131]. In preclinical and early clinical studies, the major side effects of CP 690550 have included reactivation of polyomavirus infection and anemia [14]. Subclinical pyelonephritis has also been noted along with one incidental lymphosarcoma [15]. There have been no cardiovascular or metabolic

side effects noted thus far [26]. Its use in human patients is under investigation.

2.9 AEB-071 AEB-071 (AEB) is a novel, oral compound that inhibits protein kinase C (PKC). PKC is largely restricted to T lymphocytes and mediates activation NFkB, leading to downstream IL-2 production. AEB blocks early T-cell activation independent from the calcineurin pathway. This has prompted studies on the use of this agent in place of calcineurin inhibitors. AEB has been noted to have similar antiproliferative activity to MMF and retained its inhibitory effect on IL-2 production when combined with mycophenolate [138]. Preclinical studies have reported prolonged renal allograft survival in nonhuman primates with AEB at therapeutic doses or at non-therapeutic doses in combination with cyclosporine [166]. AEB in sub-therapeutic doses has been used in combination with everolimus, mycophenolic acid or FTY720 with prolonged graft survival [13]. It does not seem to have significant drug interactions with mycophenolate or everolimus. This has prompted two clinical trials using AEB 071 in place of calcineurin inhibitors in combination with steroids and basiliximab and everolimus or mycophenolate, and assesses the incidence of biopsy proven acute rejection and graft loss. No significant side effects related to this medication have been noted. In cynomolgus monkeys, AEB was well tolerated with normal blood chemistries and normal extra-renal histology at necropsy [13].

2.9.1 LEA 29Y (Belatacept) Belatacept (LEA29Y) is an intravenously administered second-generation cytotoxic T lymphocyte antigen-4 immunoglobulin (CTLA-4Ig) that interferes with CD28 and CD 80/86 [75, 76]. CD28 is constitutively expressed by a majority of CD4+ T cells and approximately 50% of CD8+ cells [75]. CD28 helps lower the T-cell activation threshold and causes enhanced proliferation, T-cell differentiation into T helper (Th) cells, increased B-cell antibody production and increased

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proliferation of previously activated T cell [161]. In contrast to CD28, CTLA-4, binds to CD80 and CD86, but with a 10–20-fold higher affinity and inhibits T cells. Belatacept in low and intermediate doses was compared to high-dose cyclosporine and was found to have similar rates of study defined rejection in comparison to cyclosporine. Investigator treated rejection, however, was as much as twice as common in the belatacept arms compared to the cyclosporine arm (26, 32, and 16%, respectively), but there was a lower incidence of CAN and higher glomerular filtration rates in the no-cyclosporine belatacept groups compared to the high-dose cyclosporine group [160]. Similar blood pressure profiles and lipid profiles were seen with no difference in the side effect profiles of the two medications. Indication-biopsies were analyzed for infiltration of T regulatory cells [49]. Bela­ tacept did not affect the infiltration of the grafts with T regulatory cells in comparison to cyclosporine. There was a 6% incidence of PTLD at 1-year in patients treated with intermediate-dose belatacept, but no PTLD was seen in the low-dose or cyclosporine-treated patients.

2.10 Efalizumab Efalizumab is a humanized IgG1 version of a murine anti-CD11a monoclonal antibody with a noncovalently linked alpha chain (CD11a) and a beta chain (CD18). Lymphocyte functioning antigen-1 (LFA 1), CD11a/ CD18, is a classic adhesion molecule. LFA1/intracellular adhesion molecule (ICAM) interactions are necessary for T cell activation, T cell and B cell responses. LFA 1 stabilizes the major histocompatibility (MHC)/T cell receptor complex and provides an important costimulatory signal. Efalizumab is approved for the treatment of moderate to severe psoriasis [34]. In a large trial for the treatment of psoriasis, Efalizumab was more effective than placebo, well tolerated with few side effects and safe with a 5% incidence of development of anti-Efalizumab antibodies [79]. Its role in renal transplantation is still being investigated. A Phase I/II open label multicenter trial comparing low dose (0.5  mg/kg/week) and high dose (2 mg/kg/week) with Efalizumab combined with half dose cyclosporine, prednisone and sirolimus or full dose cyclosporine, mycophenolate and prednisone

A. Khurana and D.C. Brennan

regimens has been reported [162]. Complete saturation of the CD11a molecule occurred with the low dose as well as the high dose Efalizumab. There was no difference in acute rejection rates among groups and the mean GFR was similar. However, 30% of patients who received the higher dose Efalizumab combined with full dose cyclosporine regimen developed PTLD. Other drug related serious adverse events in this study included CMV infections, peritonitis and pancreatitis. No cases of PTLD were seen in trials of Efalizumab using the 1 mg/kg dose in patients with psoriasis [96].

2.11 Summary A broad range of immunosuppressive agents are now available for use in renal transplantation. The last 2 decades have seen a remarkable increase in the introduction of new agents, both pharmacological and biological. The routine use of induction therapy along with maintenance immunosuppression with calcineurin inhibitors and mycophenolate has brought about an impressive reduction in the rates of acute rejection. One-year graft and patient survival rates now exceed 90% at most centers. This improved short term benefit has not translated into improved long term graft survival with most allografts being lost to CAN. Immun­ osuppressive drugs also have cardiovascular and metabolic side effects, and cardiovascular causes continue to be the leading cause of mortality for transplant patients. With an armamentarium of new immunosuppressive drugs now available, efforts are underway to combine immunosuppressive drugs with maximal efficacy, and avoid drugs with negative cardiovascular, renal and metabolic side effects or synergistic toxicity.

References   1. A blinded, randomized clinical trial of mycophenolate mofetil for the prevention of acute rejection in cadaveric renal transplantation. The Tricontinental Mycophenolate Mofetil Renal Transplantation Study Group. Transplantation 61(7), 1029–1037 (1996)   2. Aboujaoude, W., Milgrom, M.L., Govani, M.V.: Lymphedema associated with sirolimus in renal transplant recipients. Transplantation 77(7), 1094–1096 (2004)   3. Abramowicz, D., Goldman, M., De Pauw, L., et  al.: The long-term effects of prophylactic OKT3 monoclonal antibody in cadaver kidney transplantation–a single-center,

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mycophenolate mofetil, and daclizumab induction in renal transplantation. Transplantation 79(7), 807–814 (2005) 130. Rostaing, L., Puyoo, O., Tkaczuk, J., et al.: Differences in Type 1 and Type 2 intracytoplasmic cytokines, detected by flow cytometry, according to immunosuppression (cyclo­ sporine A vs. tacrolimus) in stable renal allograft recipients. Clin. Transpl. 13(5), 400–409 (1999) 131. Rousvoal, G., Si, M.S., Lau, M., et al.: Janus kinase 3 inhibition with CP-690, 550 prevents allograft vasculopathy. Transpl. Int. 19(12), 1014–1021 (2006) 132. Sabbatini, M., Sansone, G., Uccello, F., et al.: Acute effects of rapamycin on glomerular dynamics: a micropuncture study in the rat. Transplantation 69(9), 1946–1990 (2000) 133. Shapiro, R., Jordan, M.L., Basu, A., et  al.: Kidney transplantation under a tolerogenic regimen of recipient pretreatment and low-dose postoperative immunosuppression with subsequent weaning. Ann. Surg. 238(4), 520–525 (2003). discussion 525–527 134. Sheashaa, H.A., Bakr, M.A., Ismail, A.M., et  al.: Longterm evaluation of basiliximab induction therapy in live donor kidney transplantation: a five-year prospective randomized study. Am. J. Nephrol. 25(3), 221–225 (2005) 135. Shihab, F.S., Bennett, W.M., Yi, H., Choi, S.O., Andoh, T.F.: Sirolimus increases transforming growth factor-beta1 expression and potentiates chronic cyclosporine nephrotoxicity. Kidney Int. 65(4), 1262–1271 (2004) 136. Sidhu, M.S., Nayak, K.S., Subhramanyam, S.V., Sankar, A.: Polyclonal antibodies in renal transplantation–a relook. Transplant. Proc. 39(3), 766–772 (2007) 137. Simon, J.F., Swanson, S.J., Agodoa, L.Y., et al.: Induction sirolimus and delayed graft function after deceased donor kidney transplantation in the United States. Am. J. Nephrol. 24(4), 393–401 (2004) 138. Slade, A., Vitaliti, A., Agyemang, A., Sieberling, M., Schmouder, R.: NVP-AEB071: Human pharmacodynamics in combination with mycophenolic acid. Am. J. Trans­ plant. 6(Suppl 2), 199 (2006) 139. Smith, J.M., Nemeth, T.L., McDonald, R.A.: Current immunosuppressive agents in pediatric renal transplantation: efficacy, side-effects and utilization. Pediatr. Trans­ plant. 8(5), 445–453 (2004) 140. Solez, K., Vincenti, F., Filo, R.S.: Histopathologic findings from 2-year protocol biopsies from a U.S. multicenter kidney transplant trial comparing tarolimus versus cyclo­ sporine: a report of the FK506 Kidney Transplant Study Group. Transplantation 66(12), 1736–1740 (1998) 141. Sollinger, H.W.: Mycophenolate mofetil for the prevention of acute rejection in primary cadaveric renal allograft recipients. U.S. Renal Transplant Mycophenolate Mofetil Study Group. Transplantation 60(3), 225–232 (1995) 142. Sollinger, H., Kaplan, B., Pescovitz, M.D., et  al.: Basiliximab versus antithymocyte globulin for prevention of acute renal allograft rejection. Transplantation 72(12), 1915–1919 (2001) 143. Stallone, G., Di Paolo, S., Schena, A., et al.: Addition of sirolimus to cyclosporine delays the recovery from delayed graft function but does not affect 1-year graft function. J. Am. Soc. Nephrol. 15(1), 228–233 (2004) 144. Stepkowski, S.M., Kirken, R.A.: Janus tyrosine kinases and signal transducers and activators of transcription regulate critical functions of T cells in allograft rejection and

transplantation tolerance. Transplantation 82(3), 295–303 (2006) 145. Stratta, R.J., Alloway, R.R., Lo, A., Hodge, E.: Two-dose daclizumab regimen in simultaneous kidney-pancreas transplant recipients: primary endpoint analysis of a multicenter, randomized study. Transplantation 75(8), 1260– 1266 (2003) 146. Tan, J., Yang, S., Wu, W.: Basiliximab (Simulect) reduces acute rejection among sensitized kidney allograft recipients. Transplant. Proc. 37(2), 903–905 (2005) 147. Tanabe, K.: Calcineurin inhibitors in renal transplantation: what is the best option? Drugs 63(15), 1535–1548 (2003) 148. Tanabe, K., Tokumoto, T., Ishikawa, N., et  al.: Japanese single-center experience of kidney transplantation under tacrolimus immunosuppression. Transplant. Proc. 32(7), 1696–1699 (2000) 149. Taylor, A.L., Watson, C.J., Bradley, J.A.: Immunosuppressive agents in solid organ transplantation: Mechanisms of action and therapeutic efficacy. Crit. Rev. Oncol. Hematol. 56(1), 23–46 (2005) 150. Tedesco Silva, H., Pinheiro Machado Jr., P., Rosso Felipe, C., Medina Pestana, J.O.: Immunotherapy for De Novo renal transplantation: what’s in the pipeline? Drugs 66(13), 1665–1684 (2006) 151. Tesi, R.J., Kano, J.M., Horn, H.R., Schroeder, T.: Thymoglobulin reverses acute renal allograft rejection better than ATGAM--a double-blinded randomized clinical trial. Transplant. Proc. 29(7A), 21S–23S (1997) 152. Tessmer, C.S., Magalhaes, L.V., Keitel, E., et al.: Conversion to sirolimus in renal transplant recipients with skin cancer. Transplantation 82(12), 1792–1793 (2006) 153. Theruvath, T.P., Saidman, S.L., Mauiyyedi, S., et  al.: Control of antidonor antibody production with tacrolimus and mycophenolate mofetil in renal allograft recipients with chronic rejection. Transplantation 72(1), 77–83 (2001) 154. Thistlethwaite Jr., J.R., Stuart, J.K., Mayes, J.T., et  al.: Complications and monitoring of OKT3 therapy. Am. J. Kidney Dis. 11(2), 112–119 (1988) 155. van Hooff, J.P., van Duijnhoven, E.M., Christiaans, M.H.: Tacrolimus and glucose metabolism. Transplant. Proc. 31(7A), 49S–50S (1999) 156. Verbsky, J.W., Randolph, D.A., Shornick, L.P., Chaplin, D.D.: Nonhematopoietic expression of Janus kinase 3 is required for efficient recruitment of Th2 lymphocytes and eosinophils in OVA-induced airway inflammation. J. Immunol. 168(5), 2475–2482 (2002) 157. Vieira Jr., J.M., Noronha, I.L., Malheiros, D.M., Burdmann, E.A.: Cyclosporine-induced interstitial fibrosis and arteriolar TGF-beta expression with preserved renal blood flow. Transplantation 68(11), 1746–1753 (1999) 158. Vincenti, F., de Andres, A., Becker, T., et al.: Interleukin-2 receptor antagonist induction in modern immunosuppression regimens for renal transplant recipients. Transpl. Int. 19(6), 446–457 (2006) 159. Vincenti, F., Kirkman, R., Light, S., et  al.: Interleukin-2receptor blockade with daclizumab to prevent acute rejection in renal transplantation. Daclizumab Triple Therapy Study Group. N Engl J. Med. 338(3), 161–165 (1998) 160. Vincenti, F., Larsen, C., Durrbach, A., et al.: Costimulation blockade with belatacept in renal transplantation. N Engl J. Med. 353(8), 770–781 (2005)

30 161. Vincenti, F., Luggen, M.: T cell costimulation: a rational target in the therapeutic armamentarium for autoimmune diseases and transplantation. Annu. Rev. Med. 58, 347–358 (2007) 162. Vincenti, F., Mendez, R., Pescovitz, M., et al.: A phase I/II randomized open-label multicenter trial of efalizumab, a humanized anti-CD11a, anti-LFA-1 in renal transplantation. Am. J. Transplant. 7(7), 1770–1777 (2007) 163. Vincenti, F., Monaco, A., Grinyo, J., Kinkhabwala, M., Roza, A.: Multicenter randomized prospective trial of steroid withdrawal in renal transplant recipients receiving basiliximab, cyclosporine microemulsion and mycophenolate mofetil. Am. J. Transplant. 3(3), 306–311 (2003) 164. Vincenti, F., Monaco, A., Grinyo, J., et al.: Rapid steroid withdrawal versus standard steroid therapy in patients treated with basiliximab, cyclosporine, and mycophenolate mofetil for the prevention of acute rejection in renal transplantation. Transplant. Proc. 33(1–2), 1011–1012 (2001) 165. Vincenti, F., Schena, F., Walker, R., et  al.: Preliminary 3-month results comparing immunosuppressive regimens of enteric-coated mycophenolate sodium (EC-MPS) without steroids vs short-term use of steroids vs standard steroid treatment including basiliximab and neoral C-2 in de novo kidney recipients. Am. J. Transplant. 5(Suppl 11), 548 (2005) 166. Wagner, J., Evenou, J., Zenke, G., et al.: The first-in-class oral protein kinase C (PKC) inhibitor NVP-AEB071 (AEB) prolongs renal allograft survival in non-human primates (NPH) and suppresses lymphocyte proliferation at safe exposures in human proof-of-concept studies. Am. J. Transplant. 6(Suppl 2), 86 (2006) 167. Wahba, I.M., Bennett, W.M.: Increased vascular resistance and not salt retention characterizes cyclosporine A-induced hypertension: report in an anuric patient. Am. J. Transplant. 7(8), 2042–2046 (2007)

A. Khurana and D.C. Brennan 168. Waldmann, H., Hale, G.: CAMPATH: from concept to clinic. Philos. Trans. R. Soc. Lond. B Biol. Sci. 360(1461), 1707–1711 (2005) 169. Wang, K., Zhang, H., Li, Y., et al.: Safety of mycophenolate mofetil versus azathioprine in renal transplantation: a systematic review. Transplant. Proc. 36(7), 2068–2070 (2004) 170. Watson, C.J., Bradley, J.A., Friend, P.J., et al.: Alemtuzumab (CAMPATH 1H) induction therapy in cadaveric kidney transplantation–efficacy and safety at five years. Am. J. Transplant. 5(6), 1347–1353 (2005) 171. Webster, A.C., Playford, E.G., Higgins, G., Chapman, J.R., Craig, J.C.: Interleukin 2 receptor antagonists for renal transplant recipients: a meta-analysis of randomized trials. Transplantation 77(2), 166–176 (2004) 172. Woodle, E.S., First, M.R., Pirsch, J., et al.: A prospective, randomized, double-blind, placebo-controlled multicenter trial comparing early (7 day) corticosteroid cessation versus long-term, low-dose corticosteroid therapy. Ann. Surg. 248(4), 564–577 (2008) 173. Woodle, E.S., Vincenti, F., Lorber, M.I., et  al.: A multicenter pilot study of early (4-day) steroid cessation in renal transplant recipients under simulect, tacrolimus and sirolimus. Am. J. Transplant. 5(1), 157–166 (2005) 174. Yabu, J.M., Vincenti, F.: Novel immunosuppression: small molecules and biologics. Semin. Nephrol. 27(4), 479–486 (2007) 175. Zarifian, A., Meleg-Smith, S., O’Donovan, R., Tesi, R.J., Batuman, V.: Cyclosporine-associated thrombotic microangiopathy in renal allografts. Kidney Int. 55(6), 2457–2466 (1999) 176. Zietse, R., van Steenberge, E.P., Hesse, C.J., et al.: Singleshot, high-dose rabbit ATG for rejection prophylaxis after kidney transplantation. Transpl. Int. 6(6), 337–340 (1993)

3

Clinical Aspects of Infection Rouba Ghoussoub and Daniel C. Brennan

3.1 Introduction Infections in solid organ transplant recipients continue to be a significant source of morbidity and mortality. More than 80% of recipients develop at least one infection during the first year [16]. Kidney transplant recipients have the lowest risk of infection as compared to lung, liver, and heart allograft recipients, and this is likely related to the more elective nature of the surgery and better overall status of the patient prior to transplantation. Infections in this patient population, however, can lead to graft dysfunction, allograft rejection, alterations in immune status, and affect the overall transplant outcome [11]. Moreover, chronic graft rejection will necessitate more rigorous immunosuppression which will, in turn, increase the risk of developing infections with immunomodulating viruses [16]. There is thus a fine balance between developing the adequate immunosuppressive regimen that will prevent allograft rejection while at the same time not posing a significant risk for infection. Several factors can present a risk for the development of an infection posttransplant. These can be categorized as pretransplantation, perioperative, and posttransplantation risk factors. Pretransplantation risk factors include medical comorbidities like renal failure, diabetes mellitus, autoimmune diseases and latent, untreated infections in the host or the recipient [11]. Iatrogenic immunosuppression, preoperative antibiotic exposures, and colonization with resistant bacteria such as methicillin resistant

Staphylococcal aureus (MRSA) or enterobacteriae that lower the threshold for infection [16]. The type of dialysis used prior to transplantation is also important. Peritoneal dialysis is associated with a higher risk of infectious complications compared to hemodialysis [9, 11]. Long operative times, unidentified donor infections, intricacies of surgery, and the need for reexploration are all perioperative risk factors for infection [9]. There are multiple postoperative risk factors for infection. Infection with immunomodulating viruses such as cytomegalovirus (CMV), Epstein–Barr virus (EBV), hepatitis B virus (HBV), hepatitis C virus (HCV), and human herpes virus (HHV) increase the risk for infection. Acute rejection and subsequently intensifying the immune regimen increases the risk for infection in the transplant recipient [9, 11]. If the patient develops postoperative complications and deterioration of preexisting medical comorbidities, has indwelling stents or catheters for a prolonged length of time, develops fluid collections or hematomas requiring drainage, and protracted leukopenia or thrombocytopenia the risk of infection also rises [11]. Another important but often overlooked factor is hypogammaglobulinemia. This may be acquired posttransplant and will predispose to certain infections such as those from encapsulated organisms like pneumococcus [9, 11]. The state of immunosuppression in the recipient, in general, plays a pivotal role in the likelihood of acquiring postoperative infections.

3.2 Infections and Timing of Transplant R. Ghoussoub and D.C. Brennan (*) Washington University in St. Louis, 4104 Queeny Tower, One Barnes-Jewish Hospital Plaza, St. Louis, MO 63110, USA e-mail: [email protected]

The timing of a particular infection is critical in narrowing the differential diagnosis and in shaping our strategies for prophylaxis. The majority of infections

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_3, © Springer-Verlag Berlin Heidelberg 2011

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develop in the first month after transplantation and are similar to postsurgical infections that develop in nonimmunocompromised patients, usually related to surgical wounds or the genitourinary tract [16]. There are generally three time-frames for infections occurring posttransplant; the first month, months 1–6, and greater than 6 months.

3.2.1 Month 1 As mentioned above, the infectious risk in the first month pertains to the surgery and common postsurgical or nosocomial infections. Atelectasis predisposes to bacterial pneumonias, and wounds may become infected with fungal or bacterial pathogens [11]. The incidence of surgical wound infections following kidney transplantation ranges from 2 to 25% [11]. These typically occur within 3 weeks of surgery and are linked to surgical complications as well as recipient comorbidities such as obesity and diabetes. The use of high dose corticosteroids and other immunosuppressive agents can alter mucosal integrity and increase the risk for diverticulitis, colonic perforation, and secondary sepsis [9, 11]. Urinary tract infections are ­associated with indwelling catheters. The use of trimethoprimsulfamethoxazole orally for prophylaxis has decreased the risk of urinary tract infections and blood stream infections significantly [9]. Viral infections occurring in the first month are generally related to latent virus in the recipient. The most common viral reactivation during that period is HSV 1 and 2 as well as HHV-6. EBV, CMV, and VZV may also reactivate but less frequently. It is unusual to develop new or donor derived viral infections in the recipient during the first month [9, 11, 16].

3.2.2 Months 2–6 This particular time-frame is notorious for developing opportunistic infections. Some of these infections include immunomodulating viruses such as CMV, EBV, HBV, HCV, HHV 8, and polyomavirus (BK). These infections can be either primary or a reactivation of donor or recipient disease [9, 11, 18]. Some of these immunomodulating viruses can also predispose to superimposed opportunistic pathogens such as Pneumocystis jerovecii

R. Ghoussoub and D.C. Brennan

(PCP), Aspergillus, Candida, Cryptococcus neoformans, Nocardia, Toxoplasma gondii, and Listeria monocyto­ genes [11]. Histoplasmosis, coccidiomycosis, and mycobacterial infections can also occur during this period [9, 11]. Rigorous monitoring, frequent follow-up, and adequate prophylactic regimens in this sensitive timeframe allow for improved graft and patient outcomes and less hospitalizations.

3.2.3 Greater than 6 Months Infections that develop later than 6 months after transplant are usually community-acquired pathogens such as influenza, urinary tract infections, diarrhea, and pneumococcal pneumonia. However, it is imperative to remember that there are some peculiar infections which may still occur [16]. CMV retinitis, for example, can manifest in this later time-frame and occur simultaneously with the reactivation of other viruses such as EBV, HSV, and VZV [11]. The eruption of herpetiform or zoster lesions should raise the suspicion for a possible underlying CMV infection [1, 11]. Some patients may also acquire immunoglobulin deficiencies for as yet uncertain reasons, perhaps related to the state of immunosuppression. CMV infection or vitamin B12 deficiency and this will predispose the recipient to developing infections by encapsulated organisms [9, 11, 16]. Of course, the inherent state of immunosuppression will also dictate the risk of developing opportunistic infections beyond the 6 month time-frame. Poor allograft function and episodes of acute or chronic rejection are treated with higher and more prolonged doses of immunosuppressive medications, thus inviting more opportunistic infection, in particular Nocar­ dia, Listeria, and Pneumocystis. Lymphoproli­ferative disorders also become evident at this later stage, in association with EBV, chronic hepatitis, and clinical adult immunodeficiency syndrome (AIDS).

3.3 Viral Infections 3.3.1 Cytomegalovirus CMV in renal transplant recipients is one of the most important pathogens posttransplantation and is a

3  Clinical Aspects of Infection

significant cause of increased morbidity and mortality. It usually occurs beyond 1 month posttransplant and its incidence in the renal transplant population is about 80%, while the disease itself occurs in about 8–32% only [4]. Due to the variable states of immunosuppression in patients and the various sensitivities of different detection assays, there is a significantly wide range in its incidence. Methods of transmission include the renal allograft, sexual contact or blood products [11]. Among all solid organ transplant recipients, renal transplant patients have the lowest risk of developing CMV disease, even without viral prophylaxis [7]. Pancreas and kidney-pancreas recipients have a higher risk than kidney recipients alone [7]. Risk factors for developing CMV infection also include donor-recipient mismatching, the use of lymphocyte depleting agents, high dose steroids, high dose mycophenolate, and the potent combination of mycophenolate and tacrolimus [4, 7, 16]. Infection with other herpes viruses such as HHV-6 and 7 may also be possible contributing factors. There are various clinical and subclinical entities that CMV can produce in the renal transplant recipient. The terminology may be confusing at times and is described below. Active CMV infection can be either symptomatic or asymptomatic but is defined by viral shedding and replication. Active infection can be primary, meaning it is a new infection in a previously seronegative host, or secondary, which is a reactivation of latent virus or a superimposed infection by a new CMV strain [11]. CMV disease represents symptomatic infection and is further divided into the CMV syndrome and invasive CMV disease. CMV syndrome consists of leucopenia, fever, fatigue, and occasional thrombocytopenia while invasive disease entails end-organ involvement such as pneumonitis, colitis, enteritis, hepatitis, and renal allograft infection [11]. Latent CMV infection, on the other hand, is asymptomatic and denotes lifelong persistence of the virus without replication in the host [11]. Recurrent CMV disease refers to the reemergence of disease symptoms after the interruption of CMV therapy [11]. Refractory disease is either due to viral resistance or significantly protracted disease in an excessively immunocompromised host [11]. CMV is a member of the genus herpesvirus and belongs to the family Herpesviridae. It is part of the same subfamily (beta-virinae) as HHV-6 and HHV-7 [4]. White blood cells are the main reservoir of CMV, particularly the CD-13-positive cells. However, CMV

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has been detected in most tissues in the body and can remain latent there [4]. CMV infection has been associated with several vascular injuries, including an increased rate of restenosis after cardiac revascularization, renal artery stenosis, and chronic allograft nephropathy [4]. There have been several proposed mechanisms for these effects but no all-encompassing explanation yet. Therefore, it is noteworthy that the derogatory effects of CMV infection may well stretch beyond the disease symptomatology itself to play a direct role in allograft and patient outcomes. In addition, CMV is an immunomodulating virus and can evade and suppress the immune system through various mechanisms, thus predisposing the host to super-imposed infections with other viruses, fungi, or bacteria. The most common presentation of CMV disease is fever, malaise, leucopenia, and an elevation in transaminases. CMV can affect the upper and lower gastrointestinal system and cause esophagitis, cholecystitis, duodenitis, hepatitis, and colitis [11]. Endoscopy reveals solitary and multiple mucosal ulcerations with hemorrhage [9] (Fig.  3.1a). Tissue specimens should be stained for CMV using immunofluorescent antiCMV antibodies and examined microscopically for inclusion bodies [4] (Fig.  3.1b). Pneumonitis is a severe CMV infection and is manifested by significant dyspnea, hypoxemia, diffuse interstitial infiltrates and the detection of CMV antigens, nucleic acids, or inclusion bodies on the bronchoalveolar lavage [11]. CMV retinitis is uncommon in transplant recipients but can be a late manifestation of the disease and is diagnosed by direct fundoscopy [11]. CMV can also infect the central nervous system and cause meningitis, encephalitis, and myelitis. CNS disease is more difficult to diagnose and requires cerebrospinal fluid sampling for further testing. The CMV serostatus of the donor (D) and the recipient (R) have historically been most strongly associated with the incidence and severity of the disease. The CMV D+/R– serogroup has been at greatest risk for severe primary infection during the first 3 months posttransplant and experiences double the number of symptomatic infections as compared to the D+/R+ group despite a similar incidence of infection [4, 9]. It is critical to diagnose CMV early, rapidly, and accurately. Historically, histopathology, and viral culture were used for diagnosis but these methods are not very sensitive, do not detect virus early enough and can be quite laborious. Newer techniques have been

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a

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b

Fig. 3.1  (a) CMV inclusion body in small intestine shown by black arrow. H + E stain. (b) Suspected CMV inclusion body confirmed to be CMV by immunohistochemistry

developed including shell vial, pp65 antigenemia, nucleic detection assays like polymerase chain reaction (PCR) of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), hybrid capture assay, branched DNA assay, and nucleic acid sequence-based amplification (NASBA) [4, 11]. The amount of CMV particles determined by quantitative PCR and how that correlates to clinical disease or to the severity of symptoms was analyzed in more than one study. In general, patients with CMV DNA levels of >500 copies/mg of total DNA in peripheral blood had clinical evidence of disease, although there were some patients with lower viral burdens who also displayed symptoms [4]. PCR results, however, vary according to different laboratories and this method requires further standardization. There is no optimal technique for the diagnosis and management of CMV yet, but molecular techniques are preferred [4]. Renal biopsy can reveal CMV inclusions, which are generally direct evidence of CMV nephropathy or tissue invasion. This finding, however, occurs in less than 1% of allograft biopsies and indicates a low sensitivity of routine histopathology for diagnosis [4] (Fig. 3.2). Thrombotic microangiopathy has also been associated with CMV nephropathy. Other histopathologic lesions include glomerular leucocytes, interstitial nephritis, and acute and chronic rejection. Since CMV disease significantly alters graft and patient outcomes, several strategies have been developed in an attempt to reduce its incidence and severity. Avoiding CMV sero-mismatching through organ allocation is not a

Fig. 3.2  Renal allograft biopsy showing cytomegalovirus inclusion body (arrow) H + E

feasible method. Prophylactic regimens have therefore been utilized for preventive purposes and different treatment regimens have been studied. Treatment regimens include the preemptive approach, which targets asymptomatic CMV infection to prevent the protraction of CMV disease, and the deferred approach, which treats active CMV disease. Universal prophylaxis refers to nonselective prophylactic therapy in all renal transplant recipients. Selective prophylaxis deals with the high-risk patient population only, namely the D+/R– sero-group [11]. Prophylactic therapy has been proven to reduce CMV in high-risk patients receiving antilymphocyte therapy, but the evidence in low risk patients and in those

3  Clinical Aspects of Infection

not receiving antilymphocyte agents is less solid (although established) [4]. One of the problems with prophylactic therapy is that 20–30% of high-risk patients develop lateonset CMV disease after prophylaxis is stopped, and the incidence of drug-resistance may be higher in those who received prophylaxis [4]. Universal prophylactic regimens, therefore, may be subjecting a portion of patients who would never have developed the disease to antiviral therapy with side effects, encourage viral resistance, is expensive, and may only be delaying the onset of disease rather than abolishing it. Oral ganciclovir and valganciclovir have been both shown to be extremely effective in the prevention of CMV infection and disease. Oral acyclovir may be effective in low risk patients but is generally not effective in higher risk recipients [4]. Ganciclovir can be administered orally or intravenously, but the oral formulation is much less bioavailable and achieves lower serum levels. It is, however, more convenient to administer. Valganciclovir is administered at 900 mg/ day orally for prophylaxis. A clinical study evaluated once a day valganciclovir regimen to thrice daily oral ganciclovir for about 3 months post transplantation and in high-risk D+/R– solid organ recipients. The two drugs were found to have similar effectiveness [4]. However, at 6 months valganciclovir was found to be more effective at preventing CMV infection in kidney transplant recipients [4]. Both valganciclovir and ganciclovir are effective prophylactic regimens and have been shown to delay the onset of CMV infections, decrease disease severity, reduce acute rejection episodes, and improve graft survival in general. If the donor is sero-positive, however, it may be necessary to continue prophylaxis from 6 months up to a year [4]. Preemptive therapy of CMV infection requires serial monitoring for viremia and treatment with valganciclovir or valacyclovir before the development of symptoms. This strategy reduces the cost of drug therapy in low-risk groups and treats the disease early in high-risk groups. It is labor-intensive, however, to monitor and perform frequent assays for CMV viremia. Prophylactic and preemptive strategies have been demonstrated to prevent serious CMV disease but the preemptive strategy is associated with a decreased incidence of late CMV disease [10, 11, 17]. Deferred therapy is treatment of CMV infection once disease symptoms manifest [11]. The antimetabolites MMF or azathioprine doses should be reduced

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or discontinued. The most commonly used agent for treatment is intravenous ganciclovir but there is emerging evidence that oral valganciclovir may be as effective [2, 4]. Treatment is continued until the virus is cleared and usually requires 3 weeks or longer. Hyperimmune globulin may be beneficial for patients with allograft involvement [4]. Other agents, such as foscarnet and cidofovir can be used for ganciclovir resistant CMV. They are not used first line because they are nephrotoxic. To evaluate response to therapy DNA PCR can be used. Factors that predict relapse include higher median pretreatment viral loads and persistent detectable viral DNA despite treatment with ganciclovir. This may indicate resistance to ganciclovir. Ganciclovir resistance is uncommon in renal transplant recipients but when it occurs, leflunomide, an agent used in rheumatoid arthritis, can be used instead [11]. This agent works on a different pathway and appears to target virion assembly. Another alternative would be maribavir, a novel antiviral agent that is currently being investigated for prophylaxis but may be effective in ganciclovir resistant disease [19].

3.4 Epstein–Barr Virus (EBV) and Posttransplant Lymphoproliferative Disorders (PTLD) EBV is another member of the herpesvirus family and is one of the most common human viruses worldwide. In the United States, up to 95% of adults have been infected by the ages of 35–40. Infection can be asymptomatic in about half the cases, or can cause infectious mononucleosis in 35–50% of cases [11]. Symptoms of infectious mononucleosis include enlarged and painful lymph nodes, fever, and pharyngitis. Elevation in transaminases is often seen, and splenic enlargement is common. The disease may last up to 2 months, but is rarely fatal and is self-limiting [9]. The virus is secreted into the saliva and this is the main mode of transmission. Despite resolution of symptoms of infectious mononucleosis, EBV remains latent in immune cells throughout a person’s lifetime, and may be reactivated at a later stage. There are several methods for diagnosis. Infectious mononucleosis can be diagnosed with the Paul–Bunnell heterophile antibody test result, and no further testing is necessary. Peripheral blood testing

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reveals a normal to elevated white blood cell count with greater than 10% atypical lymphocytes. In renal transplant patients, however, it is not infectious mononucleosis that develops most often but rather the reactivation of latent EBV infection and manifestation can vary from low grade fevers to rare cases of fulminant hepatitis. The diagnosis is often established through serological or PCR assays. Antibodies to several antigen complexes may be measured. A detectable EBV early antigen suggests that reactivated infection is present, but this test does not have high specificity as a number of healthy people with no symptoms have antibodies to the EBV early antigen for years after their initial EBV infection [11]. Quantitative levels of EBV exceeding 1,000 copies/mL in plasma or 5,000 copies/ mL of whole blood suggest but are not diagnostic of significant disease. Therefore in diagnosing the reactivation of EBV infection in transplant recipients, both serologic and clinical factors should be taken into account. In the nontransplant population EBV has been associated with several malignancies, including Burkitt’s lymphoma and nasopharyngeal carcinoma. In the transplant population, EBV has been closely linked with the development of posttransplant lymphoproliferative disorders (PTLD). In a database of over 5,000 transplant patients of the Cincinnati Transplant Tumor Registry, lymphoproliferative disorders were found to be the most common malignancies after transplantation, excluding nonmelanoma skin cancers and in situ cervical cancer [5]. Most PTLD are B cell Non-Hodgkin’s lymphomas, usually the large B cell type, but T cell and natural killer cell lymphomas can occur. Infection with EBV in transplant recipients seems to be the most important precipitant of PTLD, although EBV-negative disease may occur. PTLD can result from donor or recipient lymphoproliferative cells. Recipient PTLD is more common than donor PTLD but both can be seen in renal transplant recipients. Clinical manifestations of the disease depend on the origin of the lymphoproliferative cells. Recipientorigin disease appears to be more aggressive and disseminated, while donor-associated PTLD is limited in most cases to the allograft [1]. Historically, three types of EBV-related PTLDs are recognized. The first consists of a benign polyclonal B cell proliferation with no malignant transformation and manifests as an acute, mononucleosis-like illness. It

R. Ghoussoub and D.C. Brennan

occurs in 55% of cases. The second disorder presents in a similar manner as the first but is characterized by polyclonal B cell proliferation with, in this case, early malignant transformation. Thirty percent of cases are in this group. In the remaining 15% of cases, the disorder is mainly extranodal and presents with localized solid tumors which denote focal areas of monoclonal B cell proliferation with malignant abnormalities similar to the lymphomas found in AIDS patients [5, 11]. The incidence of lymphoproliferative disorders in the transplant population is significantly higher than in the general population but differs according to the transplanted organ. For instance, it ranges from 1 to 2% in liver transplants, 1 to 3% in renal transplants, 2 to 6% in heart transplants, 2 to 9% in lung transplants and up to 11–33% in intestinal or multiorgan transplants [11]. The overall degree of immunosuppression is on of the major risk factors for disease. EBV-infected cells normally are held in check through an equilibrium between cell division and death that is controlled by cytotoxic T-cells. In immunosuppressed states, impaired T-cell function can lead to unchecked proliferation and transformation leading to PTLD. Although PTLD is a B-cell disorder, primarily, it is important to remember that it results from impairment in T-cell mediated immunity. Those patients treated with higher doses of immunosuppression and particularly those exposed to induction therapy are hence at higher risk of contracting PTLD. OKT3, an induction agent, is rarely used currently as it was found to be associated with a significantly higher incidence of PTLD in a dose-dependent manner in a study on cardiac transplant recipients [17]. In a study comparing cyclosporine and tacrolimus for maintenance therapy without induction, there was a higher risk of PTLD in the tacrolimus group, although when induction was administered no significant difference was found between the two groups [5, 14]. In general, the incidence of PTLD is highest in the first year posttransplant when immunosuppression regimens are the most rigorous [5, 11]. EBV serostatus is another important risk factor for the development of PTLD. EBV seronegative recipients were found to be at a higher risk for contracting PTLD than EBV seropositive recipients (adjusting for OKT3 use and CMV seromismatch). This is likely related to a lack of preoperative immunity against EBV in the seronegative recipient who will probably acquire the infection from the donor postoperatively [1, 5].

3  Clinical Aspects of Infection

37

Thus the EBV D+/R– group is at greater risk of PTLD. Other risk factors are a history of pretransplant malignancy, younger age (pediatric, who are more likely to be EBV seronegative), CMV seromismatch and few HLA allograft matches [5, 11]. Since PTLD symptoms can be subtle, it is critical to have a high index of suspicion for this disease in transplant patients. Symptoms are often similar to those in nontransplant lymphoma patients, including “B symptoms” of night sweats, low grade fevers, and weight loss. Masses or lymphadenopathy can sometimes be found on physical exam. Central nervous system (CNS) PTLD should be suspected in patients with altered mental status or new neurologic findings. Radiologic evidence of a mass or elevations in serum lactic dehydrogenase (LDH) are suggestive of PTLD. Positron emission tomography (PET) scans can delineate metabolically active areas and

aid in diagnosis, and PET/computed tomography (CT) seems to be the most useful modality currently for staging and monitoring of PTLD [11]. Histopathologic evaluation, however, is critical in the diagnosis and classification of the disease. Optimally, a tissue biopsy is required, preferably an excisional biopsy to allow for enough tissue to completely characterize the lesion. To make the diagnosis of EBV-positive PTLD, 2 of the following three features should be found on biopsy in conjunction with a lymphoid tumor [5, 11, 14]:

When CNS lymphoma is suspected, head CT with gadolinium contrast, cerebral spinal fluid (CSF)

Fig.  3.3  PTLD – diffuse large B cell lymphoma. Malignant cells in the center of the field, have large nuclei on H+E (a) are and positive with CD20, a marker for B cells; (b) positive for

Epstein–Barr virus late membrane protein (EBER); (c) this example is a monomorphic non-Hodgkin posttransplant lymphoproliferative disorder (PTLD) × 200

• Disruption of tissue architecture by a lymphoproliferative process • Presence of mono- or oligoclonal cell populations • EBV infection of many cells (Fig. 3.3)

38

analysis for EBV by PCR, CSF cytology and flow cytometry, as well as peripheral titers of circulating EBV load are necessary for diagnosis. Enhancement of CNS lesions on gadolinium CT, positive EBV by PCR in the CSF and increased peripheral EBV load are all highly suggestive of the diagnosis, but confirmation can only be achieved through the presence of malignant lymphocytes in the CSF or by direct biopsy of the lesion [5]. Regular screening for the presence of monoclonal protein in the serum and the urine (SPEP/UPEP) may, in the future, be used to detect the presence or predict the development of PTLD as several studies in liver transplant patients have shown that its presence correlates with development of the disease [5]. Prevention of PTLD entails regular monitoring and treatment of early EBV infection, rapid tapering of tacrolimus in tacrolimus-based regimens, and possibly the use of viral prophylactic agents such as ganciclovir posttransplantation [5, 11]. The increased incidence of PTLD in the EBV D+/R– serogroup indicates that suppression of the primary EBV infection or early detection and treatment of EBV may also decrease the risk of PTLD. Treatment of PTLD varies according to the type of lymphoproliferative disease. There has been no consensus as to one treatment strategy, but it is generally agreed that PTLD of the polyclonal proliferation type, whether the benign or the malignant form, is treated with a reduction in the immunosuppressive regimen and antiviral therapy [1, 5, 13]. In those that are severely ill, reducing the prednisone dose and halting all other immunosuppressive agents is recommended [5]. In those with more limited disease, a decrease in prednisone, tacrolimus, or cyclosporine and discontinuation of mycophenolate mofetil or azathioprine seems to be sufficient to induce remission in many cases [5]. Individualization is advised. There is limited evidence that antiviral therapy is beneficial, but both acyclovir and ganciclovir have been used. In more aggressive monoclonal PTLD, anti-B cell antibodies, chemotherapy, radiation therapy, interferon alpha, intravenous immunoglobulin, or antiviral agents are used in addition to reducing immunosuppression [5, 11]. Early treatment with rituximab, an anti-CD 20 monoclonal antibody with or without chemotherapy appears to have evolved as the standard of care now in patients with CD 20 positive PTLD [5]. Patients with localized disease or CNS lymphoma may also be treated with radiation.

R. Ghoussoub and D.C. Brennan

Negative prognostic factors for PTLD include diagnosis in the first 6 months posttransplant, increasing age, and multiple involvement sites [5, 11]. Overall survival rates are 25–35%, with mortality rates in monoclonal disease as high as 80%. T-cell lymphomas have the poorest prognosis [5, 11].

3.5 BK Virus and Nephropathy Polyomaviruses are part of the Papovaviridae virus family and are small DNA viruses that infect different animals, usually in a species-specific manner. Human polyomaviruses include JC and BK viruses, which have a high seroprevalence (60 – 80%) but become clinically evident only in immunocompromised patients [3, 11]. The route of primary infection is fecal-oral, respiratory, transplacental, or from donor tissue [3]. During a viremic phase, it is presumed that the virus establishes a latent or a permissively lytic infection in target tissues such as the urothelium, lymphoid tissue, and brain [3]. JCV rarely causes nephropathy, so the discussion will focus mainly on BKV infection. BKV viruria is common and occurs in pregnancy, cancer, HIV, diabetes, and transplantation. BKV viremia and nephropathy, however, are specific to the renal transplant population. BKV viremia occurs in 13% and nephropathy in 8% of kidney transplant recipients [3]. Clinical manifestations of BK virus infection include asymptomatic hematuria, hemorrhagic and non-hemorrhagic cystitis, ureteral stenoses and subacute renal failure in HIV and renal transplant recipients [3, 9, 11]. The most important clinical risk factor associated with the development of BK virus infection is the degree of immunosuppression in the transplant recipient. There has been some suggestion that certain combinations of immunosuppressive agents, particularly tacrolimus with or without an antimetabolite, may predispose to BKV infection but a prospective study found that there was a similar incidence of BK viremia and viruria in patients randomized to tacrolimus or cyclosporine regimens [3]. Other factors include donor BK-seropositivity, older age, male gender, diabetes mellitus, and white ethnicity, but these are not universal risk factors [11]. BKV infection appears to be a donor transmitted disease, and supporting that is the finding that recipients whose donors had higher titers of BKV antibodies were more likely to develop the

3  Clinical Aspects of Infection

infection than those with low titers [3]. Another risk factor for infection seems to be injury to the allograft, including ischemic injury, mechanical stress from stent placement, or rejection injury which causes cellular damage and may allow for reactivation of a latent BKV infection [3]. Once the virus is reactivated it spreads via a cell-tocell mechanism and causes an ascending infection. A progressive lytic infection results causing lysis of infected cells and shedding of the virus into the tubule lumen, urine, interstitium, and surrounding cells [3]. This leads to tubular necrosis, cast formation and damage to the basement membrane. Tubular capillary walls may be affected and this leads to vascular spread of the virus. Infiltration with inflammatory cells, tubulitis, and necrosis/apoptosis of non-infected cells may ensue. The end result is allograft dysfunction and loss [3]. Since BKV infection begins with viruria and proceeds to viremia then nephropathy, the initial diagnosis can be made by detecting BKV in the urine through PCR for BKV DNA, RT-PCR for BKV RNA, cytology for BKV inclusion-bearing epithelial cells denoted “decoy cells,” or electron microscopy for viral particles [3] (Fig. 3.4). However, the major limitation with detection of decoy cells is their non-specificity as BKV can be shed from all cells of the urinary tract and detection in the urine does not necessarily preclude nephropathy. A better indicator of nephropathy may be the detection of plasma BKV DNA. Threshold values >10,000 copies/ mL have been suggested, but the distinction between

Fig.  3.4  Electron microscopy of polyomavirus. Round viral particles are arranged in microarrays (arrow), shown here in the cytoplasm of an epithelial cell EM × 6K

39

active BKV nephropathy, resolved BKV nephropathy, and the absence of BKV nephropathy according to these threshold values remains to be clarified [3]. A transplant kidney biopsy remains the gold standard for diagnosis [11]. BKV nephropathy can be focal or isolated, particularly, to the medulla and consequently commonly missed if only one core biopsy is available. It is thus recommended to obtain at least two core biopsies with one biopsy containing tissue from the medulla [3]. If no cytopathic changes are found on routine histology but clinical suspicion remains high, further testing with immunohistochemistry using BK-specific antibodies or antibodies against the cross-reacting SV-40 large-T antigen should be performed [3, 11]. If these tests are still non-confirmatory, a repeat biopsy should be considered. On light microscopy, characteristic findings are intranuclear basophilic and gelatinous-appearing viral inclusions in epithelial cells of the urothelium. These can be found in both the medulla and the cortex. Three histologic patterns have been described (a, b, and c) [3]. • Pattern A: viral cytopathic changes with minimal to no inflammation or tubular atrophy (Fig. 3.5) • Pattern B: viral cytopathic changes with varying degrees of inflammation, tubular atrophy, and fibrosis (Fig. 3.6) • Pattern C: cytopathic changes are less apparent in a background of increased tubular atrophy, interstitial fibrosis, and chronic inflammatory infiltrate (Fig. 3.7)

Fig. 3.5  Pattern A: viral cytopathic changes with minimal to no inflammation. BK intranuclear inclusion bodies (arrows)

40

Fig. 3.6  Pattern B: viral cytopathic changes with BK positive immunohistochemistry, associated with interstitial inflammation (arrow)

Fig. 3.7  Pattern C (courtesy Cinthia Drackenberg): polyomavirus inclusions are less obvious in the background of heavy inflammation. There are many atypical nuclei in tubular epithelial cells but none is convincing for BK. Immunohistochemistry is necessary to make the diagnosis

The degree of histologic damage corresponds with the degree of allograft nephropathy and outcome. The greatest challenge is to distinguish between BKV nephropathy, acute tubular necrosis, interstitial nephritis, and acute cellular rejection. It is thus imperative to correlate histologic findings with blood and urine BKV PCR levels. Some features, like endotheliitis and peritubular C4d deposits point more toward rejection and can be helpful distinguishing features [3]. However,

R. Ghoussoub and D.C. Brennan

they do not negate the presence of BKV nephropathy as both rejection and BKV nephropathy can be present simultaneously. Other histopathologic changes that can be found include glomerular crescents, ischemic glomerulopathy, transplant glomerulopathy, plasma cell infiltrates, and tubular microcalcifications [3, 11]. The pivotal key to treating BKV nephropathy is a reduction in immunosuppression. No specific antiviral therapy exists and therapeutic recommendations are based on anecdotal cases and small case series. However, in cases of progressive allograft dysfunction despite a marked reduction in immunosuppression, antiviral should be considered as there have been anecdotal reports that use of intravenous immunoglobulin, quinolones, cidofovir, and leflunomide have been successful [3]. In decreasing the immunosuppressive regimen, the challenge is to avoid precipitating allograft rejection. There are no standard recommendations for the systematic reduction in immunosuppression, but we tend to discontinue the antimetabolite agent first and reduce the calcineurin inhibitor dose to a level that can still presumably prevent allograft rejection. Additional interventions are based on the clinical response to these changes. Other interventions could include switching from tacrolimus to low-dose cyclosporine as that can also reduce the mycophenolate exposure. Tapering to a prednisone free regimen may also treat BKV nephropathy without triggering many acute rejections [3]. Similar to CMV a preemptive strategy based on early frequent monitoring of BK-PCR in the blood or urine to detect reactivation, prompting immunosuppression reduction can reduce morbidity and mortality. Threshold levels for presumptive disease include [3]: • Urine DNA greater than 10(log7) copies/mL • Urine VP-1 mRNA greater than 6.5 × 10(log5) copies/ng total RNA • Plasma DNA greater than 10(log4) copies/mL An allograft biopsy is recommended if one of the above tests surpasses a threshold value [3]. An international panel in 2005 recommended that screening in all renal transplant patients should be performed every 3 months for up to 2 years posttransplant, when renal allograft dysfunction occurs or when a renal biopsy is performed. We prefer to screen monthly for the first 6 months, at months 9 and 12 and as indicated. BKV nephropathy is not a contraindication to retransplantation, and should be considered in those with poor allograft function [3].

41

3  Clinical Aspects of Infection

3.6 Hepatitis C Hepatitis C viral infection in renal transplant recipients can influence graft survival and outcome. Proteinuria in the HCV positive patient can be due to HCV-induced renal disease and has been associated with a more morbid posttransplant course [19]. MPGN with or without cryoglobulinemia [6, 15], membranous [12], acute, and chronic transplant glomerulopathy as well as renal thrombotic microangiopathy may occur in the transplanted kidney of a hepatitis C positive recipient [12]. Membranoproliferative glomerulonephritis in the HCV positive recipient presents in a similar fashion as MPGN in native kidneys, with hypocomplementemia, proteinuria, microscopic hematuria, and low levels of serum immune complexes, with or without cryoglobulins [12]. Biopsy specimens are also similar in pathology. Membranous glomerulonephritis (MGN) presents with nephrotic range proteinuria and has a similar histologic picture as de novo disease in renal transplant recipients [12]. There seems to be an association, however, between HCV and MGN after renal transplant as it was reported in 18.2% of biopsies in HCV renal transplant patients as opposed to 7.7% in HCV-negative patients [12]. Glomerular lesions should be suspected in HCV positive renal transplant recipients when they exhibit persistent proteinuria or microscopic hematuria, and the gold standard for diagnosis is renal biopsy. There is no standardized treatment for HCV in renal transplant patients yet. Interferon alpha is not recommended due to its significant risk for acute rejection, renal dysfunction and exacerbation of proteinuria [12]. Ribavirin has been used in liver transplant recipients may decrease proteinuria, but has produced only mild improvement in renal transplant recipients [12].

3.7 Parvovirus B19 Parvovirus B19 causes erythema infectiosum in children, hydrops fetalis in pregnant women and aplastic crisis in chronic hemolytic anemia patients. Immu­ nosuppressed patients may exhibit prolonged or persistent viremia due to a decreased ability to clear the virus,

Fig. 3.8  Parvovirus B19 infection in a kidney transplant patient. Viral cytopathic effect is apparent in podocytes which appear smudgy (red arrow). In addition, there is collapsing glomerulopathy. Courtesy Dr. Laura Barisoni, New York University Department of Pathology

and renal transplant patients can develop symptomatic B19 infections. The virus can be acquired via the respiratory route, the allograft, or the reactivation of latent infection [18]. Red cell aplasia or other cytopenias are the most common presentation in this patient population. It should be suspected in a renal transplant patient with unexplained anemia, reticulocytopenia or pancytopenia [18]. The two glomerular lesions reported in these patients have been collapsing glomerulopathy and thrombotic microangiopathy [18] (Fig. 3.8). Diagnosis is established with serologic tests (PCR), and treatment consists of decreasing immunosuppression and/or administering immunoglobulin therapy [18].

3.8 Fungal Infections Since renal transplant recipients are immunosuppressed, they are naturally susceptible to various fungal infections which can occur in the general nontransplant population or are specific to immunocompromised hosts. Systemic fungal infections occur in 2–14% of renal transplant recipients and they preclude significant increases in morbidity and mortality as about two thirds of renal transplant recipients with systemic mycoses die. Clinical manifestations are often nonspecific so a high index of suspicion should be retained [9, 11]. Rarely fungal elements can be seen

42

Fig. 3.9  The patient had a fungal infection of the renal allograft. There is massive glomerular infiltration with yeast spores stained dark purple with PAS × 60. Courtesy of Dr. Luis Salinas Madrigal, Saint Louis University

in the renal allograft (Fig. 3.9). Therapy includes specific antifungal agents and risk factor reduction, such as removing IV catheters and decreasing immunosuppression. Candidiasis, cryptococcosis, aspergillosis, mucormycosis, histoplasmosis, coccidiomycosis, and pneumocystosis can occur. There is no standard recommendation for antifungal prophylaxis.

3.9 Pneumocystis Jiroveci (PCP) PCP is an extracellular organism that has both fungal and protozoan features. Historically, without prophylaxis for Pneumocystis jiroveci (cystis) (PCP), 6–20% developed the infection within the first year after transplantation. The clinical presentation is usually subacute with mild cough, fever, dyspnea, and interstitial infiltrates with or without cysts, and variable degrees of hypoxemia [11]. Bronchoalveolar lavage (BAL) with transbronchial biopsy and staining is a highly sensitive method of diagnosis (Fig. 3.10). Trimethoprimsulfamethoxazole is the first line of treatment, usually for 14–21 days [8]. Second-line therapy is pentamidine or dapsone-trimethoprim [8, 9, 11]. Mild to moderate PCP pneumonia can be treated with atovaquone in sulfa-allergic patients. Prophylaxis is effected with TMP-SMX, orally on a daily basis up to 1 year [8]. Dapsone, pentamidine, and atovaquone can be used as prophylactic alternatives [8].

R. Ghoussoub and D.C. Brennan

Fig.  3.10  Pneumocystis pneumonia in lung. Microorganisms are often found within frothy alveolar fluid and are oval or round membrane bound cysts containing distinct eccentric nuclei. Silver stain × 250

3.10 Summary The transplant population is at significant risk for developing infections that can directly or indirectly affect the posttransplant course, allograft outcome and overall patient survival. Risk factor delineation, rigorous monitoring in high-risk groups, accurate diagnosis and early treatment can all play a part in improving outcomes and reducing infection. Many unanswered questions persist; therefore, there continues to be a need for continued epidemiologic and randomized studies in transplant infection to improve allograft and patient outcomes posttransplantation.

References   1. Adami, J., Gabel, H., Lindelof, B., et al.: Cancer risk following organ transplantation: a nationwide cohort study in Sweden. Br. J. Cancer 89(7), 1221–1227 (2003)   2. Asberg, A., Humar, A., Rollag, H., et al.: Oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients. Am. J. Transplant. 7(9), 2106–2113 (2007)   3. Bohl, D.L., Brennan, D.C.: BK virus nephropathy and kidney transplantation. Clin. J. Am. Soc. Nephrol. 2 Suppl 1, S36–S46 (2007)   4. Brennan, D.C.: Cytomegalovirus in renal transplantation. J. Am. Soc. Nephrol. 12(4), 848–855 (2001)   5. Caillard, S., Agodoa, L.Y., Bohen, E.M., Abbott, K.C.: Myeloma, Hodgkin disease, and lymphoid leukemia after

3  Clinical Aspects of Infection renal transplantation: characteristics, risk factors and prognosis. Transplantation 81(6), 888–895 (2006)   6. Cruzado, J.M., Gil-Vernet, S., Ercilla, G., et al.: Hepatitis C virus-associated membranoproliferative glomerulonephritis in renal allografts. J. Am. Soc. Nephrol. 7(11), 2469–2475 (1996)   7. Farrugia, E., Schwab, T.R.: Management and prevention of cytomegalovirus infection after renal transplantation. Mayo Clin. Proc. 67(9), 879–890 (1992)   8. Fishman, J.A.: Pneumocystis carinii and parasitic infections in transplantation. Infect. Dis. Clin. N. Am. 9(4), 1005–1044 (1995)   9. Fishman, J.A., Rubin, R.H.: Infection in organ-transplant recipients. N Engl J. Med. 338(24), 1741–1751 (1998) 10. Khoury, J.A., Storch, G.A., Bohl, D.L., et al.: Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. Am. J. Transplant. 6(9), 2134–2143 (2006) 11. Kubak, B., Maree, C.L., Pegues, D., Hwang, A.: Infections in kidney transplantation. Handbook of kidney transplantation 4th ed. Ed Danovitch G, 2004. Lippincott Williams & Wilkins, Philadelphia

43 12. Morales, J.M.: Hepatitis C virus infection and renal disease after renal transplantation. Transplant. Proc. 36(3), 760–762 (2004) 13. Nalesnik, M.A., Makowka, L., Starzl, T.E.: The diagnosis and treatment of posttransplant lymphoproliferative disorders. Curr. Probl. Surg. 25(6), 367–472 (1988) 14. Penn, I.: Cancers complicating organ transplantation. N Engl J. Med. 323(25), 1767–1769 (1990) 15. Roth, D., Cirocco, R., Zucker, K., et al.: De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients. Transplantation 59(12), 1676–1682 (1995) 16. Rubin, R.H.: Infectious disease complications of renal transplantation. Kidney Int. 44(1), 221–236 (1993) 17. Swinnen, L.J., Costanzo-Nordin, M.R., Fisher, S.G., et al.: Increased incidence of lymphoproliferative disorder after immunosuppression with the monoclonal antibody OKT3 in cardiac-transplant recipients. N Engl J. Med. 323(25), 1723– 1728 (1990) 18. Waldman, M., Kopp, J.B.: Parvovirus-B19-associated complications in renal transplant recipients. Nat. Clin. Pract. Nephrol. 3(10), 540–550 (2007) 19. Winston, D.J., Emmanouilides, C., Busuttil, R.W.: Infections in liver transplant recipients. Clin. Infect. Dis. 21, 1077– 1089 (1995). quiz 1090–1071

4

Clinical Evaluation of Alloantibodies in Solid Organ Transplantation Gerald P. Morris and T. Mohanakumar

4.1 Introduction

4.2 Alloantibody Specificities

The importance of the humoral immune response in transplantation results from the landmark observation by Paul Terasaki and colleagues in the 1960s and has defined clinical histocompatibility testing for solid organ transplantation for over 40 years. Demonstration that hyperacute graft rejection, defined as graft dysfunction and tissue death within minutes to hours of transplantation, is associated with the presence of preformed antibodies (Abs) against donor histocompatibility antigens, underscored the critical nature of clinical testing for allotypic Abs [61, 79, 87]. Development of Abs against donor antigens, or otherwise known as donorspecific Abs (DSA), after transplantation is similarly associated with subsequent development of antibodymediated rejection (AMR) and graft failure [48, 56]. The discovery of alloantibodies and their influence in mediating rejection of allogeneic transplants spurred clinical testing to detect Abs against donor antigens to accurately define the risk of hyperacute rejection and AMR.

4.2.1 Alloantibodies Against HLA

G.P. Morris Department of Pathology and Immunology, Washington University School of Medicine, Box 8109-3328 CSRB, 660 South Euclid Avenue, St. Louis, MO 63110, USA T. Mohanakumar (*) Department of Pathology and Immunology, Department of Surgery, Washington University School of Medicine, Box 8109-3328 CSRB, 660 South Euclid Avenue, St. Louis, MO 63110, USA e-mail: [email protected]

Sensitization against Human Leukocyte Antigens (HLA) has been defined as the primary barrier to allogeneic transplantation [28]. The importance of HLA as an alloantigen results from a combination of factors, the foremost of which is the high degree of polymorphism, with as many as 200 known polymorphisms for each of the six HLA antigens [71, 90]. Additionally, HLA antigens are expressed on nearly all cells, with the class I HLA molecules HLA-A, HLA-B, and HLA-C expressed at relatively high levels on all nucleated cells in the body, and the class II molecules HLA-DR, HLA-DQ, and HLA-DP expressed constitutively on macrophages, dendritic cells, B cells, and epithelial and human T cells under inflammatory conditions. The nearly ubiquitous expression of HLA molecules provides sufficient antigen for development of a robust immune response. The influence of HLA in solid organ transplants is clear, as early transplantation prior to effective immunosuppression was limited only to HLA-identical individuals, and complete HLA matching for HLA-A, -B, and -DR demonstrated clearly superior outcomes in kidney, pancreas, and heart transplantation [66, 84, 109]. DSA against HLA-A, -B, and -DRB1 are the most frequently encountered alloantibodies correlated with AMR, and are generally considered contraindications for transplantation ([38],). Hyperacute rejection and AMR mediated by DSA against HLA-C, -DPA1, -DQA1, and -DQB1 loci have also been des­cribed [22, 29, 36, 46, 70, 121], indicating the ­importance of evaluating Abs to all HLA antigens. Additionally, alloantibodies to HLA molecules can be crossreactive to other

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_4, © Springer-Verlag Berlin Heidelberg 2011

45

46

G.P. Morris and T. Mohanakumar

distinct HLA alleles, expanding the barriers to transplantation posed by a given allotypic Ab [54, 96, 103, 104]. Several broad crossreactive groups (CREGs) have been defined (Table 4.1) and must be evaluated for potential immunologic incompatibility resulting from specific anti-HLA Abs. HLAMatchmaker, an algorithm that considers structural similarities between HLA molecules to predict crossmatch compatibility has been developed [32, 33]. This approach of attempting to match particular immunogenic epitopes has been shown to be beneficial in enhancing prediction of compatible donors for patients with large numbers of alloantibodies [34, 45, 123]. HLA sensitization, which can occur through a variety of means, affects a large percentage of potential transplant recipients. Pregnancy was identified as a means of sensitization in early characterization of HLA, which utilized serum from multiparous women with specific Abs against their children’s paternal HLA antigens [90]. Although the frequency of pregnancies that result in alloimmunization is not clearly defined, it is known that the degree of multiparity correlates with the likelihood of developing anti-HLA Abs. HLAsensi­tization has also been associated with transfusion of leukocyte-containing blood products, with over 40% of potentially sensitizing events resulting in alloimmunization [24, 124]. The high rate of sensitization, combined with the resulting difficulty with

subsequent transfusion and transplantation, has led to widespread use of leukoreduced blood products when possible [124]. Not surprisingly, the highest rates of alloimmunization result from solid organ transplantation. The rate of HLA sensitization following solid organ transplantation varies between reports, although generally reports concur that 10–20% of non-HLAmatched graft recipients develop anti-HLA Abs within the first 12 months, and nearly all (>95%) patients develop alloantibodies at some point following transplantation ([19, 37, 48, 76, 79]). Sensitization against HLA has also been reported outside of these mechanisms, as a small proportion of non-transfused men have anti-HLA Abs. It has been proposed that these anti-HLA Abs may be the result of crossreactivity between microbial antigens and HLA, but it is unclear as to the frequency with which this occurs [99, 100]. Anti-HLA Abs are of concern in transplantation not only because of their role in hyperacute rejection, but also because of their role in chronic graft dysfunction. De novo development of anti-HLA Abs following transplantation occurring in the majority of solid organ transplant recipients is associated with poor outcome in the transplant of all solid organs ([19, 48, 49, 56]). While detection of anti-HLA Abs in serum is not by itself direct evidence of concurrent AMR, the prognostic value is clear, as development of anti-HLA Abs precede rejection by months to years ([48, 52, 65, 118]).

Table 4.1  HLA-cross reactive groups CREG

HLA alleles

Population frequency (%)

A1c

A1, A3, A9 (23, 24), A29, A30, A31, A36, A80, A10c

79

A10c

A10 (A25, A26, A34, A66), A11, A32, A43, A74, A28c

20

A28c

A28 (A68, A69), A33, A34, A26

11

A2c

A2, A9 (A24, A34), A28 (A68, A69), B17 (B57, B58)

70

B5c

B5 (B51, B52), B15 (B62, B63, B75, B76, B77, B78), B18, B21, (B49, B50), B35, B46, B53, B70 (B71, B72), B73, B17 (B57, B58)

50

B7c

B7, B13, B22 (B54, B55, B56), B27, B40 (B60, B61), B41, B42, B47, B48, B59, B67, B73, B81, B82

54

B8c

B8, B14 (B64, B65), B16 (B38, B39), B18, B59, B67

38

B12c

B12 (B44, B45), B13, B21 (B49, B50), B37, B40 (B60, B61), B41, B47

44

Bw4

B5 (B51, B52), B13, B17, B27, B37, B38 (B16), B44 (B12), B47, B49 (B21), B59, B63 (B15), B77 (15), A9 (A23, A24), A2403, A25, A32

79

Bw6

B7, B703, B8, B14 (B64, B65), B18, B22 (B54, B56), B2708, B35, B39, B3901, B3902, B40 (B60, B61), B4005, B41, B42, B46, B48, B50, B62, B67, B70 (B71, B72), B73, B75, B76, B78, B81, B82

87

4  Clinical Evaluation of Alloantibodies in Solid Organ Transplantation

Anti-HLA Abs mediate rejection primarily by activation of complement, causing cell lysis and recruitment of activated immune cells. Correlation between serum anti-HLA Abs and complement activation has been demonstrated in multiple studies comparing histochemical staining for the complement component C4d and detection of rejection [15, 25, 27, 41, 86, 120]. Anti-HLA Abs binding to endothelial and epithelial cells can also directly damage allografts; binding of anti-HLA Abs directly alters cellular physiology and leads to hyperproliferation [83, 102, 107].

4.2.2 ABO Blood Group Antibodies Abs against determinants of the ABO blood group system were described over a century ago by Karl Land­ steiner as the mediators of hemolytic transfusion reactions [63]. Anti-ABO Abs have long been viewed as an uncrossable barrier to transplantation [53, 81, 89, 112] due to their ubiquitous expression on erythrocytes as well as most tissues in the body [85]. Universal sensitization results from crossreactivity between AB antigens and carbohydrate moieties of intestinal microbial flora. Early attempts at transplantation across ABO blood groups were minimally successful, with rejection of transplanted organs occurring within days with pathology similar to hyperacute rejection from antiHLA Abs [53, 81, 111, 129]. An important observation from these early studies was that a subgroup of grafts that expressed lower concentrations of the A antigen on their cell surface (blood group A2) were less likely to be rejected by recipients with anti-A Abs [18, 21]. While A2-incompatible grafts had significantly worse outcomes than fully ABO-compatible grafts, this limited success demonstrated the possibility of transplantation across ABO blood groups. Advances in immunosuppression, including the ability to remove circulating anti-ABO Abs by hemodialysis and plasmapheresis, led to increased success in transplantation of kidney, liver, and heart grafts across ABO blood groups, although splenectomy was often required as part of the immunosuppressive regimen [3, 20, 47, 97, 105, 108, 128]. Interestingly, discontinuation of anti-ABO Ab removal after transplantation results in reemergence of anti-ABO Abs, but the grafts are often unaffected, demonstrating immunologic accommodation [3, 10, 23]. Continued advancements in immunosuppression,

47

including specific targeting of B cells, have also impro­ ved outcomes in ABO-incompatible transplants and reduced the need for splenectomy [60, 119].

4.2.3 MICA While the majority of allotypic Abs mediating AMR are directed against the HLA and ABO antigens, other Abs of significance have been demonstrated. Recently, the major histocompatibility complex class I chain-related gene A (MICA), a highly polymorphic glycoproteins with over 65 known alleles, has been demonstrated as a target for allotypic Abs [57, 113]. MICA is structurally related to Class I MHC, expressed on most nonhematopoietic cell lineages, and functions in innate immunity [9, 72, 136, 137]. Several broadly reactive serological groups have been defined for the most common MICA alleles (Table 4.2) [134]. Reports have demonstrated AMR mediated by anti-MICA Abs, with evidence of complement fixation and cytotoxicity similar to anti-HLA Abs [50, 75, 113, 114, 133, 135]. The rate of sensitization to mismatched MICA antigens is high, as 6% of healthy individuals, 21% of multiparous women, and as many as 50% of kidney allograft recipients have anti-MICA Abs [77, 135]. The percentage of AMR directly attributable to anti-MICA Abs is unclear, as they often arise in conjunction with anti-HLA Abs, though large retrospective and prospective studies have clearly demonstrated infrequent, but directly attributable, anti-MICA Abs mediating AMR [5, 76]. MICA Abs should be considered in cases of AMR or crossmatch incompatibility not explained by anti-HLA DSA.

4.2.4 Alloantibodies to Non-HLA Antigens A variety of other cellular antigens have been demonstrated to be targets of allotypic Abs. While no other antigens approach the polymorphic diversity of the HLA system, several other polymorphic proteins have been reported to generate alloantibody responses mediating AMR. Examples of proteins with limited polymorphisms but reported involvement in AMR are the glutathione S-transferase T1 (GSTT1) and angiotensin type 1 receptor (AT1R). GSTT1 is expressed in the liver and kidney, making it a potential target for rejection of these grafts. Transplant of GSTT1-positive graft into a

48

G.P. Morris and T. Mohanakumar

Table 4.2  MICA serologic groups

expression, endothelial antigens are an important target for alloantibodies due to their role as the primary interface between the humoral immune system and the allograft. Anti-endothelial Abs (AECA) are a group of Abs against heterogeneous endothelial cell antigens that mediate kidney, heart, and lung allograft rejection [31, 40, 68, 93, 98, 115]. Development of AECA is relatively common in solid organ transplantation, and correlates with sensitization to other alloantigens; retrospective analyses estimate over 40% of HLAsensitized patients have AECA, with increased frequency among patients who rejected the graft [40, 62]. A multicenter study demonstrated that detection of AECA identified by cellular crossmatch has prognostic value in predicting AMR [17].

Serologic reactivity

MICA alleles

MICA-1

MICA*001

MICA-4

MICA*004

MICA-6

MICA*006

MICA-12

MICA*012

MICA-1-12-18

MICA*001, MICA*012, MICA*018

MICA-2-17

MICA*002, MICA*017

MICA-4-6-9

MICA*004, MICA*006, MICA*009

MICA-8-19

MICA*008, MICA*019

MICA-4-6-9-19

MICA*004, MICA*006, MICA*009, MICA*019

MICA-G1

MICA*001, MICA*002, MICA*007, MICA*012, MICA*017, MICA*018

4.3 Clinical Testing for Alloantibodies

MICA*004, MICA*006, MICA*008, MICA*009, MICA*019

4.3.1 Cytotoxicity Crossmatch

MICA-G2

MICA-G3

MICA*002, MICA*004, MICA*006, MICA*008, MICA*009, MICA*012, MICA*017, MICA*018, MICA*019

MICA-G4

MICA*001, MICA*002, MICA*004, MICA*006, MICA*009, MICA*012, MICA*017, MICA*018

MICA-G5

MICA*002, MICA*004, MICA*006, MICA*008, MICA*009, MICA*017, MICA*019

negative recipient leads to the development of antiGSTT1 Abs, which have been demonstrated in liver and kidney allograft rejection [1, 2, 4]. Approxi­mately 20% of the Caucasian population is negative for GSTT1, making mismatch between positive donors and negative recipients a potentially common problem. Similarly, mismatch of AT1R which occurs between relatively infrequent polymorphisms with no known physiologic relevance, were similarly described in renal allograft rejection [30, 73]. While some antigens are targets for development of Abs primarily due to polymorphisms or heterogeneous

Initially, alloantibodies were detected in pretransplant sera by the same cytotoxicity assays used to define HLA polymorphisms. Clinical detection of alloantibodies originated with the development of the complement-dependent lymphocytotoxicity assay (CDC) [117]. The CDC evaluates the presence of donor-specific alloantibodies in recipient sera by measuring lysis of donor cells with the addition of 1 mL recipient sera and rabbit complement. Cytotoxicity is assessed by visualization of uptake of fluorescent dye by killed cells (Fig.  4.1). The CDC provided a standardized methodology for testing humoral immunologic compatibility and interpretation of results (Table 4.3) [74]. The dramatic decrease in hyperacute rejection by elimination of CDC crossmatch positive transplants has made CDC the gold standard for defining immunologic incompatibility [87]. However, while testing by CDC greatly reduced the incidence of hyperacute rejection, it did not eliminate it entirely. Several modifications were made to CDC assay to improve sensitivity, including addition of multiple wash steps to reduce anti-complement factors present in sera (the Amos 3-Wash technique) [7], addition of anti-human globulin (AHG) to enhance sensitivity [58], and differential labeling of T and B cells to discriminate between anti-HLA class I

49

4  Clinical Evaluation of Alloantibodies in Solid Organ Transplantation Table 4.3  Interpretation of CDC

Fig.  4.1  Interpretation of complement-dependent cytotoxic crossmatch. Complement-dependent cytotoxic crossmatch is the gold standard assay for determining immunologic compatibility for solid organ transplantation. Briefly, 1 mL of recipient serum is incubated with donor leukocytes in the presence of complement, and cytotoxicity is measured by uptake of orange fluorescent propidium iodide. (a) A negative CDC result with less than 10% of donor leukocytes positive for cell lysis. (b, c) Examples of positive CDC reactions. Both have T cells and B cells (identified by the binding of green fluorescent FITC-labeled anti-IgG) positive for propidium iodide uptake, though (c) illustrates a much stronger reaction

Reaction

Cell death (%)

0

Un-interpretable

Interpretation

1

0–10

Negative

2

11–25

±

4

26–50

Positive

6

51–75

Positive

8

76–100

Positive

and class II Abs [43]. These refinements have markedly improved the sensitivity of CDC and are widely used in most clinical laboratories. While a positive CDC is a clear contraindication for transplantation, rare cases of hyperacute rejection occur in CDC negative crossmatch (CXM). To improve the sensitivity of crossmatching, a flow-cytometrybased crossmatch (FCXM) was developed [42]. FXCM involves incubation of donor leukocytes with recipient serum, and subsequent labeling of DSA bound to donor T or B leukocytes with fluorophore-labeled antihuman IgG for analysis on a flow cytometer. The results of FCXM are reported as the difference between the mean channel fluorescence (MCF) of the donor cells incubated with recipient serum and the MCF of donor cells incubated with control serum. Positive results are defined by an intra-laboratory developed cutoff value determined by analysis of multiple sera samples from non-alloimmunized individuals. FCXM has demonstrated improved sensitivity over CDC, with multiple retrospective and prospective studies demonstrating an improvement in detection of DSA mediating hyperacute rejection and AMR [26, 59, 69]. The improved sensitivity of FCXM has made it a replacement test for CDC in many transplant centers, though several problems exist in interpreting FCXM data. The major problem with FXCM is lack of specificity for positive results. Several reports have demonstrated that FCXM positivity, particularly with DSA specific for B cells (i.e., MHC Class II) with a low MCF shift, may not predict humoral rejection [39, 64]. This lack of predictive value of B cell positive FCXM is attributed to a variety of factors, including functionally weak DSA and the lower expression of HLA-DP and -DQ on the surface of most cell types. Many centers do not consider weak B cell positive FCXM a barrier to

50

transplantation. It has been suggested that the specificity and predictive value of FXCM can be improved by combination of FCXM with definition of alloantibody specificity determined by immunoassay [39]. More recently, a technique using flow cytometry bead coated with donor lysate, the LumXM, is shown to have improved sensitivity and specificity [13, 14]. This results from specific binding of HLA to the beads, thus increasing specificity; increased antigen density improves sensitivity.

4.3.2 Solid Phase Immunoassays for Detection of Alloantibodies While CDC and FCXM provide direct evidence for immunologic compatibility between donor and recipient, they have the limitation of requiring one or more donors of known HLA type for evaluation of alloantibodies. The highly polymorphic nature of HLA requires testing a large number of cell types to accurately screen for alloantibodies. To address this problem, immunoassays for detecting anti-HLA Abs were developed in the 1980s using ELISA to efficiently test patient sera against a large panel of cell lysates [67, 78, 132]. ELISA demonstrated improved sensitivity over CDC and FCXM [78, 132]. The use of cell lysates enables robust examination of alloantibodies, though determination of specificity remains problematic. Complexity in identification of Ab specificity led to the development of ELISA panels utilizing donor cells with HLA alleles representative of their frequency in the population, with reactivity evaluated as the percentage of donor lysates producing a positive reaction (panel reactive Ab or PRA) (Table 4.4). PRA is useful in providing a general measure of alloimmunization status and providing a general likelihood of finding an immunologically compatible donor. Additionally, PRA is useful in directing pre- and post-transplant treatment of patients likely to receive less immunologically compatible grafts (such as heart and lung graft recipients). Immunoassay specificity was greatly improved with the advent of the multiplex flow assay using recombinant individual HLA proteins, the Luminex SA [91, 92]. In addition to simplifying interpretation, with computer algorithms determining alloantibody specificity, the flow-based immunoassay demonstrated further improved sensitivity [35, 55, 88]. This increase in

G.P. Morris and T. Mohanakumar Table 4.4  Interpretation of panel reactive antibody (PRA) PRA (% panel reactive)

Interpretation

Treatment indication

<20

Non- or lowsensitized

None

20–50

Moderately sensitized

Increased posttransplant monitoring Possibly increased immunosuppression

>50

Highly sensitized

Increased immunosuppression Consider pre- and post-transplantation apheresis

sensitivity is clinically relevant, as detection of DSA by immunoassay is a strong predictor of AMR, particularly in patients with negative or equivocal crossmatch results [5, 6, 80, 125]. This discrepancy between DSA identified by highly sensitive immunoassays and those identified by cytotoxic crossmatch has raised questions about the clinical significance of Abs detected by immunoassay alone. Multiple reports demonstrated successful transplantation in the presence of low concentrations of DSA [80, 94, 101]. It has been speculated that DSA detected only by immunoassay and not mediating AMR may reflect Abs with poor complement-fixing ability, low functional avidity, or present in very low circulating concentrations, making them less likely to mediate AMR. This has led to investigation of methods to improve the specificity of immunoassays, including addition of a functional component such as complement fixation. However, data regarding increased specificity by the addition of C4d binding by DSA in a flow cytometry-based assay have been equivocal [11, 126, 127]. More commonly, attempts have been made to directly correlate DSA concentration with AMR, though no clear cutoffs between clinically-relevant and irrelevant Abs have been defined presently [5, 6, 80, 125].

4.3.3 Virtual Crossmatching and Donor Selection Significant evidence demonstrates the importance of clinical testing for DSA in solid organ transplantation. Since the first description of anti-HLA Abs detected by CXM as a defining risk factor for graft rejection,

51

4  Clinical Evaluation of Alloantibodies in Solid Organ Transplantation

improvements in the detection of DSA have nearly eliminated hyperacute rejection, and have greatly reduced AMR. While CXM remains the gold standard assay for determination of immunologic compatibility, it is not foolproof. Current use of multiplex immunoassay using single antigens has greatly improved the sensitivity for detection of alloantibodies, and reduced transplants across DSA that would not be detected by CXM but still result in rejection. The strategy of virtual crossmatching, or selection of donor-recipient pairs based upon knowledge of donor HLA type and recipient alloantibody profile, has led to improved organ allocation, directing shared organs to centers with the most likely compatible matches [12, 16, 122]. Virtual crossmatching is not infallible, however, as it is critically limited by the information used to determine compatibility. Accurate estimation of immunologic compatibility requires full knowledge for donor HLA type and recent alloantibody profile of the recipient. Knowledge of the donor organ HLA type is often limited to HLA-A, -B, and -DR antigens, while multiplex alloantibody testing is most commonly limited to antiHLA Abs, ignoring the contribution of allotypic Abs against MICA and other alloantigens. A crossmatch with donor cells, either CXM or FCXM, should be performed prior to any solid organ transplantation to minimize the risk of hyperacute rejection (Table 4.5). In the setting of living donor directed donations, it is worth considering crossmatch in the setting of weak

DSA, as multiple reports describe successful longterm transplantation against weak DSA [94, 101].

4.3.4 Posttransplant Testing Detection of alloantibodies is important not only in selection of immunologically compatible donor recipient pairs, but also to detect development of AMR. The development of de  novo DSA following allogeneic transplantation has long been associated with poor outcome [48, 56]. Despite current immunosuppressive regiments, development of alloantibodies occurs in the majority of allograft recipients; several large retrospective analyses have demonstrated high rates (14–24%) of DSA formation within 12 months following transplantation of kidney, liver, heart, and lung allografts and a majority (50–95%) developing alloantibodies at some point following transplantation [37, 52, 65, 76, 95, 118, 130]. These are primarily anti-HLA DSA, but reports have demonstrated Abs to a variety of antigens including MICA and AECA mediating allograft rejection [5, 17, 76, 135]. While development of alloantibodies is not direct evidence of AMR, detection of DSA in the presence of decreasing graft dysfunction is considered evidence for AMR [110, 116]. Multiple studies have demonstrated that detection of DSA by immunoassay is significantly more sensitive than

Table 4.5  Methods for evaluation of alloantibodies Test method

Test principle

Advantages

Disadvantages

Cytotoxic crossmatch (CDC)

Examination of recipient serum for DSA capable of mediating complementdependent lysis of donor cells

Gold standard for direct determination of donor and recipient immunologic compatibility Tests for reactivity to all allogeneic proteins found on donor cells

Time and labor intensive Technically difficult Requires viable donor or surrogate cells Only tests alloantigens on leukocytes

Flow cytometric crossmatch (FCXM)

Examination of recipient serum for DSA capable of binding to donor cells

Improved sensitivity over CDC Tests for reactivity to all allogeneic proteins found on donor cells

Requires viable donor or surrogate cells Only tests alloantigens on leukocytes May reveal weak alloantibodies not mediating humoral rejection

Multiplex immunoassay

Multiplex immunoassay of donor serum against single recombinant alloantigens

Improved sensitivity over CDC and FCXM Improved ability to identify specific alloantibodies High throughput

Only tests alloantigens in assay (usually only HLA) May reveal weak alloantibodies not mediating humoral rejection

52

detection of C4d deposition by histological examination, and has the added benefit of being performed without an invasive biopsy [15, 25, 27, 41, 86, 120]. Recent investigation of endothelial gene transcription associated with endothelial cell damage has demonstrated improved sensitivity of DSA testing at the molecular level [107].

4.3.5 Immunologic Accommodation While the link between alloantibodies and graft rejection is clearly demonstrated, it is not simply that all alloantibodies mediate AMR and preclude transplantation. The most striking example of this is transplantation in the presence of Abs against ABO blood group antigens, where removal of the antibodies by plasma exchange therapy enables transplantation across ABO groups, but anti-ABO Abs reappear following transplantation without causing graft rejection [3, 10, 23]. This disconnecting phenomenon between alloantibodies and survival of immunologically incompatible grafts is termed accommodation, and is thought to result from a combination of alterations in target cells including down-regulation or shedding of antigenic determinants, and increasing anti-apoptotic gene expression to resist killing [8, 82, 83, 106, 131]. Accommodation is not unique to anti-ABO Abs, as multiple reports demonstrated that low concentrations of circulating anti-HLA Abs may not be absolute contraindication to transplantation [44, 51, 94].

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40. Ferry, B.L., Welsh, K.I., Dunn, M.J., et al.: Anti-cell surface endothelial antibodies in sera from cardiac and kidney transplant recipients: association with chronic rejection. Transpl. Immunol. 5, 17–24 (1997) 41. Feucht, H.E., Schneeberger, H., Hillebrand, G., et  al.: Capillary deposition of C4d complement fragment and early graft loss. Kidney Int. 43, 1333–1338 (1993) 42. Garovoy, M.R., Rheinschmilt, M.A., Bigos, M., et al.: Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation. Transplant. Proc. 15, 1939–1944 (1983) 43. Gebel, H.M., Oldfather, J.W., Karr, R.W., et al.: Antibodies directed against HLA-DR gene products exhibit the CYNAP phenomenon. Tissue Antigens 23, 135–140 (1984) 44. Gloor, J.M., DeGoey, S., Ploeger, N., et al.: Persistence of low levels of alloantibody after desensitization in crossmatch-positive living donor kidney transplantation. Transplantation 78, 221–227 (2004) 45. Goodman, R.S., Taylor, C.J., O’Rourke, C.M., et al.: Utility of HLAMatchmaker and single-antigen HLA-antibody detection beads for identification of acceptable mismatches in highly sensitied patients awaiting kidney transplantation. Transplantation 81, 1331–1336 (2006) 46. Goral, S., Prak, E.L., Kearns, J., et  al.: Preformed donordirected anti-HLA-DP antibodies may be an impedimant to successful kidney transplantation. Nephrol. Dial. Transplant. 23, 390–392 (2008) 47. Gordon, R.D., Iwatsuki, S., Esquivel, C.O., et al.: Experience with primary liver transplantation across ABO blood groups. Transplant. Proc. 19, 4575–4579 (1987) 48. Hachem, R.R., Yusen, R.D., Meyers, B.F., et al.: Anti-human leukocyte antigen antibodies and preemptive antibody-directed therapy after lung transplantation. J. Heart Lung Transplant. J Heart Lung Transplant. 2010 Jun 15 (epub) 49. Halloran, P.F., Wadgymar, A., Ritchie, S., et al.: The significance of anti-class I antibody response. Clinical and pathologic features of anti-class I mediated rejection. Transplantation 49, 85–91 (1990) 50. Hankey, K.G., Drachenberg, C.B., Papadimitriou, J.C., et al.: MIC expression in renal and pancreatic allografts. Transplantation 73, 304–306 (2002) 51. Higgins, R., Hathaway, M., lowe, D., et al.: Blood levels of donor-specific human leukocyte antigen antibodies after renal transplantation: resolution of rejection in the presence of circulating donor-specific antibody. Transplantation 84, 876–884 (2007) 52. Hourmant, M., Cesbron-Gautier, A., Terasaki, P.I., et  al.: Frequency and clinical implications of development of donor-specific and non-donor-specific HLA antibodies after kidney transplantation. J. Am. Soc. Nephrol. 16, 2804–2812 (2005) 53. Hume, D.M., Merrill, J.P., Miller, B.F., et al.: Experiences with renal homotransplantation in the human: report of nine cases. J. Clin. Invest. 34, 327–382 (1955) 54. Hwang, S.H., Oh, H.B., Shin, E.S., et al.: Influence of mismatching of HLA cross-reactive groups on cadaveric kidney transplantation. Transplant. Proc. 37, 4194–4198 (2005) 55. Ishida, H., Tanabe, K., Furusawa, et al.: Evaluation of flow cytometric panel reactive antibody in renal transplant recipients- examination of 238 cases of renal transplantation. Transpl. Int. 18, 163–168 (2005)

54 56. Jeannet, M., Pinn, V.W., Flax, M.H., et al.: Humoral antibodies in renal allotransplantation in man. N Engl J. Med. 282, 111–117 (1970) 57. Jinushi, M., Takehara, T., Kanto, T., et  al.: Critical role of MHC class I-realted chain A and B expression on IFN0alphastimulated dendritic cells in NK cell activation: impairment in chronic hepatitis C virus infection. J. Immunol. 170, 1249–1256 (2003) 58. Johnson, A.H., Rosen, R.D., Butler, W.T.: Detection of alloantibodies using a sensitive antiglobulin microcytotoxicity test: identification of low levels of preformed antibodies in accelerated allograft rejection. Tissue Antigens 2, 215–226 (1972) 59. Karpinski, M., Rush, D., Jeffery, J., et al.: Flow cytometric crossmatching in primary renal transplant recipients with a negative ani-human globulin enhanced cytotoxicity crossmatch. J. Am. Soc. Nephrol. 12, 2807–2814 (2001) 60. Kawagishi, N., Satomi, S.: ABO-incompatible living donor liver transplantation: new insights into clinical relevance. Transplantation 85, 1523–1525 (2008) 61. Kissmeyer-Nielson, F., Olsen, S., et al.: Hyperacute rejection of kidney allografts, associated with pre-existing humoral antibodies against donor cells. Lancet 2, 662–665 (1966) 62. La Bas-Bernardet, S., Hourmant, M., Coupel, S., et al.: NonHLA-type endothelial cell reactive alloantibodies in pretransplant sera of kidney recipients trigger apoptosis. Am. J. Transplant. 3, 167–177 (2003) 63. Landsteiner, K.: Individual differences in human blood. Science 73, 403–409 (1931) 64. Le Bas-Bernardet, S., Hourmant, M., Valentin, N., et  al.: Identification of the antibodies involved in B-cell crossmatch positivity in renal transplantation. Transplantation 75, 477–482 (2003) 65. Lee, P.C., Terasaki, P.I., Takemoto, S.K., et al.: All chronic rejection failures of kidney transplants were preceeded by the development of HLA antibodies. Transplantation 74, 1192–1194 (2002) 66. Lo, A., Stratta, R.J., Alloway, R.R., et  al.: A multicenter analysis of the significance of HLA matching on outcomes after kidney-pancreas transplantation. Transplant. Proc. 37, 1289–1290 (2005) 67. Lucas, D.P., Paparounis, M.L., Myers, L., et al.: Detection of HLA class I-specific antibodies by the QuikScreen enzymelinked immunosorbent assay. Clin. Daig. Lab. Immunol. 4, 252–257 (1997) 68. Magro, C.M., Marshall-Klinger, D., Adams, P.W., et  al.: Evidence that humoral allograft rejection in lung transplant patients is not histocompatibility antigen-related. Am. J. Transplant. 3, 1264–1274 (2003) 69. Mahoney, R.J., Ault, K.A., Given, S.R., et  al.: The flow cytometric crossmatch and early renal transplant loss. Transplantation 49, 527–535 (1990) 70. Mahoney, R.J., Taranto, S., Edwards, E.: B-cell crossmatching and kidney allograft outcome in 9031 United States Transplant recipients. Hum. Immunol. 63, 324–335 (2002) 71. Marsh, S.G.E., Albert, E.D., Bodmer, W.F., et  al.: Nomenclature for factors of the HLA system. Tissue Antigens 60, 407–464 (2002) 72. Menier, C., Riteau, B., Carosella, E.D., et al.: MICA triggering signal for NK cell tumor lysis is counteracted by HLA-G1mediated inhibitory signal. Int. J. Cancer 100, 63–70 (2002)

G.P. Morris and T. Mohanakumar 73. Miller, J.A., Scholey, J.W.: The impact of rennin-angiotensin system polymorphisms on physiological and pathophysiological processes in humans. Curr. Opin. Nephrol. Hypertens. 13, 101–106 (2004) 74. Mittal, K.K., Mickey, M.R., Singal, D.P., et al.: Serotyping for homotransplantations. XIIX. Refinement of microdroplet lymphocytotoxicity test. Transplantation 6, 913–927 (1968) 75. Mizutani, K., Terasaki, P., Bignon, J.D., et al.: Association of kidney transplant failure and antibodies against MICA. Hum. Immunol. 67, 683–691 (2006) 76. Mizutani, K., Terasaki, P., Rosen, A., et al.: Serial ten-year follow-up of HLA and MICA antibody production prior to kidney graft failure. Am. J. Transplant. 5, 2265–2272 (2005) 77. Mizutani, K., Terasaki, P.I., Shih, R.N., et al.: Frequency of MIC antibody in rejected renal transplant patients without HLA antibody. Hum. Immunol. 67, 223–229 (2006) 78. Moore, S.B., Ploeger, N.A., DeGoey, S.R.: HLA antibody screening: comparison of a solid phase enzyme-linked immunoassay with antiglobulin-augmented lymphocytotoxicity. Transplantation 64, 1617–1620 (1997) 79. Morris, P.J., Williams, G.M., Hume, D.M., et al.: Serotyping for homotransplantation. XII. Occurrence of cytotoxic antibodies following kidney transplantation in man. Trans­ plantation 6, 392–399 (1968) 80. Morris, G.P., Phelan, D.L., Jendrisak, M.D.,et  al.: Virtual crossmatch by identification of donor-specific anti-human leukocyte antigen antibodies by solid-phase immunoassay: a 30-month analysis in living donor kidney transplantation. Hum Immunol. 2010 Mar;71(3):268–273 81. Murray, J.E., Merril, J.P., Dammin, G.J., et  al.: Study on transplantation immunity after total body irradiation: clinical and experimental investigation. Surgery 48, 272–284 (1960) 82. Narayanan, K., Jaramillo, A., Phelan, D.L., et  al.: Preexposure to sub-saturating concentrations of HLA class I antibodies canfers resistance to endothelial cells against antibody complement-mediated lysis by regulating Bad through the phosphatidylinositol 3-kinase/Akt pathway. Eur. J. Immunol. 34, 2303–2312 (2004) 83. Narayanan, K., Jendrisak, M.D., Phelan, D.L., et al.: HLA class I antibody mediated accommodation of endothelial cells via the activation of PI3K/cAMP dependent PKA pathway. Transpl. Immunol. 15, 187–197 (2006) 84. Opelz, G., Wujciak, T., Dohler, B., et al.: HLA compatibility and organ transplant survival. Collaborative Transplant Study. Rev. Immunogenet. 1, 334–342 (1999) 85. Oriol, R.: Tissular expression of ABH and Lewis antigens in humans and animals: expected value of different animal models in the study of ABO-incompatible organ transplants. Transplant. Proc. 19, 4416–4440 (1987) 86. Pascual, M., Saidman, S., Tolkoff-Rubin, N., et al.: Plasma exchange and tacrolimus-mycophenolate rescue for acute humoral rejection in kidney transplantation. Transplantation 66, 1460–1464 (1998) 87. Patel, R., Glassock, R., Terasaki, P.I.: Serotyping for homotransplantation. XIX. Experience with an interhospital scheme of cadaver-kidney sharing and tissue typing. J. Am. Med. Assoc. 207, 1319–1324 (1969) 88. Patel, A.M., Pancoska, C., Mulgaonkar, S., et  al.: Renal transplantation in patients with pre-transplant donor-specific antibodies and negative flow cytometry crossmatches. Am. J. Transplant. 7, 2371–2377 (2007)

4  Clinical Evaluation of Alloantibodies in Solid Organ Transplantation   89. Paul, L.C., Baldwin III, W.M.: Humoral rejection mechanisms and ABO incompatibility in renal transplantation. Transplant. Proc. 19, 4463–4467 (1987)   90. Payne, R., Tripp, M., Weigle, J., et  al.: A new leukocyte isoantigen system in man. Cold Spring Harb. Symp. Quant. Biol. 29, 285–295 (1964)   91. Pei, R., Lee, J.H., Shih, N.J., et al.: Single human leukocyte antigen flow cytometry beads for accurate identification of human leukocyte antibody specificities. Transplantation 75, 43–49 (2003)   92. Pei, R., Wang, G., Tarsitani, C., et al.: Simultaneous HLA Class I and Class II antibodies screening with flow cytometry. Hum. Immunol. 59, 313–322 (1998)   93. Perrey, C., Brenchley, P.E., Johnson, R.W., et al.: An association between antibodies specific for endothelial cells and renal transplant failure. Transpl. Immunol. 6, 101–106 (1998)   94. Phelan, D., Mohanakumar, T., Ramachandran, S., et  al.: Living donor renal transplantation in the presence of donorspecific human leukocyte antigen antibody detected by solid phase assay. Hum. Immunol. 70, 584–588 (2009)   95. Piazza, A., Poggi, E., Borrelli, L., et al.: Impact of donorspecific antibodies on chronic rejection occurance and graft loss in renal transplantation: posttransplant analysis using flow cytometric techniques. Transplantation 71, 1106–1112 (2001)   96. Pierres, M., Rebouah, J.P., Kourilsky, F.M., et  al.: Crossreactions between mouse Ia and human HLA-D/DR antigens analyzed by monoclonal antibodies. J. Immunol. 126, 2424–2429 (1981)   97. Pikul, F.L., Bolman, R.M., Saffitz, J.E., et  al.: AntiB-mediated rejection of an ABO-incompatible cardiac allograft despite aggressive plasma exchange transfusion. Transplant. Proc. 19, 4601–4604 (1987)   98. Praprotnik, S., Blank, M., Meroni, P.L., et al.: Classification of anti-endothelial cell antibodies into antibodies against microvascular and macrovascular endothelial cells. Arthritis Rheum. 44, 1484–1494 (2001)   99. Raybourne, R.B., Williams, K.M.: Monoclonal antibodies against an HLA-B27-derived peptide react with an epitope present on bacterial proteins. J. Immunol. 145, 2539–2544 (1990) 100. Raybourne, R.B., Williams, K.M., Cheng, X.K., Yu, D.T.: Demonstration of shared epitopes between bacterial proteins and HLA class-I proteins using monoclonal antibodies. Scand. J. Rheum. Suppl. 87, 134–138 (1990) 101. Reinsmoen, N.L., Lai, C.H., Vo, A., et  al.: Acceptible donor-specific antibody levels allowing for successful deceased and living donor kidney transplantation after desensitization therapy. Transplantation 86, 820–825 (2008) 102. Reznik, S.I., Jaramillo, A., Zhang, L., et  al.: Anti-HLA antibody binding to HLA class I molecules induces proliferation of airway epithelial cells: a potential mechanism for bronchiolitis obliterans syndrome. J. Throac. Cardiovasc. Surg. 119, 39–45 (2000) 103. Rodey, G.E., Fuller, T.C.: Public epitopes and the antigenic structure of the HLA molecules. Crit. Rev. Immunol. 7, 229–267 (1987) 104. Rodey, G.E., Neylan, J.F., Welchel, J.D., et al.: Epitope specificity of HLA class I alloantibodies. I. Frequency analysis of antibodies to private versus public specificities in potential transplant recipients. Hum. Immunol. 39, 272–280 (1994)

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105. Rydberg, L.: ABO-incompatibility in solid organ transplantation. Transfus. Med. 11, 325–342 (2001) 106. Salama, A.D.: Transplant accommodation in highly sensitized patients: a potential role for Bcl-xL and alloantibody. Am. J. Transplant. 1, 260–269 (2001) 107. Sis, B., Jhangri, G.S., Bunnag, S., et al.: Endothelial gene expression in kidney transplants with alloantibody indicates antibody-mediated damage despite lack of C4d staining. Am. J. Transplant. 9, 2312–2323 (2009) 108. Slapak, M., Evans, P., Trickett, L., et  al.: Can ABOincompatible donors be used in renal transplantation? Transplant. Proc. 16, 75–79 (1984) 109. Smith, J.D., Rose, M.L., Pomerance, A., et al.: Reduction of cellular rejection and increase in longer-term survival after heart transplantation after HLA-DR matching. Lancet 346, 1318–1322 (1995) 110. Solez, K., Colvin, R.B., Racusen, L.C., et al.: Banff 07 classification of renal allograft pathology: updates and future directions. Am. J. Transplant. 8, 753–760 (2008) 111. Starzl, T.E., Marchioo, T.L., Holmes, J.H., et  al.: Renal homografts in patients with major donor-recipient blood group incompatibilities. Surgery 55, 195–200 (1964) 112. Stock, P., Sutherland, D.E., Fryd, D.S., et al.: Detrimental effects of ABO mismatching in renal transplantation. Transplant. Proc. 19, 711–712 (1987) 113. Suárez-Alvarez, B., Lopez-Vazquez, A., Gonzalez, M.Z., et al.: The relationship of anti-MICA antibodies and MICA expression with heart allograft rejection. Am. J. Transplant. 7, 1842–1848 (2007) 114. Sumitran-Holgersson, S., Wilczek, H.E., Holgerson, J., et  al.: Identification of the nonclassical HLA molecules, MICA, as targets for humoral immunity associated with irreversible rejection of kidney allografts. Transplantation 74, 268–277 (2002) 115. Sun, Q., Liu, Z., Yin, G., et al.: Detectable circulating antiendothelial cell antibodies in renal allograft recipients with C4d-positive acute rejection: a report of three cases. Transplantation 79, 1759–1762 (2005) 116. Tan, C.D., Baldwin III, W.M., Rodriguez, E.R.: Update on cardiac transplantation pathology. Arch. Pathol. Lab. Med. 31, 1169–1191 (2007) 117. Terasaki, P.I., McClelland, J.D.: Microdroplet assay of human cytotoxins. Nature 204, 998–1000 (1964) 118. Terasaki, P.I., Ozawa, M.: Predicting graft failure by HLA antibodies: a prospective trial. Am. J. Transplant. 4, 438–443 (2004) 119. Tobian, A.A., Shirey, R.S., Montgomery, R.A., et al.: The critical role of plasmapheresis in ABO-incompatible renal transplantation. Transfusion 48, 2453–2460 (2008) 120. Trpkov, K., Campbell, P., Pazderka, F., et  al.: Pathologic features of acute renal allograft rejection associated with donor-specific antibody: analysis using the Banff grading schema. Transplantation 61, 1586–1592 (1996) 121. Vaidya, S., Hilson, B., Sheldon, S., et al.: DP reactive antibody in a aero mismatch renal transplant pair. Hum. Immunol. 68, 947–949 (2007) 122. Vaidya, S., Partlow, D., Susskind, B., et al.: Prediction of crossmatch outcome of highly sensitized patients by single and/or multiple antigen bead luminex assay. Transplantation 82, 1524–1528 (2006) 123. Valentini, R.P., Nehlsen-Cannarella, S.L., Gruber, S.A., et al.: Intravenous immunoglobulin, HLA allele typing, and

56 HLAMatchmaker facilitate successful transplantation in highly sensitized pediatric renal allograft recipients. Pediatr. Transplant. 11, 77–81 (2007) 124. Vamvakas, E.C.: Meta-analysis of randomized control trials of the efficacy of white cell reduction in preventing HLA-alloimmunization and refractoriness to random-donor platelet transfusions. Transfus. Med. Rev. 12, 258–270 (1998) 125. Vlad, G., Ho, E.K., Vasilescu, E.R., et  al.: Relevance of different antibody detection methods for the prediction of antibody-mediated rejection and deceased-donor kidney allograft survival. Hum. Immunol. 70, 589–594 (2009) 126. Wahrman, M., Exner, M., Schillinger, M., et  al.: Pivotal role of complement-fixing HLA alloantibodies in presensitized kidney allograft recipients. Am. J. Transplant. 6, 1033–1041 (2006) 127. Wahrmann, M., Bartel, G., Exner, M., et al.: Clinical relevance of of preformed C4d-fixing and non-C4d-fixing HLA single antigen reactivity in renal allograft recipients. Transpl. Int. 10, 982–989 (2009) 128. West, L.J., Pollock-Barziv, S.M., Dipchand, A.I., et  al.: ABO-incompatible heart transplantation in infants. N Engl J. Med. 344, 793–800 (1991) 129. Wilbrandt, R., Tung, K.S.K., Deodhar, S.D., et  al.: ABO blood group incompatibility in human renal homotransplantation. Am. J. Clin. Pathol. 51, 15–23 (1969) 130. Worthington, J.E., Martin, S., Al-Husseini, D.M., et  al.: Posttransplantation production of donor HLA-specific

G.P. Morris and T. Mohanakumar antibodies as a predctor of renal transplant outcome. Transplantation 75, 1034–1040 (2003) 131. Yuzawa, Y., Brett, J., Fukatsu, S., et al.: Interaction of antibody with Forssman antigen in guinea pigs. Am. J. Pathol. 146, 1260–1272 (1995) 132. Zaer, F., Metz, S., Scornik, J.C.: Antibody screening by enzyme-linked immunosorbent assay using pooled soluble HLA in renal transplant candidates. Transplantation 63, 48–51 (1997) 133. Zou, Y., Mirabha, F., Lazaro, A., et al.: MICA is a target for complement-dependent cytotoxicity with mouse monoclonal antibodies and human alloantibodies. Hum. Immunol. 63, 30–39 (2002) 134. Zou, Y., Qin, Z., Silvens, A., et  al.: Polymorphisms of MICA recognied by human alloantibodies. Immunogenetics 61, 91–100 (2009) 135. Zou, Y., Stastny, P., Süsal, C., et  al.: Antibodies against MICA antigens and kidney-transplant rejection. N Engl J. Med. 357, 1293–1300 (2007) 136. Zwirner, N.W., Dole, K., Stastny, P.: Differential expression of MICA by endothelial cells, fibroblasts, keratinocytes, and monocytes. Hum. Immunol. 60, 323–330 (1999) 137. Zwirner, N.W., Marcos, C.Y., Mirbaha, F., et  al.: Identification of MICA as a new polymorphic alloantigen recognized by antibodies in sera of organ transplant recipients. Hum. Immunol. 61, 917–924 (2000)

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Frontiers in Organ Transplantation Marc R. Hammerman

Abbreviations Alpha-gal APC E ES Gal−/− hDAF GFR MHC PAH PBMC SCID STZ UV

alpha galactosyl-transferase antigen-presenting cell embryonic day embryonic stem cell alpha galactosyl transferase deficient human decay accelerating factor glomerular filtration rate major histocompatibility complex p-aminohippurate peripheral blood mononuclear cells severe combined immunodeficiency streptozotocin urine volume

without risk of teratoma formation. Relative to transplantation of adult organs, organogenesis provides the potential for: (1) expansion of cell populations after transplantation; (2) attenuated cellular immune res­ ponse; (3) reduced susceptibility to humoral rejection after transplantation across a discordant xenogeneic barrier; and (4) selective differentiation of only desired parts of an organ. This chapter discusses challenges and progress made in organogenesis (growing new organs from transplanted embryonic primordia) in lieu of allotransplantation as a strategy for replacement of kidney and pancreas, two organs for which insufficient donors exist [2, 8, 19–21].

5.2 Growing New Kidneys 5.1 Introduction Growing new organs in situ by implanting developing animal organ anlagen/primordia (organogenesis) represents a novel solution to the problem of limited supply of human donor organs [21]. Organogenesis offers the theoretical advantage relative to transplanting embryonic stem (ES) [50] cells of intrinsically-programmed differentiation along defined organ-committed lines

M.R. Hammerman Renal Division, Departments of Medicine, Cell Biology and Physiology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8126, St.Louis, MO 63110, USA e-mail: [email protected]

The methodology for studies directed toward growing a new kidney from transplanted renal primordia derives from a literature describing the transplantation of embryonic renal metanephric kidneys. Renal primordia have been transplanted successfully to the chorioallantoic membrane of developing birds [46], the anterior eye chamber [26], beneath the renal capsule [9, 11–13, 15, 17, 32, 42, 48, 51], into the renal cortex of recipients [53–55] and into the abdominal cavity [1, 9, 27, 28, 35–39, 41–44, 49, 57]. Most studies that employed renal subcapsular transplantation, placement into the anterior chamber of the eye and onto the chorioallantoic membrane were conducted to define the immune response to fetal kidney transplants or to delineate the means by which renal primordia are vascularized. However, information emerged from these studies leading to approaches that employ transplantation to enhance renal function.

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_5, © Springer-Verlag Berlin Heidelberg 2011

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5.2.1 Immune Response to Fetal Kidney Transplants The metanephric kidneys originate in the rat on day 12.5 of a 21 day gestation period [42]. Foglia et al. [17] transplanted kidneys from adult rats or metanephroi from outbred rat embryos aged embryonic day E15– E21, beneath the renal capsule of non-immunosuppressed adult hosts. Under these conditions adult kidney transplants undergo acute rejection within 7 days [17]. In contrast, growth and survival of embryonic transplants was age-dependent in that enlargement and differentiation in situ over 15–30 days, was best for metanephroi obtained from E15 embryos and worsened progressively for those obtained on E16–E21. Primordia from E15 embryos showed maturation of renal elements when examined 10 days post-transplantation without rejection, whereas those obtained on E20 had a poor architecture and dense lymphocytic infiltrate. Liver harvested on E15 transplanted beneath the renal capsule underwent little growth and prompt rejection [17]. We [37] found a similar age-dependence for allotransplantation of rat metanephroi into the mesentery. Velasco and Hegre [51] transplanted metanephroi or liver tissue from E15, E17, E18 or E19 inbred Fisher rat embryos with rat major histocompatibility complex (RT1) RT1lvl, beneath the renal capsule of RT1- incompatible Wistar Furth adult rats (RT1u). All embryonic hepatic grafts were rejected within 10 days. In contrast, the degree of rejection of the metanephroi was age dependent, those from E15 embryos showing minimal or moderate rejection and those from older embryos showing more. If liver and metanephroi from E15 embryos were co-transplanted at different sites, metanephroi underwent a more severe rejection than if implanted without liver. It was speculated, the absence of APCs in metanephroi from E15 embryos together with their presence in liver explains the differential fate following transplantation with or without liver. Under the former, but not the latter conditions, direct presentation of donor antigens to host T cells takes place [51]. In the mouse, metanephroi arise on day 11.5 of a 20 day gestation period [36]. Statter et al. [48] transplanted metanephroi originating from E14-to-adult C57Bl/6 mice (H-2b) beneath the renal capsule of adult congenic B10.A hosts (H-2a). Expression of donor and host-specific class I (H2Kb) and class II (Abb) transcripts in E14 donor tissue was low and increased progressively in

M.R. Hammerman

renal tissue from older mice. After transplantation, surviving kidney grafts showed enhanced expression of class I and II transcripts. However, neither class I nor II protein could be detected in transplanted renal primordia. In human embryos, the metanephric kidneys arise during the first trimester [9–13]. Dekel and co-workers carried out a series of investigations in which human adult or embryonic kidney tissue is transplanted beneath the kidney capsule of immunodeficient rats (severe combined immunodeficiency (SCID/Lewis and SCID/nude chimeric rats)) [9–13]. Human adult kidney fragments transplanted beneath the renal capsule of such rats survive for as long as 2 months. Transplant architecture and normal structure of glomeruli are preserved. Intra- peritoneal infusion posttransplantation of allogeneic human peripheral blood mononuclear cells (PBMC) results in rejection of adult grafts. Human fetal kidney fragments transplanted beneath the renal capsule of immunodeficient rats display rapid growth and development. Glomeruli and tubular structures are maintained for as long as 4 months posttransplantation. In contrast to the case for transplanted adult kidney fragments, infusion of allogeneic human PBMC into hosts results in either minimal human T-cell infiltration or infiltrates that do not result in rejection or interfere with the continued growth of the human fetal renal tissue. Fetal human kidney grafts have reduced expression of tissue HLA class I and II relative to the adult grafts, consistent with reduced effectiveness in inducing an alloantigen-primed T cell response [13]. Dekel et al. showed that transcript levels for interferon gamma and interleukin-2 in fetal human kidneys grafted under the renal capsule of immunodeficient rats are markedly reduced post-transplantation relative to levels in adult human kidney tissue grafted to the same site. Peak levels of these cytokines appear late after PBMC infusion. Concomitant with these findings, interleukin 4 mRNA is upregulated during the early phase post-PBMC infusion, and interleukin 10 mRNA is expressed throughout the post-PBMC infusion interval. In addition, levels of mRNA coding for chemokines RANTES and MIP1 beta, their receptor, CCR5, and the cytolytic effector molecule, fas ligand, are suppressed in the fetal grafts relative to levels in adult grafts. Thus, fetal kidney induces the down-regulation of Th1 cytokines, chemokines and fas ligand, and the

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sparing of Th2 cytokines in the grafts. The findings suggest that the human immune response of kidney rejection is dependent on whether the target organ is of fetal or adult origin. An allogeneic immune system appears to mount a T helper 2-biased response when the target organ is fetal resulting in enhanced survival of transplanted tissue relative to adult tissue against which a T helper 1-biased response is mounted [13]. Subsequently, this group [9] showed that developing human kidneys had restricted expression of multiple factors that determine immune recognition. Thirteen of 57 genes that were significantly up-regulated in adult versus fetal human kidney tissue belonged to the HLA class I and II systems. In addition, molecules that mediate trafficking of leukocytes into the graft such as chemokines RANTES and MCP-1, adhesion molecule E-selectin, pro-inflammatory cytokines such as osteopontin and complement genes had reduced expressions in embryonic relative to adult kidneys. Reduced immunogenicity of embryonic human or pig kidneys transplanted into immunodeficient mice was confirmed by the absence of cellular rejection following infusion of human PBMCs.

5.2.2 Means by Which Renal Primordia are Vascularized The major arterial vessels supplying the kidney originate from lateral branches of the abdominal aorta that terminates in a plexus of arteries in close proximity to the renal pelvis, the renal artery rete [29]. It is a matter of controversy whether the renal microvasculature (smaller vessels and glomerular capillaries) arises exclusively via this angiogenic process, or also in part from endothelial cells resident in the developing metanephros. However, during its development, the renal primordium attracts its major arterial vessels, from the developing aorta [54]. In that its blood supply originates at least in part, from outside of the developing renal primordium, the kidney may be regarded as a chimeric organ. Its ability to attract its own vasculature in situ establishes the renal primordia as cellular transplants, capable of attracting a blood supply from an appropriate vascular bed [20, 21]. Insight into the origin of the renal microvasculature supply is provided by experiments in which developing kidneys are transplanted to ectopic sites. However,

the results of these experiments are somewhat contradictory. One explanation for the differences may be that the means of vascularization is site specific. For mouse or chick metanephroi obtained from E11.5 embryos grafted onto the chorioallantoic membrane of the quail, the vasculature is derived entirely from the host [46]. In the case of metanephroi from E11–12 mouse embryos grafted into the anterior chamber of the eye in genetically identical mice, the glomerular endothelium derives from both donor and host [26]. For metanephroi from E15 rat embryos transplanted into the abdominal cavity of mice [36], or from E28 pig embryos transplanted into the abdominal cavity of rats [10, 49] or mice [23, 44], the microvasculature is largely or entirely host. In all cases, large external vessels derive from the host.

5.3 Xenotransplantation for Kidney Replacement In that humans and pigs are of comparable size, share a similar renal physiology and because pigs are plentiful and can be bred to be pathogen free, pigs represent an ideal kidney donor for humans. [7, 56]. Unfortunately, the transplantation of whole vascularized organs such as the kidney originating from pigs into the group of primates that includes humans, the great apes and oldworld monkeys, is rendered problematic because of the processes of humoral rejection (hyperacute and acute vascular rejection directed against donor endothelial antigens) that occur across this xenogeneic barrier [7, 23, 56]. Humoral rejection following the transplantation of pig kidneys into non-human primates can be ameliorated or overcome through the use of genetically altered organs originating from pigs transgenic for the human complement activator, decay accelerating factor (hDAF) [7], or the use of organs from transgenics that do not express alpha-gal [56]. Unfortunately, neither the immunosuppressive regimens used for pig to primate kidney transplantation, nor the outcomes would be acceptable in humans. In contrast to xenotransplantation of whole vascularized organs from pig to primates, cellular transplants such as pancreatic islets from pigs can be transplanted into non-human primates [6, 25] or humans [18] without triggering hyperacute or acute vascular rejection.

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As delineated above, the renal metanephric primordium is a “candidate” for cell transplantation.

5.4 Transplantation of Renal Primordia to Enhance Host Renal Function We performed experiments in which renal primordia from E15 Sprague Dawley rat embryos were implanted within a fold of mesentery of adult Sprague Dawley hosts. Hosts received no immunosuppression. E15 renal primordia contained segments of ureteric bud and condensing metanephric blastema, but no glomeruli [42]. Under these conditions we [35–39, 42, 43] and others [1, 27, 28, 57] showed that primordia undergo differentiation and growth in hosts. Growth is enhanced if native renal mass is reduced at the time of implantation [1, 42, 57] or if the host is pregnant [1]. A renal primordium in a retroperitoneal dissection from an E15 rat embryo is shown in Fig. 5.1a. The ureteric bud is delineated by an arrowhead. If transplanted into an adult rat with its ureteric bud attached, the renal primordium enlarges and becomes kidney-shaped within 3 weeks (Fig. 5.1b). The ureteric bud differentiates into a ureter (Fig. 5.1b, arrowhead). In contrast to transplanted developed kidneys that undergo acute rejection [42] renal primordia transplanted into non-immunosuppressed hosts have a normal kidney

Fig. 5.1  (a) Photograph of retro-peritoneal dissection from an E15 rat embryo showing renal primordium or metanephros (m) and ureteric bud (arrowhead). (b) Photograph of a developed renal primordium (m) in the mesentery of an adult host rat 3 weeks post transplantation. Arrowhead shows developed ureter. Magnification is shown. Reproduced with permission [20]

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structure and ultrastructure post-development in situ and become vascularized via arteries that originate from the superior mesenteric artery of hosts and veins that originate from the host mesentery [20]. Figure 5.2a is a hematoxylin and eosin (H&E)-stained section of a renal primordia from an E15 rat embryo consisting of branched ureteric bud (ub) and undifferentiated metanephric blastema (mb). Figure 5.2b shows a renal primordia or metanephros (M), 3 weeks post-allotransplantation. An artery (a) and vein (v) originating from the host are delineated. Figure  5.2c shows a radiocontrast study that demonstrates the metanephros is supplied by the host’s superior mesenteric artery (SMA). A ureteroureterostomy (arrow) between the ureter originating from the transplanted renal primordium (M) and host ureter is shown in Fig. 5.2d. Differentiated structures at 20 weeks post-implantation are illustrated in Fig. 5.3 that shows H&E-stained sections of a developed renal primordium. The cross sectional diameter of the developed renal primordium shown in Fig. 5.3a (~1.2 cm) is about ½ the diameter of a normal rat kidney [38]. Its ureter (u) is labeled. Figure  5.3b shows a glomerulus (g) proximal tubule (pt), distal tubule (dt) and collecting duct (cd) in the cortex. A glomerulus (g) and collecting duct (arrow) are labeled in Fig. 5.3c. A glomerulus (g) proximal tubule (pt), and distal tubule (dt) are labeled in Fig. 5.3d. A collecting duct (cd) is shown in Fig.  5.3e. Electron

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Fig.  5.2  (a) H&E stained E15 rat metanephros consisting of undifferentiated metanephric blastema (mb) and ureteric bud (ub). Arrowheads show branched ureteric bud. (b) Artery (a) and vein (v) originating from the host’s mesentery supplying the developed renal primordium or metanephros (M) (c) Radiocontrast

image of kidney (K) and developed renal primordium (M), 6 weeks post-transplantation into the mesentery of a host rat. SMA superior mesenteric artery; (d) Ureteroureterostomy (arrow). Magnifications are shown for (a) and for (b) and (d) (in b). Reproduced with permission [20, 37]

microscopy of a developed renal primordium reveals normal renal structures [19] (Fig. 5.4). Kidneys contain approximately 30% as many nephrons as a normal rat kidney [35]. Developed renal primordia transplanted onto the mesentery, produce urine excreted in the normal manner following ureteroureterostomy between transplant and host (Fig. 5.2d) [28, 35, 37, 38, 42]. Levels of renal function in transplanted renal primordia (glomerular filtration rate (GFR)) were determined by measuring inulin clearance in otherwise anephric rats. In initial experiments GFRs were very low [42]. However, incubation of renal primordia with

growth factors prior to implantation increased GFRs more than 100-fold compared to those in rats with non growth factor-incubated renal primordia implanted concurrently [20]. GFRs in growth factor treated renal primordia are about 6% of normal. Others have reported even higher levels of GFR in rat-to-rat transplants [27]. Renal plasma flow, another parameter of renal function, was measured in transplanted renal primordia by calculating P-aminohippurate (PAH) clearances. The ratio of GFR/PAH clearance (filtration fraction) was 0.6, comparable to filtration fractions measured in rats with reduced renal function [20].

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Fig. 5.3  H&E-stained sections of a developed metanephros 20 weeks post-transplantation. (a) The ureter (u) is shown; (b) a glomerulus (g) proximal tubule (pt), distal tubule (dt) and collecting duct (cd) in the cortex; (c). A glomerulus (g) and collecting

duct (arrow) are labeled; (d) a glomerulus (g) proximal tubule (pt), and distal tubule (dt) are labeled; (e) a collecting duct (cd) is labeled. Magnifications are shown in a, b, c, and d (for d and e). Reproduced with permission [38]

Urine flow rates in transplanted rats are about 12% of the inulin clearance (GFR) measured in growth factortreated renal primordia. The UV/GFR of 0.12 demonstrates that transplanted renal primordia can concentrate urine [20]. Hemodialysis provides renal failure patients with GFRs that are about 10% of normal. Therefore, 6% of normal approximates a level of renal function that would be expected to preserve life. Indeed, life can be prolonged in otherwise anephric rat hosts by prior transplantation and ureteroureterostomy of one [38] or two [28] renal primordia. Using inbred congenic rats (PVG-RT1C and PVGRT1avl) we showed that renal primordia can be

transplanted across the RT1 locus into non immunesuppressed hosts. A state of peripheral immune tolerance secondary to T cell “ignorance” permits the survival of transplanted renal primordia. Most likely the “ignorance” results from the absence of APCs originating from the donor in the embryonic renal tissue, and the consequent absence of direct presentation of transplant antigen to host T cells (presentation by donor dendritic cells to host T cells) [39] as was shown previously for sub-renal capsular transplants [17, 51]. Metanephroi arise in embryonic pigs between E20– E28 [10, 44]. Transplantation of renal primordia from E28 pigs to adult pigs can be carried out without host immunosuppression [44].

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Fig. 5.4  Electron micrographs of transplanted rat renal primordium. Glomerular capillary loops show labeled: (a) mesangial cell (m); (b) endothelial cell (en); and (c) epithelial cell (ep), endothelial cell (en), podocytes (pd), and a basement membrane

(arrows); (d) A proximal tubule (pt) with a brush border membrane (arrowhead); (e) proximal tubule (pt) distal tubule (dt), and collecting duct (cd). Magnifications shown for c and e. Reproduced with permission [19]

5.4.1 Availability of Renal Primordia

from the site of harvesting and would allow time to plan the transplant procedure. To determine whether renal primordia can be stored in  vitro prior to transplantation, we transplanted renal primordia from E15 rat embryos into the mesentery of non-immunosuppressed uni-nephrectomized (host) rats either directly or suspended in ice-cold University of Wisconsin (UW) preservation solution for 3 days prior to implantation. The size and extent of tissue differentiation preimplantation of E15 renal primordia implanted directly is not distinguishable from the size and differentiation

In the case of human renal allotransplantation, there is an unavoidable delay between the time of harvest from donors and the time of implantation into recipients. Theoretically, renal primordia could be harvested immediately prior to implantation into humans. However, practically it would be best if primordia could be stored in vitro for a period of time prior to transplantation. The ability to store primordia would permit distribution to sites for transplantation, distant

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of renal primordia preserved for 3 days. By 4 weeks post transplantation, preserved renal primordia had grown and differentiated such that glomeruli, proximal and distal tubules, and collecting ducts with normal structure had developed. At 12 weeks posttransplantation, GFRs of preserved renal primordia are comparable to those of primordia implanted directly, consistent with the viability of preserved renal primordia [37]. We transplanted renal primordia from an E15 Lewis rat embryo across a concordant xenogeneic barrier into the mesentery of 10 week-old C57Bl/6J mice. In mice that receive immunosuppression, but not in its absence, the transplanted rat renal primordium undergoes differentiation and growth in situ [36]. To gain insight into the origin of the vasculature (donor vs. host) of renal primordia transplanted in the mesentery, using our rat-to-mouse model, we stained developing rat renal primordia using mouse specific antibodies directed against the endothelial antigen CD31. The vasculature of the transplanted developed rat kidney transplanted into the mouse is largely of mouse origin including glomerular capillary loops. In contrast, the capillary loops in rat renal primordia transplanted into rats do not stain for mouse CD31 [36]. Using a highly disparate model (pig to rodent) we transplanted E28 pig renal primordia consisting of undifferentiated stroma, branched ureteric bud and primitive developing nephrons into the mesentery of Lewis rats [41, 49] or C57Bl/6J mice [44]. Two–seven weeks post-transplantation, no trace of the renal primordia could be found in hosts that received no immunosuppression. Figure  5.5 illustrates E28 pig renal primordia prior to transplantation (Fig.  5.5a, b) and 6–7 weeks post-transplantation into immunosuppressed rats (Fig. 5.5c–f) [41]. The origin of the glomerular vasculature in transplants is rat (host) [49]. Dekel et al. successfully transplanted renal primordia originating from pig embryos aged E20–21 to E27–28 beneath the renal capsule of immunodeficient mice. Most transplants from the E20–25 donors fail to develop or evolve into growths containing few glomeruli and tubules, but other differentiated derivatives such as blood vessels, cartilage and bone. In contrast, the transplants originating from E27–28 pig embryos all exhibited significant growth and full differentiation into mature glomeruli and tubules [10]. Dekel et  al. found mouse CD31 expression in external vessels as well as developing glomeruli and small capillaries of

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pig renal primordium xenografts, consistent with a host origin for the vasculature of the developed renal primordium cellular transplants [10]. In addition, Dekel et  al. transplanted adult pig kidney tissue or E27–28 pig renal primordia beneath the renal capsule or onto the testicular fat of immunocompetent Balb/c mice. Some hosts were immunosuppressed. Evaluation of adult or E27–28 embryonic tissues 2 weeks postimplantation into non immunosuppressed hosts showed rejection of tissues. In immunosuppressed hosts, most E27–28 renal primordia underwent growth and differentiation. In contrast, all adult kidney grafts had a disturbed morphology, necrotic tissue and a high degree of lymphocyte infiltration. The authors interpreted these data as being consistent with an immune advantage of the developing precursor transplants over developed adult kidney transplants in fully immunocompetent hosts [10]. Yokoo et al. injected human mesenchymal stem cells (hMSC) labeled with LacZ into E9.5 mouse embryos or E11.5 rat embryos at the site of early renal organogenesis, and subjected the whole embryos to culture. After 48 h of whole culture, metanephroi were dissected from whole embryos and cultured in vitro for 6 days. It was found that hMSC-derived LacZ- labeled cells contribute to renal structures in organ-cultured metanephroi [58]. Subsequently, the investigators implanted LacZ labeled hMSC that had been transfected with glial cell linederived neurotrophic factor into the nephrogenic site of E11.5 rat embryos. Follow­ing 48  h of whole embryo culture, metanephroi containing hMSC were dissected out and transplanted into the mesentery of uninephrectomized rats. No immunosuppression was required. Transplants enlarged over 2 weeks in non-immunosuppressed rats, became vascularized by host vessels and contained hMSC-derived LacZ-positive cells that were morphologically identical to resident renal cells. These findings suggest that self-organs from autologous MSC can be generated using inherent developmental and angiogenic systems [57].

5.5 Growing New Endocrine Pancreas Hyperglycemia represents a major health problem for the diabetic patient. When inadequately controlled, chronic hyperglycemia can lead to microvascular and macrovascular complications [2]. Use of oral hypoglycemic agents

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Fig. 5.5  Photographs (a, c, d) and photomicrographs (b, e, f) of E28 pig renal primordia (a, b) or E28 pig renal primordia 7 weeks post-transplantation into the mesentery of a rat (c–f). (a) E28 primordium (ub ureteric bud) (b) E28 primordium (s stroma; ub ureteric bud); (c) E28 pig renal primordium 7 weeks post transplantation in a rat mesentery: (d) E28 pig renal primordium

after removal from the mesentery (u ureter) (e) Cortex with a glomerulus (g) proximal tubule (pt) and distal tubule (dt) labeled; (f) Medulla with collecting duct (cd) labeled. Magnifications are shown for a and b (in a); c and d (in d) and e and f (in e) Reproduced with permission [41]

and administration of exogenous insulin are cornerstones of treatment. Unfortunately adequate control of circulating glucose levels cannot be attained by most patients with diabetes and attempts at maintaining euglycemia through intensive insulin therapy lead to an increased incidence of hypoglycemia [2].

A new therapy for diabetes mellitus has long been sought through a variety of biological approaches [2]. These include whole pancreas transplantation, islet transplantation, regeneration of beta cells, and transplantation of bioengineered ES cells. Of all these approaches, only whole pancreas allotransplantation

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and islet  allotransplantation are applied in humans. Given existing technology, a major limitation to the use of either modality is the insufficient supply of human organs [6, 8, 18, 25, 52] and the need to immunosupress hosts, in effect trading one disease (diabetes) for another (immunosuppression) [2]. Islet transplantation is generally a therapy for type 1 diabetes only. Selection criteria at most US centers for pancreas transplantation dictate a conservative approach that excludes most type 2 diabetics, traditionally older and poorer surgical risks than type 1 patients [2].

5.6 Xenotransplantation Therapy for Diabetes Mellitus In that they are plentiful and because porcine insulin works well in humans, the pig has been suggested as a pancreas organ donor for human diabetics. As delineated above, islets like other cell transplants are not subject to humoral rejection [6, 18, 25]. Recent experience with pig to non-human primate islet [25] or neonatal islet [6] transplantation shows that sustained insulin independence can be achieved, but only through the use of immunosuppressive agents that are not approved for human use or result in an unacceptable morbidity in diabetic primates.

5.7 Organogenesis of the Endocrine Pancreas 5.7.1 Type 1 Diabetes Mellitus Experimental type-1 diabetes in rodent hosts has been treated successfully using embryonic rodent pancreas transplants. If rat pancreatic primordia are obtained sufficient early during embryogenesis (prior to E17), only the endocrine component differentiates post-transplantation [3–5, 24, 33, 34, 40, 41]. Selective endocrine differentiation obviates the problem of host tissue digestion by exocrine tissue that can occur post-transplantation of rodent primordia obtained at later times [3, 4]. Brown et  al. showed that a partial reversal of STZ-diabetes in rats into which fetal pancreases were

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isotransplanted beneath the renal capsule, was rendered complete following shunting of the venous drainage from the transplants to the liver [5]. Levels of circulating insulin in transplanted rats fell following imposition of the shunt as a result of increased extraction of insulin passing into the liver as well as diminished secretion by the transplanted primordia. It was proposed that intra-peritoneal transplantation is advantageous relative to sub renal-capsular transplantation for diabetes control in that the former: (1) involves more limited surgery; (2) provides a large surface area for implantation; and (3) recapitulates an orthotopic site physiologically, in that secreted insulin enters the portal system (via the superior mesenteric vein) rather than the systemic venous system (via the renal vein) [24]. In studies that compared directly the fates of E17–18 rat pancreatic primordia isografts and allografts, allo­ ransplanted non-immunosuppressed recipients showed only a transient recovery from the diabetes (3–13 days) followed by a return to the diabetic state with graft rejection [24]. However, Eloy et al. showed that xenotransplantation of pancreas from chick embryos can normalize levels of glucose in diabetic rats without the need for any host immunosuppression if the tissue is obtained sufficiently early during chick development [14]. We transplanted whole pancreatic primordia into the mesentery of streptozotocin (STZ)-diabetic rats a model for type 1 diabetes [33, 40, 41] or ZDF rats a model for type 2 disease [34]. On E12.5 the rat pancreas is relatively undifferentiated. Dorsal and ventral components remain separate [40]. By 4 weeks post-transplantation of whole pancreatic primordia from E 12.5 Lewis rats into the mesentery of a STZ-diabetic Lewis rat, the tissue had undergone differentiation and insulin-positive islets of Langerhans can be delineated amidst stroma. There is no differentiation of exocrine tissue [33, 40]. Abnormal glucose tolerance in STZ diabetic rat hosts is normalized within 2–4 weeks post-isotransplantation of pancreatic primordia as is the pattern of abnormal weight gain characteristic of diabetic animals [33, 40]. No host immunosuppression is required for isotransplantation of E12.5 rat embryonic pancreas. Xenotransplantation of E12.5 Lewis rat pancreatic primordia can be carried out in C57Bl/J6 mice. Growth and differentiation post-transplantation occurs exactly as for isotransplantation into rats if mice are immunosuppressed. Rat embryonic pancreata do not differentiate if mouse hosts do not receive immunosuppression [40].

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To establish the feasibility of pig-to-rodent xenotransplantation of pancreatic primordia, we implanted pancreatic primordia from E28–29 pig embryos into STZ-diabetic adult Lewis rats. On E28–29 the pig pancreas is relatively undifferentiated. Dorsal and ventral components remain separate [33]. Glucose tolerance in STZ-diabetic Lewis rats is normalized permanently by the physiological secretion of porcine insulin from transplanted pig pancreatic primordia [33, 41]. As is the case following transplantation of E12.5 rat pancreatic primordia into STZ-diabetic adult Lewis rats or C57Bl/J6 mice, exocrine tissue does not differentiate after transplantation of pig pancreatic primordia into STZ-diabetic adult Lewis rats. However, rather than islets surrounded by stroma that are observed following transplantation of rat pancreatic primordia into rat or mouse mesentery [40], individual alpha and beta cells engraft within the mesentery and in mesenteric lymph nodes by 6 weeks after pig to rat pancreatic primordia transplantation as demonstrated by light and electron microscopy [33, 41]. While we do not know for certain why this is the case, it may reflect the inability of individual pig islet cells – once they have migrated away from the primitive ducts – to coalesce into islets in the setting of a xenogeneic (rat) extracellular matrix [47]. Figure  5.6a is a photomicrograph of a Gomoristained pancreas from a normal rat. Islets stain purple (arrow) and exocrine tissue stains pink (arrowhead). Figure 5.6b shows a mouse mesentery 4 weeks posttransplantation of E12.5 rat pancreatic primordia. A Gomori-stained islet is delineated (arrow), but in contrast to what is shown in Fig. 5.6a, no exocrine tissue is present. Figure  5.6c depicts Gomori-positive endocrine cells (arrows) in a mesenteric lymph node of a rat 6 weeks post-transplantation of E28 pig pancreatic primordia. Since: (1) mRNA for porcine insulin and porcine insulin itself is present only the mesentery of previously-diabetic Lewis rats following pig pancreatic primordia transplantation [33]; (2) porcine insulin but not rat insulin can be detected in circulation [33, 41]; (3) destruction of beta cells and no evidence for regeneration of native beta cells is found in the native rat pancreas following administration of STZ [22]; it was concluded that glucose tolerance in STZ-diabetic Lewis rats is normalized via secretion of porcine insulin from the pig fetal pancreatic implants [33, 41]. Remarkably and consistent with the findings of Eloy et  al. [14], if obtained from E28 or E29 pig embryos, within a developmental “window” prior to E35 [41],

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Fig. 5.6  Photomicrographs of a Gomori-stained tissue (a) pancreas from a normal rat. Islets stain purple (arrow) and exocrine tissue stains pink (arrowhead). (b) mouse mesentery 4 weeks post-transplantation of E12.5 rat pancreatic primordia. A Gomori-stained islet is delineated (arrow); (c) Gomori-positive endocrine cells (arrows) in a lymph node of a rat 6 weeks posttransplantation of E28 pig pancreatic primordia. Reproduced with permission [33, 40]

pancreatic primordia engraft sufficiently well such that glucose tolerance is normalized in non-immunosuppressed immunocompetent diabetic rat hosts. In contrast, levels of glucose are not reduced post-transplantation of

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E35 pig pancreatic primordia into diabetic rats and transplanted tissue is rejected. In contrast to the case for E28 pancreatic primordia, renal primordia obtained from E28 pig embryos do not engraft unless hosts are immunosuppressed [41], and the successful transplantation of rat pancreatic primordia obtained at a comparable developmental stage (E12.5) into mice requires that hosts be immunosuppressed [40]. To determine whether Lewis rats are rendered anergic following transplantation of pig pancreatic primordia, we transplanted E28 pig renal primordia each into the mesentery of two non-immunosuppressed Lewis rats that had been rendered STZ-diabetic and into which E28 pig pancreatic primordia had been implanted previously – immediately after glucose levels were measured to confirm diabetes. The first rat was normoglycemic at 2 months post-transplantation of E28 pig pancreatic primordia, at which time the E28 kidneys were implanted. The rat was sacrificed at 8 months at which time the mesentery was examined and no trace of the implanted E28 renal primordia was observed [41]. Histology of mesentery revealed isolated alpha and beta endocrine cells [41]. Normoglycemia was maintained post-E28 kidney transplantation until the time of death. The second rat was normoglycemic at 12 months post-transplantation of pig pancreatic primordia, at which time the E28 pig renal primordia were implanted. Four weeks later a laparotomy was performed and no trace of the transplanted renal primordia could be detected. The laparotomy was closed, and the rat remained normoglycemic [41]. Thus, rats transplanted with E28 pig pancreatic primordia are not anergic as evidenced by rejection of E28 pig renal primordia transplanted subsequently. However, engrafted pancreatic primordia remain viable after kidneys are rejected and rats remain normoglycemic throughout [41]. We do not know why the host response to E28 pig pancreatic primordia transplanted into rats differs in this way from that to E28 pig renal primordia transplanted into rats or E12.5 rat renal primordia transplanted into mice. It is possible that the pattern of the growth and differentiation of E28 pig pancreatic primordia as a function of time following implantation in rats (no acinar tissue, no islets no stroma), so radically different from what occurs during normal porcine pancreas development, and different from what happens after transplantation of rat pancreatic renal

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primordia into rats or mice, results in a pattern of antigen expression that is not recognized as foreign by the host. Antigens expressed as a function of E28 pig renal primordia differentiation post-transplantation might be more widely representative of pig antigens and as such, better stimulators of the host immune response than those expressed after E28 pig pancreatic primordia implantation. Similarly, antigens expressed by E35 pig pancreatic primordia (that are rejected), perhaps some induced by relative ischemia post-transplantation, may render the process of transplanting E35 pig pancreatic primordia more immunogenic [21]. An alternative explanation is host tolerance on the basis of mixed chimerism [45] (see Figs.  5.6–5.9). Another is “T cell paralysis” on the basis host exposure to antigen plus SLA II on pig beta cells in the absence of second costimulatory signal [45]. Eventov-Friedman and co-workers implanted embryonic pig pancreatic tissues of different gestational ages beneath the kidney capsule of immunodeficient (NOD-SCID) diabetic mice and immunocom­petent diabetic mice that were immunosuppressed. Using NOD-SCID animals, they showed that pancreatic tissue obtained from E42 embryos exhibits reduced immunogenicity relative to that obtained from E56 embryos as determined by a lesser reduction in levels of circulating porcine insulin following immune reconstitution by infusion of human PBMC. In both models, it was possible to normalize level of glucose following pig pancreatic primordia transplantation [16].

5.7.2 Type 2 Diabetes Mellitus The ZDF rat is an inbred strain derived from a colony of Zucker fatty rats. ZDF and Zucker fatty animals have an autosomal recessive mutation in the gene (fa) that encodes the leptin receptor [30, 31]. Homozygous Zucker fatty rats (fa/fa) manifest hyperphagia, obesity and severe insulin resistance, but remain normoglycemic. ZDF homozygous males (fa/fa) become hyperglycemic starting after 6 weeks of age and thereafter spontaneously develop overt diabetes [30]. Homozygous females become overtly diabetic beginning at age 6–8 weeks if maintained on a diabetogenic high fat diet [30]. In ZDF males and females, hyperglycemia occurs

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Fig. 5.7  In situ hybridization was performed using pig proinsulin antisense or sense probes on tissue originating from diabetic ZDF rats into which pig pancreatic primodia had been transplanted 40 weeks previously: (a) liver stained using antisense probe; (b) liver stained using sense probe; (c and e) mesenteric lymph node stained using antisense probe; (d and f) mesenteric lymph node stained using sense probe; (g) pancreas stained with

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antisense probe and excess of unlabeled antisense probe; (h) pancreas stained with antisense probe; (i) pancreas stained with sense probe. Arrow delineates germinal centers in c and d; Arrowheads delineate cells that stain positive for porcine proinuslin RNA in a, c, e and h, and negative staining cells in d. Magnifications are shown. Reproduced with permission [34]

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Fig. 5.8  In situ hybridization was performed using pig proinsulin antisense (a, c) or sense probes (b, d) on sections of mesenteric lymph node originating from a STZ-diabetic rhesus

macaque 407 days post-transplantation of E28 pig pancreatic primordia. Scale Bars 80  um (a and b) and 30  um (c and d). Reproduced with permission [45]

concomitant with markedly elevated levels of circulating insulin and failure of insulin secretion in response to a glucose challenge, mimicking the pathophysiology of human type 2 diabetes mellitus [30]. Diet restriction (15 g/day standard rat chow) permits the use of ZDF rats as breeders, but does not reverse glucose intolerance or

insulin resistance. Homozygous dominant (+/+) and heterozygous (fa/+) ZDF rats are lean and do not become diabetic [30]. To define the utility for transplantation of pig pancreatic primordia in an animal model of human type 2 diabetes, embryonic pancreas from E28 pig embryos

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Fig.  5.9  Electron micrograph of endocrine cell (circled) in a mesenteric lymph node from macaque 3. n nucleus. Inset: n nucleus; arrowheads, endocrine granules; arrows, rough endoplasmic reticulum. Magnification: 15,000×. Reproduced with permission [45]

was implanted into the mesentery of diabetic ZDF rats. In combination with a standard diet, transplantation of E28 pig pancreatic primordia normalizes glucose tolerance in diabetic ZDF males and females and ameliorates (ZDF diabetic females) or eliminates (ZDF diabetic males) insulin resistance in formerly diabetic rats [34]. Porcine insulin is detectable in plasma of formerly diabetic ZDF rats that received pig pancreatic primordia transplants. Levels peak at 15 min after an oral glucose load [34]. To localize porcine insulin producing cells following implantation of pig pancreatic primordia into rats, in-situ hybridization was performed using a porcine proinsulin-specific antisense probe [34]. Cells expressing porcine proinsulin mRNA are present in liver, mesenteric lymph nodes and pancreas at 40 weeks post-transplantation [34]. Shown in Fig. 5.7 are cells within liver (a, b) germinal centers (c, d, arrows) and medullary sinuses (c, d arrowheads, e, f) of mesenteric lymph nodes that stain with the antisense probe (a, c, e red), but not with the sense probe (b, d and f). The cells have rounded nuclei with prominent nucleoli and abundant cytoplasm consistent with the morphology of engrafted pig beta cells in our previous studies and of pig beta cells in vivo [33,

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41]. No staining is observed in pancreas if an excess of unlabeled antisense probe is added to labeled antisense probe (Fig. 5.7g). Scattered cells within pancreas stain positive with the antisense probe (Fig. 5.7 arrowheads), but not with the sense probe (Fig. 5.7). To confirm that transcripts identified in transplanted ZDF rat tissues by in-situ hybridization are for porcine proinsulin, RT-PCR was performed using primers designed to amplify a porcine proinsulin RNA sequence different from the one recognized by the anti-sense probe. A band is amplified from RNA originating from transplanted ZDF rat liver that corresponds to a transcript present in adult pig pancreas [34]. Sequencing confirms that transcripts are for porcine proinsulin [34]. A first phase insulin release characteristic of beta cells results within 1  min of glucose addition to media in which mesenteric lymph nodes from transplanted ZDF rats are incubated [34]. We transplanted E28 pig pancreatic primordia in the mesentery of STZ-diabetic rhesus macaques. Longterm engraftment of pig beta cells within liver, pancreas and mesenteric lymph nodes post-transplantation was demonstrated by electron microscopy, positive immune-histochemistry for insulin, and positive RT-­ PCR and in-situ hybridization for porcine proinsulin mRNA [45]. Insulin requirements were reduced in one macaque followed over 22 months post-transplantation and porcine insulin detected in plasma using sequential affinity chromatography, HPLC and mass spectrometry. Of potential importance for application of this transplantation technology to treatment of diabetes in humans and confirmatory of our previous findings in Lewis and ZDF rats, no host immunosuppression is required [45]. Shown in Fig. 5.8 are sections of mesenteric lymph node from a diabetic rhesus macaque that had been transplanted 407 days previously with E28 pig pancreatic primordia. In situ hybridization was performed using pig proinsulin antisense (Fig. 5.8 a, c) or sense (Fig.  5.8 b, e) probes. Cells within medullary sinuses stain (red) with the antisense, (Fig. 5.8 a, c) but not the sense (Fig. 5.8 b, d) probe. Figure  5.9 is an electron micrograph of medullary sinus from a rhesus macaque lymph node biopsied post-transplantation. Shown (circle) is a cell with a rounded nucleus (n); rough endoplasmic reticulum, and encapsulated granules characteristic of endocrine secretory granules. Inset depicts nucleus, rough endoplasmic

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reticulum (arrow) and encapsulated granules (arrowheads). Our finding that pig pancreatic primordia engraft long-term in non-immunosuppressed STZ diabetic rhesus macaques establishes the potential for their use in human diabetics.

5.8 Summary and Conclusions We began this chapter by listing theoretical reasons why growing new organs in situ by implanting developing animal organ anlagen/primordia (organogenesis) offers advantages relative to transplanting ES cells or xenotransplantation of developed (adult) organs (Sect. 5.1). In fact, as described in the Chapter: (1) primordia differentiate along organ-committed lines. In the case of embryonic kidney, an anatomically-correct threedimensionally integrated vascularized organ develops post-implantation [19, 20] that can sustain life in otherwise anephric hosts [28, 38]. In the case of embryonic pancreas, the glucose sensing and insulin releasing functions of beta cells that differentiate from primordia are functionally linked such that glucose tolerance in formerly diabetic hosts is rendered normal and hypoglycemia does not occur [3–5, 14, 16, 24, 33, 34, 40, 41, 45]; (2) Cells populations within primordia expand after transplantation. In the case of embryonic kidney, volume increases more than a 1,000-fold [38]. In the case of embryonic pancreas, beta cell mass expands sufficiently in situ [3, 4, 24] such that a diabetic rat host can be rendered euglycemic post-isotransplantation of a single primordium [4]; (3) The cellular immune response to transplanted primordia is attenuated relative to that directed against adult organs. In the case of embryonic kidney allotransplantation is possible across the MHC without host immunosuppression [17, 39, 51]. In the case of embryonic pancreas, if obtained sufficiently early during development, xenotransplantation of pancreatic primordia (chick-to-rat [14] or pig-to-rat [33, 34, 41] or pig-to-non-human primate [45]) is possible without the need for host immunosuppression; (4) Primordia attract a vasculature from the host. In the case of embryonic kidney [10, 23, 28, 36, 49, 57] this renders them less susceptible to humoral rejection than are adult organs with donor blood vessels transplanted across a discordant xenogeneic barrier. In the case of embryonic pancreas (as for islets), this permits transplantation from pig-to-primate without humoral rejection [6, 18, 25];

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and (5) Organ primordia differentiate selectively. In the case of embryonic pancreas, exocrine pancreatic tissue does not differentiate following transplantation, obviating complications that can result from exocrine components such as the enzymatic autodigestion of host tissues [3, 4]. Some characteristics of organogenesis may depend on how primordia transplantation is carried out. For example, the nature of the vasculature in developed renal primordia appears to be transplantation site specific [23], and the absence of a host-immunosuppression requirement for successful engraftment of pancreatic primordia requires that tissue be obtained within a developmental window early during embryogenesis [14, 33, 34, 41, 45]. If successful, organogenesis could provide in essence, an unlimited supply of donor organs. This would result in a paradigm shift in how the world thinks about organ replacement: (1) there will be no need to transport organs across long distances; (2) transplantation can be done electively at a convenient time; (3) transplantation can be offered to high-risk individuals and can be repeated as needed; and (4) transplantation can be offered to patients currently not candidates including type 2 diabetics [34]. Acknowledgements  Supported by Washington University George M. O’Brien Center NIDDK P30DK07933 and by grant 1-110-2005 from the Juvenile Diabetes Research Foundation.

References   1. Armstrong, S.R., Campbell, G.R., Campbell, J.H., et  al.: Establishment of metanephros transplantation in mice highlights contributions by both nephrectomy and pregnancy to developmental progression. Exp. Nephrol. 101, e155–e164 (2005)   2. Bottino, R., Trucco, M.: Multifaceted therapeutic approaches for a multigenic disease. Diabetes 54(Supplement 2), S79– S86 (2005)   3. Brown, J., Clark, W.R., Molnar, I.G., et al.: Fetal pancreas transplantation for reversal of streptozotocin-induced diabetes in rats. Diabetes 25, 56–64 (1976)   4. Brown, J., Heninger, D., Kuret, J., et al.: Islet cells grow after transplantation of fetal pancreas and control of diabetes. Diabetes 30, 9–13 (1981)   5. Brown, J., Mullen, Y., Clark, W., et al.: Importance of hepatic portal circulation for insulin action in STZ-diabetic rats transplanted with fetal pancreases. J. Clin. Invest. 64, 1688– 1694 (1979)

5  Frontiers in Organ Transplantation   6. Cardona, K., Korbutt, G.S., Milas, Z., et al.: Long-term survival of neonatal porcine islets in rhesus macaques by targeting costimulation pathways. Nat. Med. 12, 304–306 (2006)   7. Cozzi, E., Bhatti, F., Schmoekel, M., et al.: Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts. Transplantation 70, 15–21 (2000)   8. Danovitch, G.M., Cohen, D.J., Weir, M.R., et  al.: Current status of kidney and pancreas transplantation in the United States 1994–2003. Am. J. Transplant. 5(Part 2), 904–915 (2005)   9. Dekel, B., Amariglio, F., Kaminski, N., et al.: Engraftment and differentiation of human metanephroi into functional mature nephrons after transplantation into mice is accompanied by a profile of gene expression similar to normal human kidney. J. Am. Soc. Nephrol. 13, 977–990 (2002) 10. Dekel, B., Burakova, T., Arditti, F.D., et al.: Human and porcine early kidney precursors as a new source for transplantation. Nat. Med. 9, 53–60 (2003) 11. Dekel, B., Burakova, T., Ben-Hur, H., et al.: Engraftment of human kidney tissue in rat radiation chimera: II human fetal kidneys display reduced immunogenicity to adoptively transferred human peripheral blood mononuclear cells and exhibit rapid growth and development. Transplantation 64, 1550–1558 (1997) 12. Dekel, B., Burakova, T., Marcus, H., et al.: Engraftment of human kidney tissue in rat radiation chimera: I A new model of human kidney allograft rejection. Transplantation 64, 1541–1550 (1997) 13. Dekel, B., Marcus, H., Herzel, B.H., et al.: In vivo modulation of the allogeneic immune response by human fetal kidneys: the role of cytokines, chemokines, and cytolytic effector molecules. Transplantation 69, 1470–1478 (2000) 14. Eloy, R., Haffen, K., Kedinger, M., et al.: Chick embryo pancreatic transplants reverse experimental diabetes of rats. J. Clin. Invest. 64, 361–373 (1979) 15. Eventov-Friedman, S., Katchman, H., Shezen, E., et  al.: Embryonic pig liver, pancreas, and lung as a source for transplantation: optimal organogenesis without teratoma depends on distinct time windows. Proc. Natl. Acad. Sci. U.S.A. 102, 2928–2933 (2005) 16. Eventov-Friedman, S., Tchorsh, D., Katchman, H., et  al.: Embryonic pig pancreatic tissue transplantation for the treatment of diabetes. PLoS Med. 7, 1165–1177 (2006) 17. Foglia, R.P., LaQuaglia, M., Statter, M.B., et  al.: Fetal allograft survival in immunocompetent recipients is age dependent and organ specific. Ann. Surg. 204, 402–410 (1986) 18. Groth, C.G., Korsgren, O., Tibell, A., et al.: Transplantation of porcine fetal pancreas to diabetic patients. Lancet 344, 1402–1404 (1994) 19. Hammerman, M.R.: Implantation of renal rudiments. In: Polak, J., Hench, L., Kemp, P. (eds.) Future Strategies for Organ Replacement, pp. 199–211. Imperial College Press, London (2002) 20. Hammerman, M.R.: Transplantation of developing kidneys. Transplant. Rev. 16, 62–71 (2002) 21. Hammerman, M.R.: Windows of opportunity for organogenesis. Transpl. Immunol. 15, 1–8 (2005) 22. Hammerman, M.R.: Growing new endocrine pancreas in situ. Clin. Exp. Nephrol. 10, 1–7 (2006)

73 23. Hammerman, M.R.: Strategies for cell replacement for kidney failure. Expert Opin. Biol. Ther. 6, 87–97 (2006) 24. Hegre, O.D., Leonard, R.J., Erlandsen, S.L., et  al.: Transplantation of islet tissue in the rat. Acta Endocrinol. Suppl. 205, 257–278 (1976) 25. Hering, B., Wijkstrom, M., Graham, M., et  al.: Prolonged diabetes reversal after intraportal xenotransplantation of wild-type porcine islets in immunosuppressed nonhuman primates. Nat. Med. 12, 301–303 (2006) 26. Hyink, D.P., Tucker, D.C., St. John, P.L., et al.: Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys. Am. J. Physiol. 270, F886–F889 (1996) 27. Marshall, D., Bottomley, M., Symonds, K., et  al.: Transplantation of metanephroi to sites within the abdominal cavity. Transplant. Proc. 37, 194–197 (2005) 28. Marshall, D., Dilworth, M.R., Clancy, M., et al.: Increasing renal mass improves survival in anephric rats following metanephros transplantation. Exp. Physiol. 92, 263–271 (2007) 29. Netter, F.H.: Anatomy structure and embryology. In: Becker, E.L., Churg, J. (eds.) The Netter Collection of Medical Illustrations. Kidneys Ureter and Bladder, vol. 6, pp. 2–35. Novartis, Pittsburgh (1997) 30. Peterson, R.G., Shaw, W.N., Neel, M.A., et al.: Zucker diabetic fatty rat as a model for non-insulin-dependent diabetes. ILAR News 32, 16–19 (1990) 31. Phillips, M.S., Hammond, H.A., Dugan, V., et  al.: Leptin receptor missesne mutation in the fatty zucker rat. Nat. Genet. 13, 18–19 (1996) 32. Robert, B., St John, P.L., Abrahamson, D.L.: Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mice. Am. J. Physiol. 275, F164–F172 (1998) 33. Rogers, S.A., Chen, F., Talcott, M., et al.: Islet cell engraftment and control of diabetes in rats following transplantation of pig pancreatic primordia. Am. J. Physiol. 286, E502–E509 (2004) 34. Rogers, S.A., Chen, F., Talcott, M., et al.: Glucose tolerance normalization following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic ZDF rats. Transpl. Immunol. 16, 176–184 (2006) 35. Rogers, S.A., Droege, D., Dusso, A., Hammerman, M.R.: Incubation of metanephroi with vitamin D increases numbers of glomeruli. Organogenesis 1, 52–54 (2004) 36. Rogers, S.A., Hammerman, M.R.: Transplantation of rat metanephroi into mice. Am. J. Physiol. 280, R1865–R1869 (2001) 37. Rogers, S.A., Hammerman, M.R.: Transplantation of metanephroi after preservation in  vitro. Am. J. Physiol. 281, R661–R665 (2001) 38. Rogers, S.A., Hammerman, M.R.: Prolongation of life in anephric rats following de  novo renal organogenesis. Organogenesis 1, 22–25 (2004) 39. Rogers, S.A., Liapis, H., Hammerman, M.R.: Transplantation of metanephroi across the major histocompatibility complex in rats. Am. J. Physiol. 280, R132–R136 (2001) 40. Rogers, S.A., Liapis, H., Hammerman, M.R.: Intraperitoneal transplantation of pancreatic anlagen. ASAIO J. 49, 527– 532 (2003) 41. Rogers, S.A., Liapis, H., Hammerman, M.R.: Normalization of glucose post-transplantation of pig pancreatic primordia

74 into non-immunosuppressed diabetic rats depends on obtaining primordia prior to embryonic day 35. Transpl. Immunol. 14, 67–75 (2005) 42. Rogers, S.A., Lowell, J.A., Hammerman, N.A., et  al.: Transplantation of developing metanephroi into adult rats. Kidney Int. 54, 27–37 (1998) 43. Rogers, S.A., Powell-Braxton, L., Hammerman, M.R.: Insulin-like growth factor I regulates renal development in rodents. Dev. Genet. 24, 293–298 (1999) 44. Rogers, S.A., Talcott, M., Hammerman, M.R.: Trans­ plantation of pig metanephroi. ASAIO J. 49, 48–52 (2003) 45. Rogers, S.A., Thomas, J.M., Chen, F., et  al.: Long-term engraftment following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic rhesus maca­ ques. Xenotransplantation 14, 591–602 (2007) 46. Sariola, H., Ekblom, P., Lehtonen, E., et al.: Differentiation and vascularization of the metanephric kidney grafted on the chorioallantoic membrane. Dev. Biol. 96, 427–435 (1983) 47. Slack, J.M.W.: Developmental biology of the pancreas. Development 121, 1569–1580 (1995) 48. Statter, M., Fahrner, K.J., Barksdale, E.M., et al.: Correlation of fetal kidney and testis congenic graft survival with reduced major histocompatibility complex burden. Transplantation 47, 651–660 (1989) 49. Takeda, S., Rogers, S.A., Hammerman, M.R.: differential origin for endothelial and mesangial cells after transplantation of pig fetal renal primordia into rat. Transpl. Immunol. 15, 211–215 (2006) 50. Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., et  al.: Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998)

M.R. Hammerman 51. Velasco, A., Hegre, O.D.: Decreased immunogenicity of fetal kidneys: the role of passenger leukocytes. J. Pediatr. Surg. 24, 59–63 (1989) 52. Wild, S., Roglic, G., Green, A., et al.: Global prevalence of diabetes. Estimates for the year 2000 and projections for 2030. Diab. Care 27, 1047–1053 (2004) 53. Wolf, A.S., Palmer, S.J., Snow, M.L., Fine, L.G.: Creation of a functioning mammalian chimeric kidney. Kidney Int. 38, 991–997 (1990) 54. Woolf, A.S.: Origin of the glomerular capillaries: Is the verdict in? Exp. Nephrol. 6, 17–21 (1998) 55. Woolf, A.S., Hornbruch, A., Fine, L.G.: Integration of new embryonic nephrons into the kidney. Am. J. Kidn. Dis. 17, 611–614 (1991) 56. Yamada, K., Yazawa, K., Shimizu, A., et al.: Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha 1, 3 galactosyltransferase donors and the cotransplantation of vascularized thymic tissue. Nat. Med. 11, 32–34 (2005) 57. Yokoo, T., Fukui, A., Ohashi, T., et al.: Xenobiotic kidney organogenesis from human mesencymal stem cells using a growing rodent embryo. J. Am. Soc. Nephrol. 17, 1026– 1034 (2006) 58. Yokoo, T., Ohashi, T., Shen, J.S., et al.: Human mesenchymal stem cells in rodent whole embryo culture are reprogrammed to contribute to kidney tissues. Proc. Natl Acad. Sci. 102, 3296–3300 (2005)

Part Transplant Pathology of Organ Systems

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6

Kidney Helen Liapis, Matthew J. Koch, and Michael Mengel

6.1 Introduction H. Liapis The number of patients waiting for a donor kidney is on the rise and the scarcity of organs for transplantation continues to be a problem [130]. These factors, in the last few years, brought precipitous changes in organ procurement and allocation practices in the United States and Europe. These include accepting kidneys from donors not meeting standard criteria, so called “expanded criteria donors” (ECD) [14, 48, 80]. Furthermore, there have been significant advances in the pharmacology of rejection and the management of patients with a kidney transplant resulting in improved patient and graft survival. These changes influence the pathologic findings in the allograft and the donor biopsy. For example, the frequency or even the histopathologic types of injury currently seen in the allograft kidney is different compared to 15 years ago, with acute cellular rejection declining and recurrent disease and chronic rejection rising [72]. Humoral rejection, also referred to as “antibody-mediated rejection” (AMR), occurs beyond the early posttransplant period, and is now better defined

H. Liapis (*) Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid Avenue, Campus Box 8118, Saint Louis, MO 63110-1093, USA e-mail: [email protected] M.J. Koch Renal Division, Washington University School of Medicine, St Louis, MO, USA M. Mengel University of Alberta, Department of Pathology and Laboratory Medicine, Canada

histologically, the pathophysiology better understood with the advent of C4d+ as a tissue marker of AMR and correlation with serum donor-specific antibodies (DSA) (see Chap. 4). New biopsy types, in addition to those performed for cause (indication biopsies), have emerged. These include: biopsies performed at zero-time (implantation biopsy) or at defined time intervals (protocol biopsy). Other changes in the last 15 years include implementation of standardized diagnostic criteria for pathologic interpretation of the renal allograft biopsy and adaptation of pathologic classification of rejection types [275, 319]. Wider use of the BANFF classification scheme in particular, has improved reproducibility of pathologic findings and clinical correlation. New immunosuppressive drugs in the market inferred great influences to renal allograft pathology already [320]. Biggest perhaps challenges in the interpretation of the allograft biopsy are yet to come. These include molecular approaches to diagnosis of graft dysfunction as these emerge from bench to bedside promising to improve understanding of induction of immune tolerance and renal allograft survival [224, 237]. This chapter presents an updated practical diagnostic approach to allograft pathology. Pathophysiology and immunologic mechanisms of rejection are discussed in Chap. 1. Therefore, references to these concepts are kept to a minimum and referenced only as deemed necessary to explain pathologic findings.

6.1.1 Biopsy Types, Specimen Adequacy, and Processing Clinicopathological studies in the 1980s and 1990s established the value of the renal allograft biopsy [363]. These studies demonstrated that clinical diagnosis is

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changed in ~40% of cases and therapy is changed in ~60%, following biopsy [8]. There are currently three types of renal allograft biopsies submitted for pathologic evaluation: (a) Donor, (b) Protocol biopsies, and (c) Indication biopsies. Donor biopsies are distinguished in those: (1) meeting standard criteria donors (SCD), deceased or live, and (2) expanded criteria donor (ECD). Adequacy criteria are not yet defined for donor biopsy. SCD are derived from healthy adults 18–60 years old. SCD recommended that only kidneys from healthy donors 18–60 years old with normal creatinine were suitable for transplantation. Donors with creatinine <2.0 mg/dL were often discarded because of a perceived increased risk of long-term graft failure. This practice changed with implementation of the ECD who were defined as deceased donors older than 60 years or deceased donors aged 50–59 years old who had two of the following characteristics: donor hypertension, donor history of cerebrovascular accident, or terminal serum creatinine level greater than 1.5 mg/dL. Such donor kidneys under SCD were considered “marginal.” ECD were implemented in 2002 by the United Network for Organ Sharing (UNOS) following new donor guidelines. Protocol biopsies were also introduced recently but currently performed routinely only in a few centers around the world. A standard schedule is as follows: all patients should have a biopsy at implantation (zero-hour), weekly thereafter for the 1st month, at 3rd, and 6–12 months. The main aim is to rule out subclinical rejection. Indication biopsies are performed for cause as determined clinically, for example proteinuria, acute renal failure or increased creatinine. Indication biopsy adequacy criteria were established by the BANFF consensus conferences (BANFF 1993, 1997, amended in 2005 and 2007, respectively). In BANFF 1993, a sample was considered adequate when at least 7 glomeruli and 1 artery were present; a biopsy with 1 to 6 glomeruli and 1 artery was considered marginal, and the biopsy with no glomeruli or arteries unsatisfactory [319]. According to the BANFF 1997 revision, 10 or more glomeruli and at least 2 arteries were required for adequacy [275]. A sample with 7–10 glomeruli and 1 artery was considered marginal and <7 glomeruli and or no arteries, unsatisfactory. Section thickness at 3–4  mm was recommended. Two cores with both cortex and medulla were thought adequate based on studies that examined the sensitivity and specificity of renal biopsy comparing one and two core biopsies [67]. These studies revealed that the chance of finding acute rejection in

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one core only is 82–91%. Therefore, the sensitivity of a renal biopsy on one core is about 90%, but increases to 99% when two cores are available [67]. The specificity of the renal biopsy for rejection was calculated to be 87% in a retrospective blinded study that compared core biopsy to fine needle aspiration [129]. The BANFF 97 consensus conference became the gold standard for best practices of renal allograft biopsy evaluation. Recently, a survey was performed to determine how this gold standard fared around the world and whether actual practices varied from best practices. The survey was run among members of the Renal Pathology Society (RPS), an international society with members from five continents [269]. The survey showed that the majority of the replying nephropathogists from the US, Europe, the United Kingdom, and Australia follow the BANFF 1997 criteria. Most pathology laboratories reported receiving two 16 or 18 gauge needle biopsy cores. A minority (13%) receive two 14 and 15 gauge biopsies. However, the survey showed major differences among pathology laboratories in the processing of allograft biopsies. According to best practices, the biopsy should be triaged; one core is fixed in 10% buffered formalin, a second is held refrigerated in transport media until processed for immunofluorescence, and a small fragment is fixed in 2% glutaraldehyde for electron microscopy (EM) processing to be performed at a later time and only if indicated to rule out glomerular disease. Some laboratories have trained technicians to divide the biopsies using a dissecting microscope during work hours and attending pathologists or residents after hours. However, this is not the case in all hospitals around the world [269]. For example, adequacy of the biopsy may be determined visually or with hand-held magnified glass or a conventional microscope by pathologists, radiologists or nephrologists. Some laboratories do not perform immunofluorescence (IF) or EM. Such diversions from best practices are likely due to external factors other than best intentions or philosophy of the pathologist or the hospital, but diversion from “best practices” may certainly impact diagnostic accuracy. In summary, best practices for renal allograft biopsy interpretation dictate that two cores are submitted to pathology: one core is for light microscopy, a second for immunofluorescence with a small sample for EM. A minimum of seven slides for light microscopy with at least three Hematoxylin and Eosin (H+E) stained sections, and triplicates of PAS, or silver stain and one

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6  Kidney Table 6.1  BANFF 1997 adequacy criteria for indication renal allograft biopsies Cores 2

Light microscopy Ten glomeruli Two arteries

Immunofluorescence Three glomeruli

Cortex and medulla

3 H + E slides 3 PAS (or 1 Silver) 1 Trichrome

IgG IgA IgM C3 C4d Fibrinogen Albumin

Electron microscopya One glomerulus

For glomerular disease only

a

Trichrome are deemed sufficient. A panel of seven immune stains is recommended to include IgG, IgA, IgM, C3, Fibrinogen, Albumin, and C4d (Table 6.1). Immunofluorescence is the gold standard and considered more sensitive than immunoperoxidase [319]. Immunoperoxidase is the next best alternative.

6.1.2 Expanded Criteria Donor (ECD): A Nephrologist’s Perspective Matthew Koch One of the strongest predictors of outcome following kidney transplantation is the length of time on dialysis prior to a successful transplant, with a shorter duration portending a better outcome for patient and the allograft [220]. Many patients who were initially considered transplant candidates and placed on the waiting list will eventually be ineligible for this treatment option secondary to deterioration of other health issues that occur during the prolonged time on dialysis. For those patients eventually transplanted after years on the waiting list, the inverse relationship between time on dialysis and expected transplant outcome also mitigates against improved long-term outcomes despite advances made in the current era of kidney transplantation. The aging of the population along with an expanded eligibility list for kidney transplantation has, likewise, resulted in an older population of transplant candidates as well as an older population of potential deceased organ donors. The ability to place kidneys from nonstandard donors (formerly called marginal donors) into appropriate recipients has the potential to positively impact the aforementioned issues. Given this, the Organ Procurement and

Transplantation Network (OPTN) instituted an official definition of and a policy for marginal kidney donors in 2002 [287], and these are now termed ECD. Kidneys from donors meeting this definition were predicted to have a risk of graft loss 1.7 times that of a standard donor kidney [266]. With a graft survival rate of over 90% at 1-year for a standard donor kidney, this increased risk was felt to be an acceptable option if ECD kidneys could be placed into appropriate recipients. Such patients would be expected to have a net benefit in mortality from accepting an ECD kidney following a shorter wait time on dialysis as compared to remaining on dialysis while awaiting a standard donor kidney [304]. Individual factors that are involved in determining an acceptable patient to list for an ECD kidney include age, disease process, and expected wait time for a standard donor kidney. While debate is ongoing regarding the optimal method to allocate standard and ECD kidneys to maximize the net benefit of a scarce resource, transplant centers employing a selective ECD listing process can expect a shorter time to transplantation and a benefit in life expectancy as compared to remaining on dialysis for appropriate ECD recipients [304]. The current definition of an ECD kidney includes any donor age 60 or over, and any donor from 50 to 59 years of age with at least two of the following three criteria; history of hypertension, terminal serum creatinine >1.5 mg/dL, or death from cerebrovascular event [266]. While the definition of an ECD kidney is somewhat arbitrary and many donors not meeting these formal criteria might otherwise be considered a marginal donor, it does provide consistency in policy and is open to future adjustment. The allocation process for ECD kidneys has also been simplified to help minimize cold ischemia time to further encourage increased utilization of and improved outcomes with the use of

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kidneys from this donor pool. The advent of an ECD definition and policy has had a positive impact on the number of deceased donor transplants performed. Currently, 17% of all deceased donor kidneys transplanted in the US meet ECD criteria [303] and the number of ECD transplants performed has increased by approximately 30% since the inception of the program in 2002–2005. Given the continued imbalance between supply and demand as well as aging patient and donor demographics, increasing use of ECD kidneys for transplantation is expected to continue. There is also likely room for significant improvement in utilization of this resource. It has been estimated that up to 35% of kidneys from deceased donors over age 60 may be adequate for single kidney transplantation and up to 44% of these kidneys could be used if dual kidney transplantation is included as an option [279]. This would increase the available number of kidneys for transplantation by 25–30% [279]. One of the primary difficulties in allocating ECD kidneys is the decision to rapidly determine which kidneys are suitable for transplantation. This obviously has broad clinical implications in avoiding unnecessary discard of kidneys that would provide acceptable function, but also in avoiding transplantation of an unsuitable kidney with low likelihood of success and subsequently increasing the mortality risk without potential benefit. While a preimplantation biopsy is not the only factor considered when evaluating the suitability of an ECD kidney for transplantation, it often plays a major, if not deciding role. Thus, the renal pathologist is increasingly called upon to help affirm the decision to either use or refuse an ECD kidney. Unfortunately, there is limited data regarding the clinical utility of preimplantation biopsy in the evaluation of an ECD kidney regarding prediction of graft function and graft survival, as mentioned under adequacy criteria. The incidence of biopsy and subsequent discard rate of ECD kidneys has not changed significantly since the implementation of the formal ECD policy in 2002 and remains four times that of standard donor kidneys [333–336]. An analysis of 12,536 recovered kidneys meeting the definition of ECD from October of 1999 to June of 2005 revealed that 5,139 (41%) of these kidneys were discarded. This report was an analysis of registry data, thus all of the donor information taken into account when making the decision to either discard or use a kidney from an individual ECD donor was not available. The degree of glomerulosclerosis at

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5% increments up to and then greater than 20% was available as part of the registry, but information on interstitial fibrosis, and arteriosclerosis was not. The biopsies also included a mix of wedge and core samples, further confounding the interpretation. Thus, the biopsy result may have been of significant importance in deciding whether or not to use an ECD kidney, but other unknown factors may have played a larger role. Regardless, both the performance of a biopsy and the degree of glomerulosclerosis on biopsy were significantly associated with the likelihood of discard. In this analysis, 74.8% of ECD kidneys were biopsied, compared to 18.7% of standard criteria donor kidneys. Of the 12.3% of ECD kidneys with greater than 20% glomerulosclerosis, the discard rate was 83.1%. Of the clinical ECD criteria, terminal serum creatinine greater than 1.5 mg/dL was the greatest predictor of discard, followed by a history of hypertension, and then donor age. Compared to nonbiopsied ECD kidneys, the risk of delayed graft function (DGF) was higher in biopsied ECD kidneys (likely reflecting appropriate selection bias and increased cold ischemia time), although there was no correlation between the incidence of DGF and the degree of glomerulosclerosis. The risk of graft failure was not different in biopsied ECD kidneys from nonbiopsied ECD kidneys and there was no obvious relationship for the risk of graft failure based on the degree of glomerulosclerosis, on biopsy. The estimated glomerular filtration rate was higher at 1-year in those ECD kidneys with 0–5% glomerulosclerosis compared to those with greater than five percent glomerulosclerosis, although there was no linear correlation between estimated kidney function and degree of glomerulosclerosis greater than five percent on preimplantation biopsy. Thus, based on the available data from this registry analysis, the performance and interpretation of a preimplantation biopsy appears to have a significant impact on the decision to discard an ECD kidney, but overall, was not predictive of early graft survival or function. While the emphasis on biopsy as a potential predictor of ECD allograft outcome has been discussed, it should also be noted that appropriate donor and recipient selection with a minimization of cold ischemia time may obviate the need for biopsy, as described at a single center [47]. Similarly, the Eurotransplant Senior Pro­gram experience describes a model of older donor to older recipient, which is designed to minimize cold ischemia time and incorporates relatively few biopsies

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and few resultant discards with very much acceptable results [49, 109]. In addition, simple quantitative scoring systems incorporating the most relevant prognostic clinical information could be applied to all potential deceased donors and may be a marked improvement over the current qualitative ECD description in forecasting graft outcome without the need for biopsy [29]. Despite the lack of definitive data on the utility of preimplantation biopsy in the setting of transplantation of an ECD kidney, histology will continue to be reviewed in the majority of cases in the foreseeable future. Given the overall demographics of the potential transplant population and that of potential deceased donors, ECD kidneys will continue to increase as a percentage of all deceased donor transplants. Improvement and verification of a scoring system to better evaluate nonstandard donors may obviate the need for preimplantation biopsy in many cases while still providing successful outcomes. Avoiding routine biopsy would also decrease cold ischemia time and should subsequently decrease the incidence of DGF, which may have a significant overall benefit in improving the expected outcome for an ECD kidney. Barring this development, data from a controlled study is needed to identify histologic patterns in ECD kidneys that suggest either transplantation or discard is appropriate, or that consideration of dual kidney transplantation is warranted. Current studies and retrospective reviews appear inadequate to make this determination, particularly in predicting long-term graft function. While the long-term outcome of an ECD kidney transplant is expected to be inferior to that of a standard donor kidney, the use of these kidneys is justifiable in the appropriate patient population. This includes patients whose age and/or underlying comorbidities mandate an attempt at transplantation within an acceptable timeframe after starting dialysis in order to benefit via increased life expectancy and who are unlikely to receive a standard donor kidney within that period. The increased use of ECD kidneys in this population also has the secondary effect of making available more standard donor kidneys for appropriate standard donor recipients. A systematic improvement is necessary to assist in the allocation of ECD kidneys to this patient population as is a deceased donor evaluation process that avoids unnecessary discard rates that may deprive potential recipients of kidney transplantation with an acceptable allograft, while also avoiding placement of allografts with primary nonfunction or with very poor

renal function. At present, preimplantation biopsy of an ECD kidney, histology, clinical information, and gestalt remain the determinants of allocation.

6.1.3 Donor Biopsy H. Liapis Donor biopsy, also referred to as preimplantation biopsy, is defined as biopsy of the donor kidney at harvest, as opposed to zero-time biopsy referring to biopsies taken at the time of grafting. Donor biopsies are well established, even though selectively performed in various centers. The purpose of sampling the donor kidney is to rule out pre-existing disease, be it vascular, glomerular, tubulointerstitial or malignancy. The most frequent lesions identified are vascular (arterial hyalinosis) and acute tubular injury (ATI). A list of frequent as well as some rare findings in the donor biopsy is shown in Table 6.2. Arterial/arteriolar hyalinosis is defined as accumulation of hyaline material underneath the endothelial Table 6.2  Histopathological findings in the donor biopsy Vascular disease Arteriolar Hyalinosis Arterial sclerosis Arteriolar/glomerular capillary thrombosis Parenchymal artery stenosis/segmental ischemic cortical atrophy Microthrombi (DIC) Glomerular disease Glomerulosclerosis Cystic change FSGS Diabetes IgA Thin membrane disease Fabry’s disease Other Tubular damage Acute tubular injury (ATI) Acute pyelonephritis Myoglobinuria (rhabdomyolysis) Interstitial disease Fibrosis Chronic inflammation Tumors Benign (e.g., angiomyelipoma, neurofibroma) Malignant (renal cell carcinoma)

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layer (Figs. 6.1a and 6.2c). Hyalinosis is most common in older donors and its frequency has increased with utilization of ECD kidneys. Arteriolar hyalinosis is often associated with glomerulosclerosis and many studies find that this constellation correlates with decreased graft survival (defined as recipient’s return to dialysis) [210, 239]. In spite of the presence of arterial/arteriolar thickening particularly in ECD biopsies, kidneys are often grafted because of the existing high demand. Several recent studies evaluated ECD donor biopsies and some propose standardizing the process by semiquantitative scoring of the lesions but reproducibility studies are lacking, and not one scoring scheme is currently accepted or applied routinely. Munivenkatappa et al. scoring system is an example of things to come to practice if consensus is met. Vascular lesions other than hyalinosis and arteriosclerosis, such as vasculitis or pathologic processes involving small or large vessels (interlobar or interlobular arteries) are rarely seen in donor biopsies unless a generous sample is submitted. Arteriosclerosis is characterized by intimal and medial thickening and lamellation of the internal elastic lamina (Fig.  6.2). Elastin stain distinguishes this type of injury from fibrointimal proliferation of chronic vascular rejection, which is not associated with lamellation as discussed under chronic rejection. Another not so infrequent finding in the donor biopsy is, isolated microthrombi that are often secondary to prefatal diffuse intravascular coagulation (DIC) (Fig. 6.1d arrow). These thrombi are typically not causing luminal dilatation or fibrinoid necrosis unlike thrombotic microangiopathies due to infection (HUS). Thrombi, in donor kidneys due to DIC, are reversible and not a contraindication for transplantation. On occasion, true thrombotic microangiopathy (TMA) may be found in the donor biopsy characterized by myxoid intimal proliferation. In such cases, the donor may have a reported history of fatal cerebrovascular accident but, in reality, undiagnosed thrombotic thrombocytopenic purpura (TTP), instead. TTP-induced thrombi also appear to be reversible in the recipient (anecdotal case of mine). Other unusual vascular findings include cholesterol emboli. Segmental thickening of interlobar or interlobular arteries results in grossly identified cortical scars in the donor kidney mimicking malignancy, but the corresponding microscopic pathology reveals collapsed cortical parenchyma, cortical necrosis, and thick vessels

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associated with retracted glomeruli (glomerular cysts) (Fig. 6.3). Glomerular cysts have multifactorial etiology; importantly, in young donors, may represent undiagnosed variants of hereditary cystic kidney disease. In older donors, the most frequent cause is vascular ischemia [193]. Such segmental renal vascular anomalies in donor kidneys may be surgically removed at the time of harvesting and are not an absolute contraindication for donation, particularly if double kidney transplantation is considered [33, 315]. ATI can be focal or diffuse (involving >50% of the biopsy surface area) and results from ischemic injury during transport when kidneys are kept in cold temperature (cold ischemia time). The extent of ATI is directly related to the time interval from organ harvesting to transplantation and it also depends on preservation methods. ATI is characterized by loss of brush boarder and focal or multifocal loss of tubular epithelial cell nuclei (Fig.  6.4). In contrast, acute tubular necrosis (ATN) is typically composed of extensive loss of epithelial cells with denudation of the basement membrane (Figs. 6.1a and 6.6). Ischemic reperfusion injury has a complex pathogenesis involving upregulation of lymphocyte-related genes and humoral immune res­ ponses that are directly activated during ischemia contributing to renal dysfunction [125, 192]. Preser­vation methods are also implicated including low temperature and high potassium solutions with or without continuous pulsatile perfusion [146]. Hypothermia invariably leads to altered cytoplasmic and cell basement membrane homeostasis, including mitochondrial damage in tubular epithelial and endothelial cells. These responses may have long-term effects, currently thought to enhance immunogenicity causing acute or chronic rejection, and have raised awareness and search for better organ preservations methods [19, 179]. True ATN defined as coagulation necrosis of tubular epithelial cells is not so common, currently (Fig. 6.1a). A question that often arises in the interpretation of the allograft biopsy is how best to distinguish ATN from ATI and how to avoid using the term ATN when biopsies lack diagnostic criteria. A simple rule of thumb may be to avoid the term ATN if there is no tubular epithelial cell denudation and in the absence of coagulation necrosis of the tubular epithelial cell cytoplasm. ATI, on the other hand, may coexist or be obscured by other more obvious lesions, for example, thrombi (Fig.  6.1d), white cell casts (acute pyelonephritis) or

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Fig. 6.1  Donor (preimplantation) biopsy pathology. (a) Arterial sclerosis and arteriolar hyalinosis (arrow) (H + E × 200). (b) Diabetic glomerusclerosis (asterisk) in donor kidney from 32 year old man with juvenile diabetes. Arterial sclerosis is also present (H + E × 200). (c) Focal segmental glomerulosclerosis (arrow) (FSGS) in older donor with hypertension but no clinical history of proteinuria (H + E × 300). (d) Acute tubular injury

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(ATI) concurrent with thrombosis secondary to DIC (arrow) (H + E × 200). (e) Donor biopsy containing fusiform fibrous bundle dissecting through tubules diagnosed as neurofibromatosis (Trichrome stain ×200 kidney contributed by Dr. Julie Riopel, Hôtel-Dieu de Québec, Quebec, Canada). (f) Incidental papillary renal cell carcinoma (5  mm in diameter) removed at the time of harvesting (H + E × 100)

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Fig. 6.2  Arterial nephrosclerosis and hyalinosis in wedge donor biopsy. (a) There is intimal and medial thickening with lamellation of the internal elastic lamina (×200). (b) Lamellation is

highlighted with elastin stain (×400). (c) Arteriolar hyalinosis (arrow) (H + E × 200)

myoglobin casts in donors who died from automobile crash injuries. Global glomerulosclerosis (Fig.  6.4) is a frequent finding in the donor biopsy. Studies show that even with >20% of glomerulosclerosis there is good graft survival compared to donor biopsies with <20% globally sclerosed glomeruli [17, 47, 62, 111, 131, 201], but other authors consider kidneys with >20% globally sclerosed glomeruli unacceptable for donation [239]. Overall, it appears that increased global glomerulosclerosis is a bad prognosticator of graft outcome, but the predictive ability of the donor biopsy based on this feature is often difficult to interpret because it is influenced by center practice and clinical selection

criteria. Also, it is unclear in various studies whether conclusions were drawn based on frozen section interpretation, which is not always in agreement with permanent sections. There is also variability in reporting histologic findings other than glomerulosclerosis that may influence graft outcome; the definition of successful allograft outcome varies; patient sample size is often too small for definitive conclusions. The recent study by Munivenkatappa et al. [239] at the University of Maryland proposes a scoring system (MAPI) for deceased donors most of who were ECD. The scores the authors proposed ranged from 0 (no significant changes) to 15 points (severe chronic changes). The features accounted for are: global glomerulosclerosis

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Fig.  6.3  Glomerular cysts in donor wedge biopsy. The donor was 20 year old man who died from gunshot wound to the head. The lesion was grossly described as solitary involving the lower pole of the left kidney imitating malignancy. There was no malignancy found, but glomerar cystic changes were apparent H + E × 40

Fig.  6.4  ATI and global glomerulosclerosis in donor kidney. There is diffuse tubular injury characterized by loss of proximal tubule brush boarder and focal loss of nuclei, but no tubular epithelium denudation or coagulation necrosis. A sclerosed glomerulus (asterisk) is also shown (H + E × 40)

³15% (two points), arteriosclerosis with >50% luminal stenosis (two points), presence of periglomerular fibrosis (four points), arteriolar hyalinosis (four points), and scar formation (three points). Three categories for 5-year graft survival were devised: score 0–7: low risk, score 8–11: medium risk, and score 12–15: high risk; 5-year actuarial graft survival was 90% for kidneys with low MAPI scores, 63% for medium, and 53% for high. Permanent sections prepared from paraffin blocks

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were more reliable than frozen sections in this study. In other studies, the predictive ability of global glomerulosclerosis with arteriosclerosis was evaluated [15, 83, 99, 168, 173] or glomerulosclerosis with interstitial fibrosis in regards to graft outcome [277]. These studies show that both factors are variably primary predictors. A histology-based evaluation of the donor biopsy in deciding whether or not to use a kidney is supported by a study by Remuzzi et al. that utilized a scoring system for vessels, glomeruli, tubules, and interstitium in core biopsy samples to determine discard vs. single or dual transplantation from deceased donors older than 60 years [281]. The recipients of allografts from donors over the age of 60 who underwent donor biopsy evaluation had significantly improved graft survival as compared to recipients of kidneys from donors >60 years who did not undergo preimplantation histologic evaluation. In the latter group, the risk of graft failure was 3.68 times that of those receiving a kidney from a donor over age 60 “only after histologic evaluation.” The caveat in this study is that the majority of the transplants performed in the donor biopsy group were dual transplants, thus a direct comparison to the nonbiopsy groups who were recipients of single allografts was biased. The studies discussed above, demonstrate that at this time there is no concerted effort to standardize interpretation of the donor biopsy. However, lack of directive information has not prevented authors to cite biopsy findings as the most frequent reason for discard of an ECD kidney [335]. As mentioned previously, studies using only wedge biopsy samples or multivariate analysis that includes both wedge and core samples are difficult to interpret. Although very little comparative data is available, a core biopsy is favored by pathologists [138]. A wedge biopsy has the potential to significantly overestimate the degree of glomerulosclerosis given the percentage of subcapsular material often provided as compared to a core sample. This may be even more pronounced in the setting of an ECD kidney [240] and may explain the reported lack of association of glomerulosclerosis with graft outcome in some analyses [265]. Surgeons, on the other hand, may prefer wedge biopsies because it is easy to suture the incision and control bleeding. Common primary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS), diabetic glomerulosclerosis, IgA nephropathy, thin membrane disease (TMD) are difficult to evaluate in the donor biopsy. For example, donor biopsies due to the urgency of the

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Fig. 6.5  Implantation biopsy pathology from ECD ABO compatible donor. (a) Mild interstitial fibrosis (Trichrome stain ×200). (b) Diffuse albumin positivity in tubular/glomerular basement membrane and Bowman’s capsule (IF × 300). (c) C4d+

in peritubular capillaries (PTC) without detectable serum donor specific antibodies (DSA); may reflect “accommodation” (IF × 200). (d) Electron microscopy (EM) shows focal foot process effacement (asterisk) (×4,500)

evaluation are not routinely processed for immunofluorescence and EM, but only for light microscopy. Therefore, common lesions such as FSGS appearing in a donor biopsy are difficult to interpret. In particular, true FSGS in the donor, when the biopsy also shows advanced arterionephrosclerosis is a possibility that cannot be excluded (Fig. 6.1c). Similarly, asymptomatic or mild IgA nephropathy, TMD etc., cannot be excluded. These are, on occasion, found in biopsies from live donors who were evaluated for cause. For example, the question of whether to accept a donor with so called “benign hematuria” has come up in our service. There is little experience today and no consensus as to whether the risk for a donor with TMD (or the recipient) is acceptable [158, 346]. Well-developed diabetic glomerulosclerosis is not difficult to diagnose in donor biopsies

(Fig.  6.1b). Interestingly, some transplant centers will utilize diabetic donor kidneys supported by published results from nondiabetic recipients who, having received diabetic kidneys, experienced complete resolution of diabetic glomerulosclerosis documented by allograft biopsy at a later time [3]. IgA nephropathy in the donor is another disease that does not appear to have a significant effect to graft survival [324]. Finally, asymptomatic glomerular disease in the donor (sometimes, familial) may manifest in the recipient for the first time. An extraordinary case of Fabry’s disease first diagnosed in the allograft kidney from a living related donor is such an example [268]. Interstitial inflammation and the degree and composition of the inflammatory infiltrate are thought to have a predictive value but definitive studies in the donor are

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lacking at this time. One reason may be the subjectivity in assessing interstitial inflammation and marked sampling variability [224]. Similarly, interstitial fibrosis is not accurately quantitated visually, most of the time. Several scoring systems can be applied including arbitrary grading (e.g., absent = <5%, mild = 6–25%, moderate = 25–50%, and severe = >50%) and or morphometry. Frozen specimens that are standard in this clinical setting make interpretation of anything less than severe interstitial fibrosis difficult to differentiate from artifact. Thus, permanently fixed samples are preferred in any study evaluating the utility of the donor biopsy. However, so far, there is no standard method applied universally and there is need for consensus on both methods for evaluating interstitial fibrosis as well as scoring schemes. Purely clinical parameters are not infrequently used to derive deceased donor scores to predict graft survival. For example, in a retrospective review of 34,324 patients who received cadaver renal transplants from adult donors between 1994 and 1999 were used by the UNOS Scientific Renal Transplant Registry to derive a scoring system based on five parameters: age, 10–25 points; history of hypertension, 0–4; creatinine clearance before procurement, 0–4; cause of death, 0–3; HLA mismatch, 0–3 [249]. Kidneys were stratified by cumulative donor score: grade A (0–9 points); grade B (10–19); grade C (20–29); and grade D (30–39). Donor score on renal function and graft survival was most severe if the score was >20 points. Benign and malignant kidney tumors are possible encounters in donor biopsies. Most common are small renal cell carcinomas papillary or clear cell type (Fig. 6.1f). Leiomyomas, medullary fibromas or simple cysts, render the donor kidney acceptable for transplantation after wedge tumor resection. Small epithelial neoplasms, however, can generate dilemmas. The distinction between a so-called “renal adenoma” and a “small low grade renal cell carcinoma” was arbitrarily based on the size of the lesion, although it is now increasingly recognized that lesions of any size can metastasize. If the lesion is small (less than 0.5 cm), and completely excised, the risk of residual or recurrent carcinoma in the recipient is probably extremely small. For example, six reported cases with excision of the tumor led to uneventful course 186 months posttransplantation [276]. Nonetheless, this issue requires receipient consent and some policy in place by the transplant centers.

Rare benign tumors may also be perplexing on frozen section. For example, fusiform and bizarreappearing fibrous nodules were seen in a donor kidney frozen section (Fig.  6.1e). Even though obviously benign, the diagnosis is very difficult to make if one has not encountered such a lesion before. These poorly recognized and rarely reported peritubular nodules represent hamartomatous neurofibroma involving the donor kidney (kindly provided by Dr. Julie Riopel, Canada) [203]. It is worth mentioning here that, according to OPTN, 2.2% donors have a prior history of cancer including benign and malignant neural derived tumors. Their database from 39,455 deceased donors from 2000 to 2005 revealed 1,069 donors with history of cancer [172]. In this and previous studies, OPTN and UNOS found a very low cancer transmission rate from positive donors (0.012%). The only exception was malignant melanoma. History of malignant melanoma is an absolute contraindication for organ donation [171].

6.1.3.1 Protocol Biopsies Protocol biopsies have long been implemented for cardiac allografts because subclinical rejection is common but clinical symptoms often develop late and when rejection is severe. In the kidney allograft, protocol biopsies are a recent development that is still debated and not universally accepted. Subclinical rejection in the kidney allograft is defined as the presence of histologic changes that may meet the criteria for acute cellular rejection (BANFF I or greater), in the absence of renal graft dysfunction, therefore no rise in creatinine (a marker for allograft dysfunction). Notably, it is believed that creatinine may require more extensive kidney damage prior to rising. Therefore, the proponents of protocol biopsies argue that protocol biopsies compensate for lack of rise in creatinine in subclinical rejection, but also for AMR, or other injuries that may have minor influence on creatinine values. In a series of 37 patients who had protocol biopsies performed at 1st, 2nd, 3rd, 6th, and 12th months posttransplantation, >50% had subclinical rejection. Of these, 52% had histologic evidence of acute rejection and 36% had borderline changes (suspicious for rejection). Subclinical rejection rate is reported to fall 1-year posttransplantation [291]. In spite of these findings, there is currently limited

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evidence supporting the notion that treating subclinical rejection may improve graft survival. For example, in a randomized clinical trial of 36 patients who had protocol biopsies treated for subclinical rejection, there was less chronic damage at 6 months and lower serum creatinine at 2 years compared to control group [290]. Others suggest that the incidence of subclinical rejection may be lower than reported and influenced by induction/baseline immunosuppression and HLA matching. Finally, some authors believe that immunosuppression and HLA match may affect long-term graft survival and development of chronic rejection more than subclinical rejection [273, 309]. The opponents of protocol biopsies argue that the impact on long-term graft survival is not yet clear, the risk even though small is not zero and after all, interpretation of the biopsy findings are subject to sampling error and subjectivity of the interpreter [342]. However, several recent publications demonstrate that the risk of serious complication is as low as 0.4–1.0% and biopsies very rarely lead to graft loss [226, 271]. There is agreement however, on protocol biopsies be performed in patients who are at high risk for subclinical rejection because of either previous allograft loss, history of DSA, or infection (polyoma). Protocol biopsies are performed at defined intervals as follows: (1) postimplantation (also referred to as implantation or zero hour biopsy), (2) weekly for the first month, (3) at 3 months, and (4) at 6–12 months. For stable grafts, follow-up biopsy should be obtained at 6–12 months to rule out subclinical rejection, calcineurin inhibitor (CNI) toxicity, or developing interstitial fibrosis. Biopsy at the time of renal dysfunction may be necessary beyond these time intervals. Experience with protocol biopsies so far shows variable pathology, from none to ATI or ATN, interstitial fibrosis, interstitial inflammation, arterial hyalinosis, C4d+ in peritubular capillaries (PTC), and other unexpected findings such as cellular rejection [173]. An example is shown in Fig. 6.5. The recipient was a 65-year-old man with transplant from an ABO compatible ECD donor. There was focal interstitial fibrosis, intense albumin staining and weak, but diffuse C4d+ in PTC with undetectable DSA. EM revealed minor foot process effacement (Fig. 6.5c). The significance of these findings is uncertain. In particular, the role of C4d deposits without other histologic findings in protocol biopsies is not well understood (discussed further under AMR).

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Fig. 6.6  Acute tubular necrosis (ATN) (arrow) in a biopsy from patient with delayed graft function (DGF) (H + E × 200)

6.1.3.2 Delayed Graft Function (DGF) Protocol biopsies are particularly recommended for patients with DGF. DGF is defined roughly as 20–30% drop in serum creatinine over 24–48 h period during the first week posttransplantation requiring dialysis [141]. Patients who do not meet these arbitrary criteria are often said to have “slow graft function” (SGF). DGF is a problem that often occurs in deceased allo­graft kidneys but occasionally may complicate living donor grafts; it is treated with dialysis. Graft function recovery usually occurs within a month, but it may take longer. The main difference between DGF and SGF is that in the latter, patients are not treated with dialysis. Biopsy findings of DGF include: ATI or ATN, possible early AMR, cortical necrosis/infarction, acute CNI toxicity, TMA, and drug-induced interstitial nephritis. Sometimes, acute rejection or fulminant recurrent disease (e.g., FSGS) may be present (Table 6.3). Donor disease such as severe arteriolar halinosis may also cause DGF (Fig. 6.7). When no significant histopathological findings are found, other factors such as donor age, recipient race and sensitization, preservation method, HLA mismatches, cold ischemia, or drugs used in immunosuppression protocols are possible precipitating factors [179]. Sirolimus, in particular, is reported to delay graft function [216, 263]. DGF appears to increase the probability of rejection via poorly understood immunologic mechanisms, thus adversely impacting long-term

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6  Kidney Table 6.3  Histopathological findings in delayed graft function (DGF) ATI Acute tubular necrosis (ATN) No histopathological findings Early antibody-mediated rejection Cortical necrosis/infarction Acute calcineurin inhibitor toxicity (thrombotic microangiopathy) Drug-induced interstitial nephritis Nephrocalcinosis Acute rejection Fulminant recurrent disease (e.g., FSGS) Asymptomatic glomerular disease in the donor Severe arteriolar nephrosclerosis in the donor kidney

graft survival [101, 374]. We have seen a few patients with DGF come to biopsy at our institution. In our experience, the findings on light and immunofluorescence microscopy are, with few exceptions, minor and whether related to DGF is unclear. For example, we saw an 18-year-old man who received living unrelated kidney from a healthy 28-year-old woman; biopsy showed insignificant changes on light microscopy and negative routine immunofluorescence, but on EM, segmentally thin glomerular basement membrane (GBM) was apparent. The donor did not have hematuria or proteinuria, nor was there a family history of Alport. Collagen IVa

3–5 immunofluorescence, were positive and overall, the pathology did not raise the possibility of Alport. A presumptive diagnosis of TMD was made. As mentioned earlier, the outcome of grafts with TMD is unknown and whether such donors carry higher risk for themselves after donation or whether TMD compromises graft survival is unknown [158]. Other potential causes of DGF reported in the literature include increased serum calcium levels. Calcium supplementation and vitamin D analogs are commonly used in patients on dialysis. Hypercalcemia was independently associated with DGF in a study by Boom, et al. [38]. The authors found an incidence of 31% DGF in a cohort of 585 cadaveric transplants; DGF correlated independently with serum calcium levels. Use of calcium channel blockers before transplantation in this study protected against DGF. However, most of the biopsies also had acute rejection; nephrocalcinosis was found in 12 of 71 biopsies but was not associated with serum calcium levels or the occurrence of DGF. Overall, DGF is not entirely understood when there are no significant histopathological findings but it is possible that changes in gene transcription preceding morphology underline DGF [238].

6.1.3.3 ABO Incompatible Grafts (ABOi) ABO incompatibility was once thought of as a formidable barrier to renal transplantation, but no more. The first ABOi kidney transplants were performed by Hume, Murray, and Starzl in 1955, 1960, and 1964, respectively

Fig. 6.7  (a) Biopsy performed for DGF a week after implantation. The only pathologic finding was severe arteriolar sclerosis. (b) Implantation biopsy shows donor disease. The recipient was on dialysis for a month prior to DGF resolution (H + E × 200)

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[369]. Some of the first patients lost their grafts to humoral rejection in a dramatic fashion that triggered aggressive immunosuppression measures for subsequent patients. Since then, a significant number of ABOi kidney transplantations were successful following plasmapheresis, immunosuppression, and without splenectomy or B-cell ablative (anti-CD20) therapy [232]. Eventhough ABO compatible transplantation is preferred, ABOi transplantation is often the only option, particularly in some countries with severe shortage of kidney donors (for example, Japan), or for patients with rare blood group types in western countries [157]. Most patients with ABOi grafts experience no hyperacute or acute humoral rejection, with the exception of an A2 donor into a B or O recipient, or an A2B donor to a B recipient. Studies report that unless the recipient has very low antiA titers (<1:4), there is a significant increased risk for antibody mediated rejection (AMR) [44].

The long-term outcome of ABOi kidneys is similar to that of conventional allografts at 1-year posttransplantation at least in patients who had absent serum DSA and signs of rejection. However, patients with prior evidence of humoral rejection have an increased incidence of transplant glomerulopathy (TGP) [123]. In the absence of humoral rejection, there are no significant differences in interstitial fibrosis or other findings between biopsies from ABOi and ABO compatible donors. We have seen a dozen or so ABOi kidneys transplanted at our institution. Of these, one 19-yearold man with B+ blood group and first-time kidney transplant secondary to congenital renal dysplasia, developed graft dysfunction 2 days posttransplant (Fig. 6.8). The biopsy was significant for ATN, diffuse C4d+ peritubular capillary deposits but no evidence of thrombosis (Fig. 6.8a, b). Kidney function continued to deteriorate and a second biopsy was performed on

Fig. 6.8  Allograft biopsy at 2 (a, b) and 5 days post transplant (c, d) from a 19-year-old-man with ESRD secondary to renal dysplasia who received ABOi kidney. (a) Minimal findings (mild ATI); no evidence of rejection (PAS × 400). (b) Diffuse

C4d+ in PTC (IF × 200). (c) Cortical necrosis (H + E × 400) 5 days postimplantation (H + E × 200). (d) Intense diffuse C4d+ in peritubular and glomerular capillaries (IP with alkaline phosphatase label ×200)

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day 5, which showed evolving acute AMR (Fig. 6.8c, d). This patient underwent plasmapheresis and successful resolution of this rejection episode followed by graft survival at 6 months. Most patients like this 19-year-old, who responded to initial therapy are less likely to reject the graft in the short term and as long as they maintain low antibody titers. Notably, ABOi grafts often have peritubular capillary C4d deposits on protocol biopsies without histologic features of AMR. For example, Haas et al. found that ABOi grafts have C4d+ staining in 80% of protocol biopsies compared to 59% of biopsies performed for graft dysfunction [136]. C4d+ did not correlate with neutrophil accumulation in PTC, while C3d was somewhat more predictive of neutrophil margination. The authors found no evidence that C4d+ is related to significant renal injury in most ABOi grafts. The significance of this finding

was recently revisited by the same authors who found that diffuse peritubular capillary C4d+ without rejection is, in fact, associated with a lower risk for scarring in ABOi renal allografts [137]. Whether, C4d+ is a sign of accommodation in these grafts remains to be further studied [318].

6.2 Indication Biopsy Indication biopsies are performed for clinically identified cause. Three major categories of pathologic diagnoses are common in indication renal allograft biopsies: rejection, recurrent, and de  novo disease (Table  6.4). Each of these injuries may primarily or exclusively involve glomeruli, tubules, vessels or the

Table 6.4  Histopathological findings in indication renal allograft biopsies Rejection Recurrent disease

De novo disease

Acute cell mediated Tubulitis Endotheliitis Glomerulitis Interstitial inflammation

Glomerular FSGS Diabetes Lupus IgA MPGN Membranous Vasculitis (ANCA, Anti-GBM) Amyloidosis/LCDD/Fibrillary TMA (HUS, apL) Fibronectin Glomerulopathy Other

Glomerular Transplant glomerulopathy FSGS Diabetes Anti-GBM Postinfectious Membranous Other

Chronic cell mediated Interstitial inflammation Intimal fibroplasia without fragmentation of elastin Interstitial fibrosis

Tubulointerstitial Myeloma cast nephropathy

Vascular TMA (Drug induced) Cholesterol embolism Renal artery thrombosis Renal vein thrombosis AMR

Antibody mediated (AMR) Acute Chronic Chronic/active Suspicious

Tubulointerstitial Drug reaction Oxalate crystals Calcium phosphate crystals ATN Acute interstitial nephritis

Mixed types T and B cell mediated CD20+ infiltrates Plasma cell rich (PCAR) rejection

Malignancy Malignant lymphoma PTLD Renal cell carcinoma Other Ureteral stenosis Graft Rapture Denervation Lymphocele Urinary leaks

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interstitium but not infrequently are contemporaneous (mixed). It is important for the pathologist to prioritize diagnoses and list them in order of significance, aiming at providing a clear message to nephrologists in regards to therapeutic action. In the following sections, each pathologic entity is discussed separately even though these commonly occur together. The focus is on the diagnostic criteria and scoring systems most commonly used in daily practice. Mechanisms are kept to a minimum and only when they are relevant to pathologic findings and clinicopathologic outcomes. The reader is referred to Chap. 1 for more details on the immunology of rejection.

6.2.1 Acute Rejection Acute cellular (T cell mediated) rejection has been the main target of immunosuppression therapy in the last several decades. The goal has been to achieve balance between antirejection drug levels and adverse drug effects. The results were successful with significant lowering of the rate of acute cellular rejection to approximately 5–10% in the first year posttransplant [70]. Clinical manifestations of acute rejection include: abrupt increase of serum creatinine, fluid retention, fever, graft tenderness, and occasionally nephrotic syndrome in the early posttransplantation period [5]. Acute rejection manifests both early and late posttransplantation. The key histopathological feature of symptomatic acute cellular rejection is mononuclear inflammatory infiltration of tubules (tubulitis), arteries (arteritis) or the interstitium. Renal biopsy plays a pivotal role in diagnosing rejection and distinguishing acute cell mediated vs. AMR. Severity of histopathological findings (grade) of acute rejection on renal biopsy correlates with clinical outcome. For example, 47% of grade III rejection is irreversible; in contrast, 93% of grade I rejection is entirely reversible. However, recurrent rejection is an independent factor of worse prognosis for graft outcome, irrespective of histological grade in the first rejection episode; repeat renal biopsy is the best method for assessing ongoing damage. Scoring systems for renal allograft rejection were formulated in the early 1990s by various groups. In this chapter, the most recently updated BANFF schema and the most recent scoring criteria as addressed by BANFF will be discussed (Table  6.5). The BANFF

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classification scheme now includes the Cooperative Clinical Trials in Renal Transplantation (CCTT) classification since the CCTT group merged with the BANFF group at the 1997 BANFF consensus meeting. The consensus paper was published in 1999 as the revised BANFF ’97 classification [226, 275]. The most recent BANFF recommendations of the meeting held in 2005 were released in 2007 [321]. The major change was distinguishing acute and chronic rejection in (a) T cell mediated and (b) AMR rejection (Table 6.5). In the BANFF 1997–2005 update, grading of acute cellular rejection is as follows: (1) borderline changes (suspicious for rejection) defined as 1–4 mononuclear cells per tubular cross section; (2) mild tubulitis, rejection IA: >4 mononuclear cells/tubular cross section or group of 10 tubular cells; (3) moderate tubulitis, rejection IB, >10 monoclear cells per tubular cross section or group of 10 tubular cells; (4) severe rejection IIA: intimal arteritis (endotheliitis) with <25% of vessel circumference involved and >25% interstitial inflammation; (5) severe rejection IIB: endotheliitis involving >25% of arterial circumference, and (6) severe rejection III: transmural arterial involvement with or without fibrinoid necrosis (Table  6.5). In the 2005 BANFF revision, the term acute rejection was replaced by T cell mediated rejection (TCMR), which features two subcategories: acute and chronic TCMR [321]. In this latest BANFF scheme, the g, i, t, and v lesions are quantitated as described in the first BANFF published in 1993. Briefly: g stands for glomerulitis; i = interstitial inflammation; t = tubulitis; and v = vasculitis. These are graded on a scale of 0–3+ (Table 6.6). Subclinical acute rejection also occurs. By definition, clinical symptoms are lacking and unless protocol biopsies are performed diagnosis is missed.

6.2.1.1 Tubulitis Tubulitis is the most frequent finding in T cell mediated acute rejection and is thought to contribute to the abrupt rise of creatinine. Infiltrating mononuclear cells, lymphocytes, macrophages, but not plasma cells or eosinophils invade the tubular basement membrane mainly of distal tubules causing tubulitis (Fig.  6.9). Lymphocytes are mostly CD4+ and CD8+; macrophages are common, but B cells (plasma cells) do not appear to participate. Tubulitis is diagnostic of acute rejection if it involves nonatrophic tubules only,

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6  Kidney Table 6.5  BANFF 97-update 2005 classification of renal allograft rejection 1. Normal 2. Antibody-mediated rejection Due to documented antidonor antibody (“suspicious for” if antibody not demonstrated); (may coincide with categories 3–6) Acute antibody-mediated rejection Type (grade)   I. ATN-like – C4d+, minimal inflammation   II. Capillary-margination and/or thromboses, C4d+   III. Arterial – v3, C4d+ Chronic active antibody-mediated rejectiona Glomerular double contours and/or peritubular capillary basement membrane multilayering and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries, C4d+ 3. Borderline changes: “suspicious” for acute T-cell-mediated rejection This category is used when no intimal arteritis is present, but there are foci of tubulitis (t1, t2 or t3 with i0 or i1) although the i2 t2 threshold for rejection diagnosis is not met (may coincide with categories 2, 5, and 6) 4. T-cell-mediated rejection (may coincide with categories 2, 5, and 6) Acute T-cell-mediated rejection Type (grade)   IA. Significant interstitial infiltration (>25% of parenchyma affected, i2 or i3) and foci of moderate tubulitis (t2)   IB. Significant interstitial infiltration (>25% of parenchyma affected, i2 or i3) and foci of severe tubulitis (t3)   IIA. Mild to moderate intimal arteritis (v1)   IIB. Severe intimal arteritis comprising >25% of the luminal area (v2)   III. T  ransmural’ arteritis and/or arterial fibrinoid change and necrosis of medial smooth muscle cells with accompanying lymphocytic inflammation (v3) Chronic active T-cell-mediated rejectiona “Chronic allograft arteriopathy” (arterial intimal fibrosis with mononuclear cell infiltration in fibrosis, formation of neo-intima) 5. Interstitial fibrosis and tubular atrophy (IFTA), no evidence of any specific etiologya Grade   I. Mild IFTA (<25% of cortical area)   II. Moderate IFTA (26–50% of cortical area)   III. Severe IFTA/loss (>50% of cortical area) (may include nonspecific vascular and glomerular sclerosis, but severity graded by tubulointerstitial features) 6. Other: changes not considered to be due to rejection-acute and/or chronic; may coincide with categories 2–5 Indicates changes in the updated BANFF’05 schema

a

unless perhaps in cases of tubulitis within scarred, severely damaged grafts. Injured tubular epithelial cells undergo cytoplasmic and nuclear degeneration proportionate to the degree of inflammation. These changes are now understood to be the result of effector molecules released by activated T cell lymphocytes, such as perforin, granzyme A and B, TNFa, TNFb, TNFg, TGFb, and other cytokines. Some of these also participate in chronic rejection as well, for example, TGFb causes experimental interstitial fibrosis, an invariable consequence of repeated episodes of acute rejection. Tubulitis is scored as described above. Examples of mild-to-severe tubulitis are shown in Figs. 6.9–6.11. Rejection grade as per BANFF scheme

should be routinely reported in the pathology report because it facilitates communication with nephrologists. The frequency of acute tubulitis/arteritis is about 30% in our institution, currently (Table  6.7) [174]. Most of our cases have low grade TCMR, either IA or IB. As mentioned above and previously shown by published studies, histopathological grade of acute rejection has prognostic value and also guides therapy [134, 247, 361]. For example, the initial response to antirejection therapy is significantly worse in patients with type IIB acute rejection compared to those with type IIA despite more aggressive treatment of type IIB rejection. Minimal or mild tubulitis responds better to therapy than moderate or severe tubulitis although

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Table  6.6  Quantitation of acute lesions in renal allograft biopsies according to BANFF 97 Glomerulitis (g) g0: no inflammation g1: mononuclear infiltrate ± endothelial cell swelling involving <25% of glomeruli g2: 25–75% of glomeruli g3: >75% of glomeruli Tubulitis (t1) t0:no tubulitis t1:<4 intraepithelial lymphocytes in the most inflamed tubular cross-section t2: 4–10 intraepithelial lymphocytes t3: >10 intraepithelial lymphocytes

Fig. 6.10  Borderline tubulitis: <4 mononuclear cells (arrows) per tubular cross section (PAS × 400)

Arteritis V0: no arteritis v1: intimal arteritis with decreased luminal diameter by<25% v2: >25% v3: transmural inflammation ± fibrinoid necrosis Interstitial inflammation (i) i0: no or minimal inflammation (<105 of nonscarred parenchyma) i1:10–25% of parenchyma involved i2: 26–50% of parenchyma involved i3: >50% of parenchyma involved

Fig. 6.11  BANFF IB: severe tubulitis with lysis of tubular basement membrane; lymphocytes are too many to count (>10/tubular cross section) and tubular basement membrane barely visible. Arteritis was also present (not shown) (H + E × 200)

Fig. 6.9  BANFF IB: tubulitis with >10 lymphocytes (asterisk) per tubular cross section (PAS × 400)

graft survival is not significantly affected by the tubulitis score [134]. Tubulitis and lysis of the epithelial cell cytoplasm is associated with severe damage of the tubular basement membrane, which is attenuated or broken (Fig.  6.11). Repeat tubulitis ultimately leads to chronic tubulointerstitial injury and fibrosis

participating in part in the development of chronic rejection [36]. Borderline changes (“suspicious for rejection”) (Fig. 6.10) were examined in various studies. In a study of 351 biopsies from 170 patients who had biopsy performed because of elevated creatinine, 23% were found to have borderline rejection [305]. The majority of these patients (78%) were treated for acute rejection; some with pulse steroids alone (48%), and the rest with antilymphocyte antibody (52%). Among all patients with “borderline” treated rejection, 43% had a complete response, 28% had a partial response, and 30% had no response. Follow-up biopsies were performed within 1 month of the “borderline” biopsy in 24 cases and found mild or severe rejection in the second biopsy. This and other studies

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6  Kidney Table 6.7  Histopathological diagnoses in allograft biopsies in order of frequency 2003–2007 Washington University Saint Louisa Diagnosis % T cell mediated rejection

30.2

IA/IB

22.5

IIA/IIB

7.6

III

0.4

Borderline

4.3

PCAR rejection

4.1

CNI toxicity

19.3

Interstitial fibrosis/tubular atrophy

16.5

Glomerular disease

15.5

Acute interstitial nephritis

8.7

ATI

8.3

Fig.  6.12  BANFF grade IIB: endotheliitis consists of subendothelial or mural infiltrates of lymphocytes or macrophages; arrows point to subendothelial lymphocytes involving the entire circumference of the involved artery (H + E × 200)

Kedainis et al. [175]

a

support the contention that about one third of patients with borderline changes and clinical evidence of graft dysfunction do indeed benefit from acute rejection therapy [114]. A key point in treating borderline rejection is, whether additional histological findings are present or absent. The most common concurrent lesions are chronic rejection, CNI toxicity, and ATN [115]. The borderline category in the recently modified BANFF schema includes foci of t2 or t3 tubulitis with i0 or i1 inflammation [321]. Therefore, the decision to treat or not treat borderline rejection should consider the overall pathologic findings [114].

Fig.  6.13  BANFF grade IIA: endotheliitis involves <25% of artery circumference (H + E × 200)

6.2.1.2 Arteritis Arterial rejection is currently infrequent (<5% in our institution). Arteritis may take the form of endotheliitis, transmural inflammation or thrombosing vascular rejection (Figs. 6.12–6.16). Endotheliitis is defined as subendothelial mononuclear cell inflammation involving parenchymal and, on occasion, extra-parenchymal arteries (Figs.  6.12–6.14). Mononuclear cells are either lymphocytes or macrophages. Inflammation involving the vascular wall represents more severe, acute TCMR (Fig. 6.16). An additional feature is fibrinoid necrosis characterized by fibrin deposits with or

without nuclear debris (Fig.  6.15). The exact pathogenesis of and the trigger for fibrinoid necrosis continues to be elusive. Large and small arteries may be affected. Even though endarteritis is more prevalent in small-/medium-size arteries, some authors advocate that when arterioles are involved, the significance is the same [31]. Hilar arteries (and even the ureter) may show acute rejection. Nickeleit et al. found arteritis in approximately half of their cohort of 111 biopsies in the late 1990s [247]. In our practice, arteritis is much less frequent, currently (Table  6.7) [174]. The rarest

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Fig.  6.14  BANFF grade IIB: endotheliitis involves >25% of circumference (PAS × 200)

Fig.  6.15  BANFF grade III: fibrin deposits in the wall of an artery (asterisk) without significant endotheliitis; a few subendothelial lymphocytes are noted next to fibrin deposits (H + E × 400)

among all arteritis lesions in our material is grade III, transmural arteritis, and or mural fibrinoid necrosis (Fig.  6.15). Others find fibrinoid arterial necrosis in 4% of their biopsies [247]. While tubulointerstitial inflammation (rejection) usually accompanies endo­theliitis, not all endarteritis is associated with significant inflammation (estimated <10%). For example, a biopsy may show v1 only without significant t or i as defined in Table 6.6. The significance of v1 with little or no tubulointerstitial inflammation is still under investigation. Endarteritis responds to anti-T-cell therapy but not to steroids, arguing for a pathogenetic role for T cells. However, macrophages are also important players. Foamy macrophages sometimes are the nearly exclusive cell type in endarteritis

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Fig. 6.16  BANFF IIB: circumferential endotheliitis composed of lymphocytes and foam cells. There is also fibrin accumulation (asterisk) (H + E × 200)

(Fig.  6.16). In fact, some authors have shown that applying immunohistochemistry, the majority of cells are CD68 macrophages in types II and III acute rejection [209]. Overall, prognosis of acute vascular rejection is reported. In the study by Nickeleit, graft failure at 1-year was 21% without endarteritis, 28% with endarteritis, and 100% with fibrinoid necrosis. Endo­thelial activation and interstitial hemorrhage were common in this type of vascular rejection and correlated with graft loss; however, the role of AMR was not assessed in this study. None of the other scored features including tubulitis and interstitial inflammation correlated statistically with graft outcome. More recently, molecular studies on allograft biopsies with rejection showed large groups of genes altered, either up or down regulated. But, so far, there are no distinct profiles to correlate with specific types of acute rejection as classified by BANFF. Gene transcript profile changes in acute rejection are discussed at the end of this chapter by Michael Mengel.

6.2.1.3 Glomerulitis Acute glomerular rejection (glomerulitis) is characterized by endocapillary proliferation of mononuclear and activated endothelial cells (Fig. 6.17). Infiltrating cells are CD3 or CD68 macrophages [345]. Glo­ merulitis is seen in approximately 10% of allograft biopsies most frequently in the early posttransplant

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Fig. 6.17  Glomerulitis: arrow points to infiltrating lymphocytes (HXE × 400)

period and associated with T cell mediated rejection. In 40% of all biopsies with glomerulitis, there is no rejection and 53% of all biopsies with rejection have no glomerulitis [253]. The 1-year graft survival in biopsies with early posttransplant glomerulitis was 66% in the studies by Olsen et al., but this may have been the result of acute TCMR rather than the glomerulitis. The authors thought of glomerulitis as a peculiar pattern of rejection with a pathogenesis different from that of conventional rejection and likely with no adverse effects on graft function. The role of glomerulitis in allograft outcome continues to be uncertain and poorly understood to date. In our experience, glomerulitis is more frequent in biopsies with AMR or TGP. In fact, studies show that glomerulitis correlates better with AMR and presence of anti-HLA class I antibodies, while tubulitis correlates better with TCMR [351].

6.2.1.4 Interstitial Inflammation Most allograft biopsies with acute rejection irrespective of type (tubulitis or arteritis) have some degree of interstitial inflammation. If one excludes the specific patterns of interstitial infiltrates discussed below, interstitial infiltrates may represent subclinical rejection. Interstitial rejection is, usually pleomorphic and rarely

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consists of one cell population only. CD4+ and CD8+ T lymphocytes and macrophages are the majority. Some B lymphocytes, plasma cells, eosinophils, and neutrophils may also be present but they are usually a minor component. The infiltrate can be diffuse or focal and sometimes nodular [191]. Mengel et al., found that diffuse cortical infiltrates are more common in indication biopsies with tubular rejection, but protocol biopsies have the same prevalence of inflammation [223, 224]. Persistence of inflammatory infiltrates in sequential protocol biopsies correlate with ongoing allograft damage, even though such “nonspecific” infiltrates are not accounted for in the most recent BANFF (97–2006) update. Subcapsular infiltrates are common and are usually thought not part of rejection; this however, is a matter of contention [321]. T cells (thymus derived) are the classic host defense to allograft antigens. CD8+ and C4+ cytotoxic cells bind to MHC class I molecules, secrete cytokines and participate in B (bone marrow derived) cell activation, and maturation to plasma cells [246]. Mature plasma cells synthesize immunoglobulins, lose MHC class II antigen expression, and express CD 19,20,44,45 antigens. CD20+ B cells are usually very few in allograft biopsies (Fig. 6.18) but are abundant in some biopsies with predominant plasma cells that correlate with refractory rejection or poor outcome (Fig. 6.19) [153, 191, 217]. Until recently, the participation of B cells in rejection was ignored [26], but with accumulated knowledge of AMR, the question of whether using anti-CD20 monoclonal antibody (rituximab) for steroid-resistant rejection would be appropriate was raised. If so, staining for CD20 could be a useful tissue marker for identifying allografts at risk of refractory rejection and worse graft outcome. Studies showed that CD20+ cells are present along with tubulitis and scattered T cells; C4d+ staining is negative in these biopsies [302]. These and other studies, have not provided clear evidence that intragraft plasma cells are the source of antidonor antibodies. It is argued that in some biopsies, prominent plasma cells concurrent with C4d+ may be fortuitous. For example, C4d+ seems to be better explained (or correlated) by presence of concurrent chronic rejection and TGP than cellular acute rejection (see example in Fig. 6.20). Nonetheless, it is increasingly recognized that B cells may play other roles in the immune response to the allograft kidney than simply producing antibodies. Availability of

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Fig.  6.18  Renal allograft biopsy with dispersed interstitial inflammatory cells. (a) CD3+ cells (IP × 200). (b) CD20+ are rare (IP × 200)

Fig. 6.19  Renal allograft biopsy from a 33-year-old with LRD kidney and rejection 1B contains increased interstitial plasma cells. The majority are CD20+ (IP × 200)

specific B therapies spurred quite an interest for a while and some centers routinely performed immunohistochemistry for B cells in allograft biopsies with inflammatory infiltrates. However, subsequent studies did not support the findings of significant CD20 staining B cells in allograft biopsies [22, 302]. In biopsies with a significant number of B cells, it is perhaps prudent to describe the finding in the pathology report, but for now most studies agree that B cells, either in small clusters or scattered throughout the biopsy, do not correlate with worse prognosis.

The differential diagnosis of polymorphic interstitial infiltrate without significant tubulitis includes subclinical rejection (described under protocol biopsies). Relatively, monotonous-appearing interstitial infiltrates raise the question of PTLD and drug reaction. EBV stains should be included in the evaluation of such biopsies [349]. Diagnosis of PTLD is facilitated with antibodies specific for T and B cells and will be further discussed in section 6.3. Some newer immunosuppressive agents, for example costimulatory blockage drugs, may trigger proliferation of atypical interstitial infiltrates that include activated lymphocytes (large nuclei with open chromatin) (Fig.  6.21). These drugs modulate the immune tissue response in a pattern that has not been clearly described and may not necessarily be classifiable by the existing BANFF scheme. For example, abelatacept, a recombinant fusion protein containing components of IgG and cytotoxic T-lymphocyte-associated antigen-4, inhibits cos­ timulatory signals from antigen presenting cells and prevents activation of T cell [372]. This and other similar drugs may cause fewer side effects but may not abolish rejection, which, in some biopsies, consists of predominantly interstitial inflammation (Fig. 6.21 and Chap. 2). We have seen a few biopsies from patients on belatacept (LEA29Y) develop low grade tubulitis (IA) with more than usual interstitial mononuclear cell inflammation (Fig. 6.21). We interpreted these as representing interstitial acute rejection.

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Fig.  6.20  Plasma cell rich (PCAR) rejection concurrent with transplant glomerulopathy (TGP). (a) Predominant plasma cell infiltrate (H + E × 200). (b) Peritubular and glomerular capillary

C4d+ concurrent with TGP and chronic rejection (IF ×300). In this case C4d+ is associated with TGP and chronic rejection

Fig. 6.21  Renal allograft biopsy 6 weeks posttransplant from a patient with ESRD secondary to ADPKD with no prior dialysis treated with Belatecept. Patient presented with increased serum creatinine (1.8 mg/dL). Biopsy shows abundance of interstitial

infiltrates that prompted immune phenotyping. (a) H + E × 200, (b) CD4, (c) CD20 (IP × 200 and 100). EBV was negative. Overall findings interpreted as acute rejection

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6.2.1.5 Plasma Cell Rich Rejection Plasma cells have characteristic cartwheel nuclei and a perinuclear clearing (Fig. 6.22). Plasma cells appear in aggregates in the interstitium and may be the only or the predominant inflammatory cell while lymphocytes and tubulitis are usually a minor and very focal component (arrows in Fig. 6.22). Plasma cells are usually few (<10%) in renal allograft biopsies. When abundant and in aggregates, the term “plasma cell rich” (PCAR) was coined by Lorraine Racusen who first defined this entity [54]. In the 27 cases first reported with PCAR, occurrence was 1 month to many years posttransplant. PCAR was associated with poor graft survival, and was not concomitant with chronic rejection or viral infection, including posttransplant lymphoproliferative disorder. Graft survival after a single PCAR episode was as short as 6 months. Diagnosis of PCAR requires at least 300 plasma cells per 10 high power fields (40×). There was no correlation found with antiHLA antibodies or type of immunosuppressive drugs in the early reported cases. Subsequent studies revealed diffuse circulating antibodies reactive either to donor HLA or to endothelial cells and peritubular C4d+ in some cases with PCAR, suggesting that this type of reaction may be humorally mediated [81]. More recently in 2006, a correlation of PCAR with vascular rejection and TGP was reported [116]. The concept of PCAR being of bad prognosis was recently revisited and viewed from a different angle; instead of as a bad prognostic marker, PCAR is considered an indication

Fig. 6.22  PCAR rejection: sheets of plasma cells in the interstitium (>300 plasma cells/10hpf). Tubulitis (arrow) or arteritis may be present as well (H + E × 400)

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for more aggressive therapy. Immunoglobulin (IVIG) therapy or plasmapheresis was helpful in improving graft outcome in a recent study [4]. In our institution, the frequency of PCAR is ~4% (Table 6.7). We have seen several recent PCAR cases to resolve entirely with combination immune therapy as documented by follow-up biopsy. The differential diagnosis of PCAR infiltrates in allograft biopsies includes: Epstein–Barr viral infection and PTLD. In a study of 19 allograft biopsies with tubulointerstitial inflammatory infiltrates containing abundant plasma cells, clonality of the infiltrate was determined and follow-up was available 10 and 112 months posttransplantation. Kappa and lambda light chains and polymerase chain reaction (PCR) for immunoglobulin heavy-chain gene rearrangements were performed. In situ hybridization for Epstein–Barr virus encoded RNA (EBER) was performed in 17 cases. 84% were EBER-negative. Three of 19 biopsies (16%) had EBER-negative polyclonal plasmacytic proliferation, mixed monoclonal, and PTLD, respectively at 17 months, 12 weeks, and 7 years after transplantation [217]. This study demonstrates that infiltrating plasma cells comprise a spectrum of lesions and can be predominantly monoclonal. In situ hybridization for EBER, immunoperoxidase stains for clonal plasma cell proliferation, and hematologic workup are important to rule out PTLD.

6.2.1.6 Lymphoid Neogenesis Lymphoid neogenesis is a term applied for lymphoid aggregates in renal allograft biopsies that are seen with or without acute T cell mediated (Fig. 6.23). Organized lymphoid aggregates, sometimes with follicular centers, are seen in other organs and other diseases particularly autoimmune disease (rheumatoid arthritis, for example). Their potential pathogenesis and function is intriguing. It is suggested that they may represent failure of the immune response to eradicate peripheral B cells, which then organize forming lymphoid follicles. Ectopic lymphoid tissue organization recapitulates some aspects of lymphoid organogenesis during development including lymphatic vessels; therefore, denovo formation of organized lymphoid tissue is referred to as lymphoid neogenesis [175, 343]. Chronic allograft rejection is thought to result from sustained alloimmune response against the donor’s antigens. B cells

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Fig. 6.23  Lymphoid neogenesis: isolated lymphoid aggregates associated with lymphatic vessels (H + E × 200)

may participate in local production of tissue-specific antibodies, induction of B cell tolerance, and Tcell response through antigen presentation. The question of whether lymphoid neogenesis is implicated in chronic rejection is considered but not yet resolved [343, 344]. Lymphoid neogenesis is currently not accounted for in the BANFF schema [321].

6.2.2 Chronic Rejection Chronic rejection is defined as tubulointerstitial, vascular or glomerular fibrosis due to immunologic responses to donor antigens. Antigens are thought to be primarily endothelial and of donor origin, which remain in the graft for decades after transplantation [278]. However, other factors in combination, for example pre-existing chronic conditions in the donor [233], donor age [313], as well as “immune stress” in the graft, presensitization, host response, effectiveness, and compliance with immunosuppression also play a role [142]. At the tissue level, senescence conditioned by ischemia/reperfusion may also contribute to development of chronic rejection [164]. Chronic rejection develops in grafts that undergo repeat or persistent damage from cellular and humoral injury but there are no good serum markers for chronic rejection. For example, creatinine rise may not correlate with severe biopsy findings either because the kidney has significant functional reserve or significant structural damage is required prior to creatinine increase. Therefore, the allograft biopsy is very important in

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assessing chronic damage. In support of combined immune and nonimmune mechanisms underscoring chronic rejection are the following findings: in protocol biopsies with episodes of acute rejection, interstitial fibrosis and graft failure is found 1-year posttransplantation; living related HLA-identical grafts have about 50% less chronic rejection compared to haploidentical living related grafts [184]; patients with circulating DSA experience accelerated graft loss [340]. Repeat rejection attacks on allograft cells, manifested clinically or subclinically cause specific glomerular, tubular, vascular, and interstitial damage of varying severity. Recent studies using protocol biopsies provide direct evidence for the latter [233]. Moreso et al. found that among 435 protocol biopsies performed at 6 months posttransplant, 17% had subclinical rejection and 15% had chronic inflammation and fibrosis. There was an inverse correlation between Hepatitis C and donor age; these two factors and subclinical rejection were independent predictors of graft survival and associated with chronic rejection. The specific histopathologic features of chronic rejection are: TGP, peritubular capillary, and arterial thickening (intimal fibroplasia) (Table 6.5). Nonspecific features are: interstitial fibrosis and tubular atrophy (IFTA) (Fig. 6.24). The above findings were previously lumped together and were used in numerical scoring schemes such as the Chronic Allograft Damage Index (CADI) [161]. In the CADI scheme, a scale of 0–3 was

Fig.  6.24  Severe interstitial fibrosis (Trichrome stain ×200). Renal allograft biopsy was taken 20 years posttransplant from a 58-year-old-man with ESRD secondary to Alport; patient lost the first graft at 4 years; current biopsy was performed for slowly rising creatinine (2.2 mg/dL)

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applied for six features: (1) diffuse or focal inflammation, (2) interstitial inflammation, (3) mesangial matrix increase, (4) glomerulosclerosis, (5) intimal proliferation, and (6) tubular atrophy. CADI scores were found reliable predictors of graft survival but there were no detailed definitions and standardization. The scoring system did not gain wide acceptance or validation at least in the United States [65]. The most widely used grading score, currently used, is the one devised by BANFF 1997, which incorporated the CCTT classification [275]. However, quantitation of interstitial fibrosis is a rather subjective and not as yet standardized method. In the updated 2005 BANFF schema, chronic rejection is designated as chronic active rejection, antibody mediated or T cell mediated (Table 6.5). This new BANFF classification excludes nonspecific vascular, tubular or glomerular sclerosis, which now appears under category 5 (Table 6.5). The CADI score compared to BANFF 05-update is very different and likely to have limited use at least in the US. Chronic fibrosing/sclerosing lesions in the BANFF scheme are graded as cg for glomeruli, ct for tubules, and cv for vascular sclerosis as per original classification (BANFF 1993). However, precise quantitation to include ci, ct, cv, or cg, and mm scores is rarely performed routinely and how reproduceable these are remains to be studied further. In addition, it appears that the cg score categories, may, soon be revised as we understand better the early stages of TGP. Chronic rejection affects small- and medium-size arteries, in contrast to arterioles, which are most frequently thickened secondary to chronic CNI toxicity. Arterial thickening with intimal hyperplasia, often called transplant vasculopathy, are the typical lesions (Figs. 6.25 and 6.26). Arterial wall thickening represents either repair from previous acute rejection episodes or other less well-defined causes. Thickening may be due to fibrointimal proliferation or presence of acellular matrix deposits (Fig.  6.26). Elastin stain highlights intact internal elastic lamina with no lamellation in muscular arteries (Fig. 6.26). While fibrointimal proliferation is part of the criteria for chronic vascular rejection, the typical lesion may not be found in allograft biopsies frequently but is not uncommon in transplant nephrectomies. Mixed vascular injury (acute superimposed on chronic rejection) is not infrequent (Fig. 6.27). In one allograft biopsy series, fibrointimal hyperplasia was only found in 43% of cases of chronic rejection [313]. This group of investigators examined

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Fig. 6.25  Fibrointimal proliferation with intact internal elastic lamina in transplant vasculopathy (no lamellation)) (explant allograft kidney VVG stain ×200)

Fig. 6.26  Chronic vascular rejection with thickening of arterial wall (H + E × 200)

the risk factors associated with transplant vasculopathy (group I) and compared to those with chronic rejection without vasculopathy (group II), excluding cyclosporine toxicity and other causes of interstitial fibrosis. Risk factors identified in group II were: young recipient age and donor-reactive antibodies at the time of transplantation. Acute rejection after 3 months posttransplantation was the strongest risk factor for both patient groups with chronic rejection irrespective of vasculopathy [313]. However, the pathogenesis of transplant vasculopathy is still unclear. Not infrequently, C3 deposits are present in arteries or arterioles

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Fig.  6.27  Mixed acute and chronic rejection: subendothelial lymphocytes superimposed on thick arterial wall (H + E × 200)

Fig. 6.28  C3 deposits in transplant vasculopathy (IF × 300)

in allograft biopsies with chronic rejection but the significance of locally produced C3 in the allograft kidney is not clear (Fig.  6.28). Some evidence exists to show increased local C3 expression in both AMR and TCMR, but interest in C3 seems to have eclipsed in the shadow of T and B cell mediated rejection [294, 308]. An important point in the latest BANFF (2005-update) is the elimination of the term “chronic allograft nephropathy” (CAN). When the BANFF working classification group first introduced the term CAN in 1993, it was defined as “fibrosis in the renal

allograft caused by entities other than alloimmune injury.” The rational was that, it is often difficult to define the cause of chronic allograft damage unless specific features, such as intimal fibrosis and GBM duplication are apparent; therefore, CAN was intended to emphasize that not all tubulointerstitial injury is alloimmune rejection. CAN was not meant to delete specific diagnostic categories that could be identified, but to define more precisely the lesions of chronic rejection (BANFF 1993). In the last 15 years, CAN was used synonymously with chronic rejection. This caused a controversy among BANFF researchers mainly because CAN was perceived to prevent accurate diagnoses and appropriate therapy. The eighth BANFF, in 2005, revised the classification scheme to eliminate the term CAN and replaced it with the following descriptive terms: “IFTA, no evidence of any specific etiology” [320]. It is important to note that interstitial fibrosis in the allograft kidney is the common denominator of a variety of injurious processes: chronic cyclosporine toxicity, chronic infection, hypertensive vascular disease, obstruction, and donor disease are in the differential diagnosis (Table 6.8). A distinguishing feature of interstitial fibrosis of chronic rejection from cyclosporine toxicity is the molecular composition of the scar, which cannot be assessed by currently routine pathologic evaluation of allograft biopsies, but it is possible by in situ hybridization or PCR under controlled conditions. For example, chronic rejection is characterized by more type I collagen deposited early, compared to late deposition in cyclosporine toxicity [25]. Lamb2 mRNA and TGFb expression are more specific for CsA toxicity, predicted with 87% sensitivity and 88% specificity in a separate study [182]. Trichrome stain, which detects linked fibrillar collagen deposits shows deep blue staining (Fig. 6.24) and EM, which reveals active

Table 6.8  Causes of interstitial fibrosis in the renal allograft Chronic rejection Drug toxicity (CNI) Infection (polyoma, CMV, pyelonephritis) Atherosclerosis/hypertensive vascular damage Donor disease Allergic interstitial nephritis Recurrent disease

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Fig.  6.30  Peritubular capillary lamellation in AMR (EM ×  12,000) cortecy Kim Solez, Alberta, Canada) Fig. 6.29  Electron micrograph of interstitial fibrosis in chronic rejection: black arrows point to matrix accumulation; white arrow points to peritubular capillary with lamellated wall (EM × 6,500)

matrix synthesis by proliferating interstitial cells (Fig. 6.29 black arrows); mRNA quantification may be applicable as an additional diagnostic tool in clinical practice. The vascular lesions of chronic active AMR include GBM thickening and duplication, mesangial cell and matrix increase, mesangial interposition or mesangiolysis, which are the characteristic lesions of TGP (further discussed under de  novo disease). Chronic injury to the glomerular capillaries is characterized by closure of endothelial fenestrae, subendothelial expansion, and multilayering of the basement membrane similar to the injury to peritubular capillary basement membrane (Figs.  6.30 and 6.31). This suggests that TGP and peritubular injury are likely manifestations of a generalized microvascular disorder at least in some grafts. For example, the role of AMR in this process is increasingly recognized. The similarity of the findings in glomerular and PTC has prompted some authors to name these lesions of chronic rejection “transplant capillaropathy.” The concept is supported by independent studies that show decreased density of PTC per mm2 in chronic rejection [310]. Chronic active TCMR in the BANFF-2005 schema is defined as arterial intimal fibrosis with mononuclear cell infiltration and neointima formation

Fig.  6.31  GBM duplication with partial lamelation in TGP (arrow) (EM × 8,000) courtesy Eduardo Vasquez-Martul, La Coruna, Spain)

(Fig. 6.27). Fibroplasia is not accompanied by duplication of the internal elastic lamina, as opposed to that seen in hypertensive or atherosclerotic vascular injury (Fig. 6.25 compared to Fig. 6.2). Large hilar and medium-size parenchymal arteries are involved in severe forms of T cell mediated or mixed T and B cell mediated rejection. The mechanisms of IFTA are not entirely understood and far too complex to be thoroughly discussed in this chapter. Lack of better understanding may be reflected in the nonuniformity of scoring criteria and ambiguous reporting of fibrosis in the allograft biopsy. There is certainly more to be done in this area, but generally speaking, the process is, in part, mediated by cytokine release

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from damaged tubular epithelial cells, metalloproteinases, and infiltrating immune cells [90]. TGFb in particular, and its antagonist decorin, expression of smooth muscle alpha-actin, and increased collagen types in the interstitium are major players. Some of these can be easily detected by immunoperoxidase or histochemical stains (Trichrome or Sirius red) [77]. TGFb was originally thought of as a fibrogenic cytokine, but recent studies show that TGFb may in fact play a protective role. For example, investigators found higher TGFb, decorin, and collagen IV mRNA in allograft biopsies taken early posttransplantation with evidence of acute rejection but absent histopathological evidence of chronic rejection. These studies suggest that TGFb may, in fact, play a role in tissue repair and may be protective against chronic rejection [91]. In addition, other investigators find that non-HLA, AT1-receptor-mediated pathways may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors [88].

6.2.3 Antibody-Mediated Rejection AMR, also called “humoral” rejection, was recognized early in the history of transplantation as hyperacute rejection caused by the presence of preformed antibodies (“humors”) to blood group ABO or HLA antigens. Initially, it was thought to occur exclusively within hours of transplantation [267, 367]. Introduction of the pretransplant crossmatch techniques and use of ABO compatible donors essentially eliminated hyperacute rejection, but humoral rejection continues to be an enemy to be reckoned with and it may occur months or years, posttransplantation. The terms acute AMR, “accelerated acute rejection,” or “delayed hyperacute” rejection were introduced to describe AMR beyond rejection within hours from implantation. The above terms are now rarely used and mostly replaced by the term acute AMR. The overall incidence of AMR is estimated to approximately 5–7%; in biopsies taken for acute rejection AMR was identified in 12–37% [211]. AMR currently manifests in many forms and a spectrum of histologic findings (Table 6.9). Typically, acute AMR occurring within hours or days after transplantation is resistant to standard therapies, causes rapid functional deterioration, and is associated with a

Table 6.9  Diagnostic criteria of AMR in renal allografts Acute AMR Neutrophil/lymphocyte margination (capillaritis) Thrombosis/necrosis C4d+ in PTC Circulating antidonor antibodies (DSA) T cell mediated rejectiona ATNa Chronic AMR Histologic evidence of chronic damage (2/4 required) Tubulointerstitial fibrosis/atrophya Arterial intimal thickening without elastosis GBM/TBM duplication C4d+ in PTC DSA Suspicious for AMR C4d+ in PTC with capillaritis No DSA No histopathological findings These may accompany AMR but are not specific AMR features

a

poorer graft prognosis than pure acute TCMR. Clinical symptoms of acute AMR are: renal dysfunction with oliguria, rapid rise in creatinine, and increased DSA. In severe acute AMR, the graft undergoes cyanosis caused by massive neutrophil accumulation in peritubular and glomerular capillaries, vascular thrombosis, and cortical necrosis (Figs.  6.32 and 6.33). Cortical necrosis may, in some cases, be the only finding without the presence of neutrophils. Thrombosis usually affects the arterioles (TMA) but it may involve larger arteries as well (Fig. 6.34). In addition, ATN and vascular rejection may be concurrently present, or be “pure” with no arteritis or tubulitis (T cell poor) (Fig. 6.35). ATN may be the earliest feature of AMR (Fig. 6.8). Most importantly, current AMR diagnostic criteria include C4d+ staining in PTC (Fig. 6.36). At least, three criteria of those listed in Table  6.9 are required for diagnosis. The differential diagnosis of AMR includes other causes of TMA; however, these are not associated with rising DSA in which HLA antibodies and C4d are negative (described below). Notably, in AMR C4d+ is not always concurrent with detectable DSA (see below). In recent years, it became clear that acute AMR may occur not only weeks posttransplantation, but also months or years later (also referred to as late acute AMR) in patients with HLA mismatched grafts [143]. In our material, the overall incidence of acute AMR is about 4%, currently. This percentage includes late

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Fig. 6.34  Arterial thrombosis in acute AMR. There is also diffuse peritubular capillary congestion and glomerular capillary thrombosis (H + E × 50)

Fig.  6.32  Acute antibody mediated rejection (AMR) in ABO compatible transplant recipient with preformed DSA shows diffuse peritubular neutrophils, tubular necrosis, severe arteritis and thrombosis (H + E × 100)

Fig.  6.33  Diffuse hemorrhage in acute AMR (H + E × 200). Patient presented with creatinine 4.6 mg/dL and 0.5 gm proteinuria 2 weeks posttransplant. First biopsy 3 days earlier showed ATN

AMR. Late acute AMR may be clinically unexpected (subacute), not always associated with detectable DSA at the time of biopsy, and may not be associated with previous sensitization (de  novo AMR) (Fig.  6.37). AMR >1 year posttransplant is caused by reduced immunosuppression, noncompliance and or other not yet known factors. Histologic criteria in late acute AMR

are similar to early acute AMR, for example TMA, ATN, varying degrees of capillaritis, and concurrent TCMR. Clinical manifestations are heterogeneous; patients may present with increased creatinine, serum sickness (fever, muscular and jaw pains), or nonspecific and mild symptoms. Subclinical AMR is documented in the literature and correlates with chronic rejection developing later [135]. Risk factors for de novo AMR include Hepatitis C treated with interferon alpha. Seventeen percent of such patients developed de novo DSA and C4d+ within 6 months of interferon therapy, according to one study [24]. A subcategory of AMR is chronic as defined in Table 6.9; two of the following findings are required: arterial intimal thickening, interstitial fibrosis, tubular atrophy, GBM duplication, and C4d+ or DSA [68, 339]. T cell component varies and some cases are T cell poor (Fig. 6.38). In this context, the term chronic denotes a slow but active process over a long time period. As noted earlier, under chronic rejection, IFTA are nonspecific features and can be seen in chronic AMR, chronic TCMR, and other conditions (Table 6.8). GBM or TBM lamellation is considered a characteristic feature of chronic AMR and one of the criteria; the drawback is that EM is required, which is only selectively performed in renal allograft biopsies and not available in every renal pathology laboratory. Furthermore, the specificity of GBM or TBM duplication is debated as this finding can be seen in other chronic kidney diseases (CKDs) (lupus, for example)

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Fig. 6.35  T cell poor acute AMR in a 63-year-old-woman not previously sensitized. She presented with graft dysfunction day 10 posttransplant. (a) Focal hemorrhagic necrosis (H + E × 200).

(b) Focal interstitial fibrosis but no capillaritis or tubulitis (Trichrome × 400). (c) C4d+ has varying intensity (IF × 400). Arrow points to focal C4d+

Fig.  6.36  Diffuse C4d+ (green) in PTC (tubules are counterstained with Evans blue (red); same patient as in Fig.  6.33 (IF × 400)

if sought after enough. Interstitial mononuclear inflammatory aggregates, plasma cell aggregates, and transplant glomerulitis are some additional findings in chronic AMR. An association of DSA-HLA with late AMR is found in many but not all cases [79, 174, 273], but most authors agree that AMR plays a role in both acute and chronic TCMR [280, 339]. Suspicious AMR is a category defined by PTC C4d+ but absent histopathological findings and DSA. BANFF 2007 introduced capillaritis in the histopathological definition of AMR. Capillaritis is defined as dilation of PTC by marginating inflammatory cells, either neutrophils or mononuclear cells referred to as the PTC score (Fig.  6.39) [121]. Neutrophils are the hallmark of severe acute AMR (Figs. 6.32 and 6.40), but lymphocytes and or macrophages may be the exclusive inflammatory population in acute AMR. The scoring scheme of capillaritis is similar to other scores adapted by BANFF. It focuses on the most involved

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Fig. 6.37  Denovo acute AMR. Biopsy is from a 52-year-old-man 2 years posttransplant with living related donor kidney. He presented with increased serum creatinine and fever. (a) Biopsy shows capillaritis (PAS × 400) and diffuse (b) C4d+ in PTC (IF × 300)

Fig. 6.38  T-cell poor chronic AMR. Forty-nine year old woman with ESRD and allograft kidney 15 years prior to this biopsy presented with slowly rising creating to 1.8 mg/dL. (a) There is

moderate interstitial fibrosis, but no tubulitis or glomerular disease (H + E × 200). (b) Diffuse C4d+ in PTC (IF × 400)

areas in the cortex and medulla; scores are as follows: 0 = no significant or <10% of PTCs with inflammation; score 1 = ³10% of PTC containing a maximum of 3–4 luminal inflammatory cells; score 2 = ³10% of PTC involved with maximum 5–10 luminal inflammatory cells; score 3 = ³10% of PTC with >10 luminal inflammatory cells (Fig. 6.39). The pathology report should mention both the composition (mononuclear cells vs. neutrophils) and extent (focal, £50% vs. diffuse, >50%) of capillaritis. Capillaritis can be mistaken for interstitial inflammation unless examined carefully and on PAS or silver-stained sections that highlight basement membranes, thus facilitating recognition of the exact

location of the inflammatory cells. Inflammatory cells within veins and medullary vasa recta should not be scored. At this time, there is limited experience with the capillaritis score and it remains to be seen whether is specific for AMR. For example, we have seen capillaritis with C4d- and DSA- in a patient who presented with fever and symptoms of rejection and was later found to have been exposed to tick bites and his blood grew Ehrlichia chaffeensis (Fig. 6.41). Therefore, it is presumed that capillaritis is not unique to AMR, but the combination of capillaritis associated with allograft dysfunction, diffuse C4d+ and or elevated serum DSA are features diagnostic of AMR (Table 6.9).

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Fig. 6.39  Capillaritis score (a). Score 0 = no significant inflammation <10% PTC involved (PAS × 200) (b). Score 1 = ³10% of PTC involved with maximum three to four luminal inflammatory cells (PAS × 200) (c). Score 2 = ³10% of PTC involved with

maximum five to ten luminal inflammatory cells (H + E × 200) (d). Score 3 = ³10% of PTC involved with >10 luminal inflammatory cells; arrows point to capillaries containing lymphocytes (PAS × 200)

Fig. 6.40  Neutrophilic capillaritis (PAS × 400)

Fig. 6.41  Lymphocytic capillaritis (arrows) without C4d+ or DSA(Trichrome ×400). Patient had Ehrlichiosis, a tickborne disease

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6.2.3.1 C4d Pathogenesis, Detection Methods, and Scoring Even though C4d+ is not to be taken as the single mechanism of AMR, it is an easy marker to use in tissue. Numerous studies support a role C4d+ in AMR pathogenesis. C4d is a cleaved product of the complement C4 activation pathway that binds to vascular endothelium when C4 is activated [104, 105, 227]. C4 is split into C4a and C4b; C4b is converted to C4d. A unique feature of C4d is that it binds covalently to endothelial and collagen basement membranes avoiding removal and therefore, raising the possibility of serving as an immunologic footprint of complement activation. In normal kidneys, C4d is detectable in the glomerular mesangium suggesting that there is constitutive turnover of immune complexes. When the burden of immune complexes increases (for example, with immune complex-mediated glomerular diseases) C4d overflows to the glomerular capillaries. C4d deposition in the PTC is mostly described in renal allografts, and is felt to represent anti donor humoral activity. Diffuse C4d+ is defined as bright linear staining along the wall of PTC capillaries involving >50% of the biopsy area, performed either by immunofluorescence of immunoperoxidase (Figs. 6.35–6.38 and 6.42). Focal linear C4d+ is defined as involving <50% of PTC (Fig. 6.43). Granular staining seen sometimes is of unknown significance. Good correlation of PTC C4d+ with donorspecific HLA antibodies is demonstrated in many studies. For example, Mauiyyedi et  al. found 18 of their 20 cases of C4d+ acute rejection to have serum

Fig. 6.43  Focal C4d+ in PTC (arrows) involving <50% of PTC (IF × 300) The biopsy is from a 17-year-old-man with ESRD secondary to interstitial nephritis and kidney transplant 2 years prior, now presenting with renal dysfunction

DSA+, compared to 1/47 C4d- controls, supporting the notion that C4d antibodies are a manifestation of allosensitization [211]. High levels of DSAs are indeed associated with acute AMR and low DSA levels with chronic AMR [124]. However, the reason for the specificity of C4d staining in the PTC is not entirely clear. Donor-specific antibody directly engages HLA antigens, which are present in the glomerulus as well as the PTC. It is known that anticomplement protection in the PTC is weaker than in the glomerulus. Part of the reason for this is that, the glomerulus has at least four cell-surface complement inhibitors: decay accelerating factor-CD55, membrane cofactor protein-CD46, CR1CD35, and protectin-CD59. Only CD59 is actively expressed in PTC. Protectin inhibits the formation of complement membrane attack complex (C5–9) and therefore, the generation of C4d is relatively uninhibited [64]. Approximately, 10% of biopsies thought to have damage associated with AMR are C4d-. There are at least three possibilities to explain negative C4d in Table 6.10  BANFF 2007 C4d scores (% of biopsy with PTC+) IF IHC C4d score % of cortex or medulla

Fig. 6.42  C4d+ in PTC by the immunoperoxidase method (IP × 400)

0 Negative

None

–

–

1 Minimal

<10

–

Unknown

2 Focal

10–50

?

Positive

3 Diffuse

>50

Positive

Positive

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allograft biopsies with clinical and serologic evidence of AMR: (a) biopsy was performed early in the process, prior to diffuse PTC C4d+ antibody binding, (b) antibody previously bound may have degenerated at the time of biopsy, and (c) factors other than anti HLA antibodies and C4d may participate in AMR [69, 174]. A scoring system for C4d staining was proposed by BANFF 2005 (Table 6.10) [320]. The scoring system is based on percentage of peritubular capillary staining (PTC score), or five high power fields, as follows: >50% of biopsy tissue staining = positive irrespective of method; no staining in PTC = negative; 10–50% PTC C4d+ = focal, and <10% = minimal when staining by immunofluorescence (IF) only. Less than 50% PTC C4d+ by the immunoperoxidase (IP) method is still not clear whether should be considered positive (Table 6.10). C4d antibodies, currently in the market, include a monoclonal antibody suitable for IF performed on frozen tissue; a polyclonal works on formalin fixed paraffin embedded tissue and with IP detection (Serotec, Brentwood, New Hampshire, US). The affinity of the two antibodies and methods are comparable. The false negative rate of IF is 5–10% and that of IP 10–20% [241]. The significance of diffuse C4d+ and its correlation with AMR is well established, but the significance of focal C4d+ (Fig.  6.43) has been debated. Since the publication of the 2005 BANFF update [320], we published our experience with focal C4d+ detected by immunofluorescence. Our data demonstrated that focal PTC C4d+ in allograft biopsies: (a) correlates with serum DSA and (b) is associated with decreased graft survival [174]. Our results are in agreement with the experience of the pathologists at Pittsburg and others. Table 6.11  Capillaritis scoring system Score 0

No significant or <10% of PTCs with inflammationa

1

³10% of cortical PTC with capillaritis, with maximum 3–4 luminal inflammatory cells

2

³10% of cortical PTC with capillaritis, with maximum 5–10 luminal inflammatory cells

3

³10% of cortical PTC with capillaritis, with maximum >10 luminal inflammatory cells

Mononuclear cells vs. neutrophils

a

6.2.3.2 C4d+ Without Histopathological Findings of AMR and or DSA In a subcategory of C4d+ allograft biopsies, there are no histopathological correlates with either acute or chronic rejection. In fact, C4d+ protocol biopsies from stable ABO compatible grafts may show nil histopathological findings (including absent capillaritis) but have diffuse C4d+ staining (Fig. 6.44). C4d+ in these biopsies is thought to represent accommodation. This term denotes acceptance of the graft, which continues to function in spite of the presence of C4d antibodies. Furthermore, biopsies from ABOi donors performed for graft dysfunction show diffuse C4d+, but only 54% of stable grafts have PTC C4d+ [273]. ABOi grafts usually survive an acute episode of AMR with appropriate therapy, but whether the same mechanism applies for HLA incompatible grafts remains to be determined [68, 70]. It is also of interest that ~1.2% of HLA identical grafts have C4d+ [63]. 10–40% of patients with C4d+ have no detectable DSA antibodies. This may be caused by adsorption of the antibody to the graft or C4d+ caused by non-HLA antibodies [206].

6.2.3.3 PTC C4d+ in Chronic AMR C4d+ in some late graft biopsies may not, and usually is not, associated with other histopathologic features of AMR (capillaritis) or serum DSA+. In fact, C4d+ is perhaps the best in situ evidence of active humoral rejection and is associated with poor prognosis unless treated aggressively [368]. For example, in Fig. 6.38, there is very mild capillaritis but strong C4d+ developing 2 years posttransplant. This patient had very low titers of DSA but responded well to aggressive plasmapheresis.

6.2.3.4 C4d+ in Various Locations Other than PTC C4d+ may also be present in the glomerular capillaries without other pathology. This finding was initially thought nonspecific and was taken as an internal positive control in the C4d assay, but this may not be the definitive answer. For example, one hypothesis is that C4d+ in the glomeruli may precede TGP [314]. Indeed, many if not most cases of TGP show glomerular C4d+

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Fig. 6.44  C4d+ (a) with normal histology in (b) ABO compatible donor consistent with accommodation (IF and H + E × 200)

Fig. 6.45  TGP with C4d+ in glomeruli and PTC (a); TGP with C4d- in PTC (b) (IF × 200)

deposits; but C4d in PTC is not present in all TGP cases (Fig. 6.45). C4d+ may also be detected in damaged arterioles with nodular hyalinosis due to chronic cyclosporine toxicity. The role, if any, of C4d+ in this process is unknown. Graft ischemia, as discussed under donor ATI, is considered an important factor mediating both T cell and AMR and may underline C4d deposition in some biopsies with no definitive histopathologic evidence of either [179, 339].

Finally, a role for PTC C4d+ PTC outside transplantation may emerge in the future. For example, there is now some evidence that glomerular C4d+ may predict TMA in lupus patients [61]. In conclusion, it should be emphasized that not all C4d+ reactions are equal, but in the context of renal allograft biopsy, C4d+ has become almost synonymous with AMR. The classic pattern is PTC C4d+ in biopsies from patients with renal dysfunction. Diagnosis of (a) definitive AMR can be made when microvascular thrombosis, capillaritis, C4d+ or DSA

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Fig.  6.46  PTC C4d+ in renal allograft biopsies is multi­ factorial

are present or (b) suspicious for AMR if ATN is present and two of the definitive features are absent. Results should be interpreted in context and in combination with histological and laboratory findings (Table  6.9). There are many questions remaining particularly on the meaning of accommodation defined as normal histology, normal graft function in spite of persistent antigens, and donor-specific antibody in serum [69, 245, 364]. A hypothetical scheme on the potential pathogenesis of C4d is drawn in Fig. 6.46. Accommodation and ischemic insults are diagnoses of exclusion.

6.2.3.5 C3 and AMR C3 production may be caused by activation of both the classic and the alternative pathway of the complement cascade, but is not clear whether when a particular C3 fragment (C3c, d) deposits in allograft PTC indicates a distinct form of acute rejection or is part of C4d+ AMR. Some allograft biopsies with PTC C4d+ and DSA+ also have PTC C3d+ (Fig. 6.47). Studies show that concurrent C4d+ and C3d+ have significantly less tubulitis but conspicuous intraluminal accumulation of polymorphonuclear leukocytes in PTC. Thrombi, fibrinoid necrosis, and ATN are not more prevalent compared to pure PTC C4d+ [186]. In the study by Kuypers, 19% of biopsies had C3d deposition without C4d. In the remaining biopsies, C3d and C4d deposits were found simultaneously. Those with exclusive C3d+ developed permanent graft failure compared to those with exclusive C4d+ that recovered with therapy. The

Fig. 6.47  Biopsy shows C3d+ in PTC (IF × 300). Patient was 19 year old man with ABOi kidney with high anti-A titers within a week posttransplantation. C4d+ was also diffusely present

authors concluded that C3d+ in PTC indicates a variant of acute AMR with worse clinical outcome. A different study found C3c glomerular deposits concurrent with C4d+ in PTC in early AMR. Biopsies associated with macrophage accumulation, in addition to C3c and C4d+, had a particularly aggressive course [230]. Other authors have compared biopsies with C4d+ PTC and C3d+ PTC and find C3d (PTC) in 57% of biopsies with acute rejection, but no association with polymorphonuclear cells in PTC, no clear association with chronic rejection, and no association with DSA or graft outcome [149]. Finally, using laser microdissection and real-time RT-PCR for C3c of RNA extracted from tubules and glomeruli from protocol and indication biopsies with humoral and/or cellular rejection increased IFN gamma expression was detected suggesting that allograft-infiltrating T-cells may be a stimulus for local C3 production [308]. There is currently no clear direction from these studies, perhaps because they are small and not yet validated by a wider group of investigators.

6.2.4 Recurrent be Glomerular Disease In spite of improvements in short- and long-term renal allograft survival in the last decades, there have been no breakthroughs in prevention of recurrent or de  novo

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disease. In approximately 30–50% of patients with endstage renal disease (ESRD), the underlying cause is glomerulonephritis either primary or secondary. Biopsyproven, recurrent glomerular disease occurs in 36% of allografts according to a recent Canadian study of 2,026 patients [50]. In this study, the meantime interval of recurrence was 5 years but some cases did manifest much earlier; incidence was 5.5% at 5 years , 10.1% at 10 years, and 15.7% at 15 years posttransplantation [50]. Other studies report an overall 6–20% recurrence rate but the true incidence in various studies is difficult to determine for a number of reasons: many patients have not had a native kidney biopsy; recurrent and de  novo disease are often lumped together; data are usually retrospective, pooled from clinical databases without biopsy review or extracted from registries subject to collecting unit variations; biopsies are usually performed for cause, therefore recurrent disease may be underrepresented; there are overlapping features between recurrent and de  novo glomerular diseases, which can only be distinguished by thorough biopsy evaluation including EM [58]. In one center, among 1,156 patients glomerular disease was diagnosed in 132 cases (11.4%) and recurrent glomerulonephritis in 86 transplants (7.4%) [357]. A summary of reported incidence of recurrent disease is shown in Table  6.12. In our retrospective review of 1,200 allograft biopsies performed between 1997 and 2007, the overall incidence is similar; glomerular disease is diagnosed in 11.1% of allograft biopsies (134/1,200 unpublished data, Table  6.12  Rate of recurrence and estimated graft loss in various glomerulonephritides from registry data Glomerulonephritis Recurrence rate Graft loss (%) FSGS

20–50

13–20

IgA

13–46

2–16

MPGN   Type I   Type II

20–25 80–100

−15 15–30

2–9

2–4

Membranous

10–30

10–15

ANCA vasculitis

17

SLE

Table  6.13  Incidence of recurrent and de  novo glomerulo­ nephritis at Washington University Saint Louis 1997–2007 Glomerulonephritis Recurrent De novo FSGS

17 (12.5%)

15 (1 collapsing) (11%)

Diabetes

12 (8.8%)

–

Lupus

9 (7.5%)

–

IgA

8 (5.8%)

4 (2.9%)

MPGN

8 (5.8%)

–

Membranous

6 (4.4%)

1 (0.7%)

ANCA vasculitis

1 (0.7%)

–

Anti-GBM

1 (0.7%)

1 (0.7%)

LCDD

1 (0.7%)

–

Transplant glomerulopathy

–

42 (31.4%)

Other

8 (5.8%)

–

Total

71 (52.9%)

63 (47%)

Table  6.13). Nonetheless, with prolongation of graft survival, recurrent glomerulonephritis appears more frequent than before and the third leading cause of allograft loss following chronic dysfunction and death with a functioning graft [41]. The risk increases from 0.6% in the first year to 8.4% at 10 years posttransplantation, even though the clinical course, rate of recurrence and outcome varies among the various glomerulonephrititides. FSGS is the most frequent recurrent glomerulonephritis [144] and our unpublished data), followed by immunoglobulin A nephritis. Less frequent are: membranoproliferative glomerulonephritis (MPGN), membranous nephropathy, diabetic nephropathy, vasculitis, TMA (hemolytic uremic syndrome, antiphospholipid syndrome [rAPLS]), lupus nephritis, anti-GBM disease, and recurrent amyloidosis of the Congo red negative types (light chain deposition disease, fibrillary glomerulopathy). In the following segments, recurrent glomerular disease is discussed in relation to the differential diagnosis including de novo and “donor” disease with similar morphology.

6–8

Diabetes

8–30

Variable

Amyloid

5–30

Variable

HUS

1–100

40–80

Baum et al. [30] Hariharan [145], Briganti [40], Choy [59]

6.2.4.1 rFSGS Recurrent FSGS is, generally speaking, more likely to occur in the early posttransplant period, in contrast to

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de  novo disease, which usually occurs late (>1 year posttransplantation). Histological criteria include segmental sclerosis of glomerular capillaries with associated hyaline and/or lipid deposition and with variable adhesion to Bowman capsule (Fig. 6.48). The exception to this easily recognized histological pattern is early fulminate rFSGS, which may show absent glomerular scarring but cellular increase (cellular FSGS)

Fig. 6.48  Renal allograft shows focal segmental hyalinosis consistent with rFSGS (arrow) (H + E × 200). Patient was a 55-yearold African American man with ESRD secondary to hypertension induced FSGS. He developed significant proteinuria 2 years posttransplant

Fig. 6.49  Fulminate rFSGS in a 6-year child with living related kidney. Patient developed nephrotic range proteinuria 3 days post transplant. (a) There is marked glomerular enlargement

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and glomerular enlargement, instead. The case shown in Fig.  6.49 is of a 6-year-old white boy with living related donor kidney from his mother. Prior to transplantation, he had mild proteinuria. The boy developed massive proteinuria within 3 days posttransplant. The glomeruli were diffusely enlarged with no evidence of sclerosis. There was no tubulitis or arteritis. EM revealed foot process effacement and podocyte lifting and detachment (Fig.  6.49b) consistent with rFSGS. There was no evidence of familial disease in this child but genetic studies to exclude podocyte gene mutations were not performed. The relative risk for recurrence in primary FSGS is estimated to be 20–50 and 13–20% for graft failure [58]. Kidneys from living donors were thought as being at higher risk but this is not, however, substantiated by recent data [30]. We have reviewed transplant biopsies performed at Washington University in Saint Louis over a 10-year period (1997–2007) and found that rFSGS is the most frequent recurrent glomerulonephritis in our material making up to 12.5% of all recurrent glomerular diseases (Table 6.13). Patients with rFSGS have a higher graft failure and shorter half-life compared to patients with other glomerular diseases. The differential diagnosis of rFSGS includes de  novo FSGS discussed below and in patients with early recurrence, the possibility of “donor kidney associated FSGS.” The latter is defined as occult FSGS in the donor manifesting in the recipient. Such cases are rare and difficult to diagnose, but raise important questions particularly in certain populations with high

(H + E × 400). (b) Foot process effacement and lifting of podocytes (EM × 8,000)

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prevalence of FSGS (e.g., African Americans with genetic variations in the MYH9 locus). Factors implicated in increased risk of recurrence include: young age, mesangial hypercellularity in the original disease, white race, and previous recurrence, which increases the risk in the subsequent graft by 80%. It is also possible, that susceptibility genes that predispose some patients to recurrence exist, but currently, not well studied. Also, gene-environmental interactions, for example, cold ischemia may, in the appropriate genetic background, act as a trigger for rFSGS. In any event, the pathogenesis of rFSGS is still debated and is likely multifactorial. Factors thought of as mediators include: a circulating permeability factor detected in about 30% of patients with primary FSGS, and podocyte dysfunction due to genetic mutations of the slit diaphragm components. In the early 1990s, a test was developed by Savin et al., which used plasma of patients with FSGS injected into rats that developed proteinuria and FSGS. The hypothesis was that, the permeability factor increases albumin permeability of glomerular capillaries and allows for transmembrane escape of intraglomerular albumin (Palb). Patients with pretransplant sera that induced a Palb ³0.5 were at greater risk of rFSGS after transplantation. Other investigators have used Savin’s bioassay and/or direct infusion of fractions of FSGS sera in rodents in an attempt to purify and identify the permeability factor. However, a reproducible test is still lacking and the nature of the permeability factor is still under investigation [300]. In the meantime, recent developments on molecular defects of the components of the glomerular slit diaphragm suggest that multiple factors may precipitate the heterogeneous entity called FSGS. Mutations in NPHS1 coding for Nephrin and or cytoskeletal components such as NHPS2 coding for Podocin are implicated in the most common forms of familial FSGS. Polymorphisms in podocin or nephrin are potential risk factors for rFSGS [366]. Treatment with plasmapheresis or cyclosporine, cyclophosphamide, and other newer medications (e.g., rituximab or mycophenolate mofetil [MMF]) are effective in reversing acute renal dysfunction [6, 370]. Patients with NHPS2 mutations may have lower risk for recurrence, particularly if heterozygous. For example, in a study of 44 patients with familial FSGS, 32 of who had two mutations, only one developed recurrence [366]. There are five distinct histologic variants of FSGS as described by the Columbia group. These include,

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cellular FSGS, perihilar, tip lesion, not otherwise specified (NOS), and collapsing. All types may occur as rFSGS or de novo FSGS; the incidence among the two types is about equal (Table  6.12). CNIs, viral infections, decreased nephron number, hyperperfusion, and immunologic factors that are hard to prove in vivo may underline de novo FSGS. The distinction is important whenever possible because therapy for rFSGS is different (plasmapheresis) compared to de  novo FSGS (immunosuppression, drug discontinuation, etc.). In many of our cases, the etiology of de novo FSGS was unclear, but an association with CNI was not uncommon (Fig. 6.50). Among other causes of de novo FSGS, we saw an interesting case occurring in a young patient transplanted for end-stage IgA nephropathy. The patient presented with nephrotic range proteinuria associated with CMV viremia. There were no IgA deposits found in the allograft biopsy. Subendothelial tubuloreticular inclusions were identified on EM along with severe foot process effacement. There were no CMV inclusions on light microscopy, but the possibility of CMV-induced FSGS is an intriguing question. Viruses are a bona fide cause of collapsing FSGS, but whether they may also cause other types of FSGS (e.g., FSGS NOS) in the transplant kidney remains to be further investigated. Sirolimus is implicated in both recurrent and de  novo FSGS. Therefore, some authors recommend that sirolimus should be avoided in the treatment of rFSGS, particularly in children [84].

Fig. 6.50  De novo FSGS associated with calcineurin inhibitor toxicity (CNI); asterik points to hyalinosis of afferent arteriole characteristic of CNI toxicity (H + E × 400)

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Finally, the diagnosis of FSGS NOS recurrent or de novo developing in the allograft kidney should only be made to the exclusion of other glomerular diseases complicated by segmental sclerosis, and also to the exclusion of proteinuria derived from the native kidneys. The latter can be a challenging diagnosis, but if proteinuria is derived from the native kidneys after transplantation, the amount is usually minor. The pattern of FSGS in patients who had both native and allograft biopsies available for review was examined by a Dutch group asking the question of whether recurrent disease may be a morphologically different one from the primary disease, as described in the Columbia classification [75, 159]. Twenty one cases of rFSGS were evaluated; 81% occurred in the same pattern as in the native kidney, but four cases switched to a different variant. For example, two FSGS NOS recurred as the collapsing variant, two collapsing recurred as FSGS NOS, and one cellular variant as FSGS NOS. Some biopsies with the cellular variant were preceded by minimal change like disease diagnosed in a previous biopsy. Therefore, the authors propose three categories of FSGS recurrence: type I of the same pattern, type II proceeded by minimal change, and type III of a different pattern [159]. The collapsing variant of FSGS (cFSGS) is of interest because of its frequent association with viral infection (HIV, Parvovirus B19, Hepatitis C, CMV, etc.) [9, 338, 348]. cFSGS is defined by wrinkling and retraction of the glomerular capillary wall, hyperplasia, and

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hypertrophy of the overlying visceral epithelial cells often accompanied by prominent intracytoplasmic protein reabsorption droplets (Fig. 6.51). Diagnosis can be made if at least one glomerulus demonstrates typical changes. Collapsing glomerulopathy (as some authors prefer to call it) is well known to recur in transplants, even though not as frequent as childhood primary FSGS. In general, collapsing glomerulopathy is associated with worse proteinuria, renal insufficiency, and higher rate of graft loss compared to other FSGS patterns, except when a virus or drug is identified and appropriately eradicated or discontinued, thereby achieving reversal of proteinuria in many cases [9, 338, 348]. However, transplanted HIV patients have no significant adverse allograft complications directly related to the HIV virus and therefore, are as good candidates for transplantation as any [326]. Collapsing glomerulopathy can also develop de novo, but is rare. In a cohort of 892 allograft biopsies, from 1,079 recipients who received renal transplants between 1978 and 1996, five cases of de novo collapsing glomerulopathy were identified, an incidence of 0.6% of biopsies. All occurred after 1993 (3.2% since 1993). The patients were 31–66 years of age and presented 6–25 months posttransplantation, 24-h urinary protein ranged from 1.8 to 11.8 g and all patients and donors were HIV negative. Diffuse or focal, global or segmental collapse of glomerular capillaries, hyaline arteriolosclerosis, and interstitial fibrosis were present. Two cases had concomitant glomerular immune complex deposits. All five patients

Fig. 6.51  De novo collapsing glomerulopathy. (a) There is GBM wrinkling and circumferential epithelial cell proliferation (asterisk) (Trichrome ×400). (b) EM shows diffuse foot process effacement and lipid vacuoles in podocyte cytoplasm (arrow)

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developed allograft dysfunction and loss 2–24 months after diagnosis [219]. Collapsing glomerulopathy is often accompanied by severe chronic vascular changes; the possibility of CNI inhibitors acting as mediators is proposed. The case shown in Fig.  6.51 is that of a 45-year-oldman with ESRD secondary to Alport who developed heavy proteinuria 21 years after transplantation. He was HIV, Hepatitis C, CMV, polyoma, and Parvovirus B19 negative. The biopsy showed collapsing glomerulopathy and chronic CNI toxicity. Glomeruli did not show GBM duplication excluding TGP. EM showed diffuse foot process effacement and lipid vacuoles in podocyte cytoplasm (arrow). The latter is a feature of dysregulated podocyte phenotype. This case represents de novo disease of unknown etiology, even though drug effect remains possible (Fig.  6.51). Another example of cFSGS is shown in Fig.  6.52. This patient was a 70-year-old African American with ESRD secondary to hypertension. He presented with nephrotic range proteinuria and increasing creatinine, no evidence of HIV or other viral infection. Typical collapsing FSGS was present in two glomeruli with proliferating podocytes (arrow in Fig.  6.52) overlaying collapsed glomerular capillary loops. The original disease was not documented by renal biopsy, therefore, is unclear whether this is recurrent or de novo FSGS.

Fig.  6.52  Collapsing FSGS characterized by segmental loop collapse with overlaying proliferating podocytes (arrow) PAS × 200

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A final word may be added here to note that podocyte injury presenting with nephrotic syndrome in the transplant may be reversible and may not be associated with adverse prognosis in all cases. This tends to be true in patients with original disease diagnosed as minimal change [352].

6.2.4.2 rDiabetes The second most common recurrent disease in our material is Diabetes [145] (Table 6.13). An estimated recurrence rate of 11.4 and 53% graft failure is reported by Hariharan et al. [145]. Others report a 26.8% recurrence rate based on serum glucose levels 2 months post transplant [213]. A retrospective review of all allograft biopsies performed between 2004 and 2007 in our own institution, identified 12 cases of recurrent diabetes (8.8%) (Table  6.13). Our data are closer to those reported by Kasiske et al. who found an overall incidence of posttransplant diabetes of 9.1% from the US Renal Data System [169]. In the last 3 months, we have seen at least three allograft biopsies with welldeveloped rDiabetes, suggesting that the incidence may be increasing. Histopathological findings vary from mild mesangial hypercellularity to KimmelsteilWilson nodules (Fig.  6.53). Diagnosis of rDiabetes requires EM to exclude TGP, which in late stages may mimic diabetic nodules. Immunofluorescence typically shows linear capillary staining with IgG and albumin and thick lamina densa is identified in the GBM by EM. Other characteristic lesions of diabetes are stiff capillary loops (arching over the mesangium) and extensive podocyte damage with foot process disruption and detachment of the cell body. The differential diagnosis includes denovo diabetes posttransplantation. Factors that influence development of diabetes in the transplant patient are: age, African American race, ethnicity, male donor, increasing HLA mismatches, hepatitis C infection, body mass index over or equal to 30 kg/m2, and treatment with Sirolimus as the initial maintenance immunosuppression [169]. Why some patients develop diabetes posttransplant while others do not, is unclear, but a recent study found that the R325W polymorphism in the islet-specific zing transporter gene SLC30A8 confers resistance against development of diabetes in transplant patients [166]. It is also possible that patients may

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6.2.4.3 rLupus

Fig.  6.53  rDiabetes. (a) Allograft biopsy with KimmelsteilWilson nodules (H + E × 400). (b) Linear albumin staining (IF × 400)

carry dormant (previously undetected) mutations that are triggered by transplantation to predispose them to develop diabetes (for example, HNF1b [193]. Factors that reduce the risk of de novo diabetes include use of MMF, azathioprine, young recipient age, glomerulonephritis as a cause of kidney failure, and college education. Sirolimus has a unique mechanism of action that is distinct from CNIs. The proposed mechanism for Sirolimus-induced diabetes implicates chronic inhibition of the mammalian target of Rapamycin (mTOR), which leads to insulin-resistant hyperglycemia [108]. In contrast to other donor glomerular diseases that may be transmitted to the allograft (for example, latent IgA), donor diabetes is reported to resolve with transplantation and therefore donor kidneys with diabetic nephropathy are now acceptable (see under ECD).

A recurrence rate of ~30% is reported in lupus nephritis but in a single study of 106 lupus patients with clinically and serologically quiescent disease at the time of transplantation, only nine patients (8.4%) had pathologic evidence of rLupus [328]. Recurrence occurred between 3.1 and 9.3 years after transplantation; the shortest was 5 days. Histopathologic diagnoses included focal or diffuse proliferative glomerulonephritis, membranous, and mesangial glomerulonephritis. In four patients, rLupus contributed to graft loss. Three of the patients with recurrence had serologic evidence of active lupus, but only one had symptoms of active lupus (arthritis). Three patients who lost their grafts secondary to rLupus underwent second renal transplantation and had functioning grafts at 7, 30, and 35 months, respectively. rLupus was often present in the absence of clinical and serologic evidence of active systemic disease. In our material rLupus is the third most common recurrent glomerular disease (7.5%) and the most common histologic type is mesangial lupus. In a study of 50 patients with lupus and at least 3 months of followup posttransplantation, rLupus was found in 30% of all patients: mesangial lupus nephritis (WHO class II), focal proliferative (class III), and membranous (class V) were reported. Overall, patient survival was 96% at 1-year and 82% at 5 years, and graft survival was 87% at 1-year, and 60% at 5 years. One patient had graft loss (class II) at 10.5 years. Graft loss due to rLupus is rather rare [128].These studies did not consider lupus patients with antiphospholipid antibodies who fall in the category of TMA and have higher increased risk of recurrence (see below).

6.2.4.4 rIgA Nephropathy IgA nephropathy accounts for 15–40% of renal failure among transplant patients. Recurrence rate is reported in the range of 50–60% in protocol biopsies and 13–50% in indication biopsies [41]. The latter is thought to be an underestimate, since asymptomatic patients are usually not biopsied. Clinical presentation is similar to primary IgA: microscopic hematuria, low grade proteinuria, and slow decline in renal function. Histological findings of mesangial hypercellularity

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and IgA mesangial deposits are variable and usually milder compared to the disease in native kidney. Sometimes crescents and other chronic changes, for example CNI toxicity or TCMR are concurrent. Crescentic IgA in the allograft biopsy is rare but it should be noted that is associated with accelerated graft loss [183]. Similarly, about 70% of adults transplanted for crescentic Henoch-Schonlein purpura have much higher incidence of recurrence (50%) compared to patients without crescents (12%) and eventually experience graft loss [235]. Glomerular C4d+ or C4D+ in PTC in biopsies with rIgA may not be directly related to IgA (our own experience). Concurrent findings need to be assessed and diagnosed separately and clearly described in the pathology report so that not all graft dysfunction is attributed to IgA deposits. Allograft survival at 5 years posttransplant is reported to be better in patients with primary IgA nephropathy compared to other glomerulonephritides. It is proposed that reactive allo-reactive IgA antibodies against HLA epitopes may block the effect of IgG or IgM allo-antibodies to the graft, thus decreasing the possibility of graft dysfunction [197]. However, at 10 years graft survival in patients with IgA becomes similar to other glomerular diseases suggesting that other factors intervene long term [59]. The exception to this is previous graft loss, which increases the risk in the second transplant. Interestingly, latent IgA in the donor is a significant risk factor for recurrent IgA that compromises graft survival more than any other factor (age, gender, race, HLA status) [234]. There is no effective treatment for rIgA. Overall, rIgA with longer and better patient follow-up, IgA is no longer considered as benign as before. Graft loss is ~10% at 10 years post transplantation, according to the latest registry [58].

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biopsy shows lobular glomeruli with mesangial hypercellularity and double contours in the glomerular capillaries. Graft loss is estimated at 15% at 10 years posttransplant, but the risk increases to 80% following a previously lost graft [58]. The risk of recurrence is reduced if underlying causes such as hepatitis B and C are treated. The main differential diagnosis of rMPGN is TGP, which is very similar morphologically. C4d+ in TGP may help distinguish the two. rMPGN type II (dense deposits disease (DDD)) is characterized by dense ribbon-like deposits on EM, similar to native biopsies (Fig.  6.54). Crescents are variably present. IF is variable and three patterns are described: globular mesangial deposits, linear capillary loop deposits with mesangial C3, and prominent linear loop deposits with few mesangial deposits [16]. DDD is a rare disease with a high rate of recurrence, up to 80–100%. Patients present with nonnephrotic proteinuria within the first year. There is no correlation with pretransplantation symptoms or complement C3 levels but there is a strong association of recurrence with proteinuria and of crescents with allograft survival. Graft loss is around 15–30%, 5 years posttransplantation [39]. In cases of absent history of DDD prior to transplantation, the possibility of asymptomatic disease in the donor should be considered in the differential diagnosis, however rare this may be.

6.2.4.5 rMembranoproliferative Glomerulonephritis (MPGN) Both type I and type II MPGN recur in the transplant. MPGN type I, in adults, is usually secondary to infection or systemic disease. Recurrence in 20–50% of patients is reported. Risk factors for recurrence include HLA-B8DR3, living related donor and previous graft loss. Patients with rMPGN type I present with proteinuria and declining renal function. Renal allograft

Fig. 6.54  rDDD in a 26-year-old with LR kidney transplant a year prior to this biopsy presenting with proteinuria. There are ribbon-like dense deposits in lamina densa diagnostic of recurrent disease (EM × 2,600)

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6.2.4.6 rMembranous Glomerulonephritis Membranous glomerulonephritis in the transplant kidney may be primary, secondary or develop de  novo. Histopathological findings in the allograft are similar to the native kidney, e.g., thickening of the capillary loops, IgG, and C3 immune deposits by immunofluorescence and subepithelial deposits by EM (Fig. 6.55). Idiopathic membranous, according to registry data, recurs in 10–30% of patients with a mean onset ~10 months, in contrast to de novo disease, which usually presents late. Nephrotic range proteinuria is the main presenting symptom in primary disease and more insidious proteinuria in de  novo. Graft failure is reported in 10% of recurrent membranous; it occurs late and at about 10 years posttransplant [40].

6.2.4.7 rANCA Vasculitis and rAnti-GBM Disease Recurrence in pauci-immune ANCA vasculitis is estimated to around 7.7% in cumulative databases [40]. The optimum time to transplant patients and the risk of renal or nonrenal disease recurrence was recently evaluated in 35 patients with ANCA vasculitis [119]. Nonrenal relapse occurred in three patients who responded to treatment. There was no risk factor for relapse found and no detrimental effect to renal function. Overall, prognosis on graft survival is similar to other diseases and the diagnosis of vasculitis should not preclude transplantation [248]. The diagnostic finding in allograft biopsies is crescents in >50% of glomeruli and absence of or minimal deposits on IF. None of the pretransplant features (cANCA vs. pANCA) or underlying disease (Wegener’s vs. other) predicts recurrence. Crescents may respond to steroids, cyclophosphamide, or plasma exchange with subsequent disease reversal. Hematuria is common in rANCA vasculitis and may be considered a red flag for biopsy. Anti-GBM disease is characterized by rapidly progressive glomerulonephritis and or pulmonary hemorrhage (Goodpasture’s syndrome) mediated by antibodies against collagen IVa3 in the GBM. In the transplant kidney, the disease may be recurrent or de novo, the latter occurring in patients with Alport. Typical immunofluorescence finding is linear diffuse and global IgG deposits along the glomerular capillary loops. Recur­ rence is approximately 50% in patients with circulating antibodies at the time of transplantation, but if

Fig. 6.55  rMembranous. (a) Light microscopy shows minimal capillary loop thickening and no “spike” formation (H + E × 400). (b) Diffuse granular deposits in capillary loops are seen by IF (×300). (c) EM reveals small subepithelial electron dense deposits (×4,500)

transplantation is deferred until antibodies are undetectable, recurrence is very rare and only a handful of cases are reported (estimated 0–5%) [176]. The differential diagnosis of vasculitis on light microscopy includes rIgA and rMPGN presenting with crescents,

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but these entities are easily excluded by IF and EM, as previously described. De novo anti-GBM in Alport syndrome occurs in patients with X-L Alport who carry truncating mutations in collagen IVa5. IVa5 is necessary for insertion of IVa3 in the GBM and formation of the triple helix within the lamina densa. Patients with X-L Alport have defective triple helix with collagen IVa5 and a3 being absent in the GBM of the native kidney. When a nonAlport donor kidney is transplanted in an Alport patient, antibodies are elicited against the a3IV epitope located in the NC1 terminal domain (also called antiGBM epitope) [167]. For reasons not well understood, anti-GBM linear IgG deposits manifest only in a minority of patients with X-L Alport (~5%). The onset can be days to months, posttransplantation [55]. An example of anti-GBM developing in an Alport patient 5 years posttransplant is shown in Fig. 6.56.

6.2.4.8 rHUS and the Spectrum of Thrombotic Microangiopathy (TMA) in the Transplant Kidney TMA is a major cause of graft loss in the first-year post renal transplantation with a reported incidence of 3–15% [306]. Causes include both recurrent and de novo disease. rTMA in children is primarily due to rHUS; in adults, the main primary causes are TTP and rAPLS. Drug reaction complicated by TMA may affect all ages. In addition, a rare but possible complication is graft versus host disease (GVHD) affecting the

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allograft kidney, whilst more often GVHD is seen in native kidneys of patients who had bone marrow transplantation (Table 6.14). TMA is the histopathologic term used for syndromes characterized by thrombosis of small arterioles or glomerular capillaries. Familial TMA is due to factor H deficiency, vWF protease activity or complement deficiency. Acquired TMA is mostly due to infections or drugs (E. Coli, HIV, CMV). Disease recurrence varies with underlying etiology. Transplantation should be considered with careful preliminary appraisal of the risk for recurrence. Studies in the early 2000 indicated that the risk of rHUS was approximately 20% in pediatric-onset HUS and at least 50% in adult-onset HUS. More recent studies show much less recurrence in nonfamilial types such as the diarrhea- associated HUS (D+HUS) [200]. The risk of recurrence in D+HUS is less than 1%. The underlining cause is toxin-producing Escherichia coli, the most frequent form in children. In familial HUS there are specific genetic defects in

Table 6.14  Recurrent vs. de novo TMA in the renal allograft rTMA De novo TMA D + HUS (<1%)

CNI inhibitors

Familial HUS (60–100%)

Rapamycin

HIV

OKT3

CMV

Interferon alpha

Antiphospholipid syndrome

AMR CMV Graft versus host disease

Fig. 6.56  rAnti-GBM. (a) Glomeruli are hypercellular and both contain crescents (arrow) (H + E × 200). (b) Linear IgG along the capillary loops is found by IF (×400)

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endothelial cell protection. Approximately 60% of patients have mutations in the genes of five proteins that regulate the complement alternative pathway and protect host cells from complement activation: CFH, MCP (or CD46), a noncirculating transmembrane protein anchored in cell membranes and IF [200]. Patients with IF and CFH mutations have an 80% recurrence risk, MCP mutations carry a 20% risk. Complement genotyping is now possible and permits a more precise evaluation of the risk for rHUS; patients with very high risk should not be considered for transplantation [57, 200]. TTP defects include abnormal vWF and ADAMTS 13 metalloproteinase. Typical findings in renal allograft biopsies are microthrombi in glomerular capillaries or afferent arterioles, mesangiolysis and ischemic retraction with glomerular obsolescence. Mucoid intimal degeneration is typically present (Fig. 6.57 arrow). Recurrent disease may occur within days posttransplantation usually limited to the allograft kidney. The differential diagnosis of rHUS includes de novo TMA due to (a) drug toxicity (acute CNI toxicity/OKT3/ sirolimus), (b) AMR, and (c) infection (Table  6.14). rHUS is treated with plasmapheresis and or other newer modalities. Also, the majority of patients with de novo TMA presenting with hemolysis and thrombocytopenia (62%) require plasmapheresis or dialysis and are likely to experience graft loss. A subset of patients have kidney localized disease (38%) without systemic hemolysis (transplant TMA) and respond to drug reduction,

Fig. 6.57  rHUS in a young child 6 weeks posttransplant. There is diffuse hemorrhage and microvascular thrombosis including glomeruli; arterioles show mucoid intimal degeneration (arrow) (H + E × 200)

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Fig. 6.58  TMA due to CNI toxicity. Fibrin thrombus (arrow) in arteriole (H + E × 200)

temporary discontinuation of CNI or switch to another regimen [306]. The incidence of cyclosporine-induced TMA ,when first introduced, was reported ~8%, but has diminished greatly in recent years. The typical findings are arteriolar or glomerular capillary thrombosis (Fig.  6.58). TMA-like changes are reported in patients treated with tacrolimus with a prevalence of 1.0–4.7%. CNIinduced TMA is reversible with discontinuation or reduction of CsA/tacrolimus (or switch to sirolimus), plasmapheresis, and or intravenous immunoglobulin administration. Graft salvage rate is 80–90%. Recently, rapamycin was reported to induce de novo TMA [74]. Rapamycin is coadministered with CNI because it decreases long-term CNI nephrotoxicity and facilitates early CNI withdrawal [250]. Both rapamycin and CNI inhibitors induce endothelial cell activation and thrombosis but not in the same patient. Therefore, paradoxically, when one drug is complicated by TMA, thrombosis reverses upon switching to a different agent [74]. Younger recipient age, older donor age, female recipient, and initial use of sirolimus are risk factors for de  novo TMA [282]. Patient survival after TMA was approximately 50% at 3 years in the study by Reynolds et al. In recent decades, AMR-induced TMA is more frequent. It is distinguished from other TMA causes by C4d+, which is present in ~90% of AMR. Serum drug levels and DSA are helpful in excluding CNI toxicity and AMR, respectively (see below). De novo TMA was also reported in Hepatitis C+ patients with kidney transplant. The reported incidence

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is 4.8%. TMA occurred 5–120 days posttransplant. Interestingly, all patients had anticardiolipin serum antibodies [23]. They all lost their graft. APLS is a hypercoagulable state that predisposes patients to systemic thromboses. About 50% of lupus patients have apL antibodies and about half of these experience complications linked to apL. However, APLS may occur without lupus stigmata. Current diagnostic criteria for APLS require presence of detectable antiphospholipid antibodies, evidence for thrombotic complications, and fetal loss or thrombocytopenia. Patients with APLS are at very high risk for thrombosis [354]. APLS is unique since it can cause both arterial and/or venous thromboses in both large and small vessels. Stroke, pulmonary embolism, myocardial infarction, and ocular ischemia are some of the vascular complications. The allograft kidney may be involved with recurrent disease, even though rarely reported. In a study of 96 consecutive patients with lupus who underwent renal transplantation, 25 patients had at least one apL+ test. Ten patients (10.4%) had graft thrombosis within 3 months of transplantation or died from thrombotic complications [327]. Why some lupus patients develop apL antibodies is not understood. What can be done to stratify these patients and assess the risk of graft thrombosis is also not adequately studied or understood [215]. We have seen a lupus patient whose renal allograft failed 5 days after transplant due to both large and small vessel thromboses. Initially, the renal artery was thrombosed on day 3 posttransplant, but by day 5, the graft was nonfunctioning. The explant kidney demonstrated hemorrhagic cortical necrosis, glomerular, and arteriolar microvascular thrombosis; C4d was negative and DSA absent; cyclosporine levels were within normal limits (Fig. 6.59). The patient had history of a morbid pregnancy, which is part of the diagnostic criteria for APLS, but no apL serum antibodies at the time of transplantation (seronegative APLS) [7]. The graft was removed and the patient was returned to dialysis. Because of absent apL pretransplant, the patient did not receive anticoagulation. Recent studies demonstrate that patients with APLS are at risk for thrombosis even in the absence of serum antibodies [360] justifying the old saying absence of proof is not proof of absence. In our most recent experience, the majority of TMA in allograft biopsies is de novo, more frequently associated with AMR, and less so with drug toxicity. The incidence described in the literature, is not seen in our

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Fig. 6.59  rAPLS. Glomerular thrombi and arteriolar endothelial swelling 5 days posttransplant in lupus patient with history of APLS (H + E × 300)

patient population. Some authors have questioned the reported high incidence of TMA and suggest that the reported numbers may be derived from databases or retrospective chart reviews, which have no pathology documentation. Alternatively, the reported high incidence may be systemic TMA with laboratory abnormalities but uncertain kidney involvement [282].

6.2.4.9 rAmyloidosis/rLCDD/rFibrillary Amyloidosis recurs in 10–30% of renal allografts primarily in patients with cardiac amyloidosis, which is thought of as a contraindication for transplantation. The onset varies from 1 to10 years posttransplantation. Diagnostic features are similar to those in the native kidney when enough amyloid deposits are present. However, when small amounts are deposited the diagnosis can be missed on light microscopy. Congo red stain is not sensitive enough when deposits are sparse; Thiflavin S and EM may be most sensitive methods and should be sought after routinely in allograft biopsies for patients with primary amyloidosis presenting with proteinuria. De novo amyloidosis is rare and similar to recurrent disease can be missed unless diligent evaluation is performed and adjunct techniques are sought to prove amyloid deposition (Fig. 6.60). Monoclonal immunoglobulin deposition disease, either light or heavy chain (LCDD/HCDD) and fibrillary glomerulopathy are, by definition, Congo red negative amyloidoses, characterized by monoclonal linear

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Fig. 6.60  De novo Amyloidosis in a 45-year-old-woman with unknown primary disease who presented with proteinuria 8 years post transplant. Biopsy showed kappa light chains (b) and filamentous GBM deposits (a) (IF × 300 and EM × 6,000)

deposits along the GBM or TBM (or both). Fibrillar deposits on EM 12–18 nm in diameter are key to diagnosis of recurrent fibrillary GN. Both LCDD/HCDD and fibrillary glomerulopathy can be associated with hematologic malignancy [127]. In the allograft kidney, LCDD recurrence is associated with underlying disease activity (multiple myeloma) and persistent production of monoclonal light chains [10] (Fig.  6.61). Multiple myeloma affects the kidney in various ways: myeloma casts, amyloidosis, nephrocalcinosis, renal stones, and LCDD [71]. On occasion, cast nephropathy may be associated with benign paraproteinemia, undetectable myeloma or precede clinical myeloma [12, 311]. Some patients present with combined LCDD and cast nephropathy [127]. Although exceptional patients survive with functioning grafts over 10 years after transplantation, renal allograft survival is poor in LCDD [194]. It is proposed that renal transplantation should not be an option for LCDD patients unless measures are taken to reduce light chain production. Overall rate of recurrence is 50% [311]. We have seen at least two cases of recurrent MIDD, one concurrent with cast nephropathy shown in Fig. 6.61. Pure light chain cast nephropathy in the allograft may have an indolent course or manifest with rapid decline of renal function and heavy proteinuria. Clinical presentation correlates with pathologic findings. Three patterns are possible: nodular glomerulosclerosis, tubular casts, or crescentic glomerulonephritis. At least 12 cases are published; recurrent and native disease was similar.

Patients with proliferative glomerulonephritis had fast graft loss. Treatment for recurrent LCDD is limited. Heavy immunosuppression for acute rejection may trigger recurrence [311]. Fibrillary glomerulopathy is rare, but recurrence is documented in case reports. An interesting case of patient with recurrent fibrillary glomerulopathy is described presenting with kidney involvement concurrent with lung deposits identical to those in the kidney [46]. Similarly, de  novo fibrillary glomerulonephritis occurring in a patient with lupus is published [160]. The findings in the allograft are similar to native kidney, with IgG and C3 deposits detected by immunofluorescence and typical fibrillar glomerular deposits by EM. Fibronectin glomerulopathy, a rare familial glomerulopathy, is characterized by massive mesangial fibrillar deposits of fibronectin. Most families develop ESRD late and a few are in need of transplantation. Recurrent disease is described in the literature in case reports. An example, a 46-year-old man with living unrelated allograft kidney 5 years prior to biopsy is shown in Fig.  6.62. Patient presented with increased creatinine and low grade proteinuria.

6.2.4.10 Miscellaneous Glomerular Disease Recurrence Other glomerular diseases also recur in the transplant. We have seen some cases of cryoglobulinemic Hepatitis

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Fig. 6.61  rLCDD in 47 year old man with history of multiple myeloma, 5 years posttransplant. He presented with nephrotic range proteinuria. Biopsy shows: (a) Nodular glomerulosclero-

sis, (b) tubular paraprotein casts and (c) Lambda monoclonal deposits by IF (×400, 300 and 400 respectively)

C and hereditary Oxalosis; others have described Fabry disease, Cystinosis, and other recurrent metabolic diseases. Recurrence may be early posttransplant with catastrophic effects. The pathology is similar to native kidney, however, less typical at times and diagnosis may be missed if the material is inadequate or the biopsy is not thoroughly examined including EM. Dual disease (recurrence and superimposed second disease) is known to occur and can be a diagnostic challenge. For example, recurrent Hepatitis C may be complicated by TMA in patients treated with interferon alpha [23]. rOxalosis should be differentiated from nonhereditary causes of hyperoxaluria particularly early posttransplant when patients may damp oxalates stored prior to transplantation in the urine. Hyperoxaluria type I is the most common type. The primary defect is in hepatocyte

peroxisomes and common complications are kidney stones and nephrocalcinosis leading to ESRD. A combined liver and kidney transplantation is the current treatment option with good results when strict dietary restrictions, stone inhibitors, and living donors are used. In contrast, deceased donors have generally poor prognosis [162]. New therapies using probiotic bacteria to restore defective enzymatic activity and hepatocyte cell transplantation are currently employed presenting exciting new modalities that may alleviate the dangers of organ transplantation [35]. The case shown in Fig. 6.63 is that of a 21-year-oldwoman with hereditary oxalosis-induced ESRD transplanted 2 weeks prior to biopsy. Clinically, she presented with a large lymphocele but the biopsy showed numerous calcium oxalate deposits (Fig. 6.63).

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Fig.  6.62  rFibronectin glomerulopathy. Biopsy shows (a). Thickened glomeruli with occlusion of capillaries, (b) bulky fibrillar electron dense deposits, (c) fibronectin positive deposits

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(H + E and IP × 400 and EM × 8,000 (kindly provided by Dr. Anjali Satoskar, Ohio state University)

Fig. 6.63  Recurrent oxalosis. (a) Computed tomography (CT) shows classic bilateral cortical calcification in the native kidneys (arrows). (b) Multiple oxalate crystals in tubules are seen under polarized light (×200)

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6.3 Malignancy

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In the early posttransplant period, the most frequent malignancy is lymphoproliferative disorder (PTLD). PTLD consists of B-lymphocyte proliferations ranging from benign polyclonal lymphoid hyperplasia to monomorphic lymphoma (Fig. 6.64). PTLD, in the allograft kidney, is generally very rare. Many, if not most, of these proliferations are Epstein–Barr virus positive. Two of the most frequent risk factors for PTLD in transplant recipients are: EBV seronegativity and excessive immunosuppression. Induction immunosuppression with polyclonal or monoclonal antilymphocyte globulins is associated with an increased risk of PTLD and mycophenolate maintenance immunosuppression with a reduced risk [56]. Furthermore, the incidence of PTLD has decreased dramatically in the

last few decades during which new immunosuppression agents and routine drug monitoring was implemented. In addition, the prognosis of patients who develop PTLD improved and is excellent upon retransplantation [163]. Interestingly, a case report of PTLD developing in the native kidney is published [18]. An even rarer de  novo lymphopoetic malignancy other than PTLD is intravascular B cell lymphoma. It is characterized by preferential growth of malignant lymphocytes within the renal microvasculature. Patients present with systemic (fever, malaise) and renal symptoms (proteinuria or renal failure). Malignant cells may involve glomerular and PTC (Fig. 6.65). When glomeruli are involved, the presenting symptom is proteinuria mimicking glomerular disease. Isolated peritubular capillary infiltrates can present without proteinuria [301]. Bilateral renal enlargement is common, if not

Fig. 6.64  PTLD. (a) Renal biopsy shows massive infiltrates in the cortex obscuring tubular and glomerular profiles (PAS × 200). (b) Higher magnification shows that the infiltrate is monomor-

phic and atypical (H + E × 400). (c) The majority of atypical cells are positive for B cell marker (CD 45) and (d) negative with CD20 (T cells) (IP × 400 and 250)

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Fig. 6.65  Intravascular lymphoma. (a) Large malignant cells infiltrate glomeruli and PTC (H + E × 400). (b) Immunohistochemical stains with CD45 (mature B cells) stain glomerular and (c) interstitial infiltrates (IP × 400) (courtesy David Thomas, Nephrocor)

typical [347]. In native kidneys, 15 intravascular B cell lymphomas were reported between 1985 and 2007, all were B cell type [375]. A case report of an 18-year-old renal allograft recipient who developed renal dysfunction and intermittent hematuria was diagnosed as a T-cell intragraft lymphoma. At the time of biopsy, there was no evidence of systemic lymphoma [120]. Intragraft lymphoma resolved completely after chemotherapy, but the patient died 6 months later as a result of cerebral hemorrhage caused by brain involvement by intravascular cerebral lymphoma. An immunohistochemistry with CD45, CD20 and CD79a (B cells), and CD3 (T cells) was helpful in defining this rare neoplasm. The patient in Fig. 6.65 was a 43-year-oldwoman who developed ESRD secondary to preeclampsia. A year later, she received a living related allograft kidney, which was lost immediately after grafting due to thrombosis. Her

second allograft kidney was also living related and had no complications posttransplant. Six years later, she presented with rapid creatinine increase over 1-month period and trace urine protein and blood. The renal biopsy revealed large malignant cells infiltrating the glomerular and PTC. Diagnosis was facilitated by immunohistochemical stains with CD45 (mature B cells) and CD79a (precursor B cells). In contrast to PTLD, non-lymphoid malignancies occur in a percentage as high as 50% at 20 years posttransplantation and are a main cause of mortality in renal allograft recipients [330]. In a single center study in northern Italy, among 400 renal allograft recipients, 30 patients developed malignancy (7.5%). Only three were lymphomas, the remaining were nonmelanoma skin cancer (12), and solid organ malignancies (15): seven genitourinary, three lung, two

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breast, two gastrointestinal, and one sarcoma. Mean age at diagnosis was 55 years and mean time from transplant was 27 months. Six patients died and two lost their graft. As life expectancy of transplant recipients has increased in the last few decades, the incidence of malignancy has also increased and is expected to increase further. In a review of 35,765 patients with first-time renal allograft from deceased donors (1995– 2001) at the University of Minnesota, an increased incidence compared to the general population was found for the following malignant neoplasms: colon, lung, prostate, stomach, esophagus, pancreas, ovary, and breast cancers were increased twofold. Melanoma, leukemia, hepatobiliary, cervical, and vulva carcinoma increased by fivefold. Testicular and bladder cancers increased by threefold, but kidney cancer was approximately 15-fold higher. Kaposi’s sarcoma, nonHodking’s lymphoma and skin cancer other than melanoma were 20-fold higher compared to the general population [170]. This study also found that certain immunosuppressive drugs had a lower association with cancer; tacrolimus had 35% lower incidence of skin cancer and antibody induction 17%. Some cancers like Kaposi’s sarcoma are linked to viruses that may be present in some transplant recipients, but there is no good hypothesis proposed to explain the high incidence of renal cell carcinoma. Single-center studies are usually too small to draw conclusions [28], but results from large centers and registry data have now led to the development of guidelines for cancer screening and prevention. Noninvasive resection of renal cell carcinoma from grafted kidneys is frequently performed with good residual kidney function [208].

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6.4.1 Transplant Glomerulopathy (TGP) An overall incidence of 20–30% of TGP is reported in sequential renal allograft biopsies. Clinical presentation includes graft dysfunction and or an insidious and subclinical course [124, 174]. Patients with graft dysfunction usually present late posttransplant (>5 years) with a mean of 8 years [314, 317]. However, based on recent findings in protocol biopsies, TGP may start long before symptoms are apparent. Renal biopsy findings vary from mild hypercellularity, vague lobular appearance in early stages, and or nodular pattern in late stages resembling MPGN. The characteristic late findings are thickened capillary loops with double contours (Figs. 6.66 and 6.67). On EM, thickened capillary loops have a distinct widening of the lamina rara interna (subendothelial edema), which is considered the hallmark of TGP (Fig. 6.67c asterisk). Other findings include mesangiolysis, intramembranous electron dense deposits, inflammatory cells or matrix accumulation [41, 156]. Multi-layering of the GBM, first described by Monga et  al. [231], is the basis for the term transplant capillaropathy [68, 69, 86]. GBM multilayering can be focal and or segmental and unless sought after can be missed (Figs. 6.31 and 6.68). Even though some have debated whether this is an exclusive feature of TGP, is nonetheless, characteristic and involves not only the GBM but PTC as well, as shown in Fig. 6.30. TGP, on IF, shows mild usually low intensity IgM, IgG, C3 deposits. The exception is C4d, which in the majority of cases is strongly positive

6.4 De Novo Disease De novo disease may be primary or secondary to drug toxicity, infection, malignancy or other insults. It can involve glomeruli, the tubulointerstitium or vessels and includes malignancy (Table 6.4). Glomerular disease combining recurrent and de novo disease, is the fourth most frequent diagnosis accounting for 15.5% in our material (Table  6.7). The majority of de  novo glomerular disease is TGP (31.4%) (Table  6.13) followed by de novo FSGS (11.1%) [174].

Fig. 6.66  TGP with double contours (arrows) (EM × 2,800)

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Fig. 6.67  TGP: (a) thick capillary loops with segmental double contours (PAS). (b) Diffuse C4d+ in PTC and glomerular capillaries. (c) Massive subendothelial edema (asterisk) (EM × 4,500); biopsy was taken 5 years posttransplant (H + E, IF × 300)

Fig. 6.68  TGP with GBM lamellation (arrows) (EM × 8,000)

within the capillary loops or the mesangium. Late TGP mimics idiopathic MPGN and TMA. In fact, rarely, TGP may be complicated by TMA. The example in Fig.  6.69 is from a 21-year-oldman with ESRD secondary to obstructive nephropathy 5 years posttransplant. C4d and DSA were negative, but the patient was known to miss his medications (Fig. 6.69). This occurrence is rare but perhaps, points to the important role endothelial injury plays in TGP pathogenesis. For example, there is significant evidence that closure of endothelial cell fenestrae and subendothelial edema is an early finding of TGP and can even be found in clinically stable grafts [365] (Fig.  6.70). Endothelial cell damage includes loss of fenestrations, luminal microvilli formation, apoptosis, and inflammatory cell aggregation. In addition, an immunologic mechanism

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Fig. 6.69  TGP with concurrent TMA. (a) Glomerular thrombi (H + E × 400). (b) Fibrin thrombi are highlighted by IF (×300). C4d was negative

Fig. 6.70  Renal biopsy from a patient biopsied for low grade proteinuria and renal dysfunction; (a) shows closure of endothelial fenestrae (black arrows). Three months later, he developed

nephrotic range proteinuria. (b) EM demonstrates diffuse subendothelial edema (white arrows). The case is an example of progressing TGP

of endothelial injury mediated by donor-specific antibody binding is proposed. For example, the specific histopathological and ultrastructural TGP features and peritubular basement membrane multi-layering were recently correlated with DSA-HLA and C4d+ [317]. Multilayering of PTC was present in 91% (48/53) biopsies with TGP; C4d+ was present in 36% and DSA-HLA in 70%. Overall, 73% of TGP was associated with DSA. Previous studies found 50–60% of TGP associated with peritubular capillary C4d+ deposition and in the majority of cases, with serum DSA

HLA [212, 280]. Very frequently strong C4d+ deposits are present in TGP glomeruli (Fig. 6.67). In the study by Sijpkens et al., the great majority of biopsies with TGP had glomerular C4d+. Peritubular capillary C4d deposits and donor-specific anti-HLA antibodies were demonstrated in approximately half of their cases. Presensitization and late acute rejection episodes were identified as risk factors [314]. In our material, the overwhelming majority of TGP had DSA+ [174]. Subclinical TGP is not a well-recognized cause of antibody-mediated chronic injury. In the study of

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Gloor et  al. although TGP was associated with both acute and chronic histolopathological findings, 14.5% of TG biopsies showed no interstitial fibrosis or tubular atrophy, while 58.0% of biopsies with severe TGP showed only minimal abnormalities. Nonetheless, TGP often coexists with chronic tubulointerstitial or vascular rejection and is considered part of chronic rejection as previously alluded. TGP is also associated with pretransplant hepatitis C and anti-HLA antibodies (Class II, in particular). Prognosis is worse compared to recurrent glomerular disease and causes accelerated graft loss in spite of modern immunosuppression. Prognosis of subclinical TGP is equally poor to symptomatic TGP.

6.4.2 Miscellaneous De Novo Glomerular Diseases Other de  novo glomerular diseases of common and some uncommon entities are discussed with recurrent disease. An exception is postinfectious glomerulonephritis, which is a rare complication in the renal allograft [264]. The first case was reported in 1983 [297]. Only a dozen cases are reported in the literature. Staphylococcus aureus, mycotic aortic aneurysm, and other bacteria are reported [244]. Many patients were diabetic presenting with acute renal failure and history of infection [264]. Biopsy revealed immune complex glomerulonephritis and histologic findings typical of postinfectious glomerulonephritis. We have seen

one case, recently. The patient was a 74-year-oldwoman with ESRD secondary to MPGN, admitted for acute renal failure and proteinuria. History revealed recent staphylococcal infection. Renal biopsy had lobular and hypercellular glomeruli with prominent neutrophils, typical of postinfectious glomerulonephritis (Fig. 6.71). IF was positive for glomerular IgG and C3 (2+) granular deposits. EM showed small subepithelial bell-shaped deposits (humps), confirming the diagnosis of de novo postinfectious glomerulonephritis in the allograft kidney.

6.4.3 CNI Toxicity CNI, namely cyclosporine and tacrolimus have been responsible for improved short-term outcomes and diminished acute rejection rate. Cyclosporine (CsA) was introduced in clinical practice in the 1980s. It was isolated from extracts of soil fungi screened for novel antifungal agents in the 1970s and soon found to reduce proliferation of immunocompetent T lymphocytes. The molecular mechanisms of action of CNI inhibitors were extensively studied in the last decade or so. Briefly, calcineurin (CN) is a complex ubiquitous intracellular enzyme activated by calcium binding to calmodulin, which engages the inhibitory domain of CN and removes it from its active site. CN inhibitors (CNI) prevent cytokine transcription interfering with the CN-NFAT pathway and prevent immune response. However, the exact pathways via which CNI promote

Fig. 6.71  De novo postinfectious glomerulonephritis. (a) Glomerular neutrophils (H + E × 400). (b) Subepithelial small bell-shaped deposits (arrows) (EM × 6,500)

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immunosuppression are not entirely clear. In spite of acceptable results, there is sufficient clinical evidence to show that immunosuppression by CNI is incomplete [140, 242]. CsA causes no bone marrow toxicity, but both acute and chronic renal injuries and systemic complications. CsA nephrotoxicity clinically presents with symptoms of acute renal failure (increased creatinine, decreased glomerular filtration, increased sodium, etc.). Acute nephrotoxicity is dose-dependent inducing afferent arteriolar vasoconstriction and decreased glomerular blood flow. Several factors are implicated even though the exact mechanisms are not fully understood. These include imbalance of prostaglandin E2 and thromboxane A2 and increased endothelin-1. Histologically, the lesions of acute CsA toxicity are tubular and or vascular. The tubular lesions are: isometric vacuolization defined as cytoplasmic vacuoles of similar size in proximal tubular epithelial cells (Fig.  6.72), and or ATN. Giant mitochondria corresponding to tubular epithelial cell vacuoles are found by EM [228]. Prolonged vasoconstriction induces ATN, tubular epithelial vacuolization or sloughing. These lesions are reversible with dose adjustment (Fig. 6.73). Arterioles and very small arteries are the CsA-targeted vessels [228, 229]; Acute CsA toxicity manifests with TMA (discussed under TMA). Acute CNI toxicity is rarely seen (<1% of allograft biopsies) currently because of low CsA dose is used in maintenance immunosuppression often ­combined with less toxic agents such as sirolimus [189]. Arterial wall subendothelial deposits (beaded

Fig.  6.72  Isometric vacuolization in acute CNI toxicity (PAS × 400)

H. Liapis et al. Reversible lesions

Renal Pathology of cysclosporine toxicity

Tubulopathy: Proximal tubules

Isometric vacuolization Giant mitochondria Single cell necrosis Microcalcifications

Irreversible lesions

Vasculopathy: arterioles

Endothelial cell damage Smooth muscle thickening Arteriolar thrombosis

Local ischemia Glomerular obsolescence Tubular atrophy Striped insterstitial fibrosis

Fig. 6.73  Lesions of CNI toxicity

Fig.  6.74  Arteriolar beaded hyalinosis (arrow) induced by chronic CNI immunosuppression (PAS × 400)

hyalinosis) are characteristic of CNI toxicity (Fig. 6.74). Such lesions are irreversible, in contrast to reversible tubular damage, which is mainly due to impaired renal blood flow [178]. This is amply demonstrated in animals in which CsA induces renal vasoconstriction followed by decreased GFR [43, 122, 285]. The differential diagnosis of isometric vacuolization includes osmotic nephrosis (ATI with accumulation of lysosomes) due to many exogenous agents. In native kidneys, radiocontrast preparations, low-molecular-weight dextrans, and intravenous immune globulin administration are implicated. The amount and duration of exposure to these agents, pre-existing kidney injury, ischemia, nephrotoxic drugs, diabetic hyperglycosuria, and old age are thought of as risk factors. In the renal allograft, the most common etiology

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of osmotic nephrosis is drugs. Tubular changes of osmotic nephrosis are usually reversible, but not in all patients [82]. Chronic nephrotoxicity is not dose dependent, but results from prolonged vascular injury and arteriolar hyalinosis leading to tubular atrophy and interstitial fibrosis along ischemic segments, known as strip or skip fibrosis (Fig. 6.75). Late in the course, interstitial fibrosis may be diffuse. Pathogenesis of chronic CsA toxicity includes decreased production of nitric acid and increased expression of TGFb1 [177]. Chronic CsA appears to, also be influenced by hyperuricemia. The mechanism appears to involve activation of the renin angiotensin system and inhibition of nitric oxide production in the kidney [214]. Rarely, acute superimposed on chronic CNI-induced vascular injury is present concurrently (Fig. 6.76).

Fig. 6.75  CNI toxicity induced striped fibrosis (Trichrome × 200)

The new generation of CNI such as tacrolimus is also nephrotoxic. Comparison of histologic and molecular changes induced by CsA and tacrolimus show no difference in nephrotoxicity 12 months posttransplantation (overall, 24%) or interstitial fibrosis (~13%)) [284, 289]. Similar increase in TGFb and collagen a1 and a3 was found in protocol biopsies of patients treated with CsA or tacrolimus at 1-year posttransplantation [177]. Preliminary evidence suggests that therapeutic drug monitoring has little value in the diagnosis or management of CNI nephrotoxicity [106]. In addition, CNI induce de novo diabetes, dyslipidemia and hypertension, increasing the cardiovascular risk in renal transplant recipients. In the past few years, alternative agents emerged with specific advantages over CNIs. For example, randomized clinical trials with Sirolimus demonstrated an advantage of rapamycin (Rapamune, Wyeth Pharmaceuticals, Collegeville, PA) over CNI in regards to nephrotoxicity [113, 132]. New drugs are often used in combination with lower CNI dose in certain subpopulations of renal transplant recipients who are at high risk of acute rejection and allograft loss. These include African Americans, patients with elevated titers of panel-reactive antibodies (PRAs), or patients with history of previous allograft loss. Data from the UNOS documented lower graft survival at 1-year among black transplant recipients (88.0 vs. 91.5% in nonblacks), among patients with PRAs ³80.0% (87.2 vs. 91.3% for low PRAs), and among retransplantations (88.5 vs. 91.2% for primary transplants) (UNOS 2006). Beyond nephrotoxicity, CsA also has other side effects including: fibrocystic breast disease and fibroadenoma [298], urolithiasis (see below), and lymphocele formation in the allograft kidney [188].

6.4.4 Crystal Deposition Disease 6.4.4.1 Oxalate Crystal Deposits

Fig.  6.76  Acute thrombosis (arrows) superimposed on thick arterioles due to long standing CsA immunosuppression (H + E × 400)

Blood oxalate derives from erythrocytes, dietary sources, the liver, and ascorbate metabolism. Oxalate is filtered in the glomerulus and excreted in the urine as the terminal metabolite of the above sources or actively reabsorbed in proximal tubules to maintain homeostasis [205]. Many factors influence oxalate metabolism: intake of foods high in oxalate, malabsorption (short

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bowel syndrome), reduced activity of liver enzymes (hereditary hyperoxaluria), drugs (CNI), and decreased urine output; all lead to high serum oxalate. As a result, high serum levels of oxalate are common in ESRD. Transient hyperoxaluria lasting from 3 days to 3 weeks occurs in the early period posttransplantation. Because, the kidney is the only organ through which oxalate is excreted and dialysis removes a fraction only of the daily intake, excess oxalate stored pretransplantation is rushed to clearance after transplantation. Most patients achieve oxalate balance without experiencing renal dysfunction. A minority of patients have CaOx deposits in renal biopsies performed for dysfunction (incidence ~4%) [21, 353]. Others find an incidence of 50%, 3 months posttransplant [261]. This may represent posttransplantation clearance or drug toxicity. CNI treatment can lead to significant hyperoxaluris or hypocitraturia, especially in patients receiving the highest dose of medication; both conditions may cause urolithiasis or nephrocalcinosis, an association that may not be obvious [322]. CaOx crystals are typically colorless on hematoxylin-eosin stained sections but multicolored under polarized light (Fig.  6.77). Oxalate deposits may occur immediately after transplantation or years after. They may represent recurrent primary hyperoxaluria or be secondary to nonhereditary causes. CaOx deposits, seen early posttransplantation, are usually few, often associated with ATN or acute rejection. These are reversible and resolve with recovery of allograft function. Within the first year of transplantation an average

Fig.  6.77  CaOx crystals (arrow) associated with ATN early posttransplantation (H + E × 400); multicolored crystals under polarized light (insert)

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number of 1.3 ± 1.2 crystals per biopsy tissue mm2 are reported [21]. Deposits are associated with ATN but not with interstitial inflammation or rejection. Increased tubulointerstitial fibrosis is present in repeat biopsies but there is no significant graft loss at 2 years compared to controls. ATN may facilitate CaOx deposition; alternatively, the deposits participate in tubular injury. Previous studies have found pronounced CaOx deposits associated with recurrent primary hyperoxaluria at variable time intervals after transplantation, but usually within weeks. We have seen a couple of recurrent hyperoxaluria cases occurring within the first month, posttransplantation; CaOx deposits were abundant and involved both the tubules and the interstitium (Fig. 6.63). Multifocal CaOx crystals in allograft biopsies are also seen with chronic tubulointerstitial injury and are associated with graft loss in contrast to reversible acute renal failure caused by CaOx deposits secondary to increased intestinal absorption or oxalate-containing drugs (naftidrofuryl oxalate) [353].

6.4.4.2 Calcium Phosphate Crystal Deposits In renal transplantation, abnormal calcium metabolism may be a residual effect from preimplantation dialysis or develop de  novo, posttransplant. Serum calcium levels follow a biphasic pattern with significant decline during the first postoperative week, followed by a significant increase. High pretransplantation parathyroid hormone levels protect against hypocalcemia within the first postoperative week, but later patients are at risk for hypercalcemia [100]. Hyperparathyroidism and hyperphosphatemia are treated with phosphate-binding agents [73]. After transplantation, cessation of calcium-containing phosphorus binders and vitamin D intake affect serum calcium levels. Hypercalcemia directly and indirectly leads to a reduction in glomerular filtration rate by causing renal vasoconstriction and decreasing extracellular volume. Decreased glomerular filtration rate impairs the kidney’s ability to excrete calcium. Experimental studies show that crystal retention in the distal nephron is limited to regenerating tubular cells expressing hyaluronan and osteopontin at their luminal surface. In transplant kidneys, luminal expression of hyaluronan and osteopontin precedes renal distal tubular retention of crystals; it is proposed that this crystal-binding phenotype may play a general role in renal calcification [355].

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Calcium deposits are often found within acutely damaged tubules and or within atrophic tubules, in areas of interstitial fibrosis, and rarely in glomeruli. These deposits stain dark blue with Hematoxylin-Eosin (Fig. 6.78), do not polarize, and are easily identified; von Kossa stain specifically stains calcium phosphate. Calcium phosphate deposits predispose to stone formation (nephrocalcinosis). Nephrocalcinosis and crystalluria in addition to abnormal metabolism may also be due to poorly soluble drugs. These include acyclovir, indinavir, and triamterene [165]. Older drugs

known to cause crystalluria are sulfonamides. Foscarnet (phosphonoformate) is a relatively newer drug used as a second-line agent for cytomegalovirus infection (CMV) after ganciclovir therapy. Foscarnet inhibits the DNA polymerase of all herpes viruses and the reverse transcriptase of retroviruses. It is often used to treat HIV infection as well. Calcium deposits can be either in glomeruli or tubules (Fig. 6.79). The mechanism of nephrotoxicity due to Foscarnet is debated but increase of urine flow rate is effective in reversing kidney damage in some patients. Irreversible damage is reported when crystal deposits involve the glomeruli [165].

6.4.4.3 Cholesterol Embolism

Fig.  6.78  Calcium phosphate deposits associated with focal tubular atrophy. Nephrocalcinosis was diagnosed by CT in this 75 year old woman who presented with creatinine of 6.4 mg/dL 2 years posttransplant (H + E × 400)

Less than 50 cases are reported in the literature, but this may be an underestimation of the true incidence (reportedly <0.5%). Arterioles and small arteries are the most frequent loci, but this may be a sampling error, because renal biopsy is usually more representative of the outer than the deep cortex where large vessels lie. Glomerular capillaries are less frequently involved. The classic presentation is spindle-shaped, colorless crystals engulfed by giant cells causing luminal obstruction (Fig. 6.80), but cholesterol crystals can be inconspicuous and not associated with inflammatory reaction, thus are easily missed because of their small size and focal distribution; for example, diagnosis may depend on a single focus (Fig. 6.81). Elderly patients with severe atherosclerosis are at high risk. In a study by Lai CK and Randhawa, among 5,435

Fig. 6.79  Froscanet induced nephrocalcinosis in a young patient treated for CMV. (a) Deposits fill many tubular profiles (H + E × 200); (b) highlighted with von Kossa stain (×200)

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Fig. 6.80  Typical cholesterol embolus engulfed by a giant cell in a small arteriole (H + E × 200)

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source of cholesterol emboli appears to be more important than the time of insult. In the study by Lai and data by others, the great majority of patients were men; most donor kidneys were also from male donors. The mean age of both donors and recipients was 55–57 years. Hypertension and diabetes were the two most common reported diseases. Risk factors listed in order of frequency were: procurement process, recipient atherosclerosis, heparin anticoagulation, vascular catheterization, and trauma. Grafts that failed because of cholesterol embolism presented with primary nonfunction. Pathogenesis of cholesterol embolism is attributed to dislodging of cholesterol-rich portions of atherosclerotic plaques when these are exposed to arterial blood flow because of endothelial ulceration (fibrous cap). Dislodged cholesterol emboli are usually caused by trauma; for example, angiography or vascular surgery. However, spontaneous renal embolism is not uncommon with main source being the abdominal aorta. Extrarenal manifestation may also occur with emboli in the lower extremities causing digit necrosis (blue toes). Systemic manifestations include fever, hypereosinophilia, thrombocytopenia, and increased creatinine [185, 307]. With increased utilization of older donors currently, cholesterol embolism appears to be on the rise.

6.4.4.4 Rhabdomyolysis

Fig.  6.81  Cholesterol emboli in afferent arteriole (arrow); patient presented with rising creatine 2 years post transplantation from deceased donor (H + E × 200)

allograft biopsies, 12 patients with cholesterol embolism were identified. All recipients received grafts from deceased donors. In nine, emboli were likely derived from recipient atherosclerotic vessels and three were of donor origin. Five patients developed acute renal failure without cellular rejection and in two, cholesterol emboli were the sole or predominant finding. In a 2-year follow-up, graft survival was worse in donor-derived cholesterol emboli with early posttransplant occurrence [187]. Cholesterol embolism may manifest early or late posttransplantation, but the

Myoglobin deposits are due to skeletal muscle cell damage secondary to a long list of medications, illicit drugs, alcohol, trauma, excessive exercise, hereditary muscle defects, and infections [243]. Transplant recipients are not immune to these conditions; clinical presentation includes acute renal failure, increased CK and transaminases, increased creatinine, and pigmented urine. Myoglobin casts are found in distal convoluted tubules, which appear dense and red in color (Fig. 6.82a), in contrast to other pigments in the urine. For example, Tamm-Horsfal protein has a bluish tint on H + E, bilirubin casts are green (see below under cholemic nephrosis), and hemoglobin casts (secondary to ABOi, malaria, etc.) are pink. Antibodies to myoglobin are commercially available and confirm the diagnosis. On EM, myoglobin deposits stain black with osmium (Fig. 6.82b).

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Fig. 6.82  (a) Myoglobin casts appear dense and deeply red with H + E (× 200) and (b) stain black with osmium (EM × 2,500)

In the transplant recipient numerous medications may cause rhabdomyolysis. For example, statins are often prescribed for hyperlipidemia, antibiotics (Bactrim) for prophylaxis of urinary tract infection (UTI), cyclosporine for rejection, and calcium channel blockers for heart disease; all these medicines can be complicated by rhabdomyolysis [325]. Importantly, statins interact with other drugs and their metabolism is affected by the liver cytochrome P450 enzyme CYP3A4. Cyclosporine and clarithromycin, for example, are CYP3A4 inhibitors, thus inferring an increased risk [254]. The mechanisms involved are poorly understood but drug effect may not be the sole reason. For example, studies in animals have shown that hypovolemia, renal vasoconstriction, or urine acidification is additionally required for myoglobin cast formation. Cessation of the suspected drug, intravenous fluid administration, forced diuresis, and urine alkalinization are the therapeutic options.

complete loss of the tubular lining, and denudation of the tubular basement membrane. Clinically, ATN is defined by increased serum creatinine reflecting changes in glomerular filtration rate. However, for many reasons, creatinine is not an accurate marker of GFR and GFR is a late marker of ATN. Histologically, true ATN is characterized by desquamation of tubular epithelial cells, denudation of the tubular basement membrane, and tubular dilatation. PAS stain highlights loss of brush boarder and necrotic tubular epithelial cells, which stain dark pink (Fig.  6.83); necrotic tubules autofluoresce under fluorescent light

6.4.5 ATN The incidence of ATN in the allograft kidney depends on its definition, which is currently under discussion [286, 376]. ATN is pathologically defined as loss of proximal epithelial cell brush boarder in early stages of injury, followed by coagulation necrosis of the cytoplasm of tubular epithelial cells and subsequently

Fig.  6.83  ATN with dramatic coagulation necrosis of tubular epithelial cells in a 7-year-old-man whose creatinine increased from 2.5 to 9 mg/dL over a short time (H = E × 200)

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Fig. 6.84  Necrotic tubules in ATN autofluoresce under fluorescence light (courtesy Luis Salinas-Madrigal, Saint Louis University)

(Fig. 6.84). This is a simple method using H + E stained sections or paraffin unstained sections; necrotic tubules show bright yellow autofluorescence [296]. True ATN is not very common in the allograft kidney. To the contrary, ATI defined as multifocal loss of brush boarder and loss of tubular cell nuclei is frequent. ATI is associated with numerous conditions including preservation injury (ischemia), drug toxicity, proteinuria, acute interstitial nephritis (AIN), pyelonephritis, rhabdomyolysis, crystal deposition, rejection, etc. Reversible injury and recovery of renal function is the rule in most cases treated early with rehydration and elimination of inciting agents, usually drugs or infections. We have seen just about a handful of patients presenting with acute renal failure who had “true” ATN as the only finding to explain the clinical presentation (Fig. 6.83). In contrast, ATI is not infrequent in allo­ graft biopsies.

6.4.6 Infections: Bacterial, Fungal, Viral Posttransplantation UTI are common in kidney transplant patients and can precipitate pyelonephritis and graft dysfunction, sepsis, and even death. The reported incidence varies from 10 to 98% depending on the definition of UTI. The terms bacteriuria, bacteremia, and pyuria are now clearly defined. UTI is pathological invasion of urothelium that causes inflammatory response and can be defined by culture and microscopy. The incidence of

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UTI, in the first 6 months after transplantation, reported in 1992, was 7–40% [359]. Currently, the true incidence is much lower because it is standard practice to administer prophylactic antibiotics for the first 6 months and in some centers, even after 6 months posttransplant, based on recent data that show that late onset UTI is complicated with graft loss and death. Factors predisposing to UTI include: female sex, diabetes, deceased donor kidney, infected donor kidney, prolonged catheterization, retransplantation and excessive immunosuppression, instrumentation of the urinary tract, and infection of the native kidneys. In addition, the anatomic relationship of the grafted kidney in the iliac fossa may cause reflux and urine stasis predisposing the kidney to infection. Native kidneys left in situ (for example, polycystic kidneys) may be a reservoir for pathogens [78]. The most common organisms are bacteria with Enteroccoccus species accounting for 70% of UTI. Pseudomonas, Klebsiella and Proteus mirabilis are frequent. The latter is associated with stone formation and can cause relapsing UTI. Symptomatic UTI is treated with antibiotics for 2 week, or longer as indicated and or with additional measures to remove source of infection (for example, stent removal). It is now customary to administer a single dose of second or third generation cephalosporins prophylactically, before transplantation and before removal of catheters. Long-term prophylaxis, after transplantation, is practised in most centers. Without antibiotic prophylaxis, the incidence of UTI is still very high (up to 98%) [78]. However, this practice is complicated by emergence of resistant bacteria plus side effects of long-term antibiotic therapy to the graft, for example interstitial nephritis. The most frequent finding on allograft biopsies is AIN with eosinophils secondary to prophylactic antibiotics with an incidence of 8.7% in our material (Table 6.7). Overall, in spite of the benefit of antibiotics in controlling UTI, there is concern that graft or patient survival may not be equally benefited. Better approaches to estimate the risk are proposed [288]. A retrospective study found that UTI, at 6 months posttransplantation, were associated with worse graft and patient survival [2]. Patient education, early detection, prevention, and use of probiotic bacteria are promising to alter this grim outlook. Fungal infections account for about 5% of infections in renal transplant recipients and rank fourth after esophagitis, pneumonia, and meningitis [1].

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Oppor­tunistic organisms are the majority including Candida, Aspergillus, Cryptococcus, and Zygomyces species. Although rare, fungal infections have worse mortality compared with bacterial and viral infections for various reasons; for example, they are difficult to diagnose early, there is low index of suspicion clinically, limited antifungal prophylaxis and therapy, and toxicity of antifungal medications is high (discussed also in Chap. 1). The prime time for fungal infections in transplant recipients is 1–6 months posttransplantation. Risk factors include diabetes, tacrolimus, prolonged pretransplantation, dialysis time, and induction with muromonab-CD3 [258]. Clinical presentation includes fever and may be nonspecific (increased creatinine or acute renal failure). The histological findings of fungal infection in the allograft kidney are: white cell casts (pyelonephritis), granulomatous inflammation, and crescentic glomerulonephritis. Fungal colonies may be obscured by inflammation or be very focal (Fig. 6.85). Diagnosis is not infrequently delayed and many patients lose their graft, develop septicemia, and eventually succumb to disseminated infection. Diagnosis may be made only in the nephrectomy specimen or at autopsy. In some recipients, the source of infection may be from the donor [89]. Interstitial nephritis containing multiple noncaseating granulomas are rare findings in the allograft kidney. In a systematic review of 1,574 renal allograft biopsies obtained from 514 patients in the period 1993–1998, only three cases (0.6%) were recovered [218]. One biopsy was from a 44-year-oldwoman with a 6-day history of systemic candida infection and showed multiple

Fig.  6.85  Fungal spores (arrow) in a transplant patient who died from sepsis (courtesy Isaac Stillman, Deaconess Hospital, Harvard) (GMS × 400)

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Fig.  6.86  Acute interstitial nephritis (AIN) with granulomas (arrow) secondary to Bactrim prophylaxis (H + E × 200)

granulomas containing budding yeasts. The second biopsy was from a 33-year-oldman who presented with miliary spread of mycobacterium tuberculosis, and the third was from a 23-year-oldwoman who presented with Escherichia coli urinary infection and bacteremia. Granulomatous inflammation in response to infectious agents (virus, bacteria or fungi) or drugs can cause interstitial nephritis that is distinct from acute rejection (Fig. 6.86). Cryptococcal UTI is a serious complication that can be disseminated and may cause not only graft failure but death (Fig.  6.85). The incidence of cryptococcal infection in renal transplant recipients ranges between 0.8 and 5.8% and carries a treated mortality rate of 36.0–59.1% [151]. Most common sites of infection in the transplant patient are the lung and skin. UTI is infrequent and typically, silent. In renal transplant patients, the incidence of disseminated disease involving at least two organs is more common and typically occurs more than 6 months posttransplantation. Most cases are primary infections rather than reactivation. Risk factors for cryptococcal infection are exposure to birds, geographic location, male gender, and steroid treatment. Cryptococcus is more prevalent on the East coast of the US. Histologically, there is interstitial granulomatous inflammation often with necrosis- containing fungal colonies. Absence of hyphae and pseudohyphae favor Cryptococcus neoformans or Histoplasma capsulatum (Fig.  6.87). Since cryptococcus species are the only pathogenic fungi with a mucinous capsule, mucicarmine stain (or Fontana Mason) facilitates the diagnosis.

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In endemic countries, schistosomiasis and tuberculosis may be reactivated after transplantation and cause UTI. These are rare in the rest of the world. Viral infections discussed further in Chap. 1, are only briefly mentioned here. Viral complications in the renal allograft include ureteral obstruction caused by polyoma virus (BK and JC) and granulomatous and nongranulomatous interstitial nephritis secondary to Adenovirus, EBV [87, 349, 362] or CMV [196]. CMV is the only virus among the herpes viruses that infects both the nucleus and cytoplasm of affected cells. An example of CMV presenting with severe interstitial inflammation is shown in Fig. 6.88. CMV involves epithelial and endothelial cells and

very rarely mononuclear hematopoietic cells, which are the preferred target of EBV (discussed under PTLD). Viral cytopathic effect is typically owl-eye nuclei with clear halo and enlarged cytoplasm of infected cells. Tubular and or glomerular in addition to endothelial cells can be infected. Similarly, polyoma virus infects tubular epithelial cells typically distal tubules, or endothelial cells causing endarteritis. Polyoma virus intranuclear inclusions are homogeneous (ground glass), basophilic, or granular or bubbly (Chap. 1). Patients acquire polyoma from donors or reactivate dormant virus of their own. Shedding of infected tubular or transitional epithelial cells in the urine is an indication of carrier state. Decoy cells (named so because they mimic cancer) found in urine cytology are not diagnostic of active infection unless associated with neutrophils (Fig.  6.89). However, our perception of urine cytology and its ability to predict BK nephropathy may change in the near future as cast-like, three-dimensional polyoma virus aggregates so called “Haufen” become recognized better (Nickeleit preliminary data). Polyoma virus nephropathy findings vary from an acute inflammatory process, glomerular crescents, immune complex-like tubular deposits, and chronic sclerosing nephropathy, which may resemble or coincide with acute or chronic rejection. Infection can be productive or quiescent. The latter may be impossible to diagnose with histology and immunohistochemistry; in situ hybridization or PCR are required. Histologic classifications for polyoma virus nephropathy have been devised but not consistently applied in routine

Fig.  6.88  Typical owl eye CMV inclusions (arrow) amidst heavy interstitial inflammation (H + E × 200)

Fig. 6.89  Urine cytology: decoy cells for active polyoma infection require also presence of inflammatory cells; otherwise they represent asymptomatic shedding (courtesy Lourdes Ylagan, Washington University St. Louis)

Fig.  6.87  Cryptococcal colony (arrow) in an autopsy kidney from a patient with disseminated infection who succumbed to sepsis (H + E and PAS × 200) (insert)

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Fig. 6.90  (a) Homogeneous and granular intranuclear polyoma virus inclusions (arrows) (PAS × 400). (b) Stained with antibody to SV-40 (IP × 200)

biopsy evaluation [85]. Polyoma (BK) viral inclusions resemble adenovirus and immunostaining is essential for definitive diagnosis (Figs. 6.90 and 6.91). Helpful histologic features to distinguish the two are: dirty necrosis, hemorrhage, granulomatous inflammation or edema; these are associated with adenovirus but not polyoma or CMV infection. EM is also helpful. Both polyoma and adenovirus present with virion assemblies, but adenovirus viral particles are larger (80 nm) than polyoma virions (40 nm) (Fig. 6.92). For further discussion on polyoma, see Chap. 1.

Fig. 6.91  Ultrastructure of polyoma virus: microparticle crystal-like intranuclear arrays (courtesy Eduardo Vazquez-Martul, La Coruna, Spain)

6.4.7 Acute Interstitial Nephritis (AIN) AIN is characterized by presence of interstitial inflammation with eosinophils in renal allograft biopsies. Eosinophils are due to allergic drug reaction typically associated with prophylactic (trimethoprim-sulfamethoxazole) antibiotics. Inflammation is reversible with drug discontinuation [98]. Usually, there is insignificant (borderline) tubulitis present, but when a significant number of eosinophils (>10 eosinophils/mm2) are seen in biopsies with rejection II or III, these according to some authors,may be part of rejection as well [202, 221]. Therefore, interstitial eosinophils in allograft biopsies should be interpreted in context. Granu­lomatous inflammation is in the spectrum of allergic drug-induced interstitial nephritis (Fig. 6.86), but in most cases, granulomas are associated with infection [218]. As previously discussed under rejection, mononuclear, polymorphonuclear, eosinophils, plasma cells, and other (dendritic, mast cells, etc.,) polymorphic infiltrates may be part of ejection [246]. The differential diagnosis of pleomorphic infiltrates includes drug reaction, infection, PTLD, and rejection depending on the location and predominance of a particular cell population. For example, neutrophils in tubular lumens (white cell casts) indicate bacterial infection/pyelonephritis (Fig. 6.93), or viral infection. Neutrophils in PTC are characteristic of hyperacute AMR and often coincide with C4d+ staining in PTC, as discussed under AMR.

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Fig. 6.92  (a) Adenoviral infection with ground glass and granular inclusions in tubular epithelial cells (H + E × 400). (b) Antibody to adenovirus highlights infected tubular epithelial cells (IP × 400)

Fig.  6.93  Acute pyelonephritis; neutrophils are the predominant infiltrate and form white cell casts (arrow) (H + E × 200)

Neutrophils, in glomerular capillaries, can also be seen with renal vein thrombosis [295].

6.4.8 Graft Versus Host Disease (GVHD) in the Allograft Kidney Acute GVHD is usually a manifestation of bone marrow stem cell transplantation occurring early posttransplantation in 20–50% of patients and represents an immunologic reaction of the host against donor lymphoid cells. When GVHD involves the kidney, it

manifests as TMA or ATN (see below). Chronic GVHD affects as many as 50% of patients who survive stem cell transplantation longer than 6 months. Among solid organ transplant recipients, patients with liver and intestinal allografts are complicated by GVHD, but patients with kidney transplants very rarely develop GVHD. Clinically, chronic GVHD resembles vasculitis and involves skin, the GI tract, and lung. GVHD may clinically mimic infection and or allograft rejection [262]. Skin vasculitis with or without elevated serum creatinine are the most frequent findings. Renal symptoms may mimic renal allograft rejection [251] and are attributed to passenger lymphocytes that persist in the host (chimerism) thought to participate in sustained graft tolerance [259]. Lethal GVHD is reported after simultaneous kidney and pancreas transplantation [180]. The patient reported by Kimball was similar to the report by Ohtsuka et al.; the main findings were vasculitis affecting the skin and liver but not the allograft kidney. GVHD may affect native kidneys of patients with other solid organ transplants as discussed under Sect. 6.5.1 below.

6.4.9 Other Complications Some complications to the renal allograft rarely come to pathology but are not infrequently diagnosed by imaging studies. These include renal artery thrombosis

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Lymphocele defined as fluid collection lacking epithelial lining, may result from injured lymphoid channels in the donor kidney hilar vessels during grafting, and other factors including acute rejection, ATN, retransplantation, transplant biopsy, and native polycystic kidney disease. An overall incidence of 0.6– 22.0% is reported. Maintenance immunosuppressive drugs, for example, Sirolimus/MMF/Prednisone, are also reported to cause lymphocele [188].

Fig. 6.94  Renal artery thrombosis in allograft kidney

6.5 Kidney Damage Secondary to Nonrenal Transplantation The kidney is at risk for CKD in nonrenal solid organ and hematopoietic stem cell transplantation. Typically, kidney biopsy shows severe global glomerulosclerosis and tubulointerstitial atrophy and fibrosis (Fig. 6.96). The reported incidence of CKD secondary to bone marrow stem cell transplantation varies from 13 to 62%. CKD may occur as early as 2 months but is more common 5–10 years posttransplantation. Nonrenal transplant recipients who develop CKD have a twofold higher mortality risk compared to patients without chronic renal failure [329]. The risk of renal failure increases with years posttransplantation and is very common after 5–10 years. Kidney disease is the highest in patients with intestinal transplantation (~20%),

Fig. 6.95  Renal vein thrombosis with global hemorrhagic kidney necrosis in allograft kidney

(Fig. 6.94), renal vein thrombosis (Fig. 6.95), and lymphocele [295]. Renal artery thrombosis may be precipitated by trauma during surgical anastomosis or late posttransplantation caused by vascular rejection and systemic thrombotic diseases such as rAPLS (see under TMA). Renal vein thrombosis is due to endothelial damage from surgical anastomosis and typically manifests early posttransplantation. A rare case of fibromuscular renal artery dysplasia recurrence is reported [32]. This anomaly is mostly seen in young women, involves the distant two-thirds of the renal artery and when bilateral, may cause ESRD. It is questionable whether such patients benefit from transplantation.

Fig.  6.96  Chronic kidney disease (CKD) in heart transplant recipient; biopsy shows increased glomerulosclerosis and tubulointerstitial atrophy and fibrosis (PAS × 200)

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followed by liver (18%), lung (15%), heart (11%), and heart–lung transplantation (7%) [148]. About 30% of nonrenal transplant patients require renal replacement therapy [252]. CNI renal toxicity is the most common complication following chronic use. Risk factors other than CNI vary with type of the transplanted organ. For example, for heart recipients, pretransplant dialysis, African American race, hypertrophic cardiomyopathy and pretransplant diabetes or extracorporeal membrane oxygenation are putting patients at high risk for CKD. In liver allograft recipients, risk factors include: older age, female sex, acute renal failure, Hepatitis C, pre-existing diabetes, and low glomerular filtration rate [152]. The most common kidney complications are: chronic CNI toxicity (Table 6.15), followed by glomerular disease de  novo or recurrent including IgA nephropathy, diabetes, FSGS, amyloidosis, membranous glomerulonephritis, and Hepatitis C [52, 126]. Other less frequent complications are: infectious TMA, drug-induced rhabdomyolysis, drug-induced interstitial nephritis (secondary to antibiotics or NSAID), cholemic nephrosis (jaundice-related renal insufficiency) [34], pyelonephritis, and BKV nephritis [52]. A list of kidney complications secondary to nonkidney solid organ allografts and stem cells is shown in Table 6.15. Liver transplant recipients are particularly at high risk for CKD with >60% of patients experiencing increased serum creatinine, postoperatively. Dialysis may be required in as many as 10% of patients [198]. Two-thirds of patients who develop hepatorenal syndrome recover, but it may take 3 months or longer [358]. ATN is the most common histopathologic Table 6.15  Kidney injury in nonrenal transplantation CNI toxicity Glomerular disease (diabetes, FSGS, MCD, hepatitis C, membranous, amyloidosis) Thrombotic microangiopathy ATN Infection (CMV, BK, other) Interstitial nephritis (NSAID, other drugs) Tubular casts (bilirubin, myoglobin) Glomerulosclerosis and tubulointerstitial atrophy and fibrosis (ESRD)

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finding in these situations. The overall risk of CKD increases with time from 18% at 5 years to 25% at 10 years. Postoperative risks for CKD in liver recipients are ATN, CNI toxicity, and Sirolimus and the most common cause of kidney disease in liver transplant patients is CNI toxicity [373] and ATN. ATN may be secondary to CNI therapy, rapid immunoglobulin infusion, other nephrotoxic drugs, hepatorenal syndrome or infection [257]. Management of CKD in nonrenal transplantation includes prevention and treatment of renal complications. Kidney transplantation is a viable option for patients who develop ESRD.

6.5.1 Renal GVHD Following Bone Marrow Stem Cell Transplantation Both acute and chronic GVHD may affect the kidney function but there is confusion in the literature with the term GVHD when it is applied to the kidney; some authors have even questioned whether GVHD occurs in the kidney [117]. The term GVHD is used interchangeably with TTP, HUS or TMA, entities that have in common endothelial damage. However, TMA is only one of the manifestations of GVHD in the kidney and obviously, the most severe. It can be associated with systemic disease (HUS) or be limited to the kidney. The clinical definition of GVHD requires: >4% schistocytes, de novo, prolonged or progressive thrombocytopenia, sudden and persistent increase in LDH, and decreased Hb, haptoglobin requiring increased transfusions. The sensitivity and specificity of this definition exceeds 80% [292]. However, it is not unusual to find TMA in a kidney biopsy performed for renal failure, with no evidence of systemic hemolysis or thrombosis suggesting a subacute illness. The confusion in the literature derives from nonstandardized criteria for posttransplant TMA. Apparently, 28 different sets of criteria have been applied [118]. Therefore, HUS or TTP are terms that better be avoided in the setting of kidney disease secondary to nonkidney solid organ transplantation. A better term is proposed instead when acute thrombosis is identified in the kidney and this is posttransplant TMA. A characteristic example is shown in Fig. 6.97. Glomerular and arteriolar thrombi as well as mesangiolysis (C) are present. Biopsy was

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Fig.  6.97  Posttransplant TMA following bone marrow stem cell transplantation. (a) Subendothelial edema in arteriole; arrow points to luminal occlusion (Trichrome × 400). (b) Glomerulus

with mesangiolysis (H + E × 400). (c) Glomerular and arteriolar thrombosis (arrows) (H = E × 200), (d) fibringogen highlights rhrombi (immunofluorescence ×200)

from a 48-year-old woman with leukemia postchemotherapy who presented with renal failure. Clinical symptoms were acute in this case but TMA may be subacute and present a diagnostic challenge because of lack of diagnostic thrombotic lesions on biopsy. An example is shown in Fig. 6.98. Renal biopsy was performed for acute renal failure in a 52-year-old man treated for CLL and showed no evidence of thrombi but there was extensive ATN. IF was negative for immune deposits. EM demonstrated massive subendothelial edema and distorted GBM with lamina densa irregularities and lucencies. The findings were suggestive of active endothelial damage and perhaps evolving TMA but without thrombosis. The EM findings even

though not specific along with ATN on light microscopy argued for GVHD involving the kidney, but admittedly these are subtle findings. It should be reminded that the spectrum of renal pathology following hematopoietic stem cell transplantation includes TMA, glomerulonephritis, ATN, interstitial nephritis, and hematopoietic disease recurrence involving the kidney [350]. GVHD, histologically, manifests with at least three distinct patterns: (a) TMA, (b) glomerulonephritis, and (c) ATN. Membranous glomerulonephritis, minimal change disease, and FSGS are the commonest entities associated with late GVHD in the kidney; amyloidosis also; occurs late and it is rare [51– 53, 312] (Table  6.15). CNI inhibitors and total body

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Fig.  6.98  Subacute GVHD after stem cell transplantation for CLL (a). Diffuse ATN; glomeruli appear unremarkable (H + E × 200) (b). CD3+ infiltrating lymphocytes (IP ×400) (c).

Massive subendothelial edema (EM × 4,000) (d). GBM irregularity and wrinkling suggestive of GBM damage/repair (EM ×2,500)

irradiation are risk factors for endothelial cell damage but GVHD is likely multifactorial and includes drugs, high-dose chemotherapy, and angioinvasive fungal or viral infections. These factors, directly or indirectly release IL-1, TNF-a, IFN-g leading to microvascular endothelial cell damage and apoptosis, leukocyte adhesion, activation of platelets, coagulation factors, and thrombi formation [181]. A role for antigen presenting cells interacting with recipient’s thymus derived T cells is also thought to precipitate in GVHD pathogenesis via production of autoreactive T cells and impaired negative selection [60]. In the biopsy shown in Fig. 6.98, CD3+ infiltrating lymphocytes were identified, perhaps participating in tubular and glomerular damage. Treatment is not well established but prednisone, MMF, and rituximad were tried (350).

6.5.2 Cholemic Nephrosis Patients with obstructive jaundice are at risk for renal failure particularly after transplantation. The diagnosis is made on light microscopy when typical bilirubin casts are present. Diagnosis can be missed when there are no histopathological abnormalities of tubular necrosis and bilirubin casts are sparse. Bilirubin stains confirm the diagnosis (Fig. 6.99). The term cholemic nephrosis was coined for this complication, which is now attributed to altered hemodynamics and bile toxicity to tubular epithelial cells. Jaundice-related nephropathy is currently the preferred term [34]. Patients usually have hepatorenal syndrome, reduced glomerular filtration rate and increased concentration of bile salts, and bilirubin in urine.

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Fig. 6.99  Cholemic nephrosis (a). Tubular casts stain red–brown on H–E, green with bile stain (b)

6.6 Molecular Correlates of Renal Allograft Pathology Michael Mengel, MD University of Alberta Department of Pathology and Laboratory Medicine

6.6.1 Background Histopathology is an empirical method. Sections from diseased tissue are assessed and lesions are identified, which are absent in normal tissue, i.e., which are pathological. The underlying biology causing the transition from normal to pathological is not necessarily taken into account to consider a lesion as diagnostically useful. Further, not all lesions observed in a certain disease state are specific/pathognomonic for this entity. Thus quantitative thresholds are necessary, separating variations in the biological spectrum of “normal” from disease states, which need therapeutic intervention. Defining the diagnostic lesions and thresholds is usually accomplished by generating consensus among “specialists.” But, consensus does not necessarily mean correctness. Since 1991, the BANFF consensus process, led by the experience of a small group of specialists, has defined the criteria for diagnosis of renal allograft pathology [226, 319]. It was agreed that interstitial

infiltrates with tubulitis and endotheliitis are the lesions observed in allograft biopsies from transplanted kidneys with dysfunction that respond to therapy. Already, it was known that these lesions are not pathognomonic for rejection because they can be observed in nonransplanted kidneys and disease processes other than rejection [226, 319]. Hence, arbitrary minimum thresholds were established by consensus to prevent over-diagnosing and over-treating rejection. The clinical relevance and value of the BANFF consensus has been repeatedly shown by correlating the lesions and diagnostic classes with response to treatment and outcome [236, 361]. Such iterative reassessment between pathology and clinic enabled continuous refinement of the classification system [226, 274, 275, 320, 321]. But neither the lesions nor the thresholds were biologically/mechanistically validated, because there was no independent external standard for validation. With the advent of molecular high-throughput technologies, we now have an independent external measurement of disease states in the tissue on a sub-microscopic level. Assessing the transcriptome by cDNA-microarrays is similar to a low-power view through the microscope, i.e., it allows for pattern-recognition between physiological and pathological processes. Furthermore, the richness in transcriptome data has the potential to provide mechanistic insights and thus, definite diagnostic categorization – the indispensable prerequisite for development of adequate therapy. The following paragraphs are aimed to summarize current state of the art in the field of molecular renal

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allograft pathology. In keeping with this book, the primary focus is to give an overview of how molecular measurements can enhance diagnostics as a complementary tool to histopathology, similar to the addition of other techniques such as immunohistochemistry (e.g., C4d staining). Therefore, the included data are restricted to those from studies, which were conducted on tissue specimens and in close correlation to histopathology.

6.6.2 Molecular Correlates of Tissue Injury in Renal Allografts Tissue injury is an inevitable feature to every renal transplant beginning with the surgical procedure of organ harvest. Furthermore, numerous immunological and nonimmunological insults occur during the posttransplant course to injure the kidney: parenchyma eventually resulting in irreversible atrophy of functional units (glomeruli, capillaries, tubules) and loss of allograft function. However, in general, histopathology is not very good at assessing and quantifying tissue injury. Describing necrosis would just capture the worst stage of injury. And there is no BANFF consensus for assessing and grading tubular epithelial or endothelial injury before these cells become obviously necrotic. It can also be questioned whether light microscopy is capable of reliably detecting specific sub-lethal cell changes. Nevertheless, some studies done on zero-hour biopsies and sequential protocol biopsies have suggested that signs of acute tissue injury (i.e., ATN/injury), by morphology, are of clinical relevance in terms of immediate, short-term, and/or long-term allograft function [133, 281]. However, protein and gene expression data are expected to make significant contributions in terms of improved assessment of tissue injury. Herewith, the major questions are: Is injury present?; what is the cause of injury?; what is the degree of injury?; and will the tissue recover? The most obvious approach towards addressing these questions is to analyze zero-time biopsies taken at transplantation where pathology is usually restricted to tissue injury and no other confounding disease processes (e.g., rejection, infection) are operating. Several groups have applied cDNA microarrays or rtPCR to zero-time biopsies and correlated the transcriptional changes with future allograft function with the

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aim to identify transcriptional biomarkers being predictive for DGF and thus impaired long-term function [20, 147, 155, 207, 238]. All studies were able to discriminate between living and deceased donor organs. That sounds trivial but indicates that differences, in terms of the extent of tissue injury (less in living donation and more in deceased donation), can be assessed on a transcriptional level. Furthermore, all studies were able to identify lists of several dozen transcripts discriminating between those grafts with immediate and delayed functions. Most transcripts on these lists are annotated to proteins being part of the immune response in particular, complement components in addition to cell-cycle and signaling molecules. However, in none of the studies was it possible to reveal distinct histological features corresponding to the molecular phenotypes of injury. In general, the significant changes described in the transcriptome are quantitative and rarely qualitative, i.e., the allografts with delayed function have increased or decreased expression of these transcripts that are also expressed in the functioning allografts but to a different extent. In one study, it was demonstrated that the transcriptional changes represent a continuum from living donor organs via those deceased organs with immediate function to those with delayed function [238]. These findings indicate that analysis of large scale transcriptome data can provide relatively small sets of transcripts, which can be used as a measurement for the degree of tissue injury. The Edmonton group of Philip Halloran developed the system of pathogenesis-based transcript sets (PBTs) in kidney transplantation. Applying a priori biological knowledge to well-defined experimental models, the group generated sets of transcripts, which reflect major biological processes and pathological disease states in renal allograft tissue: interstitial inflammation by cytotoxic T cell-associated transcripts (QCATs) [95], macrophage-associated transcripts [103], B cell-associated transcripts [93, 96, 97] immunoglobulin transcripts [97], as well as transcript sets being interferon-g inducible [102]. Additionally, by filtering for the transcripts associated with infiltrating cells, PBTs were generated comprising transcripts restricted to the kidney parenchyma with decreased expression during rejection [92, 94], and those with increased expression in injury and repair [97]. Probe sets for each published PBT are available at http:// transplants.med.ualberta.ca/. The PBT score represents the geometric mean of fold changes across all probe

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sets within each PBT. By this approach, large scale and cumbersome microarray gene expression results can be collapsed into single PBT scores representing a measurement of the respective biological/pathological process. Vice versa, the PBT annotation of a probe set acts as a rapid way of understanding the biological process represented by changes in that probe set. In terms of tissue injury, the Edmonton group defined a set of 790 transcripts, which show increased expression in the kidney parenchyma induced by injury and during consecutive repair processes as observed in time-course series done in mouse isografts [101]. More detailed analysis of these 790 transcripts revealed that they show a time-dependent heterogeneity with early (day 1 after transplantation), intermediate (day 2–5), and late (>day 5) expressed subsets. These subsets could be associated with different types of injuries: (1) a more systemic/general injury response on posttransplant day 1, which is similar to the isograft and the contralateral native kidney, (2) a more transplant stressrelated response observed in iso- and allografts, (3) and an allo-induced injury response, which is sustained and more severe in allografts and evolves here into fibrosis and atrophy but resolves in isografts. The injury and repair induced PBTs comprise virtually all transcripts, which are extensively described by single transcript analysis approaches (i.e., PCR-based studies) in ischemia-reperfusion models. One of the best described injury biomarkers is KIM-1 [37]. But, remarkably, within a particular injury PBT, virtually all transcripts behave in a stereotyped fashion indicating that each of them has the potential to be a suitable diagnostic biomarker for this disease process. Surprisingly, numerous transcripts generally associated with fibrosis and scarring can be found in the injury PBT indicating that fibrogenesis is a necessary part of the repair process. Collagens and TGF-b-dependent transcripts are all transiently expressed in injured isografts, but without any morphological correlate of scarring; isografts always show a “restitution ad integrum.” Furthermore, although more than 700 biologically meaningful transcripts are differentially expressed in the isografts, histology was normal. This highlights the limitations of histopathology to assess parenchymal injury in kidneys that have undergone serious stress, i.e., transplantation with ischemia and reperfusion. Assessment of tissue injury and its potential of recovery become even more challenging when rejection is present simultaneously. Counting lymphocytes

per tubular cross-section is probably a measurement of limited feasibility for tubular epithelial injury during rejection, at least it is poorly reproducible [112]. The Edmonton group was able to define PBTs with parenchymal transcripts that show mostly decreased expression during rejection [92, 94]. A large group of these also demonstrates stereotyped behavior including the solute carrier transcripts that are usually expressed in the various nephron segments. It makes sense and corresponds to clinical features (e.g., rise in creatinine) and morphological signs (e.g., loss of brush border) that tubular epithelial cells cease their transport functions as a reaction to injury. Microarray and pathology results from a large series of human allograft biopsies for cause show that the degree of aberrant expression of these PBTs correlates with the severity of histopathological lesions of allograft pathology and allograft function [45, 237] (Fig. 6.100). Thus, sets of transcripts with restricted expression in kidney parenchyma can serve as a robust and sensitive measurement of tissue injury. This represents a diagnostic asset to histopathology since morphological signs of parenchymal injury and their potential for recovery cannot, as yet, reliably be assessed by light microscopy. However, using molecular expression values as a standard can lead to the establishment of corresponding immunohistochemical markers and morphological features of tissue injury.

6.6.3 Molecular Correlates of Renal Allograft Rejection Histopathology is the current diagnostic gold standard for diagnosing allograft rejection. Two types of allograft rejection can be discriminated by histopathology: TCMR and antibody-mediated rejection (ABMR). Empirically, interstitial infiltrates, tubulitis, and endothelialitis are the morphological hallmarks of TCMR [68], while ABMR is characterized by antibody-mediated lesions to the microcirculation, i.e., capillaritis, glomerulitis, glomerulopathy, and C4d deposition [66]. However, the described rejection lesions are not pathognomic and can be found in disease states other than rejection (e.g., interstitial nephritis, glomerulonephritis) and overlap of cellular and antibody-mediated mechanisms in generating the lesions is also possible. Therefore, at BANFF, consensus was generated that combinations of lesions

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exceeding arbitrary minimum thresholds allow for the diagnosis of rejection [272, 275]. Thus, there is no specific histological entity of “rejection,” but rather it represents experts’ consensus. Robust transcriptome measurements of the respective pathological processes/lesions can be used as an independent standard to validate and reassess biological specificity and diagnostic sensitivity of lesions and thresholds. Transcripts representing the burden of interstitial T cell, macrophage, B cell, and plasma cell infiltration, of systemic interferon-g effects on the tissue, of parenchymal injury (see above), and endothelial stress can provide a molecular phenotype against which corresponding histopathology can be re-evaluated. In iterative approaches between histopathology

and transcriptome measurement, a complementary diagnostic system can be developed. Analyzing, which transcripts correlated with the histological lesions of TCMR revealed large numbers of stereotyped T cell, macrophage-associated, and interferon-g dependent transcripts [236]. Using a refined set of just 25 QCATs allowed robust quantification of the T cell burden (i.e., the extent of interstitial infiltrate = the BANFF i-score) in human allograft biopsies [150] (Fig. 6.101). The QCATs were derived from T cell cultures and include cytotoxic molecules (granulysin, granzymes A, B, and K, perforin (1), signaling molecules (CD3D, CD8A, LCK, ITK, and STAT4), the NK receptor NKG2D/KLRK1 as well as the effector cytokine interferon-g. The association of

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Fig. 6.100  Relationship between PBT scores, histopathologic lesions and clinical diagnosis. Biopsies for cause are sorted based on the cytotoxic T cell associated transcript (CAT1) score (from lowest to highest). According to this order, scores for all PBTs (CAT1, CAT2 (=cytotoxic T cell associated transcripts), GRIT1, GRIT2 (interferon-g inducible transcripts), KT1, KT2 (=kidney parenchymal transcripts with decreased expression during rejection and injury) are illustrated for each individual

biopsy for cause (n = 143). The panel above the graph illustrates the relationship of the PBT scores to the presence of ATN, the degree of interstitial infiltrate (i score), tubulitis (t score), intimal arteritis (v score), histopathology diagnosis, retrospective clinical-pathologic diagnosis, and the probability of rejection (%), predicted from a classifiers. (Republished with permission from Wiley International from reference 40: Mueller et al. [238])

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a CD2 CD3D TRA@ LCK IL2RB GPR171 CD8A KLRK1 ITK CST7 PRF1 NKG7 GZMA GZMB STAT4 CXCR6 GZMK GNLY IFNG NELL2 TRGV9 KIAA0101 RRM2 NUSAP1 HOP

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Fig.  6.101  Principal component analysis of quantitative cytotoxic T cell associated transcripts (QCATs) in biopsy samples. Individual biopsies and CD8+ cytotoxic T lymphocytes were analyzed using a set of QCATs n = 25. (a) Clustering was based on distance. Branch lengths represent the degree of similarity between individual samples or transcripts. Black, green and red tiles indicate no change, decrease in expression and increase in expression, respectively. Colors for top branch lines and categories at bottom of heatmap correspond to the groups of the sam-

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ples: Turquoise, blue, orange, yellow, and red color indicates nephrectomy, ATN, TCMR, treated TCMR, and CD8+ CTL samples, respectively. (b) Principal component analysis of nephrectomies, biopsies with ATN, treated TCMR or TCMR, and CTL. (c) Principal component analysis of individual QCATs based on their expression in nephrectomies, biopsies with ATN, treated TCMR or TCMR, and CTL. Circles highlight the main subgroups formed by this analysis. (Republished with permission from Wiley International from reference 28: Hildago [151])

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expression of most of these individual genes with the histopathological diagnosis of rejection has been shown by many groups [13, 190, 195, 199, 293, 331, 332, 337]. Therefore, there is no TCMR-specific ­transcript. Large “herds” of transcripts behave in a stereotyped fashion when histopathological signs of rejection are found in a biopsy. However, using a gene set score rather than individual markers such as granzyme B may be an advantage in that random variation between biopsies is reduced by averaging over the gene set [11]. Assessing the molecular burden of interstitial infiltrates in allograft biopsies showed that what pathologists call rejection per consensus (i.e., infiltrate above a certain quantitative threshold) also has a continuous molecular phenotype [237]. Comparing protocol biopsies without infiltrate with those with subclinical rejection and those with clinical overt rejection revealed just quantitative but no qualitative differences between these biopsies in terms of expression of numerous infiltrateassociated transcripts [154]. Currently, there is not a single transcript identified, which is exclusively expressed during rejection. Active BK nephritis has qualitatively an identical transcriptional phenotype as TCMR, and significant differences are only of quantitative nature for a few transcripts [204, 237]. Today, neither pathology nor the transcriptome can discriminate whether a T cell enters the allograft due to rejection, infection, or nephritis. But the likelihood that the transcriptome will hold the capability to do so is greater than for light microscopy. Now that the transcriptome from a large number of renal allograft biopsies is available [107, 237, 256, 299, 323], further more detailed exploration of the transcriptome-wide data will reveal more specific expression patterns of subsets of transcript sets. For example, a small set of transcripts associated with endothelial activation (including PECAM (CD31), factor VIII, selectin E, CD34) correlates with the pathology of microcirculatory injury, i.e., capillaritis, glomerulitis, C4d, and antibody presence [317]. And it is conceivable that low expression scores for transcripts associated with alternative macrophage activation and those induced by interferon-g together with increased expression of the endothelial activationassociated transcripts indicate a high probability for ABMR, despite the fact that some of these cases lack C4d. Vice versa, cases with high T cell-associated transcript burden and signs of alternative macrophage activation but no endothelial activation strongly suggest

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TCMR, even if advanced tubular atrophy makes it impossible to diagnose sufficient BANFF t-scores. Furthermore, virus-specific sequences can be easily added to any transcriptome array and thus further increase the differential diagnostic power of the technique. Similar to current histopathology, combining single lesions/PBT scores will generate the final diagnosis, whereas the transcriptome measurements have the advantage to be technically robust and more objective. Furthermore, in animal models, it has been shown that changes in the transcriptome precede corresponding histopathological lesions [94–96].

6.6.4 Molecular Correlates of Interstitial Fibrosis and Tubular Atrophy (IFTA) of Renal Allografts IFTA is the morphological correlate of functional deterioration of renal allografts and represent the common final pathway of multiple simultaneously or sequentially operating mechanisms of tissue injury. However, in the majority of IFTA cases, one or several underlying causes for the chronic changes can be identified by histopathology [321]. Thus, molecular footprints of various disease processes should be detectable in cases with progressive injury eventually evolving to IFTA. But scar (fibrosis and atrophy) will not be transcriptionally very active. Therefore, the aim is to identify molecular correlates indicating ongoing epithelial deterioration and fibrogenesis, the harbingers the active components of IFTA, and extracellular matrix turnover [222]. The problem is that, it has been shown that virtually all generic “fibrogenesis” transcripts (e.g., collagens, CTGF, TGF-b dependent transcripts) are actually more involved in repair processes and thus seem to be essential to reconstitute the tissue after acute injury [101]. Therefore, transcripts associated with epithelial deterioration have the potential to be a molecular correlate indicating the risk for progression of IFTA. Even having annotated those transcripts, which are associated with dedifferentiation and thus deterioration of tubular epithelial cells [92–94], it is still challenging to identify the point of no return at which a nephron cannot be saved and proceeds to shut down and become atrophic. In this regard, histopathology, even looking at sequential protocol biopsies, has not provided clues. Looking at the currently available

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transcriptome data suggests some candidate molecules, which can serve as measurements for the extent of tubular epithelial injury and deterioration, and thus loss of specific epithelial function. For example, reexpression of embryonic genes and fruit fly pathway genes might be an indicator of dedifferentiation into a more primitive cell type [92, 101]. Up-regulation of tubular epithelial-specific integrins might indicate the starting point of epithelial-to-mesenchymal transition and herewith loss of nephron-specific function, and eventual replacement of a highly specific tubular epithelial cell by a primitive surface epithelium lining an atrophic nephron [101, 139, 356]. But, also, simply measuring the extent of epithelial injury by a single molecule like Kim-1 [5] or a set of injury-related epithelial transcripts [92, 94, 101] and correlating the results to follow-up data, might be sufficient to provide an estimate about the probability whether a kidney will recover or deteriorate. However, it has become more obvious that the probability of recovery, in general, depends on the balance between the extent of injury and capacity to repair. Thus, simultaneous molecular assessment of extent of injury and extent of IFTA (the equivalent of lost capacity to repair) would provide a reasonable assessment of the current functional status of a kidney. The molecular correlates of the extent of IFTA have recently been described by the Edmonton group [225]. The analysis revealed that the molecular burden of mast cell-, plasma cell-, and B cell-associated transcripts represents a robust correlate of the histological extent of IFTA. These results raise the question whether these cell types play a role in the pathogenesis and progression of IFTA. Accumulation of mast cells in IFTA was described 30 years ago [68], but their role has still not been elucidated. Confirming this, histopathological observation, by genome-wide analysis, takes it to the next level: the extent of a histological feature showing robust correlations with four mast cell-associated transcripts out of 45,000 analyzed, warrants further mechanistic consideration of this cell type [275]. Furthermore, the molecular link between inflammation and onset and progression of fibrosis is well described [255, 371]. However, identifying the underlying disease entity causing IFTA should still be the major aim in renal allograft pathology. Revealing the all-inclusive IFTA pathway being amenable to one therapy is not only very appealing but also very challenging, even with “omics” approaches.

6.6.5 Future Perspectives in Molecular Transplantation Pathology From a methodological point of view, transcriptomics is ready for prime time for clinical application. The various platforms (microarrays, low-density RT-PCR) are technically robust, and provide comparable results. They also have reasonable turn-around-times (24– 48 h) and cost per test (500–1000$), both of which will significantly decrease with increase in application [11, 76, 256, 270, 364]. Commercially available systems are highly standardized by using internal controls and normalization processes and thus show very robust inter- and intra-laboratory reproducibility [27, 283], which is definitely greater than for the assessment of some histopathological lesions [112]. The challenge is to establish valid diagnostic consensus. Since numerous transcripts have the potential to provide similar diagnostic information, consensus is necessary to make results comparable in multicenter approaches [42]. The BANFF-process should be the platform for consensus generation in molecular transplant pathology as it was for histopathology [226]. Suitable transcripts and sets of transcripts have to be identified and cross validated for their diagnostic robustness in multicentre approaches to become part of a revised BANFF classification. Inclusion of gene expression scores into BANFF will entail acceptance by regulatory agencies [110, 260]. The validation process of potential molecular markers for diagnostics has to be done by iterative approaches among expression analysis – pathology – laboratory medicine – and clinic. Currently, the diagnostic “truth” is unknown. Histopathology has, in some cases, an error rate of up to 50%. For example, potentially 50% of all true ABMR cases are C4d negative. They have antibody in the serum, ABMR pathology in the biopsy, and molecular signs of endothelial activation but do not fit the current diagnostic criteria for ABMR by lack of C4d [316, 317]. But these cases behave clinically like those with C4d positivity. Thus, the only possibility to improve diagnostic accuracy, overall, is to include all levels of information, i.e., pathology, gene expression, laboratory data, and clinic. Future diagnosis in transplantation will be more of a systems pathology approach including the different levels of information as is already partly done for the diagnosis of ABMR, which is per BANFF consensus based on lesions, C4d positivity, and detection of antibody. Adding

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transcriptome findings is the next logical step for increasing diagnostic accuracy, specificity, and sensitivity. Following the PBT approach where stereotypically behaving transcripts are collapsed into a single score, represents a feasible approach towards a complementary diagnostic tool in daily practice.

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167 nosis after kidney transplantation. Pediatr. Nephrol. 20, 652–656 (2005) 323. Stegall, M., Park, W., Kim, D., Kremers, W.: Gene expression during acute allograft rejection: novel statistical analysis of microarray data. Am. J. Transplant. 2, 913–925 (2002) 324. Steiner, R.W., Spital, A.: Evaluating living kidney donor candidates with minor medical abnormalities. Kidney Int. 72, 378 (2007); author reply 378 325. Stirling, C.M., Isles, C.G.: Rhabdomyolysis due to simvastatin in a transplant patient: are some statins safer than others? Nephrol. Dial. Transplant. 16, 873–874 (2001) 326. Stock, P.G., Roland, M.E., Carlson, L., Freise, C.E., Roberts, J.P., Hirose, R., Terrault, N.A., Frassetto, L.A., Palefsky, J.M., Tomlanovich, S.J., Ascher, N.L.: Kidney and liver transplantation in human immunodeficiency virus-infected patients: a pilot safety and efficacy study. Transplantation 76, 370–375 (2003) 327. Stone, J.H., Amend, W.J., Criswell, L.A.: Antiphospholipid antibody syndrome in renal transplantation: occurrence of clinical events in 96 consecutive patients with systemic lupus erythematosus. Am. J. Kidney Dis. 34, 1040–1047 (1999) 328. Stone, J.H., Millward, C.L., Olson, J.L., Amend, W.J., Criswell, L.A.: Frequency of recurrent lupus nephritis among ninety-seven renal transplant patients during the cyclosporine era. Arthritis Rheum. 41, 678–686 (1998) 329. Stratta, P., Canavese, C., Quaglia, M., Balzola, F., Bobbio, M., Busca, A., Franchello, A., Libertucci, D., Mazzucco, G.: Posttransplantation chronic renal damage in nonrenal transplant recipients. Kidney Int. 68, 1453–1463 (2005) 330. Stratta, P., Morellini, V., Musetti, C., Turello, E., Palmieri, D., Lazzarich, E., Cena, T., Magnani, C.: Malignancy after kidney transplantation: results of 400 patients from a single center. Clin. Transplant. 22, 424–427 (2008) 331. Strehlau, J., Pavlakis, M., Lipman, M., Shapiro, M., Vasconcellos, L., Harmon, W., Strom, T.B.: Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation. Proc. Natl. Acad. Sci. U S A 94, 695–700 (1997) 332. Strom, T.B.: Rejection–more than the eye can see. N. Engl. J. Med. 353, 2394–2396 (2005) 333. Sung, R.S., Christensen, L.L., Leichtman, A.B., Greenstein, S.M., Distant, D.A., Wynn, J.J., Stegall, M.D., Delmonico, F.L., Port, F.K.: Determinants of discard of expanded criteria donor kidneys: impact of biopsy and machine perfusion. Am. J. Transplant. 8, 783–792 (2008) 334. Sung, R.S., Galloway, J., Tuttle-Newhall, J.E., Mone, T., Laeng, R., Freise, C.E., Rao, P.S.: Organ donation and utilization in the United States, 1997-2006. Am. J. Transplant. 8, 922–934 (2008) 335. Sung, R.S., Guidinger, M.K., Lake, C.D., McBride, M.A., Greenstein, S.M., Delmonico, F.L., Port, F.K., Merion, R.M., Leichtman, A.B.: Impact of the expanded criteria donor allocation system on the use of expanded criteria donor kidneys. Transplantation 79, 1257–1261 (2005) 336. Sung, R.S., Guidinger, M.K., Leichtman, A.B., Lake, C., Metzger, R.A., Port, F.K., Merion, R.M.: Impact of the expanded criteria donor allocation system on candidates for and recipients of expanded criteria donor kidneys. Transplantation 84, 1138–1144 (2007)

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7

Lung Anja C. Roden and Henry D. Tazelaar

7.1 Introduction 7.1.1 Historic Perspective Experiments with animals in the 1940 and 1950s demonstrated that lung transplantation was technically possible [33]. In 1963, Dr. James Hardy performed the first human lung transplantation. The recipient survived 18 days, ultimately succumbing to renal failure and malnutrition [58]. From 1963 through 1978, multiple attempts at lung transplantation failed because of rejection and complications at the bronchial anastomosis. In the 1980s, improvements in immunosuppression, especially the introduction of cyclosporin A, and enhanced surgical techniques led to renewed interest in organ transplantation. In 1981, a 45-year-old-woman received the first successful heart–lung transplantation for idiopathic pulmonary arterial hypertension (IPAH) [106]. She survived 5 years after the procedure. Two years later the first successful single lung transplantation for idiopathic pulmonary fibrosis (IPF) [128] was reported, and in 1986 the first double lung transplantation for emphysema [25] was performed. Over the following years, the number of lung transplants rapidly increased, and the operation became an accepted treatment for an end-stage lung disease.

A.C. Roden (*) Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected] H.D. Tazelaar Department of Laboratory Medicine and Pathology, Mayo Clinic, Scottsdale, AZ, USA

Today, there are four major surgical approaches to lung transplantation: single and bilateral lung transplantation (BLT), heart–lung transplantation, and transplantation of lobes of lungs from living donors. In 2007, 2,708 lung transplantation procedures were reported worldwide to the Registry of the International Society for Heart and Lung Transplantation (ISHLT) in adults, the highest number for any year until then [21]. In the same year, 93 lung transplantations were reported in children, the majority in adolescents (12–17 years old) [6]. Although the number of single lung transplantations has been relatively stable, BLTs have continuously increased within the past 15 years. In fact, in 2007, BLT was the most common lung transplantation procedure performed with 69% of all lung transplantation procedures, largely due to transplantation for cystic fibrosis and chronic obstructive lung disease/ emphysema which made up for 26.6 and 25.7% of all BLTs between 1995 and 2008 [21]. The mean age of transplant recipients has consistently increased since 1989 rising to an all time high of 50.8 years in 2008 [21].

7.1.2 Native Disease in Explanted Lungs The most common indications for lung transplantation  in adults are chronic obstructive pulmonary disease (COPD)/emphysema, IPF, cystic fibrosis and alpha-1 antitrypsin deficiency emphysema (AAT) (see Table 7.1) [21]. Indications for pediatric lung transplantation vary by age (see Table  7.1). In children over 5 years old, cystic fibrosis is the most common indication [6], followed by IPAH. In contrast, in infants and preschool children, lung transplantations are usually performed

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_7, © Springer-Verlag Berlin Heidelberg 2011

171

172

A.C. Roden and H.D. Tazelaar

Table 7.1  Distribution of diagnoses among adult and pediatric lung transplant recipients (January 1995–June 2008) [6, 21] Diagnosis Adult transplants (%) Pediatric transplants Age <5 years 6–17 years COPD/emphysema

35.8

3.9

0.8

IPF

20.8

6.6

3.5

Cystic fibrosis

15.9

3.9

65.4

AAT

7.1

IPAH

3.3

18.2

8.3

Sarcoidosis

2.6

Bronchiectasis

2.7

LAM

1.0

Congenital heart disease

0.7

16.0

1.4

OB

0.9

5.0

3.8

  OB

1.2

3.3

3.2

  Non-OB

0.9

2.8

2.4

Connective tissue disease

0.8

Interstitial pneumonitis

0.3

9.4

0.6

Cancer

0.1

Eisenmenger syndrome

3.3

1.1

Surfactant protein B deficiency

8.3

Bronchopulmonary dysplasia

2.2

0.6

17.1

7.6

1.3

Retransplant

Other

6.0

IPF idiopathic pulmonary fibrosis; AAT alpha1-antitrypsin deficiency; IPAH idiopathic pulmonary arterial hypertension; LAM lymph­ angioleiomyomatosis; OB obliterative bronchiolitis

for IPAH, congenital heart disease, idiopathic interstitial pneumonitis, and surfactant protein deficiency. Well-selected patients with systemic diseases such as sarcoidosis, lymphangioleiomyomatosis, and pulmonary Langerhans’ cell histiocytosis have also had satisfactory results after lung transplantation [27, 71, 91, 99, 119] as have selected patients with scleroderma [84, 110, 113]. Multiple cases of incidental T1N0M0 or even Stage IIIA non-small cell carcinoma in the excised native lungs of transplant recipients have been reported [14, 30, 124]. Although one patient with Stage IIIA poorly differentiated squamous cell carcinoma died 6 months after transplantation of a neoplastic thromboembolus, patients with T1N0M0 carcinoma are ­generally free of recurrence.

7.1.3 Allograft Selection and Procurement Currently, only patients with near end-stage lung disease and a limited life expectancy should be considered for lung transplantation [95]. However, since lung transplantation is a rapidly evolving field, there are no hard and fast rules about who may be transplanted. When choosing a transplantation procedure, several issues are considered including the shortage of organ donors, the original disease, and the center’s experience with graft and patient survival. General guidelines for the selection of the procedure have been proposed [36] and are based on the nature of the underlying lung disease. While BLTs are mandatory for cystic fibrosis

173

7  Lung

[35], this procedure has also become more popular for indications such as AAT, COPD, IPF, and IPAH. Singlelung transplantation is usually performed in patients with restrictive fibrotic lung disease, Eisenmenger syndrome with reparable cardiac anomaly, and older patients with COPD. Heart–lung transplantation is considered in patients with Eisenmenger syndrome with irreparable cardiac defect, pulmonary hypertension with cor pulmonale, or end-stage lung disease with concurrent severe cardiac disease [83, 89]. Transplantation of lobes from living donors is a recently developed technique involving bilateral implantation of the lower lobes usually from two blood group–compatible living donors. The procedure has been performed in patients with cystic fibrosis, although the indications have been recently broadened. The functional and survival outcomes are similar to those achieved with conventional transplantation of cadaveric lungs. Donation of a lobe decreases the donor’s lung volume by an average of approximately 15%, which is not associated with long-term functional limitation. Other factors of the recipient that must be taken into consideration on an individual basis include ventilator dependence, previous cardiothoracic surgery, and preexisting medical conditions (e.g., hypertension, diabetes mellitus, osteoporosis) since posttransplantation medical regimen can worsen these illnesses. Severe coronary artery disease is a contraindication to lung transplantation. However, coronary artery bypass grafting at the same time as lung transplantation has been performed with a reasonably good outcome in some centers, although less invasive preoperative interventions, such as percutaneous transluminal coronary angioplasty and stenting, are preferred. Although the donor selection criteria may vary amongst centers, generally acceptable donor criteria include age of donor <65 years for lung transplantation and <45 years for heart–lung transplantation. In 2008, the average donor age was 35.5 years [21]. Other donor criteria include the absence of severe chest trauma or infection, no prolonged cardiac arrest (heart–lung transplantation only), minimal pulmonary secretions, negative screens for HIV, hepatitis C, and hepatitis B and blood type (ABO) compatibility. A close match of lung size between donor and recipient, PaO2 >  300 mmHg on 100% fraction of inspired oxygen (FiO2), clear chest radiograph and no history of malignant

neoplasms are also required. Most transplant centers will use lungs from a cytomegalovirus (CMV)-positive donor for transplantation into a CMV-negative donor given an adequate postoperative CMV prophylaxis. With the current techniques, satisfactory graft function can be obtained after an ischemic interval of as long as 6–8  h. For pulmonary preservation, systemic heparinization of the donor and hypothermic flush perfusion of the allograft are most commonly used in clinical practice. Most flush solutions are administered at a temperature of 4°C, while topical cooling is carried out by filling the pleural cavity with iced crystalloid solution. The harvested lungs are then immersed in crystalloid solution, packed in ice, and transported at a temperature of 1–4°C. The infusion and transport is performed during active ventilation and static inflation with O2, respectively.

7.2 Allograft Rejection 7.2.1 Overview Acute and chronic alloreactive injury to the donor lung affects both the vasculature and the airways [123]. Usually, rejection is evaluated on transbronchial biopsies (see below Sect.  7.3). On only rare occasions, wedge biopsies are performed. Other specimens might include explants for retransplant or autopsy specimens. Acute rejection is characterized by perivascular mononuclear cell infiltrates, which may be accompanied by sub-endothelial chronic inflammation (e.g., endotheliitis or intimitis), and also by lymphocytic bronchiolitis. In contrast, chronic rejection is manifest by fibrous scarring, involving the bronchioles and sometimes associated with accelerated fibrointimal changes affecting pulmonary arteries and veins. The presence of presumed irreversible dense eosinophilic hyaline fibrosis in airways and vessels remains the key histologic discriminator between acute and chronic rejection of lung. The histologic changes are divided into grades based on intensity of the cellular infiltrate, and the presence and absence of fibrosis.

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7.2.2 Hyperacute Rejection Hyperacute rejection occurs within minutes to a few hours after the newly transplanted organ begins to be perfused. It is a type II hypersensitivity reaction, mediated by preexisting antibodies to ABO blood groups, human leukocyte antigens (HLA) class I, or other antigens on graft vascular endothelial cells. Preexisting antibodies can result from previous pregnancies, blood transfusions, or a previous transplant. Antibody binding provokes complement and cytokine activation leading to endothelial cell damage and platelet activation with subsequent vascular thrombosis and graft destruction. The outcome is usually fatal. In the lungs, hyperacute rejection grossly presents by edema and cyanosis of the graft. Histologically, platelet thrombi, neutrophilic infiltration, fibrin thrombi, necrosis of vessel wall, and morphologic features of diffuse alveolar damage (DAD) are observed [29]. Although hyperacute rejection is a well-known complication in kidney and heart transplantations, in lung transplantation it appears to be rather rare with only five cases reported. One patient reported presented with severe hypoxia, high fever, hemodynamic instability and developing acute renal failure 1 h after completion of the anastomoses [29]. Chest radiograph displayed a completely opacified left lung, with homogenous infiltrates. Bronchoscopy revealed abundant pink frothy fluid draining from the allograft. Mean pulmonary artery pressure increased to 29 mmHg. The patient died 24 h later. At autopsy, the vascular and bronchial anastomoses appeared patent without signs of injury. The transplanted lung showed red hepatization and a firm consistency. Microscopically, signs of acute lung injury were evident. Although a pretransplant panel-reactive antibody (PRA) was negative, flowcytometry revealed 56 and 45% reactivity against HLA class I and II, respectively with anti-A2 detected among the preformed antibodies. Three other reported patients with hyperacute rejection died within 4 h to 13 days after transplantation [11, 19, 43, 116]. Although in three of the five reported patients pretransplant PRAs were negative, crossmatch was positive in all cases with antiA2 the most common identified antibody. Collectively, although hyperacute rejection is rare after lung transplantation, one should keep this reaction in mind given that false-negative PRAs may

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occur and pretransplantation cross match is not often possible [29].

7.2.3 Acute and Chronic Rejection 7.2.3.1 Overview

Acute rejection is the host’s response to the recognition of the graft as foreign. Most patients develop at least one episode of acute rejection within the first 3 weeks following transplantation, typically in the first 5–10 days, with 36% of patients experiencing at least one episode in the first year [21]. Obliterative bronchiolitis (OB) is the most common late cause of mortality and morbidity after lung transplantation occurring in 28% by 2.5 years and 74% by 10 years in patients who survive at least 14 days [21]. It also has a significant negative impact on quality of life parameters. Risks for acute rejection include HLA mismatching, type of immunosuppression, infection, and recipient factors. It is generally thought that the intensity of host alloimmune response is related to recipient recognition of differences with the donor HLA antigens and that this process drives acute lung allograft rejection. A higher degree of mismatch increases the risk of acute rejection [101, 115, 141]. However, this effect is not consistent across all HLA loci or studies. Mismatches at the HLA-DR, HLA-B [115], and HLA-A [101] loci, as well as a combination of all three loci [141], appear important. In addition, the ISHLT registry has not found a correlation between HLA mismatching and survival [130]. Thus, while HLA mismatching between donor and recipient likely contributes to the immunologic basis for acute rejection, it is difficult to discern if a mismatch at a particular locus or if different degrees of mismatch significantly alter the overall risk for acute rejection. Viral infections have been thought to modulate the immune system and heighten alloreactivity. Indeed, a high incidence of acute rejection has been found in lung transplant recipients after community-acquired respiratory tract infections with human influenza virus, respiratory syncytial virus (RSV), rhinovirus, coronavirus, and parainfluenzavirus [44, 73, 137]. Although CMV is considered a potential risk factor for OB, studies directly linking CMV infections or CMV

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prophylaxis strategies with acute rejection have been inconsistent [118]. In one study, Chlamydia pneumoniae infection was linked to the development of acute rejection and OB [50]. Several host genetic characteristics have been suggested to modulate acute lung rejection. For instance a genotype leading to increased IL1- production may protect against acute rejection [147] and a multidrug-resistant genotype (MDR1 C3435T) appears to predispose to persistent acute rejection resistant to immunosuppressive treatment [148]. The effect of age on acute rejection appears to be bimodal, with the lowest incidence of acute rejection in infancy (
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acute rejection or infection. However, although early small studies attempted to demonstrate the usefulness of chest X-rays and chest CT scans in the diagnosis of rejection, more recent data show very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes [51]. Exhaled nitric oxide (NO) is also an attractive marker of lung injury; it has been correlated with lymphocytic bronchiolitis [31] and acute rejection [120]. Furthermore, in a study of inert gas single breath washout, the slope of alveolar plateau for helium had a sensitivity of 68% for acute rejection [133]. Although lung transplantation has come of age, the development of OB remains the biggest hurdle preventing long-term survival in many patients [136]. Because of its low sensitivity (28%) and specificity (75%), OB remains difficult to prove pathologically with transbronchial biopsy [24, 72]. As a consequence, a clinical definition, called bronchiolitis obliterans syndrome (BOS) was proposed [24]. This is based on pulmonary function criteria, initially FEV1 evolution, and more recently, mid-expiratory flow rate (FEF25–75) [38]. The onset of BOS-symptoms is usually insidious, with progressive exertional dyspnea, often accompanied by cough, which may be dry or productive. The incidence of BOS is highest after the first year following lung transplantation. However, the risk of BOS increases to 60–80% 5–10 years after the lung transplantation procedure. It remains the leading cause of morbidity and death after lung transplantation, accounting for about 19–29% late mortality and some 35% of patients affected by the condition 5 years after transplantation [21]. As discussed above, an acute immunologic event (acute rejection) may trigger the onset of OB [63]. However, nonalloimmune injury such as a respiratory tract infection (CMV or non-CMV viral infection) is increasingly recognized as also having an important impact on the development of OB [41, 46]. In fact, CMV pneumonitis affects over 20% of lung transplant recipients. Despite treatment, it increases the risk for OB and death. Early detection of OB in a preclinical stage is ideal so that aggressive attempts can be made to prevent a fully developed syndrome. However, to date, no particular marker to indicate OB, either from the peripheral blood or bronchoalveolar lavage (BAL) fluid, has predicted a risk for this disease. An association between chronic aspiration and OB after heart–lung transplantation has also been observed

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[105], and there is a high frequency of gastroesophageal reflux with resultant aspiration following lung transplantation [28, 57, 97, 105, 108, 143]. For instance, one study described gastrointestinal complications including gastroesophageal reflux in 51% of patients who underwent lung transplantation [80]. Other than leading to an increase in acute rejection by exacerbating the alloimmune response, aspiration of gastric contents might act independently of the alloimmune response to promote allograft injury and the development of OB. Experiments in rats have further substantiated the association between chronic aspiration and OB [79]. Despite the common clinical impression that lymphocytic pleural effusions are a hallmark of acute rejection, published data are inconclusive [65]. Collectively, although the presentation of the patient and several ancillary studies might suggest a transplant rejection process, tissue diagnosis is necessary for definitive diagnosis.

7.2.3.2 ISHLT Classification In 1990, the ISHLT sponsored the Lung Rejection Study Group (LRSG), a workshop to develop a “working formulation” for the diagnosis of lung rejection by transbronchial biopsy [9]. The proposed grading scheme found wide acceptance. Due to developments

in the field and experience with its use, LRSG has remet twice since to assess the merits of the grading scheme. The revisions were published in 1996 [145] and 2007 [123], respectively. The grading scheme is strictly pathologic and does not consider any clinical parameters. Due to overlapping histologic features between acute rejection and infection, the grading scheme relies on the absence of concurrent infection. The most recent classification of lung allograft biopsies is the 2007 ISHLT revised consensus classification of allograft rejection [123] (see Table 7.2). This classification was revised from the 1996 ISHLT consensus classification. There were no significant changes in the grading of acute rejection in the latest classification, but it was acknowledged that minimal acute rejection is not solely a high power diagnosis but can also be recognized at low power in an adequately alveolated biopsy. In the 1996 grading system, small airways rejection was graded in a four-tiered system, B1 through B4, respectively (see Table 7.2). The 2007 classification consolidated those into low grade and high grade small airways inflammation to make the evaluation easier. The “R” behind B1 and B2 denotes the revised 2007 classification. Chronic airways rejection is not divided into active and inactive anymore but only into present and not present. There are no differences in the classification of chronic vascular rejection between the 1996 and 2007 classification.

Table 7.2  Classification of allograft rejection according to 2007 revised and 1996 ISHLT consensus classifications of lung allograft rejection [123] Grade 2007 Grade 1996 Acute rejection

A0 A1 A2 A3 A4

None Minimal Mild Moderate Severe

A0 A1 A2 A3 A4

None Minimal Mild Moderate Severe

Small airways inflammation

B0 B1R

None Low grade

B2R

High grade

BX

Ungradeable

B0 B1 B2 B3 B4 BX

None Minimal Mild Moderate Severe Ungradeable

Chronic airways rejection

C0 C1

None Present

C0 Ca Cb

None Active Inactive

Chronic vascular rejection

D0 D1

None Present

D0 D1

None Present

R denotes revised

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An attempt should be made to accurately distinguish the grade of rejection since treatment is largely dependent on the histologic grade. Furthermore, an effort should be made to review the report of the previous biopsy and to comment on whether rejection is ongoing or resolving. Although in general, patients with grade A1 biopsies will not receive increased immunosuppression, surveillance might be intensified and the time to the next biopsy shortened. In contrast, A2 biopsy patients will receive an increase in immunosuppression. Grade A3 and A4 patients will be treated similar and receive more immunosuppression. Inter- and intraobserver variability in grading can impact treatment and outcome [16, 22]. Studies have evaluated the inter- and intraobserver variability of the 1996 grading scheme. Two studies found relatively good interobserver agreements for the A-grades (kappa of 0.65 and 0.73) [16, 22], but this could not be replicated in another study in which the kappa was 0.47 in spite of dichotomization of the A-grades to A0/A1 vs. A2–4 [122]. Intraobserver agreement for acute rejection has been found to be good: kappa values of 0.65 and 0.795 [16, 122]. Infection can complicate the diagnosis of acute rejection. Viral infection in particular can cause mononuclear inflammation [126]. In addition, alveolar damage with macrophage and fibrin accumulation was found to be present in 80% of transbronchial biopsies in the first 6 months after transplantation, further increasing interobserver pathologist discordance. Therefore, in general, the LRSG recommends grading rejection only after the exclusion of infection. In contrast to the A-grade, the interobserver variability for grading airways inflammation (B-grades) was only fair with kappas of app 0.3 [16, 122]. For this reason, the LRSG has now simplified B-grading to two possible grades. This nomenclature is to be used for grading of noncartilagenous small airways only after rigorous exclusion of infection [123]. Eosinophils can accompany acute lung rejection and are recognized in the 2007 ISHLT classification in high grade rejection (grades A3/A4) [123]. The presence of eosinophils has been suggested as a risk factor for BOS in small single-center studies [114]. Furthermore, high proportions of B cells have been shown to accompany steroid-resistant rejection in lung transplant patients [146]. The mechanisms by which B cells contribute to refractory rejection are not entirely clear, although they may reflect ongoing humoral rejection, perhaps

explaining the diminished responsiveness to standard immunosuppression. Mast cells have been identified in acute rejection biopsies of increasing A-grade but their role has not been elucidated [144]. Rejection and chronic airways inflammation should be distinguished from bronchiolar associated lymphoid tissue (BALT). BALT is found in the vicinity of airways, usually contains black anthracotic pigment and presents as a rather nodular collection of chronic inflammatory cells which does not surround a vessel.

7.2.3.3 2007 ISHLT Revised Consensus Classification of Lung Allograft Rejection Acute Rejection: A Grade Acute rejection is defined by the presence of perivascular mononuclear cell infiltrates with or without endotheliitis. With progression, this infiltrate becomes more widespread and extends into the alveolar septa and, subsequently, into the alveoli. The majority of the mononuclear cells in acute rejection are T cells, although a few studies have described increased populations of B cells or eosinophils [104, 123, 146]. No Acute Rejection (ISHLT Grade A0) In grade A0 normal pulmonary parenchyma is present.

Fig. 7.1  Minimal rejection (ISHLT Grade A1). A single venule is surrounded by a thin layer of chronic inflammatory cells (H&E, magnification × 400)

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Minimal Acute Rejection (ISHLT Grade A1) Scattered infrequent blood vessels, particularly venules, in the alveolated lung parenchyma are surrounded by a relatively thin chronic mononuclear infiltrate (see Fig. 7.1). The lymphocytic rim is rather small and does not spill into the adjacent interstitium. Endotheliitis and eosinophils are absent. Although previous classification systems commented on a certain number of lymphocyte layers, the current ISHLT classification only requires a complete rim of vessels by lymphocytes. In adequately alveolated and artifact-free specimens, the lymphocytic infiltrates might be detected at low magnification.

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may be associated with hyperplastic or regenerative changes in the endothelium, i.e., endotheliitis. Concurrent lymphocytic bronchiolitis may be seen in association with mild acute rejection. Moderate Acute Rejection (ISHLT Grade A3) Grade A3 acute rejection shows easily recognizable cuffs of venules and arterioles by dense perivascular mononuclear cell infiltrates which are commonly associated with endotheliitis (see Fig.  7.3). Eosino­phils

Mild Acute Rejection (ISHLT Grade A2) Although in mild acute rejection the perivascular infiltrate of lymphocytes is essentially confined to the perivascular adventitia without infiltrating the interstitium, there are more layers of lymphocytes surrounding the vessel and lymphocytes might focally, minimally spill into the adjacent interstitium (see Fig. 7.2). More frequent perivascular mononuclear infiltrates are seen surrounding venules and arterioles. They are readily recognizable at low magnification. These infiltrates usually consist of a mixture of small round lymphocytes, activated lymphocytes, plasmacytoid lymphocytes, macrophages, and eosinophils. There is frequently subendothelial infiltration by mononuclear cells which a

Fig.  7.2  Mild rejection (ISHLT Grade A2). (a) Low power view with readily apparent multiple small vessels surrounded by lymphocytes. The interstitium is normal and lacks chronic

Fig. 7.3  Moderate rejection (ISHLT Grade A3). Mononuclear cells surround several small vessels and infiltrate into the adjacent interstitium. Scattered eosinophils are also present (H&E, magnification × 200)

b

inflammation. (b) On high magnification, the perivascular infiltrate has a thicker cuff than in Grade 1A (H&E, magnification × 100 (a), 400(b))

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and even occasional neutrophils are common. This grade is defined by the extension of the inflammatory cell infiltrate into perivascular and peribronchiolar alveolar septa which broadens the interalveolar septa and airspaces are associated with collections of intraalveolar macrophages in the zones of septal infiltration. Type II pneumocyte hyperplasia and histologic features of acute lung injury may become apparent.

a

Severe Acute Rejection (ISHLT Grade A4) In severe rejection there are diffuse perivascular, interstitial, and air space infiltrates of mononuclear cells with prominent alveolar pneumocyte damage and endotheliitis (see Fig.  7.4). This may be associated with intra-alveolar necrotic epithelial cells, macrophages, eosinophils, hemorrhage, and neutrophils and usually some evidence of acute lung injury in form of organizing pneumonia or hyaline membranes. Parenchymal necrosis, infarction, or necrotizing vasculitis might be identified; however, these features are more evident on surgical rather than transbronchial lung biopsies. It should be noted that a paradoxical diminution of perivascular infiltrates can occur as cells extend into alveolar septa and spaces where they are admixed with macrophages. This grade can sometimes be difficult to distinguish from an infectious process, harvest/reperfusion injury, or drug toxicity, however, the presence of perivascular inflammation is helpful in establishing the diagnosis.

b

c

Acute Small Airways Rejection: B Grade This grade applies only to small airways such as terminal or respiratory bronchioles. Bronchi, if present, should be described separately. It is important to mention in the pathology report whether or not small airways are present. The R behind grades 1 and 2 denotes the revised 2007 version.

No Airways Inflammation (ISHLT Grade B0) The small airways appear unremarkable without evidence of bronchiolar inflammation.

Fig.  7.4  Severe rejection (ISHLT Grade A4). (a) Low power view reveals perivascular lymphocytic infiltrate (arrow), thickening of the interstitium due to chronic inflammatory cells, and intraalveolar fibrin indicative of acute lung injury. (b) On high magnification, scattered eosinophils are identified. (c) Small vessels with features of endotheliitis characterized by the presence of lymphocytes between endothelial cells and basement membrane are also present (H&E, magnification × 100 (a), 400(b, c))

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a

b

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High Grade Small Airways Inflammation (ISHLT Grade B2R) In high grade small airways inflammation there is marked lymphocytic infiltrate of the airway epithelium and airway wall. The mononuclear cells in the submucosa appear larger and activated with greater numbers of eosinophils and plasmacytoid cells. In addition, there is evidence of epithelial damage including necrosis, metaplasia, and marked intra-epithelial lymphocytic infiltration. In its most severe form, high grade airways inflammation is associated with epithelial ulceration, fibrino-purulent exudate, cellular debris, and neutrophils. It is important to exclude an infectious process. Ungradeable Small Airways Inflammation (ISHLT Grade BX) In this grade the changes are ungradeable due to sampling problems, infection, tangential cutting, artifact etc. Chronic Airways Rejection C-Grade

Fig.  7.5  Low grade small airways inflammation (ISHLT Grade B1R). (a) On low magnification a small airway is present with mild chronic inflammation of the airway wall. (b) Chronic inflammatory cells are in the submucosa and mucosa. Only rare neutrophils and lymphocytes are present (H&E, magnification × 100 (a), 400(b))

The working formulation for pulmonary rejection has equated OB with one type of chronic rejection, the C-grade. The term as used in the consensus classification is restricted to submucosal and intraluminal scarring of membranous and respiratory bronchioles. When large tissue sections of lung are examined, the process of OB is pan-lobar but patchy. No Chronic Airways Rejection (ISHLT Grade C0)

Low Grade Small Airways Inflammation (ISHLT Grade B1R) Low grade inflammation is characterized by lymphocytes within the submucosa of the bronchioles (see Fig. 7.5). The lymphocytic infiltrates can be infrequent and scattered or forming a circumferential band, however, intra-epithelial lymphocytic infiltration is not present. Although occasional eosinophils may be seen within the submucosa, there is no evidence of epithelial damage, neutrophils, necrosis, ulceration, or significant amount of nuclear debris. This grade combines and replaces the 1996 working formulation B1 and B2 grades.

The small airways appear similar in size to the accompanying artery with a ragged inner surface. Fibrosis is not present. Chronic Airways Rejection (ISHLT Grade C1) Narrowing of the airways due to fibrosis in the airway wall is seen. The fibrosis is usually eccentric (see Fig. 7.6). OB may be manifest by a histopathologic spectrum of changes depending on the acuteness of the process, the degree of organization and the amount of accompanying inflammation. In the acute phase there is usually loose myxoid granulation tissue with variable numbers of

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a

b

inflammatory cells filling or partially obstructing the airway lumen, resembling “organizing pneumonia.” In later phases, OB may consist of eccentric, occasionally confluent plaques of dense hyalinized collagen applied to the wall of bronchioles. Metaplastic squamous or cuboidal epithelium may cover these bronchiolar scars. In other airways a slit-like lumen may remain as a result of a confluent submucosal scar or intraluminal polyps of scar tissue. Capillaries supplying these intraluminal masses of collagen are occasionally prominent. In the most severe cases the bronchiolar lumen can be entirely occluded by dense scar tissue and be recognizable only with the aid of an elastic stain, its location adjacent to an artery, and by the presence of residual circumferential smooth muscle. In the later phases, inflammation may be minimal. Usually, the scarring process is confined exclusively to respiratory bronchioles and terminal bronchioles, although it may occasionally involve adjacent alveoli.

Chronic Vascular Rejection D-Grade No Chronic Vascular Rejection (ISHLT Grade D0)

c

The pulmonary arteries appear of a similar size as the accompanying airway. The intima is slender, the media not thickened.

Chronic Vascular Rejection (ISHLT Grade D1)

Fig. 7.6  Obliterative bronchiolitis (IHSLT C1). Autopsy slides from a patient who underwent bilateral lung transplantation 15 months prior. Her posttransplant course was complicated by PTLD, CMV and Pseudomonas aeruginosus pneumonia. She presented with cough and progressive dyspnea shortly before her demise. The H&E images, (a) and (b) show complete obliteration of the airway by fibrosis. A VVG stain (c) outlines the elastic layer indicative of the original airway diameter (H&E, magnification × 40 (a), 100 (b))

Chronic vascular rejection rarely is identified on biopsies since they usually lack vessels of sufficient size. Wedge biopsies, explants, or autopsy material may reveal it. In the typical case, pulmonary arteries and more often veins are thickened by fibrointimal connective tissue (see Fig. 7.7). Also, thickening is usually concentric. Chronic vascular rejection may be patchy and typically involves smaller vascular arteries and veins. In the early stages of chronic vascular rejection, the intimal proliferation occurs on the luminal aspect of an intact elastic lamella. Subsequently, the internal elastica may become fragmented and discontinuous. Occasionally the underlying muscular wall becomes thinned. In approximately half of the reported cases a concurrent endovasculitis has been observed. The process is similar in pulmonary veins, although the

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b

Fig. 7.7  Chronic vascular rejection (ISHLT D1). (a) At high power, there is a small vein with eosinophilic, waxy fibrointimal thickening as also evaluated on VVG stain (b) (H&E, VVG, magnification × 400)

intimal deposits may be less cellular and more waxy, eosinophilic, and sclerotic. Chronic vascular rejection should be distinguished from recanalizing thrombi. Unlike the situation with heart transplant recipients, chronic vascular rejection in lung transplants has not resulted in graft loss; however, some patients develop pulmonary hypertension particularly those with BOS [92, 111].

7.2.3.4 Mimickers of Severe Acute Cellular Rejection Mimickers of severe acute rejection include conditions that might present with acute lung injury or DAD. These conditions include infection, drug toxicity, antibody mediated rejection (AMR), or harvest/reperfusion injury. Therefore, careful slide review for perivascular inflammation should be performed, stains for microorganisms including Gomori-Grocott methenamine silver stain (GMS) and acid fast bacilli (AFB) might be added and careful search for viral inclusions should be undertaken. Although perivascular mononuclear infiltrates are helpful to identify rejection, these are not entirely specific for acute rejection and many other conditions may simulate or mimic alloreactive lung injury [126]. Differential diagnostic considerations include CMV pneumonitis, Pneumocystis jiroveci pneumonia ­and posttransplantation lymphoproliferative

disease (PTLD). Further differential diagnosis of perivascular and interstitial infiltrates include recurrent primary diseases.

7.2.3.5 Antibody-Mediated Rejection AMR or humoral rejection is well established in other solid organ transplantation. It was originally recognized in kidney transplant patients who presented with acute allograft rejection, antidonor antibodies, and poor prognosis [125]. AMR is thought to be due to circulating antibodies that are either preformed because of pregnancy, blood transfusion, or previous organ transplantation or arise de novo after transplantation due to HLA-mismatch. Although AMR is an increasingly recognized entity in lung transplantation, it is still under investigation. Early observations were based on the phenomenon of hyperacute rejection, where preexisting donor-specific antibodies lead to complement activation and rapid graft loss. Later it has been recognized that lung transplant recipients also can develop antibodies after transplantation to the allograft that might lead to AMR. Evidence suggests that AMR occurs to donor major histocompatibility (MHC) antigens, although other endothelial and epithelial antigens expressed in the lung may become antibody targets as well. The recent development of very sensitive and specific solid phase flowcytometry and Luminex-based methodologies has allowed for accurate detection of

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antibody specificities in sensitized recipients and it has become clear that more patients than previously expected present with preformed anti-HLA antibodies. Immune stimulation by prior infections or autoimmunity might contribute to the development of antibodies to alloMHC in those patients with no identifiable risk factors. These preexisting antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection) or accelerated humoral rejection and BOS [23]. Furthermore, recent studies have consistently demonstrated an increased incidence of acute rejection (a threefold increase in one study) [48], persistent rejection, increased BOS [96] or worse overall survival [56] in patients with anti-HLA antibodies. This effect is apparent both with pretransplant HLA sensitization and with the development of de novo anti-HLA donorspecific antibodies after transplantation [96]. About 10–15% of lung transplant recipients are presensitized to HLA antigens [3]. Even though “unacceptable antigens” are avoided during the virtual crossmatch, patients with positive pretransplant PRA are at higher risk for posttransplant complications. Their posttransplant PRA can stay stable or increase via generation of either donor-specific or nondonor-specific anti-HLA antibodies. Similarly, patients that had negative PRA screening tests before transplantation can develop de  novo nondonor-specific or donor-specific anti-HLA antibodies after transplantation. The mechanisms by which antibody promotes lung allograft injury remain poorly understood. Antibody binding to alloMHC or other endothelial or epithelial targets in the lung could lead to activation of the complement cascade with complement deposits leading to endothelial cell injury, production of proinflammmatory molecules, and recruitment of inflammatory cells. Com­plement independent antibody-mediated mechanisms can also induce endothelial cell activation without cell injury, leading to increased gene expression and subsequent proliferation [23]. As demonstrated by in vitro studies, anti-HLA antibodies can cause proliferation of airway epithelial cells as well, producing fibroblast-stimulating growth factors [64], potentially contributing to the generation of obliterative airway lesions. Recent studies have attempted to evaluate immunoglobulins (Ig) and complement deposits in the subendothelial space. Septal capillary deposits of Igs and complement products such as C1q, C3d, C4d, and

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C5b-9 have been described in association with antiHLA antibodies [62, 90] as well as allograft dysfunction and BOS [82, 139]. The concept of specific histopathologic features associated with humoral rejection remains controversial in lung transplantation. Recent studies question the relation between complement or Ig staining and allograft rejection [112, 138]. Others demonstrate that C3d and C4d staining can occur in lung transplant recipients with nonalloimmune lung injury such as infection and primary graft dysfunction (PGD) with no evidence of anti-HLA antibodies [139]. Differences in staining techniques between different laboratories may further explain some of the inconsistencies in the published data. The 2007 ISHLT revised consensus classification did not agree upon any histopathologic features that might be specific for AMR in lung [123]. Although capillary injury/small vessel intimitis might raise the suspicion of AMR, these are nonspecific findings that can also occur in severe acute rejection, infection, or harvest/reperfusion injury. Furthermore, although often the term “capillaritis” is mentioned, ISHLT recommends to use the term “capillary injury” which allows for a broader spectrum of morphological changes of the vessels including intimitis, whole wall thickness inflammation, neutrophilic infiltration (which should be distinguished from neutrophilic margination), thrombosis, and necrosis. Furthermore, signs of DAD and intra-alveolar hemorrhage can occur with AMR although again, they are not specific. Given the lack of specific histologic findings of AMR in lung transplantation, a multidisciplinary approach to diagnosis is recommended that includes the following: (1) The presence of circulating antibodies (HLA antibodies, antiendothelial and antiepithelial antibodies), (2) Focal or diffuse C4d deposition (see Fig. 7.8), (3) Histologic features of acute lung injury or hemorrhage (DAD, capillary injury with neutrophils and nuclear debris) and (4) Clinical signs of graft dysfunction. If AMR is clinically, immunopathologically, or histologically suspected, one might perform immunostains for C3d, C4d, CD68, and CD31. However, these stains are extrapolates from kidney and heart transplantation and although there have been several studies advocating their use [47, 62, 123, 139], there are no large scale studies validating their use. C4d has been studied extensively in kidney and heart transplant and has been identified as a useful adjunct in the diagnosis of AMR in these organs.

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Fig. 7.8  Antibody mediated rejection (AMR). The high power view shows capillaritis in a case of AMR characterized by focally thickened interstitium due to neutrophils and nuclear debris with capillary destruction. The insert reveals C4d immunoperoxidase stain (see arrow) focally lining the endothelium of capillaries and small vessels (magnification ×  400, insert × 600)

However, in lung that is not the case. One of the reasons for the difficulties in lung is the relatively high background that is encountered in immunohistochemistry (IHC) as well as immunofluorescence (IF). Often, C4d binds to elastic laminas or shows other nonspecific binding. Staining is commonly only focal and therefore sensitivity and specificity have not been established yet given the limited sample size of transbronchial biopsies. Only linear, continuous subendothelial staining of capillaries, arterioles, and/or venules count as positive by IHC. Also, C4d is not specific to AMR but also can be seen in infection, harvest/ reperfusion injury, or even in severe acute rejection, basically in any process that is associated with complement activation. There is no IHSLT recommendation at this time regarding to the coexistence of AMR and acute rejection.

7.3 Transbronchial Biopsy 7.3.1 Background and History Although clinical evaluation of the patient may suggest the possibility of rejection, tissue evaluation is necessary for a definitive diagnosis. Initially, investigators were hesitant to routinely utilize transbronchial biopsy

A.C. Roden and H.D. Tazelaar

to monitor the graft as it was thought that the amount of tissue obtained would be too small to make a definitive diagnosis. There was also concern about passing a bronchoscope over the anastomosis. However, transbronchial biopsy is now a routine procedure and has become the gold standard to evaluate the graft for acute and chronic rejection, infection, and possible recurrent disease since currently, no surrogate markers have been sufficiently validated as means to reproducibly identify patients with acute rejection. Specifically, there is no serological marker to indicate rejection is occurring. After lung transplantation the total BAL fluid cell count is constantly increased even in periods with no evidence of infection or rejection [127]. In the early posttransplantation period (first 4 weeks), there is a dominance of neutrophils in the BAL fluid (up to 25–50% of total cell count) until about 3 months later when the cell count normalizes [127]. Acute rejection has been associated with elevated CD8+ T cells, activated CD4+ T cells, a trend toward increased NK T cells, increased B cells, and decreased NK cells in the BAL [53]. Nevertheless, no study has proven the BAL cellular composition to be adequately sensitive or specific in the discrimination of rejection from infection [107]. Small studies have found a correlation between acute rejection and elevation of IL17 [134], IL15 [10], and IFN gamma in the BAL [10]. Recent advantages in genomics offer the potential for more specific means of diagnosing rejection in lung transplantation. A pilot study of gene expression in the BAL of lung transplant recipients found that gene expression signatures related to T-lymphocyte function, cytotoxic CD8 activity, and neutrophil degranulation correlate with acute rejection [98]. However, these studies have no clinical use at the present time. Recently published studies in heart transplantation describe the use of peripheral blood gene expression profiling to identify future risk of cardiac allograft rejection [88]. A similar study is now underway in lung transplantation, known as the lung allograft rejection gene expression observational (LARGO) study. Preliminary data from almost 900 patients, similar to the CARGO results, show differential gene expression in the lymphocyte priming and neutrophil homeostasis pathways for A0 vs. ³A2 acute lung rejection [66]. Such testing may hold promise for a noninvasive technique to monitor the status of the transplanted organs. Another study described marked serum elevations of hepatocyte growth factor (HGF) in lung transplant

7  Lung

recipients with acute rejection. However, smaller elevations in HGF also occurred with lung infection, and additional studies are needed to validate specificity and sensitivity of HGF for acute rejection [1]. In conclusion, although there are a few promising noninvasive techniques, these are early studies that need to be confirmed in larger studies before being considered for widespread clinical use. Therefore, microscopic tissue evaluation remains the gold standard for diagnosing rejection.

7.3.2 Timing of Posttransplantation Biopsies Bronchoscopy and biopsy postlung transplantation are generally performed if there is any clinical suspicion of rejection, infection, recurrent disease, or PTLD. In addition, many transplant centers now follow protocol biopsies. However, the pros and cons of surveillance bronchoscopies with biopsy are still debated and therefore, there are no guidelines established in regards to the timing of postlung transplantation surveillance bronchoscopy and biopsy. One institution reports surveillance biopsies in pediatric lung transplant recipients at 1 week and 1, 3, 6, and 12 months posttransplantation [8]. The rational for surveillance biopsies includes the occurrence of clinically silent acute rejection, inadequate surrogate markers for acute rejection, and the relatively low risk of the bronchoscopy procedure. The yield for acute rejection by transbronchial biopsies was reported 6.1–31% and 25% or greater in studies performing surveillance transbronchial biopsies [17, 60], clinically indicated and follow-up bronchoscopies [18]. Grade A2 and higher acute rejection has been found in a relatively high percentage of asymptomatic patients, ranging from 22 to 39% [54, 131]. Silent acute rejection appears most common within the first 3 months of transplantation (24.8% at 0–3 months; 16.7% at 3–12 months; 2.7% after 1 year) [87]. The rate of unsuspected but clinically significant infection was highest between 3 and 12 months posttransplantation but a relatively high rate (18.9%) was also detected after 1 year. One small study questioned the benefits of performing surveillance bronchoscopy with biopsy, suggesting that the benefits do not outweigh the procedural risks [132]. In this study, changes in management based on transbronchial biopsy and BAL results were

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significantly higher in the clinically indicated group (65%) compared with the surveillance group (13%) and were mostly related to infection. Interestingly, no silent acute rejection episode requiring treatment was detected in the surveillance group of this study. Collectively, these findings may reflect differences in programs and operators. Although it has been shown that transbronchial biopsy is often the only mean to reveal silent acute rejection of the allograft, definitive evidence that treatment of such episodes have a positive impact on survival or prevention of BOS has yet to be demonstrated. However, based on the link between acute rejection and development of BOS, surveillance transbronchial biopsies in asymptomatic lung transplant recipients has become common practice in many large lung transplantation centers because evidence suggests that patients who have multiple episodes of low grade (A1) lesions within the first 12 months posttransplantation develop early onset BOS. However, there were no differences in overall survival in patients with and without A1 acute rejection [59]. There is also evidence that A1 rejection increases the risk of higher-grade subsequent rejections (³A2) [13, 34]. The finding of a solitary perivascular monocytic infiltrate was followed by worsening acute rejection in four untreated patients in one study, while the treatment of such a solitary infiltrate in nine patients resulted in improvement of the rejection score [67]. The presence of severe lymphocytic bronchiolitis also has been associated with increased risk of BOS and death after lung transplantation, independent of the presence of acute rejection [49]. A study [49] in which surveillance transbronchial biopsies were performed at 3, 6, 9, and 12 weeks posttransplantation, at the time of symptoms, and for follow-up of acute rejection or CMV pneumonia showed that patients who develop acute small airways rejection within the first year after transplantation are at risk of development of BOS at 1.76, 3.3, and 5.5 years after detection of B3/ B4 lesion (by 1996 ISHLT criteria, see Table 7.2), B2 lesion or B0/B1 lesion, respectively. This study strongly suggests that the main utility of surveillance biopsies may be to use lymphocytic bronchiolitis as a surrogate predictor of long-term outcome. However, the impact of therapy for lymphocytic bronchiolitis has yet to be assessed. Although complications related to bronchoscopy occur, including a small risk of severe complications, the majority of cases have a favorable outcome. Adverse events reported with bronchoscopy in lung

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transplant recipients include transient hypoxemia, bleeding, pneumothorax, arrhythmia, and anesthesia related complications [74, 87]. Bronchoscopy may have contributed to faster deterioration of one patient’s already critical condition; however, in the posttransplantation setting, -bronchoscopy has no reported mortality [18, 60], although there are anecdotal reports of death related to transbronchial biopsy.

7.3.3 Specimen Adequacy and Handling The 2007 ISHLT revised consensus classification of acute allograft rejection [123] requires the evaluation of at least five pieces of well-expanded alveolated parenchyma. However, the bronchoscopist may need to submit more than five pieces to provide this minimum number. Further biopsies may improve the detection of OB, although there is no specific number of small airways required by the ISHLT consensus classification to exclude this diagnosis. Gentle agitation in formalin to open up the alveoli may improve the histologic appearance of the fragments. Histologic examination should include three levels from the paraffin block for hematoxylin and eosin (H&E) staining [123]. Connective tissue stains such as Trichrome or Verhoeff-Van Gieson (VVG) stain to evaluate airways for the presence of submucosal fibrosis are essential for the diagnosis of OB, and atherosclerosis. Silver stains such as GMS can be performed for fungi, including pneumocystis, but have not been routinely mandated by the 2007 ISHLT revised consensus classification, given the numerous microbiologic, serologic, and molecular techniques available for the diagnosis of opportunistic infections. BAL may be performed at the time of biopsy and is useful for the exclusion of infection, but currently has no clinical role in the diagnosis of acute rejection (see above).

A.C. Roden and H.D. Tazelaar

immunosuppression, reduced mucociliary clearance, decreased cough reflex resulting from denervation, and interruption of lymphatic drainage.

7.4.1.1 Bacterial/Viral Pneumonia Bacterial pneumonias are the most common infections following lung transplantation [26] and occur in more than 35% of patients during the first year after transplantation (highest incidence is during the first month posttransplantation) (see Fig.  7.9). Furthermore, bacterial pneumonia remains a major infectious complication throughout the patient’s life. The donor lung is affected most often. Gram-negative organisms are most common, especially Enterobacter and Pseudomonas. Bronchitis secondary to Pseudomonas species or Staphylococcus aureus infection also is observed. Bacterial pneumonia typically manifests radiographically as a lobar or multilobar consolidation. Viral pneumonias develop in approximately 11% of patients who have undergone lung transplantation. They occur at any time following transplantation. 7.4.1.2 CMV Infection CMV is the second most common cause of pneumonia in patients who have received lung transplants, and it

7.4 Complications of Immunosuppression 7.4.1 Infection Infection is the leading cause of death in lung transplant recipients. Factors that increase a patient’s susceptibility to infection after transplantation include

Fig.  7.9  Acute Bronchopneumonia, likely bacterial. On low magnification, some of the alveoli are filled with clusters of cells and fibrin. High magnification view (insert) reveals that the intraalveolar infiltrate is largely comprised of neutrophils, findings consistent with acute bronchopneumonia (H&E, magnification × 100, insert, × 400)

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7  Lung

is the most common opportunistic infection (35–60% of opportunistic infection) [26]. It represents the most significant viral infection, and usually occurs 1–4 months after transplantation. Primary infection is the most serious form and is observed in 50–100% of seronegative patients who received a graft from a seropositive donor. In patients who are seropositive, secondary CMV infection develops from reactivation of latent disease following the institution of immunosuppressive therapy or from infection with a different strain of CMV. Infected patients may be asymptomatic or may develop a fulminant pneumonia, possibly with extrathoracic findings such as retinitis, hepatitis, and gastritis. Presenting symptoms include dyspnea, fever, and cough. The most common finding on chest radiographs in patients with CMV infection is diffuse parenchymal haziness. CT scan findings include areas of ground-glass attenuation; reticulation; multiple, small, ill-defined 1- to 3-mm nodules; and, even less commonly, areas of dense consolidation. The diagnosis of CMV pneumonia can be made by bronchoscopy with lavage and biopsy. Cytopathic changes associated with CMV infection include cytomegaly, multiple small basophilic cytoplasmic inclusions, and a large nuclear inclusion surrounded by a halo with thickened nuclear membrane (see Fig. 7.10).

Prophylactic therapy with acyclovir and immune globulin has not reduced the incidence of CMV infections in patients who have undergone transplant procedures.

7.4.1.3 Herpes Simplex Virus Infection A less common cause of viral infection includes the herpes simplex virus (HSV) infection. Patients with HSV infection present with fever, cough, and dyspnea, but they demonstrate symptomatic improvement after therapy with intravenous acyclovir. Radiographic findings may be absent or may demonstrate diffuse groundglass opacities. Histopathologically, the classic HSV inclusion consists of a dense, eosinophilic mass within the nucleus that is surrounded by a clear halo and peripherally marginated, beaded nuclear chromatin. HSV may form multinucleated cells.

7.4.1.4 Fungal Infections Opportunistic fungal infections are less common than viral infections, but they are associated with higher mortality. Fungal pneumonias usually occur 10–60 days following transplantation and more commonly involve the transplanted lung. However, they also can involve the native lung in single lung transplantation cases, especially in patients with COPD. CT imaging studies most commonly reveal a combination of nodules (multiple, variable sizes, irregular margins), consolidation, and ground-glass opacification. Pleural effusions are also common (63% of cases). GMS will help to identify fungal organisms. However, even in the absence of identifiable organisms on GMS, infection might be considered and cultures and serology should be attempted.

7.4.1.5 Aspergillus Infection Fig.  7.10  Cytomegalovirus pneumonia. The biopsy shows intraalveolar plugs of mucopolysaccharide rich proliferating fibroblasts of organizing pneumonia and scattered large atypical cells characterized by eosinophilic nuclear and cytoplasmic inclusions (insert right upper corner) diagnostic of CMV infection. Immunohistochemistry highlights CMV-infected cells (insert lower right corner) (H&E, magnification ×100, inserts, × 600)

Locally invasive or disseminated Aspergillus infection accounts for 2–33% of posttransplantation infections and 4–7% of deaths in patients who undergo lung transplantation. Aspergillus infection most commonly is characterized by local invasion of a necrotic bronchial anastomosis (i.e., ulcerative

188

tracheobronchitis), which typically occurs within 4 months of transplantation. Aspergillus infection, when involving the lung parenchyma, tends to cavitate and has an upper-lobe predominance. Histopathologically, the hyphae of Aspergillus sp are septated and branching. They appear uniform and grow in parallel fashion with septae at regular intervals. The branching is dichotomous and usually at 45°angle. Inhaled amphotericin B is often used in the immediate posttransplantation period to help eliminate this complication [26]. Patients are also discharged on voriconazole as daily prophylaxis for the first year.

7.4.1.6 Pneumocystis jiroveci Pneumonia Patients who have undergone lung transplant procedures have an increased susceptibility to P jiroveci infection, but prophylaxis with trimethoprim-sulfamethoxazole is effective in preventing the infection (incidence is nearly 0%). Without prophylaxis, the incidence of P jiroveci infection approaches 90%. Histologically, cysts of P jiroveci are round to oval, measure 5–7  mm in diameter, and often have very prominent grooves or folds. P jiroveci pneumonia classically consists of an intra-alveolar foamy exudate in which the organisms appear as small “bubbles” in a background of proteinaceous exudate, although a variety of other reactions can occur as well [129].

7.4.2 Posttransplant Lymphoproliferative Disorder The incidence of PTLD is significantly higher after thoracic organ transplantation compared with any other solid-organ transplant [94]. They develop in 4–10% of lung transplant recipients, as opposed to an approximate 2% incidence in other solid organ recipients, with 60% of the PTLDs presenting in the allograft. In adults, PTLDs are the most common neoplasms at 1 year posttransplantation (peak, 3–4 months posttransplantation) [21]. In children [6], lymphomas are by far the most common posttransplantation malignancy at any time. In fact, PTLD is a major cause of morbidity and mortality in pediatric lung transplant recipients [12]. Risk factors for PTLD after solid organ transplantation include patient’s age, type of organ transplanted,

A.C. Roden and H.D. Tazelaar

EBV infection status of host before transplantation, and the immunosuppressive regimen [4, 77, 93, 140]. PTLDs are a clinically, morphologically, and molecularly heterogeneous spectrum of lymphoproliferative disorders, ranging from early, EBV-driven, polyclonal, polymorphic proliferations (infectious, “mononucleosis-like”) to EBV-positive or EBV-negative monomorphic tumors [15, 69, 70, 100]. PTLDs most commonly are associated with EBV infection of B-cells either by reactivation of latent virus or primary EBV, most commonly acquired from donor organs [12]. A recent study showed [37] that five of seven lung-transplanted children who developed PTLD were EBV-negative recipients who received EBV-positive lungs. In a series [81] of 988 heart and/or lung transplant recipients, 17 PTLDs were identified. Amongst the 17 PTLD cases, there were two B-cell monoclonal polymorphic PTLDs and 15 B-cell monomorphic PTLDs (see Fig. 7.11) (13 diffuse large B-cell lymphomas (DLBCL) and two Burkitt lymphomas). EBV was detected in 9 of the 17 patients. All cases showed monoclonal IGV gene rearrangements; IGV somatic hypermutation was found in 88% of cases, indicating a prevalent origin from germinal center-experienced B cells. Given the association between EBV infection and PTLD, elevation in EBV viral load measured in peripheral blood has been proposed as a marker for the development of PTLD [109]. In addition, monitoring the degree of immunosuppression after diagnosis of PTLD with immune function assays and immunosuppressive drug levels may aid clinicians in assessing the risk for PTLD and monitoring treatment after diagnosis [45]. T-cell PTLDs tend to occur later and tend not to be associated with EBV infection. T-cell PTLDs are associated with a worse prognosis than B-cell PTLD. Patients with PTLDs may be asymptomatic, or they may have nonspecific complaints such as fever, weight loss, dyspnea, and lethargy. Solitary or multiple pulmonary nodules ranging in size from 0.1 to 5 cm are the most common pulmonary manifestation of patients with PTLDs. Mediastinal and hilar adenopathy also can be observed in 22–50% of cases. Patients who present with a solitary pulmonary nodule have a better overall prognosis. However, most PTLDs have a rapid onset, a predilection for extranodal sites, and an aggressive clinical behavior with poor outcome [15, 69, 70, 78, 100]. As a result of the high mortality associated with PTLD, treatment often poses a challenge. The most common treatment of PTLD has included decreasing immunosuppression in combination with

189

7  Lung

a

b

EBVish

Kappa Fig. 7.11  Post transplant lymphoproliferative disorder (PTLD). Wedge biopsy from a patient who underwent bilateral lung transplant 14 months prior. (a) Low power view reveals a cellular infiltrate infiltrating blood vessels and focally causing necrosis. (b) High power view shows abnormal large lymphoid cells with rounded nuclei, dispersed chromatin, prominent nucleoli and abundant amphophilic cytoplasm. The cytoplasm is often

Lambda eccentrically distributed and there are many cells with perinuclear clear zones. These atypical lymphoid cells are strongly and uniformly positive for EBV by in situ hybridization and show kappa light chain restriction. This immunoarchitecture is consistent with PTLD, monomorphous type (H&E, magnification × 40 (a), 400 (b))

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surgical resection, antiviral agents, radiation, and chemotherapy [45, 103, 109]. The reduction or withdrawal of immunosuppressive therapy may result in a partial or complete regression of the PTLD [69]. A higher risk of graft rejection associated with PTLD treatment is one reason for the need for early detection of the disease [45, 102, 103, 109].

7.4.3 Solid Organ Neoplasms In adults, malignancy remains a common complication after lung transplantation [21]. Among lung transplantation survivors, 3.5, 12.6, and 28.1% have a malignancy at 1, 5, and 10 years posttransplantation, respectively. Although lymphoproliferative disorders are most common at 1 year posttransplantation, nonmelanoma skin malignancy is the leading malignancy at 5 and 10 years posttransplantation. In children [6], the incidence of posttransplantation malignancy is higher than in adults early after transplantation with 5.9% of children developing them within the first year after transplantation. At 5 years posttransplantation, 13.1% were found to have a malignancy. At any time posttransplantation, lymphomas are by far the most common posttransplantation malignancy in children. The risk for cancer is higher following lung transplantation than after kidney transplantation [110, 121]. Lung transplant patients have unique characteristics, as their baseline genetic and environmental background, and immunosuppressive regimens are different from patients receiving kidney transplant, all of which may contribute to the increased risk [2]. Besides nonmelanoma skin cancer and lympho­ proliferative disorders, other malignancies repor­ted to be associated with solid organ transplantation include: Kaposi’s sarcoma, anogenital cancers, oral cavity malignancies, esophageal and urinary bladder cancer, hepatocellular carcinoma, and sarcomas [110, 121].

7.4.4 Graft vs. Host Disease Acute graft vs. host disease (Gvhd) is an uncommon and usually fatal complication of lung transplantation for which no effective therapy exists. Among the ten

A.C. Roden and H.D. Tazelaar

reported patients, eight had grade 3 to 4 acute Gvhd and died within 208 days. There is one recent case report [42] of a patient with grade 3 to 4 acute Gvhd after BLT who was successfully treated with highdose corticosteroids after basiliximab and extracorporeal photopheresis were unsuccessful. After 26 days the patient had developed low grade fever, chills, and pruritic maculopapular rash on the chest that spread to the proximal extremities in the next 4 days. Liver function tests were elevated. A skin biopsy showed dermal inflammation, patchy loss of basal layer with vacuolar degeneration, apoptosis, and cell necrosis suggesting Gvhd. A few days later, profuse, green, watery diarrhea developed with abdominal pain and paralytic ileus. Colonoscopy showed ulceration of mucosa and destruction of intestinal crypts, features compatible with Gvhd. There was leucopenia, severe anemia, and deterioration of liver function. The diagnosis was confirmed by chimerism study that revealed a recipient/donor lymphocyte ratio of 50:50. A second case of acute Gvhd after lung transplantation was successfully treated with corticosteroids [5]. This patient presented with mild symptoms (pruritic rash and blurry vision) suggesting low-grade Gvhd. The diagnosis was confirmed when a small dose of prednisone resulted in the resolution of Gvhd within 3 weeks.

7.5 Nonrejection Related Allograft Pathology 7.5.1 Harvest/Reperfusion Injury Lung harvest/reperfusion (ischemia/reperfusion, I/R) injury after transplantation remains the most common cause of early posttransplantation respiratory failure and manifests typically during the first 72 h after transplant [75]. Reported rates are as great as 41% [52]. The 30-day mortality of patients with I/R injury is about 40%, compared with 7% in patients without I/R injury [86]. The clinical equivalent to I/R injury is PGD. The ISHLT Working Group on PGD proposed a four-tiered grading scheme of PGD based on PaO2/FiO2 (P/F) ratio and radiological chest infiltrates assessed at time points up to 72 h: T (0-within 6 h of reperfusion, 24, 48, and 72 h) [20]. I/R injury usually presents with the immediate impairment in lung function after transplantation

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accompanied by rapid development of pulmonary edema, increased pulmonary vascular resistance, and decreased airway compliance. Patients with I/R injury require prolonged mechanical ventilation with greater hospital stays and are at an increased risk of multiorgan failure. Lung I/R injury has long-term consequences and is a risk factor for late graft failure (OB) [39, 40]. I/R injury can occur due to prolonged ischemia during transplantation. Ischemic injury to the pulmonary vascular endothelium increases permeability and results in pulmonary edema. Histologically, I/R injury presents as acute lung injury pattern including DAD (see Fig. 7.12). Perivascular infiltrates are usually not present and distinguish it from acute rejection. However, infection can present similarly and needs to be excluded with stains, cultures, and serology. The pathophysiology of lung I/R injury remains incompletely understood. The lungs are particularly susceptible to I/R injury, likely owing to the rich vascularity and relatively large surface area over which blood-borne components interact with the endothelium. The mechanisms of I/R injury are diverse and include generation of reactive oxygen species (ROS), leukocyte activation/recruitment, complement and platelet activation, abnormalities in pulmonary vascular tone, and increased procoagulant activity. The production of proinflammatory cytokines is increased considerably in the lung after I/R. Expression of

cytokines in the lung after I/R may not only cause immediate tissue injury, but may predispose the lung allograft to rejection. Several studies suggest that lung I/R injury is biphasic, with a distinct, acute injury characterized by macrophage activation followed by a later, neutrophildependent injury [39].

7.5.2 Recurrent Native Disease Although recurrent disease in the allograft has been reported in some of the transplantation cases, in general, this has not been clinically significant. Diseases for which recurrences in the allograft have been reported include giant cell interstitial pneumonia, sarcoidosis, lymphangioleiomyomatosis, Langerhans’ cell histiocytosis, allergic bronchopulmonary aspergillosis, desquamative interstitial pneumonia, bronchioloalveolar carcinoma, alveolar proteinosis, and diffuse pan bronchiolitis. Morphological features are identical to the disease in nontransplanted lung.

7.5.3 Anastomotic Complications in Airways 7.5.3.1 Bronchial Dehiscence Bronchial dehiscence is the most common anastomotic airway complication in the early postoperative period. It occurs in 2–3% of cases. Ischemia at the anastomotic site is the major factor in the development of this complication. Dehiscence probably is best assessed by bronchoscopy; however, CT scans typically demonstrate the presence of extraluminal gas, which is 100% sensitive and 72% specific for dehiscence. Patients with telescoping anastomoses also may develop small anastomotic diverticula, which appear as smooth rounded air collections at the inferior-medial aspect of the anastomosis.

Fig.  7.12  Harvest/reperfusion injury. In this biopsy taken 2 days after transplant, there is interstitial thickening due to proliferation of mucopolysaccharide rich fibroblasts and hyperplastic type II pneumocytes. Eosinophilic hyaline membranes are also present. No perivascular inflammatory infiltrates are identified. These morphological features of diffuse alveolar damage and the history, are consistent with harvest/reperfusion injury (H&E, magnification ×100) (Case contributed by Dr. Andras Khoor)

7.5.3.2 Stricture Anastomotic stricture occurs in approximately 10% of cases, and the risk for stenosis may be increased with a telescoping anastomosis. Stenoses often manifest with

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progressive airflow obstruction that can be difficult to differentiate from other causes, such as acute rejection or BOS. Stricture probably is best evaluated by bronchoscopy; however, CT scans often demonstrate the area of narrowing. Treatment is stenting, typically with an expandable metallic stent. More recently, balloon dilatation has obviated the need for stents in some centers.

7.5.4 Pathology in the Remaining Native Lung App 43% of lung transplantations between 1995 and 2008 were single lung transplants (SLT) [21]. COPD, AAT, and IPF accounted for app 85% of the indications for SLTs [21]. Advantages of SLT over BLT include a technically easier procedure, shorter surgery time, and the ability to transplant two patients from one donor. However, there are disadvantages to SLT. For instance, survival rates for SLT and BLT recipients are different, diverging most obviously in later years after transplantation. 5-year survival for SLT was 46% compared with 54% for BLT [130]. Other disadvantages of SLT include less pulmonary reserve, and the propensity for complications related to the residual native lung. Native lung complications have previously been reported in 13.8–50% of patients. They may be associated with significant morbidity and mortality [85, 135] and may partly explain why outcomes with SLT are inferior to those of BLT. A recent study [68] reported the occurrence of pneumothoraces, malignancy, aspergilloma, pneumonia, bronchopleural fistulas, and pulmonary embolism. Some forms of advanced lung disease, including COPD and IPF, are associated with increased risk for lung cancer, a complication that can arise in the native lung after transplantation [76, 142]. Patients also often have structural damage to the native lung from their underlying disease process, which can increase the risk of other complications such as aspergilloma and pneumothoraces. The median time from transplantation to major native lung complication was 1.28 years (range, 0.04–5.1 years). In one study [68], 8 of 18 patients with native lung complications died thereof. Median posttransplant survival was lower in SLT recipient with significant native lung complications (3.2 vs. 5.3 years, p = 0.004). Native lung pneumonectomy was performed in 11 patients. In patients

A.C. Roden and H.D. Tazelaar

with native lung complications there was a trend toward improved posttransplant survival in patients who underwent native lung pneumonectomy compared with those who did not undergo pneumonectomy. However, there was no difference in survival between patients who underwent native lung pneumonectomy to those who had no native lung complication.

7.5.5 Bronchiectasis The majority of patients with OB also have severe bronchiectasis. By specimen bronchograms, the bronchial tree shows alternating areas of dilatation and constriction. Microscopically, the bronchiectasis may be associated with areas of mucous plugging, goblet cell hyperplasia, squamous metaplasia, denudation of the bronchial epithelium, submucosal scarring, and acute and chronic inflammation of the bronchial wall. Occa­ sionally foreign body giant cells are present, probably representing a manifestation of aspiration. Obliteration of the terminal respiratory bronchioles is often observed distal to these areas. The bronchiectasis of lung allografts is probably the result of several factors including immune-related injury, infection, mucostasis, aspiration, and loss of innervation.

7.6 Outcomes Despite advances in operative management, lung preservation, critical care, and immunosuppression, longterm survival in lung transplantation remains limited. In fact, outcomes for lung transplantation are the worst of any solid organ transplant [75]. The main obstacles to present day lung transplantation involve: (1) Lack of donor organs, (2) I/R injury, (3) Acute rejection, and (4) Development of OB. The increased susceptibility of the lung to injury, infection, and constant environmental exposure with local innate immune activation likely contributes to the high rates of rejection. The ISHLT Registry reports a 1-year survival rate of 78% and 5-year survival rate of 52% [21]. Mortality is highest in the first year, which consistently decreases across subsequent time periods (see Table 7.3). In the first 30 days, graft failure, non-CMV infections, cardiovascular complications, and technical problems account for most of the mortality. After the first year,

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7  Lung Table 7.3  Causes of death after lung transplantation in adult lung transplant recipients (January 1992–June 2008) [21] Cause of death <30 days 31 days–1 year 1–5 years (%) >5 years OB

0.4

4.6

26.6

23.8

Acute rejection

4.3

1.8

1.3

0.6

  Lymphoma

0.1

2.6

2.0

2.7

  Other

0.2

2.7

7.1

9.7

0

3.0

0.7

0.1

  Non-CMV

20.0

35.4

21.6

17.8

Graft failure

28.8

17.6

18.9

18.8

Cardiovascular

11.0

4.3

4.0

5.2

8.0

2.2

0.5

0.9

17.3

25.9

17.3

20.4

Malignancy

Infection   CMV

Technical Other OB obliterative bronchiolitis

BOS and non-CMV infections were the predominant causes of death. By 5 years, malignancies and cardiovascular causes account for almost 17% of reported causes of death. However, compared with data beginning in 1988, overall survival has consistently improved by era. The improvement in survival is largely due to improvement in the 1 year survival. Long term survival has improved as well, although to a seemingly lesser degree. Evidence suggests that age, pretransplant diagnosis, and donor CMV status play important prognostic roles. For instance, the survival half-life for patients older than 65 years of age was 3.2 years compared with 6.3 years for those aged 35–49. Overall survival rates at 3 months after transplantation are lowest for IPF (86%) and IPAH (78%) and highest for CF (91%) and COPD (91%), most likely due to differences in early complications, including PGD [21]. CMV seropositivity of the donor is associated with worse survival; however, the underlying reasons for this association are not entirely clear. Inducing a state of immune suppression is the key to successful clinical lung transplantation. The immunosuppressive regimens used for lung transplantation are based on successful protocols that have evolved for renal and heart transplantation. Posttransplantation morbidities (see Table 7.4) present at 5 years are those commonly caused or exacerbated by immunosuppressive medicines, including hypertension, renal dysfunction, and dyslipidemia. BOS and malignancies are other common posttransplantation

Table  7.4  Morbidity after lung transplantation in surviving adult recipients (follow-up: April 1994–June 2008) [21] Outcome £1 year (%) £5 years Hypertension

52.4

85.2

Renal dysfunction

25.0

36.6

  Creatinine <2.5 mg/dL

17.4

24.1

  Creatinine >2.5 mg/dL

5.9

9.0

  Chronic dialysis

1.6

3.0

  Renal transplant

0.1

0.5

Hyperlipidemia

23.2

55.5

Diabetes

26.1

37.0

9.5

35.3

OB OB obliterative bronchiolitis

morbidities. For instance, BOS had developed in 28% of patients by 2.5 years after transplantation and in 74% by 10 years. However, most surviving patients reported no activity limitations at 1, 3, 5, and 10 years (>80% at each time point). Furthermore, 13% of survivors reported at least one malignancy at 5 years after transplantation, and 28% were affected by malignancies at 10 years. Survival after pediatric lung transplantation is similar to that reported in adults with a median survival of 4.5 years for the period 1990–June 2007. But, results are clearly improving [6]. One and 5-year survival rates for pediatric recipients transplanted in the most

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recent era (2002–6/2007) are 83 and 50%, respectively, compared with 67 and 43% for recipients transplanted between1988 and 1994. Graft failure, technical issues, cardiovascular failure, and infection are the most common causes of pediatric death in the early posttransplant period whereas infection, graft failure and BOS are the most common causes of late death. The prevalence of BOS steadily increases with time posttransplantation. As expected, the cumulative incidence of malignancy also increases with time after transplantation, with lymphoproliferative disorders making up the great majority of reported malignancies in children. Despite the complications, the functional status of the great majority of long-term pediatric survivors is very good, with 84% of 5-year survivors reporting no limitations in activity. A total of 57 pediatric retransplant procedures were reported between January 1994 and June 2008. The majority of these procedure were performed >12 months after the initial transplantation. Survival over this period was slightly poorer than for primary transplantations, being 41% at 5 years.

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198 126. Tazelaar, H.D.: Perivascular inflammation in pulmonary infections: implications for the diagnosis of lung rejection. J. Heart Lung Transplant. 10, 437–441 (1991) 127. Tiroke, A.H., Bewig, B., Haverich, A.: Bronchoalveolar lavage in lung transplantation. State of the art. Clin. Transplant. 13, 131–157 (1999) 128. Toronto Lung Transplant Group: Unilateral lung transplantation for pulmonary fibrosis. N. Engl. J. Med. 314, 1140– 1145 (1986) 129. Travis, W.D.: Surgical pathology of pulmonary infections. Semin. Thorac. Cardiovasc. Surg. 7, 62–69 (1995) 130. Trulock, E.P., Christie, J.D., Edwards, L.B., et al.: Registry of the International Society for Heart and Lung Trans­ plantation: twenty-fourth official adult lung and heart-lung transplantation report-2007. J. Heart Lung Transplant. 26, 782–795 (2007) 131. Trulock, E.P., Ettinger, N.A., Brunt, E.M., et al.: The role of transbronchial lung biopsy in the treatment of lung transplant recipients: an analysis of 200 consecutive procedures. Chest 102, 1049–1054 (1992) 132. Valentine, V.G., Gupta, M.R., Weill, D., et  al.: Singleinstitution study evaluating the utility of surveillance bronchoscopy after lung transplantation. J. Heart Lung Transplant. 28, 14–20 (2009) 133. Van Muylem, A., Melot, C., Antoine, M., et al.: Role of pulmonary function in the detection of allograft dysfunction after heart-lung transplantation. Thorax 52, 643–647 (1997) 134. Vanaudenaerde, B.M., Dupont, L.J., Wuyts, W.A., et  al.: The role of interleukin-17 during acute rejection after lung transplantation. Eur. Respir. J. 27, 779–787 (2006) 135. Venuta, F., Boehler, A., Rendina, E.A., et al.: Complications in the native lung after single lung transplantation. Eur. J. Cardiothorac. Surg. 16, 54–58 (1999) 136. Verleden, G.M., Vos, R., De Vleeschauwer, S.I., et  al.: Obliterative bronchiolitis following lung transplantation: from old to new concepts? Transpl. Int. 22, 771–779 (2009) 137. Vilchez, R.A., Dauber, J., McCurry, K., et al.: Parainfluenza virus infection in adult lung transplant recipients: an emergent clinical syndrome with implications on allograft function. Am. J. Transplant. 3, 116–120 (2003)

A.C. Roden and H.D. Tazelaar 138. Wallace, W.D., Reed, E.F., Ross, D., et al.: C4d staining of pulmonary allograft biopsies: an immunoperoxidase study. J. Heart Lung Transplant. 24, 1565–1570 (2005) 139. Westall, G.P., Snell, G.I., McLean, C., et al.: C3d and C4d deposition early after lung transplantation. J. Heart Lung Transplant. 27, 722–728 (2008) 140. Wilkinson, A.H., Smith, J.L., Hunsicker, L.G., et  al.: Increased frequency of posttransplant lymphomas in patients treated with cyclosporine, azathioprine, and prednisone. Transplantation 47, 293–296 (1989) 141. Wisser, W., Wekerle, T., Zlabinger, G., et al.: Influence of human leukocyte antigen matching on long-term outcome after lung transplantation. J. Heart Lung Transplant. 15, 1209–1216 (1996) 142. Yang, P., Sun, Z., Krowka, M.J., et al.: Alpha1-antitrypsin deficiency carriers, tobacco smoke, chronic obstructive pulmonary disease, and lung cancer risk. Arch. Intern. Med. 168, 1097–1103 (2008) 143. Young, L.R., Hadjiliadis, D., Davis, R.D., et  al.: Lung transplantation exacerbates gastroesophageal reflux disease. Chest 124, 1689–1693 (2003) 144. Yousem, S.A.: The potential role of mast cells in lung allograft rejection. Hum. Pathol. 28, 179–182 (1997) 145. Yousem, S.A., Berry, G.J., Cagle, P.T., et al.: Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J. Heart Lung Transplant. 15, 1–15 (1996) 146. Yousem, S.A., Martin, T., Paradis, I.L., et al.: Can immunohistological analysis of transbronchial biopsy specimens predict responder status in early acute rejection of lung allografts? Hum. Pathol. 25, 525–529 (1994) 147. Zheng, H.X., Burckart, G.J., McCurry, K., et  al.: Interleukin-10 production genotype protects against acute persistent rejection after lung transplantation. J. Heart Lung Transplant. 23, 541–546 (2004) 148. Zheng, H.X., Zeevi, A., McCurry, K., et al.: The impact of pharmacogenomic factors on acute persistent rejection in adult lung transplant patients. Transpl. Immunol. 14, 37–42 (2005)

8

Liver Hanlin L. Wang, Christopher D. Anderson, Sean Glasgow, William C. Chapman, Jeffrey S. Crippin, Mathew Augustine, Robert A. Anders, and Andres Roma

8.1 Introduction Hanlin L. Wang Since first performed in 1963 by Starzl and colleagues [627], orthotopic liver transplantation (OLT) has evol­ ved to become the gold standard therapy for end-stage liver diseases, acute liver failure and selected primary hepatic malignancies. The progress made in the field over the last 25 years has been logarithmic, with a 1-year survival rate approaching 90% and a 5-year

H.L. Wang (*) Department of Pathology and Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, CA 90048, USA e-mail: [email protected] C.D. Anderson, S. Glasgow, and W.C. Chapman Department of Surgery, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA e-mail: [email protected] J.S. Crippin Liver Transplantation Program, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA e-mail: [email protected] M. Augustine Department of Surgery, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21231, USA R.A. Anders Department of Pathology, Johns Hopkins School of Medicine, 1550 Orleans Street, Baltimore, MD 21231, USA e-mail: [email protected] A. Roma Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA

survival rate of 75% [551]. In spite of an increasing percentage of chronically ill patients, liver transplantation generally provides a good quality of life. In fact, the focus has moved from getting a patient through the transplant and the perioperative period, to prolonging long-term survival through the minimization of immunosuppression and its side effects, dealing with the medical complications seen with time, and preventing and treating diseases in the liver allograft, including the same diseases that initially led to the need for transplantation. Nearly 17,000 patients in the United States are currently on liver transplant waiting lists, but only about 6,500 liver transplants are performed annually. Among them, 95% of the organs are from deceased donors and the remaining from living donors (based on OPTN data as of January 2008). The United Network for Organ Sharing (UNOS), the organization that administers the nation’s policies on organ transplantation, designates institutions as living donor liver transplant centers, which allows family members or other volunteers to donate a part of their liver, usually the right lobe, to save a patient’s life. The liver begins to regenerate within 24 h and is usually of normal size within 3 months post-surgically. In general, patients are categorized into three groups. Those who are very ill are at the top of the waiting list and are likely to receive organs from deceased donors. Those who are not especially ill can safely wait. Those in the middle group are generally considered as good candidates for living donor transplantation. They need to become very ill in order to possibly receive a deceased donor organ. There are a wide variety of complications associated with liver transplantation (Table 8.1). Most lifethreatening complications during perioperative and early postoperative period may include primary graft nonfunction, hyperacute rejection, and technical complications such as hepatic artery thrombosis (HAT) and

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_8, © Springer-Verlag Berlin Heidelberg 2011

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Table 8.1  Liver allograft complications Time Common complications period Zero

Donor liver diseases (e.g., steatosis) Preservation injury Hyperacute rejection Primary nonfunction

1–7 days

Preservation/reperfusion injury Hepatic artery thrombosis Portal vein thrombosis Bile duct leak Acute cellular rejection

1 week to 2 months

Acute cellular rejection Recurrent viral hepatitis Preservation/reperfusion injury Biliary complications Drug reactions

2 months and beyond

Acute cellular rejection Chronic rejection Recurrent liver diseases Acquired liver diseases Biliary complications Drug reactions Opportunistic infections

Less likely complications

Hyperacute rejection Recurrent viral hepatitis Sepsis

Chronic rejection Opportunistic infections

Persistent preservation/ reperfusion injury Posttransplant lymphoproliferative disorder

biliary leaks. Long-term complications mainly include recurrent liver diseases, acute and chronic rejections, and the adverse effects of immunosuppressive medications. In a study that examined causes of death among 299 adult liver transplant recipients who survived more than 3 years [528], hepatic failure caused by recurrent hepatitis C virus (HCV) infection and chronic rejection accounted for the majority of hepatic causes of death, seen in 8 (21%) and 7 (18%) of 38 patients, res­ pectively. Interestingly, nonhepatic causes (mainly de novo malignancies and cardiovascular complications) accounted for more than one half of the deaths in this study, seen in 22 (58%) patients. These patients were significantly older than those who died of hepatic causes. Therefore, the long-term management of liver transplant recipients should comprise not only preservation of graft function, but also prevention and treatment of metabolic complications as well as regular screening for malignancy [58].

Many of the complications result in morphologic changes in the allografts and are reversible with appropriate therapy. Histopathologic evaluation of liver biopsy thus serves an extremely important role in correctly identifying the problems in order to determine the proper therapy in all stages of patient management. This is so because a number of important posttransplant complications cannot be differentiated from one another clinically and biochemically and treating one condition may jeopardize the patients to another problem, such as recurrent hepatitis C vs. acute rejection. On the other hand, different complications may share similar histologic findings. It is thus imperative for pathologists to integrate all pertinent clinical, laboratory, imaging and histopathologic data before the final diagnosis is generated. It is particularly helpful to be aware of transplantation time, original liver disease, immunosuppression status and liver enzyme profile. Comparison with previous biopsies may be necessary and communication with hepatologists, transplant surgeons or other caring physicians is essential. Needle biopsies of the allografts are usually carried out when patients develop symptoms of deterioration of liver function or discovery of abnormal liver tests during clinical follow up. Some centers may perform protocol biopsies on a planned schedule irrespective of clinical presentations or liver enzymes. This latter approach may identify important morphologic changes before the diseases become clinically evident [7, 586]. However, protocol biopsies are not advocated by many hepatologists because many of the histologic changes do not warrant additional immunosuppression or other clinical intervention if the patient is clinically well with stable graft function. This issue thus remains unsolved [49, 62].

8.2 Surgical Perspectives of Liver Transplantation Christopher D. Anderson, Sean Glasgow, and William C. Chapman Advances in immunosuppression as well as technical achievements have combined to make modern liver transplantation safe and effective. Successful clinical liver transplant programs require a multidisciplinary approach to the evaluation and selection of recipients

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and donors as well as aggressive diagnosis and treatment of posttransplantation complications. Consulting pathologists and laboratory medicine specialists plays an important role in all phases of clinical liver transplantation. This section is designed to give an overview of recipient and donor selection, organ allocation, organ procurement, recipient implantation, and posttransplant complications.

8.2.1 Overview The framework for modern liver transplantation began in 1955 when the first article appeared in the literature from Welch, who described full size heterotopic auxiliary liver transplantation in non-immunosuppressed dogs [243]. Further research over the next decade led to the first clinical attempt at OLT in 1963 by Starzl. The patient was a 3-year-old child with biliary atresia [627] who did not survive the operation. After technical improvements and minor success in other attempts, Starzl and colleges performed eight successful transplants in children in 1967 [625]. The research carried out during these years laid the groundwork for modern liver transplantation. The evolution of organ

Year End Waiting List

preservation techniques, better understanding of the immunologic mechanisms of engraftment and impro­ ved immunosuppression continued to advance liver transplantation and ultimately resulted in its widespread acceptance for the treatment of end-stage liver disease [411, 625, 626, 628, 652]. In addition to better preservation and immunosuppression, modern liver transplantation has been advanced by innovations in surgical technique and improved appreciation of hepatic anatomy and regeneration [269]. Liver transplantation and liver resection surgery have evolved with overlapping influence. Expertise in oncologic liver resection based on the segmental anatomy of the liver was the absolute prerequisite for the initiation and advancement of partial liver transplantation. Conversely the improved anatomic and hemodynamic knowledge of the liver gained via the evolution of partial and full size OLT, ultimately led to multiple advancements in major liver resection including in situ and ex situ resections [501, 519, 520]. Over 4 decades of experience with OLT has ultimately resulted in a 1-year and 5-year overall survival of 87 and 73% for all recipients [1]. However, many limitations remain including the relatively fixed pool of cadaveric organ donors. Techniques such as living donor liver transplantation, deceased donor split liver

Deceased Donor Liver Transplants

Live Donor Liver Transplants

16000 14000 12000 10000 8000 6000 4000 2000

0 1996

1997

1998

1999

2000

Fig. 8.1  The number of liver transplant candidates on the waiting list grew steadily during the late 1990s and is currently stable at almost 13,000 patients. However, the number of liver transplantation performed has grown much slower over time due to donor shortage. The total number of liver transplantation performed in 2005 from both deceased and live donors was 6,441. Source: OPTN/SRTR 2006 Annual Report Tables 8.9.1a, 9.4a,

2001

2002

2003

2004

2005

9.4b. The data and analyses reported in the 2006 Annual Report of the U.S. Organ Procurement and Transplantation Network and the Scientific Registry of Transplant Recipients have been supplied by UNOS and Arbor Research under contract with HHS. The authors alone are responsible for reporting and interpreting these data [1]

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transplantation, and donation after cardiac death all have evolved as methods to extend the donor pool. However, as demonstrated in Fig. 8.1, a severe disparity still exists between patients awaiting a liver transplant and the number of available donors. In 2005, 12,822 candidates were listed for liver transplantation, but only 6,441 total liver transplants were performed from all available donors [1]. While non-cholestatic liver disease remains the most common diagnosis of patients on the waitlist, the indications for liver transplant continue to evolve placing further strain on the donor pool. This chapter presents a broad overview of liver transplantation including the common criteria for recipient and donor selection, standard operative techniques, and indications for liver transplantation.

8.2.2 Recipient Selection Decision making during the listing of patients for liver transplantation is sometimes very difficult. A limited supply of donor organs must be rationed to larger pool of eligible recipients. While liver transplant is a curative therapy, it essentially exchanges a patient’s terminal illness with a chronic condition. In addition, the transplant procedure is a complicated operation that carries potential morbidity and mortality risks. Listing patients for transplantation not only requires evaluation of an individual patient, but also consideration of other patients currently on the waitlist. For example, allocation of a liver to a patient who is too sick to survive or too well will result in either graft loss or an inappropriate use of a graft. This indirectly harms patients on the waitlist who may have received greater benefit from that organ. Once a patient is found to have an indication for liver transplantation, the evaluation process must ensure that the patient is fit for surgery from a medical standpoint. This includes cardiovascular and pulmonary evaluations as well as evaluations and screenings for malignancy (colonoscopy, mammogram, etc.). In addition, the patient should be evaluated for his or her psychosocial preparedness. An adequate social support structure must be in place to help ensure compliance with a relatively rigorous medication and rehabilitation regimen. Patients must be able to refrain from addictive behavior such as recidivism to alcohol

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or drug abuse. Upon completion of the transplant workup, the entire transplant team including surgeons, hepatologists, psychologists, nurses, transplant coordinators, social workers and financial advisors must make a uniform objective decision regarding the patient’s suitability for liver transplantation.

8.2.2.1 Waitlist Prioritization Once a patient is placed on a transplant center’s OLT waitlist, the patient is registered with the UNOS which maintains a centralized computer network that includes all waitlists in the United States and links all Organ Procurement Organizations (OPOs). Organs are generally allocated within an individual OPO, followed by a region (group of several OPOs), and then nationally. Before 2002, donor livers were allocated based on the Child-Turcotte-Pugh score (Table 8.2), time on the waiting list, and patient location (e.g., intensive care unit); however it became clear that these parameters were suboptimal measures of disease severity [210]. Organs are currently allocated based on medical urgency using the Model for End-Stage Liver Disease (MELD) and the Pediatric End-Stage Liver Disease (PELD) mathematical regression models. These models have been found to accurately predict 3 month mortality on the waitlist [405, 713]. MELD is based on a logarithmic calculation of the patient’s INR (international normalized ratio), bilirubin and creatinine, while PELD includes albumin, bilirubin, INR, age and the presence of growth failure. These systems have more effectively allowed transplant surgeons to determine a patient’s temporal probability for OLT based on their short-term prognosis. Since these models were implemented for organ allocation, there have been decreases in waitlist mortality and removal from the waitlist secondary to health decline [207, 211]. It was

Table 8.2  Child–Turcotte–Pugh classification Points 1 2

3

Encephalopathy (grade)

None

1 or 2

3 or 4

Ascites

Absent

Slight

Moderate

Bilirubin (mg/dL)

1–2

2–3

>3

Albumin

>3.5

2.8–3.5

<2.8

Prothrombin time (sec beyond control)

1–4

4–6

>6

Class A: 5–6 points; Class B: 7–9 points; Class C: 10–15 points

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recognized early following implementation of the MELD and PELD systems that they prioritize adult and pediatric patients with primary liver tumors lower on the waitlist. As discussed more in-depth below, these patients currently receive MELD or PELD exception points to overcome this disparity.

8.2.3 Indications and Outcomes There are essentially four main indications for OLT: chronic liver disease, acute liver failure, malignancy, and metabolic disorders. Of these, chronic liver disease accounts for the vast majority of liver transplants performed each year. Over the last decade, the most common indication for OLT has been non-cholestatic cirrhosis which represents approximately 72% of the waiting list [1]. For patients with chronic liver disease or cirrhosis, the difficulty is not deciding if the patient would benefit from OLT, but rather, when they would benefit. On the one hand, the more fit the patient, the less the morbidity and mortality of the procedure; on the other hand, transplantation too early would place the patient at risk of death and complications for less benefit. In general, patients with a MELD of less than fifteen have a greater risk of death from OLT than they would from remaining on the list [444]. The general indications for listing patients for liver transplantation are life expectancy (due to liver disease) of less than 1 year or quality of life (due to liver disease) which is unacceptable.

8.2.3.1 Specific Indications Viral Hepatitis It is rare for acute hepatitis A virus infection to lead to OLT. When it does, patients present with fulminate liver failure. Hepatitis B virus (HBV) may present as fulminate liver failure or as cirrhosis. The advent of effective antiviral therapy for HBV has revolutionized therapeutic options for HBV. High pretransplant viral loads are very predictive of HBV recurrence in the transplanted organ and are associated with poor outcomes [208]. Hepatitis B immunoglobulin (HBIG) prophylaxis combined with antiviral therapy can prevent HBV reinfection of the graft. Thus, patients transplanted using variants of this protocol have extremely

low rates of significant HBV recurrence following transplantation. In addition, these protocols have allowed the safe use of HBV core antibody positive donor organs in these patients. HCV infection is one of the major causes of cirrhosis and accounts for approximately 30–40% of OLTs performed worldwide [662], and it accounts for the largest portion of patients on the current liver transplant waitlist (based on OPTN data, 2007). Since 1991, the number of patients listed for OLT secondary to HCV has grown, and this trend is expected to continue for the next 20 years [138]. Recurrence of hepatitis C in the transplanted graft is nearly universal; however, the short and medium term patient survival following transplantation does not differ significantly from HCVnegative patients who receive transplants for nonmalignancy related indications [73, 236]. Most patients will develop chronic hepatitis that has a similar clinical course to non-transplanted HCV infected patients; however, progressive HCV disease after transplantation represents the leading cause of death, graft failure, and retransplantation in this group of patients [182]. There is no standard antiviral regimen which prevents HCV recurrence. Whether to treat clinically significant recurrence, which agents to use and timing of therapy are unanswered questions.

Alcoholic Liver Disease Alcohol use and abuse is a very common finding in the history of patients undergoing evaluation for OLT. It often serves as a cofactor in the development of liver disease in patients who undergo transplantation for other indications (e.g., hepatitis C). In addition, alcohol alone is a significant cause for liver disease requiring transplantation, and the results following transplantation are essentially equivalent to the results in nonalcoholic liver disease [54, 390, 391, 564]. The major concern surrounding transplantation for alcoholic cirrhosis is a recidivism rate of approximately 20–30%. There is little evidence suggesting that a return to drinking adversely affects graft or patient survival, but there are legitimate ethical concerns over allocating scarce organs to a substance abuser [376]. A period of abstinence prior to transplantation is a strong predictor of non-relapse following transplant, and most transplant centers require a period of 6 month abstinence before listing a patient with alcoholic liver disease for transplantation [448].

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Autoimmune Hepatitis Autoimmune hepatitis is a chronic, necroinflammatory hepatic disease of unclear etiology. It is characterized by the presence of various circulating autoantibodies, hypergammaglobulinemia, and lymphoplasmacytic necroinflammatory infiltration on liver biopsy [88, 129–131]. Autoimmune hepatitis tends to occur more frequently in females and has been shown to have an association with the human leukocyte antigen A1-B8DR3 or DR4 [129, 459]. It tends to respond to immunosuppression, and patients who do not achieve remission are at risk for developing chronic liver disease and cirrhosis. OLT is the treatment of choice for patients with cirrhosis secondary to autoimmune hepatitis, and these patients have excellent 5-year survival rates (83–92%) [17]. Following OLT, these patients have a higher rate of acute cellular rejection (ACR), and recurrence of the disease is not uncommon. Because of these factors, it is sometimes difficult to wean immunosuppression in these patients.

Cholestatic Liver Disease Primary biliary cirrhosis is an autoimmune disorder of unknown etiology. It is typically characterized by serum antimitochondrial antibodies (AMA). These patients typically have good long-term survival following OLT, but they are more prone to chronic rejection and often require slower weaning from immunosuppression [399, 403]. Recurrence of the disease is uncommon, but long-term data are lacking. The diagnosis of recurrence is made by observation of granulomatous destructive cholangitis on liver biopsy [187]. Primary sclerosing cholangitis (PSC), a chronic cholestatic disease, is frequently (70–80%) associated with inflammatory bowel disease and is a known risk factor for developing cholangiocarcinoma. The only cure for this disease is OLT, and long-term outcomes are good with 5-year survival rates ranging from 80 to 85% [246, 547, 615]. However, approximately 10% of these patients will have recurrence in the transplanted graft. A subset of these patients will have cholangiocarcinoma discovered in the explant. It remains unclear what effect this discovery has on patient survival. Biliary atresia, a neonatal progressive cholangiopathy of unknown etiology, is the most common reason for OLT in children [499, 514, 614]. Left untreated,

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biliary atresia usually leads to death by the age of 2 years [514]. A timely portoenterostomy (Kasai procedure) improves survival of the native liver, although OLT remains the ultimate treatment for most (60–80%) patients, many of these in infancy [48, 107, 317, 401, 499, 514, 608, 620, 684]. OLT is a very effective and durable treatment option for children with biliary atresia. Recent large cohort reviews of both the Studies of Pediatric Liver Transplantation registry [684] and the UNOS Organ Procurement and Transplant Network liver transplant database [48] have revealed encouraging long-term survival rates following OLT (5-year actuarial survival >85%). The improvement of live donor and split liver techniques has expanded the donor pool and extended OLT to an increased number of children with biliary atresia.

Fulminate Hepatic Failure Fulminate hepatic failure is a clinical entity defined by encephalopathy, coagulopathy, and jaundice in patients without a history of chronic liver disease. This may result from a number of etiologies and the exact cause is often never determined. Commonly drugs, toxins, viruses, and other liver injuries are to blame [288]. Although many liver support systems are under development, the only definitive therapy is OLT. Due to the relatively limited donor availability, patients with acute liver failure have very high waitlist mortality rates. In addition, patients with fulminate hepatic failure have a below average 5-year survival [185].

Malignancy Cirrhosis, in general, and especially HCV-related cirrhosis are risk factors for the development of hepatocellular carcinoma (HCC). Transplantation for this malignancy is based on the oncologic premise of performing a complete resection (total hepatectomy) with wide surgical margins. OLT in the setting of extrahepatic disease is contraindicated, but OLT for early stage HCC in the setting of cirrhosis has become the standard of care. The guidelines for selection of patients with HCC for transplantation are still largely governed by the Milan Criteria based on the results of Mazzaferro and colleagues [433]. This group obtained a 75% 4-year survival in patients with a solitary HCC

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under 5 cm in diameter, or 3 or fewer nodules all measuring less than 3 cm in diameter. Other groups have validated these results, routinely achieving 5-year survival rates between 60 and 75% [242, 689, 732]. Improved survival in such patients receiving neoadjuvant transarterial chemoembolization has been reported [64]. In addition, there are trials currently examining multimodality therapies to clinically down stage a HCC to within the Milan criteria and subsequently perform OLT. There is no effective medical therapy for cholangiocarcinoma, and less than 30% of patients are surgically resectable [23]. Patients who are able to undergo a potentially curative resection have reported 5-year survivals between 8 and 44% [23]. Transplantation for cholangiocarcinoma is controversial, and most centers have abandoned this as an indication for liver transplantation [239, 309, 445]. The largest review available reported 1-, 2- and 5-year survivals of 72, 48, and 23%, respectively, with 50% 2-year recurrence rate in 207 patients who underwent liver transplantation for cholangiocarcinoma [445]. However, recent trials in highly selected patients using specific neoadjuvant protocols have shown encouraging results. Heimbach et al. have reported 56 patients with unresectable, stage I and II hilar cholangiocarcinoma of which 34 ultimately underwent OLT. All 34 transplanted patients had a negative staging laparotomy and underwent neoadjuvant therapy with external-beam irradiation, systemic 5-FU, brachytherapy with 192Ir plus oral capecitabine prior to liver transplantation. The actuarial posttransplant 5-year survival was 82% [279, 280]. Further prospective trials are underway at other centers to validate these results. Hepatic malignancies account for a small portion of pediatric liver transplantation. Hepatoblastoma is by far the most common followed by HCC and undifferentiated embryonal sarcoma. In contrast to adults, there is usually no predisposing chronic liver disease. The traditional teaching has been to consider transplantation only in cases in which the tumor is unresectable [504]. However, improvements in outcome following pediatric OLT in general, improved success in pediatric patients transplanted for hepatic malignancies with or without chemotherapy, and the substantially diminished outcome following salvage transplant are beginning to change the algorithm of treatment for these patients so that OLT may be considered as primary therapy at some centers [36, 113].

Metabolic Disease There are a number of inborn errors of metabolism that may be cured by OLT. These rare indications for liver transplantation are sometimes not recognized by nontransplant physicians and surgeons leading to delays in potentially curative therapy. In general, these diseases are caused by a liver specific enzyme or may be corrected by correcting a liver enzyme only. A list of the metabolic disorders most commonly treated by OLT can be reviewed in Table 8.3.

Uncommon Indications In addition to inborn errors of metabolism, there are other more rare indications for liver transplantation which should be mentioned. Cystic fibrosis is the most commonly fatal, autosomal recessively inherited disease in the white population. Approximately one third of patients with cystic fibrosis will have significant liver disease from secondary biliary cirrhosis. Many of these will require OLT with or without simultaneous lung transplantation [118]. Budd-Chiari syndrome is caused by focal or diffuse hepatic vein thrombosis. It is associated with congenitally present webs in the inferior vena cava (IVC), thrombophilic syndromes, and myeloproliferative disorders

Table  8.3  Inborn errors of metabolism in which liver trans­ plantation is often used for curative therapy a1-Antitrypsin deficiency Wilson’s disease Tyrosinemia Urea cycle defects   Ornithine transcarbamylase deficiency   Carbamoyl-phosphate synthetase deficiency   Argininosuccinate synthase deficiency Disorders of amino acid metabolism   Maple syrup urine disease   Methylmalonicacidemia   Propionicacidemia Disorders of carbohydrate metabolism   Galactosemia   Fructosemia   Glycogen storage disease type IV Hyperoxaluria type 1

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[589]. OLT is required in those patients presenting with acute Budd-Chiari syndrome and fulminate hepatic failure. In addition, long standing Budd-Chiari syndrome leads to chronic liver failure and cirrhosis. Many of these patients will require OLT. These patients should be evaluated for hypercoagulable states when possible before OLT and, in general, they should all receive anticoagulation following transplantation. Other conditions such as sarcoidosis, polycystic liver disease, hepatic adenomatosis, neuroendocrine tumors, and massive hepatic hemangiomas or hamartomas are all extremely rare indications for OLT and should be evaluated on a case-by-case basis.

Retransplantation The percentage of patients on the waiting list who have had a previous OLT decreased over the last decade to approximately 3% in 2004 (based on OPTN data as of 2007). This decrease is primarily because the total number of patients listed has risen over the same time period. One large single center study has recently estimated that up to 15% of patients will ultimately require retransplantation [306]. The most common indications are primary nonfunction (46%), HAT (29%), and acute rejection [306]. Survival following retransplantation is significantly lower with reported 5-year survival rates of approximately 50% [417]. Factors portending a worse outcome following retransplantation include preoperative mechanical ventilation, HCV infection, acute renal failure, elevated bilirubin, and prolonged donor cold ischemia [416, 732]. The results of retransplantation for recurrent HCV are poor and most centers consider this a contraindication to retransplantation.

8.2.4 Donor Selection The theoretical ideal donor is an otherwise healthy, hemodynamically stable young person who has suffered an irreversible traumatic head injury resulting in brain death. However, the increase in the need for donor organs has resulted in the increasing use of less than ideal donors. While there are some absolute contraindications to the use of donor livers such as extracranial malignancy, overwhelming sepsis, and cirrhosis; “marginal” donors are increasingly being used. There

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are no absolute criteria to define a marginal liver donor, but most authors consider age >60, greater than 30% macrovesicular steatosis, presence of viral hepatitis, organ damage, ongoing acidosis, and/or need for vasopressor support to constitute a marginal liver in which the risk of delayed graft function or primary nonfunction to be greater [204]. There are several reports of successful use of selected older donor livers, and these organs do not seem to affect recipient outcomes if selected properly [178, 249, 730]. Hepatic steatosis is the most prevalent underlying condition found in potential donor livers, and is the most important donor graft variable affecting posttransplant graft function [490]. Studies have shown that graft and recipient survivals are not influenced by the degree of microvesicular steatosis [301, 681], but the quantitative estimate of macrovesicular steatosis can be used to estimate the risk of primary nonfunction. In general, donor livers with <30% macrovesicular steatosis have no increased risk of primary nonfunction (3%), and moderately steatotic livers have an increased risk (13%) [301]. Grafts with greater than 60% macrovesicular steatosis should not be used as the risk of primary nonfunction is exceedingly high [592]. The proper selection of donors who are marginal secondary to steatosis does represent a safe and effective method of increasing the donor pool [650]. Positive hepatitis serologies in the donor represent relative contraindications. Before the effective use of HBIG and antiviral therapy for HBV, the use of a hepatitis B core antibody (HBcAb) positive donor resulted in a high risk of transmission of the virus to the recipient [189]. In addition, recipients with HBV were more likely to have recurrent disease if the donor was HBcAbpositive [313]. At many centers HBcAb-posi­tive donor organs that have no evidence of HBV parenchymal disease are routinely offered to patients whose indication for transplantation is HBV-related. HBV antiviral prophylaxis is employed in all of these patients and essentially prevents HBV recurrence. Transplantation of HCV antibody positive donors into HCV antibody positive recipients appears to have no detrimental effect on graft or patient survival [560, 690]. HBV and HCV positive donors represent a viable and safe option to increase the donor pool. However, histologic evaluation of the graft is mandatory during the donor evaluation phase. Only those grafts with minimal fibrosis and minimal inflammation on histology should be used for transplantation.

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Other strategies to increase the donor pool include split liver donation, live donor liver transplantation and non-heart-beating donation (or deceased cardiac donor). Split liver donation is a surgical technique that creates two usable allografts from one deceased donor organ. This technique can provide grafts for two patients from one donor while still providing outcomes comparable to whole organs [545, 729]. Living donor transplantation has been used for transplant in small children and infants since the early 1990s and has become well standardized and accepted. The first living donor liver transplant for an adult was performed in 1996 and gained rapid acceptance. Living donor transplantation is appealing because it allows elective transplantation after optimizing the health of the recipient. The disadvantages include a significant morbidity and rare mortality in the donor population [82]. Despite the theoretical advantages, outcome following live donor transplantation in adults is slightly worse than expected. Reported 1-year graft survivals are 15% lower than in patients receiving deceased donor organs [659]. Living donor liver transplantation for adults continues to evolve. However, with the current decea­ sed donor allocation system favoring the more urgent patients, it has a limited role at most centers. Currently, a National Institutes of Health multicenter study evaluating live donor liver donation is underway. Donation after cardiac death (DCD) donors are generally patients with severe neurologic injury with no chance of meaningful recovery, but who do not meet brain death criteria. Family members and the treating physicians must elect to withdraw support independent of and before an OPO coordinator approaches the family regarding consent for organ donation. Following withdrawal of support and declaration of death, which must be made by the treating physician, a rapid organ recovery operation is mandatory. The experience with DCD grafts is still in evolution and donor selection criteria are not fully defined. Several single center reports suggest that appropriate selection of DCD livers results in 1-, 3-, and 5-year patient survivals similar to brain death donors [117, 213, 363, 410]. However, there may be a slight decrease in graft survival [213, 410]. Early reviews reported the primary nonfunction rate as high as 12%, but more recent series showed a primary nonfunction rate and a vascular thrombosis rate equivalent to standard brain death donors [9, 117, 132, 213, 410]. The overall biliary complication rate is similar to brain death donors,

but the sequelae in the DCD liver is much greater and more often leads to graft loss [213, 363]. These biliary complications are related to the donor warm ischemic time and often present with diffuse cholangiopathy (based on OPTN data, 2007). While DCD livers do successfully expand the donor pool, their use at this point should be limited to non-steatotic livers with short (<20–30  min) donor warm ischemic time and short cold ischemic time (based on OPTN data, 2007).

8.2.5 Operative Techniques 8.2.5.1 Donor Hepatectomy There are many well-described techniques used to procure donor livers in standard brain death donors [546, 632]. In brief, there are two portions to the donor operation. The first is the warm dissection phase in which the abdomen and chest are entered via a long midline laparotomy and sternotomy. The liver is mobilized by dividing the umbilical, falciform and left triangular ligaments. The porta hepatis is dissected carefully to inspect the hepatic arterial anatomy and identify any replaced or accessory arteries which may be present in up to 50% of cases. The distal common bile duct is identified and ligated, and the biliary system is flushed with saline via a cholecystostomy. Next, the right colon and small intestine mesenteries are mobilized and reflected medially to expose the aortic bifurcation, the IVC, the left renal vein and the superior mesenteric artery. Control of the distal aorta for subsequent cannular placement is obtained during this step. The inferior mesenteric vein is identified, controlled and cannulated to prepare for flushing of the portal system. Next, attention is turned to the diaphragmatic crus and control is obtained of the supraceliac aorta. The warm dissection phase usually takes 30–45 min. The cold dissection phase occurs after the liver, pancreas, and kidney mobilization phases are complete and in coordination with the thoracic organ procurement team (if present). Just prior to organ flushing with preservation solution, intravenous heparin is given to the patient (400 U/kg IV). Following heparin administration, the distal aorta is cannulated and the supraceliac aorta is crossclamped. The patient is exsanguinated via the supradiaphragmatic vena cava and the aortic

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and portal cannulas are flushed with cold preservation solution (typically University of Wisconsin (UW) solution or histidine-tryptophan-ketoglutarate (HTK) solution). At this point cold slush is applied topically to the organs for cooling. Following satisfactory cooling, the porta hepatis is further dissected by dividing the gastroduodenal artery, splenic artery and left gastric artery (assuming normal hepatic arterial anatomy) and the celiac trunk is divided off of the aorta preserving a cuff of aorta. This maneuver must be modified in the presence of aberrant arterial anatomy. When an accessory left hepatic artery is present, this should remain in continuity with the left gastric artery and the celiac axis. An accessory right hepatic artery is maintained in continuity with the superior mesenteric artery and is removed with a carrel patch from the aorta. Next, the portal vein is dissected and divided at the level of the pancreas, and the IVC is divided above the renal veins. The diaphragm is dissected around the suprahepatic vena cava and all tissues between the right kidney and liver are divided. The liver is then removed and taken to a cold back table preparation area where it is further flushed and packaged in ice.

8.2.5.2 Recipient Hepatectomy While the recipient operation will not be presented in detail in this chapter, there are many excellent descriptions of the procedure [238, 296, 336]. Removal of a diseased liver from the transplant recipient can be a very challenging technical exercise. It is often complicated by marked portal hypertension, coagulopathy, and extensive collateralization of venous drainage, a friable yet firm liver, portal venous thrombosis and prior abdominal operations. The operation is usually carried out via a bilateral subcostal incision with an upper midline extension. After mobilization of the liver from its diaphragmatic attachments, the right and left hepatic arteries and common bile duct are ligated in the hilum of the liver. The portal vein is skeletonized from the hilum to the confluence of the superior mesenteric vein and the splenic vein. Next, the retrohepatic dissection is carried out which requires meticulous control of many short hepatic veins, which drain directly into the vena cava. This portion of the operation is often assisted by division of the portal vein with or without portal venous decompression (e.g.,

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portacaval shunt or venovenous bypass). We prefer an end-to-side anastomosis between the divided portal vein and the IVC, which is then taken down upon implantation of the donor graft. With the liver dissected off the IVC, an angled vascular clamp is placed across the hepatic veins at the point of entry into the suprahepatic vena cava and the liver is removed sharply leaving cuffs of hepatic vein present on the vena cava (Fig. 8.2). This is the “piggyback” technique, which is a popular alternative to the traditional technique, which requires complete occlusion and resection of the infrahepatic vena cava. Back table preparation of the donor liver is carried out in cold preservation solution. This step removes excess connective tissues and delineates the liver vessels. Preservation of aberrant arterial anatomy is essential and reconstruction of these vessels should be carried out to create one inflow artery when they are present. The traditional OLT procedure requires four vascular anastomoses: suprahepatic vena cava, infrahepatic vena cava, portal vein, and hepatic artery. The piggyback technique allows for a single caval anastomosis (donor suprahepatic vena cava to recipient confluence of hepatic veins) and subsequent oversewing of the infrahepatic vena cava. Before creating the infrahepatic vena caval anastomosis (or oversewing) the donor graft should be flushed with cool saline or lactated ringers via the portal vein to flush the preservation

Fig. 8.2  In the piggyback method, the recipient total hepatectomy is completed leaving the inferior vena cava intact. The confluence of the left, middle and right hepatic veins is clamped and the cuff prepared from this venous confluence will be anastomosed to the suprahepatic vena cava of the donor liver. In some instances, a temporary portal vein to vena cava anastomosis is created to prevent mesenteric congestion as well as to improve recipient hemodynamics during the anhepatic phase

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solution, which is often high in potassium and may perpetrate cardiac arrhythmias following reperfusion. A portal anastomosis is usually performed end-to-end and the liver is reperfused via the portal system. There are several methods to perform the arterial anastomosis, but the key principle is to ensure pulsatile inflow through a large caliber vessel over a short length. After completion of the vascular anastomoses, biliary continuity is restored via a choledochocholedochostomy or a Roux-en-Y choledochojejunostomy. The procedure of choice depends on the size match of the bile ducts, the viability of the ducts, and any ductal pathology (e.g., recipient PSC).

Table 8.4  Known risk factors for graft primary nonfunction Donor factors   Age >65   >30% macrovesicular steatosis   Peak sodium >155 mEq/L   Use of multiple pressors   Long ICU stay  Prolonged interval between brain death and organ procurement Procurement factors   Cold ischemia time >12 h   Deceased cardiac donor (DCD) Recipient factors   Retransplantation   Severely ill/high MELD   Use of high dose of multiple pressors   Renal failure

8.2.6 Complications Complications following OLT can generally be divided into three groups including technical complications, complications arising from immunosuppression, and disease recurrence. Complications that are generally considered technical in nature include HAT, portal venous thrombosis, biliary leak or stricture, and to some extent primary nonfunction. Infection and metabolic disturbances are generally considered complications of immunosuppression. Finally, acute and chronic graft rejection and recurrence of certain causes of original liver dysfunction (e.g., HCV) ultimately may lead to graft loss.

8.2.6.1 Primary Nonfunction (PNF) When early graft failure occurs and no causal factor can be found, the diagnosis of exclusion is PNF. Over the last 2 decades, the rates of PNF have remained relatively consistent between 4 and 7% [306]. It is the indication in between 38 and 46% of patients who require retransplantation [30, 306]. The diagnosis of PNF is usually made within 72 h of OLT, although the signs and symptoms usually are recognized earlier. Ominous signs include persistent lactic acidosis, worsening coagulopathy, and absence of bile production during the transplant procedure. While there are no absolute laboratory values indicative of PNF, serum transaminases in the tens of thousands imply severe organ damage and unlikely recovery. A PNF rate of greater than 40% has been reported with aspartate

transaminase (AST) levels greater that 5,000 [556]. Rather than any one test, the trends over time are more important in estimating graft viability. There is no clear etiology of PNF and most authors believe it to be multifactorial. A list of known risk factors can be found in Table 8.4. Knowledge of the risk factors and avoidance of grafts with multiple risk factors are likely the best strategy for prevention of PNF. Biopsy of grafts with PNF usually shows massive necrosis. It usually requires urgent retransplantation.

8.2.6.2 Hepatic Artery Thrombosis (HAT) HAT is estimated to occur in 2–10% of adult OLT patients with a similar rate of occurrence in the pediatric population [290, 378, 421, 683]. The need to reconstruct multiple hepatic arteries, the use of aortic conduits, small caliber vessels, and the use of prosthetic vascular grafts are all risk factors for HAT [421, 522, 701]. HAT can be broadly divided into early (acute) and late (delayed) HAT. In general, HAT within the first 1 or 2 months following OLT is defined as early. Because the transplanted liver has no collateral arterial circulation, early HAT can result in massive hepatocellular and biliary injury. In particular, the biliary tree receives its entire blood supply via the hepatic artery. Consequently, bile leak following OLT, especially those presenting with cholangitis or sepsis, should raise suspicion for early HAT. Although there is frequently a marked elevation of transaminases and

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impaired hepatic synthesis, these findings are not universal. It has been estimated that one third of early HATs are asymptomatic, one third develop ischemic biliary syndromes (i.e., cholangiopathy), and the remainder experience fulminate hepatic failure [521]. The diagnosis can be confirmed in virtually all cases with duplex ultrasonography and visceral arteriography is the “gold standard.” The diagnosis can also be confirmed via exploratory laparotomy. In addition, attempted revascularization is indicated in patients who are experiencing graft failure secondary to acute HAT. Hepatic thrombectomy and revascularization has been reported to have patient survival rates of approximately 90% [521]. However, successful graft salvage is dependent on early recognition and diagnosis. Patients who show continued signs of graft failure following revascularization should be listed for urgent retransplantation. Late HAT usually has a milder course than early HAT. Generally, late HAT presents with fever secondary to a biliary abscess, biliary strictures, or recurrent cholangitis, but rarely with acute graft failure [259]. The treatment for late HAT requires control of the biliary sepsis via drainage or stenting and retransplantation when acute liver failure or secondary biliary cirrhosis occurs. The prevailing opinion is that late HAT is less likely a technical complication [510] , but more likely related to nonsurgical factors such as tobacco abuse, intrinsic coagulation disorders (e.g., factor V Leiden), and the presence of circulating antiphospholipid antibodies [509, 530]. In addition, cytomegalovirus (CMV) infection has been implicated as an inciting factor for late HAT [259]. However, it remains unclear if continued CMV antiviral prophylaxis reduces the HAT rate. The routine use of low dose aspirin has been reported to decrease the incidence of HAT with minimal to no side effects such as hemorrhage [278, 701]. It is our practice to routinely give low dose aspirin therapy to all OLT recipients at our center.

8.2.6.3 Portal Vein Thrombosis (PVT) PVT occurs less frequently than HAT, occurring in less than 2% of adult recipients and 10% of pediatric recipients [24, 369, 450]. Low velocity portal flow, small diameter veins, pretransplant PVT, preduodenal portal vein, and the use of vascular grafts are known risk factors for PVT [24, 114]. PVT is typically asymptomatic, but can present with acute fulminate

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graft failure if it occurs early following OLT. Asymp­ tomatic PVT can lead to sequelae of portal hypertension such as ascites or variceal hemorrhage. As with HAT, the diagnosis is easily confirmed with duplex ultras­onography. The treatment of late PVT generally only requires the management of portal hypertensive complications, but PVT presenting with acute graft failure requires urgent exploration for an attempt at revascularization. Acute graft failure from PVT that cannot be revascularized requires retransplantation.

8.2.6.4 Biliary Complications Biliary complications are reported to arise in between 7 and 29% of patients following OLT. Biliary strictures occur twice as frequently as biliary leaks [45, 531]. There is a clear association between HAT and biliary complications as discussed above, and HAT should be ruled out in any patient with a biliary complication. Biliary complications are also reported to occur more frequently in partial and live donor grafts [450]. The method of biliary reconstruction has been reported to influence the biliary complication rate with Roux-en-Y choledochoduodenostomy reported to have higher complication rates than choledochocholedochostomy [495]. The use of T-tube decompression has also been reported as an independent risk factor for biliary complications [531]. It is argued that these serve as markers of patients at high risk of biliary complications rather than independent causes. For example, these maneuvers are often used in patients with small caliber bile ducts, recipient biliary pathology, or high risk grafts (e.g., non-heart-beating donor). The treatment of biliary complications can usually be managed non-operatively using percutaneous or endoscopic procedures. Intraabdominal biliary collections should be percutaneously drained and patients with symptoms of cholangitis should be treated with broad spectrum antibiotics and undergo urgent biliary decompression. If endoscopic and percutaneous methods fail or if there is complete anastomotic disruption, operative revision to a Roux-en-Y choledochojejunostomy is the treatment of choice. Retransplantation is rarely required for appropriately managed biliary complications in the absence of HAT. A multimodality approach to the treatment of post OLT biliary complications results in patient and graft survival rates of 83 and 80% [695].

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8.2.6.5 Infection Infection is the leading cause of death following OLT in both the adult and pediatric populations [24, 306]. It is estimated that two thirds of all OLT recipients will experience at least one serious infectious episode despite prophylactic regimens [718]. OLT patients are necessarily immunosuppressed, but other risk factors for infections include malnutrition, chronic illness, and multiple blood transfusions. These patients undergo lengthy and complex surgical procedures which further places them at risk. Appropriate antimicrobial prophylaxis can reduce the postoperative infection rate. The current prophylactic regimen employed at Washington University in St. Louis is shown in Table 8.5. Bacterial pathogens are the most common infectious agents in the early postoperative period. Diabetes mellitus which is exacerbated by steroid administration in the post OLT period is an independent risk factor. Our center employs a tight glucose control regimen in these patients and studies are underway to determine the efficacy of these regimens on the prevention of bacterial infections. Gram-negative biliary tract and gut derived pathogens are frequent culprits following OLT, but methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE) are becoming increasingly prevalent at transplant centers [506]. Risk factors for these infections include indwelling vascular catheters, and other sources include surgical site infections, biliary complications, and post­ operative pneumonia. Table 8.5  Antimicrobial prophylaxis for liver transplantation at Washington University in St. Louis Bacterial  Routine use of broad-spectrum antibiotics prior to incision and for 24 h postoperatively Fungal   Fluconazole once weekly for 6 weeks postoperatively Pneumocystis carinii   Trimethoprim-sulfamethoxazole for 1-year postoperatively  Pentamidine or dapsone in patients with sensitivity or contraindications to Bactrim Viral  Ganciclovir (Cytovene) or Valganciclovir (Valcyte) postoperatively for 6 months in the setting of CMV-positive donor transplanted into a CMV-negative recipient (high risk patients)  Acyclovir (Zorivax) postoperatively for 3 months in CMV-positive recipients or who receive a CMV-negative donor (low risk patients)

CMV is the most common viral pathogen encountered by OLT recipients. Without prophylaxis, the overall incidence in this population is 50–60% with most clinically apparent infections occurring 3–12 weeks post OLT [238]. CMV infections can present as fever, gastrointestinal complaints, or transaminitis. The diagnosis is made with detectable viral replication by polymerase chain reaction (PCR). The routine use of ganciclovir has been shown to dramatically curtail the incidence of CMV infection in OLT patients and other agents are currently being investigated [718]. Prolonged treatment can be very costly with little efficacy in low risk patients (seronegative donor and recipient). Our current strategy is to preserve prolonged therapy for seronegative patients receiving a seropositive graft or in patients with active CMV infection (Table 8.5). Invasive fungal infections are usually caused by Candida or Aspergillus species, cryptococcus, and non-Aspergillus mycelial fungi. About 9% of OLT recipients will experience an invasive fungal infection [612]. Risk factors for Candida infection include biliary complications, renal failure, and retransplantation. Routine prophylaxis with fluconazole significantly decreases the candidal colonization following OLT, but Aspergillus is not sensitive to fluconazole. Currently there is no literature support for routine Aspergillus prophylaxis [612].

8.2.6.6 Rejection As novel immunosuppressive regimens have been advanced, the lifetime risk of rejection following OLT has significantly decreased. The frequency of an ACR episode within the first year following OLT is approximately 50% which rarely affects graft function. However, about 10% of these patients will ultimately develop chronic rejection, which can lead to graft failure and potentially to retransplantation. In addition there are clear subpopulations of patients who are successfully weaned entirely from immunosuppression following OLT [429, 430]. It remains an area of great research interest to determine which factors allow such weaning of immunosuppression. In depth discussions of rejection in liver transplantation can be found elsewhere in this text. In general, rejection following OLT can be divided into hyperacute, acute and chronic rejection. Hyper­ acute rejection is rarely seen and is classically thought

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to be caused by circulating antibody to the graft endothelium. This usually results from transplantation of incompatible blood groups without proper desensitization protocols. This type of rejection usually leads to rapid graft destruction and requires retransplantation. ACR is usually seen early in the posttransplant course (e.g., 7–30 days) although it can be seen later. Generally, this type of rejection is detected by elevations in the hepatic transaminases and confirmed with liver biopsy. It is usually easily treated with bolus steroids and does not reduce overall graft survival. However, acute rejection can be difficult to distinguish clinically and pathologically from recurrent HCV and before increased immunosuppression is given the diagnosis should be confirmed. Repeat biopsy may be useful in unclear cases. Chronic rejection is characterized by a progressive loss of bile ducts, obliteration of small and medium sized arteries. Today, less than 2% of patients experience chronic rejection and it often leads to graft loss.

8.2.6.7 Renal Dysfunction Almost every recipient who undergoes OLT will experience some degree of renal impairment. This is usually encountered in the early postoperative period as oliguria and transiently increased serum creatinine. Risk factors are pre-existing renal dysfunction, delayed graft function, intraoperative hypotension, and primary nonfunction. Some patients will require short term hemodialysis, but renal recovery generally occurs within 2–3 weeks. Chronic renal dysfunction is increasingly recognized following liver transplantation and is a source of considerable morbidity. This occurrence is largely attributed to nephrotoxic chronic immunosuppressive therapy. There are ongoing clinical trials examining nephron-sparing immunosuppression regimens. While some have encouraging results, others have shown little improvement in renal function following calcineurin inhibitor withdrawal [564, 603]. Further studies in this area are needed.

8.2.6.8 Metabolic Complications As increases in overall survival following OLT have been achieved, it is increasingly recognized that recipients are predisposed to many long-term medical

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illnesses. A common sequela of immunosuppression is the development of hypertension and hyperlipidemia. Hypertension is observed in 70% of OLT patients at 1 year, and almost 40% of patients develop hyperlipidemia during the same time frame [526]. De novo diabetes mellitus occurs in at least one third of previously nondiabetic OLT recipients [601]. HCV is a known risk factor, but the mechanisms governing this association are not understood. In most instances, the diabetes resolves over the first posttransplant year, but the need for diabetes education and tight glucose control in all OLT patients is paramount. Almost all OLT patients will experience some osteodystrophy related to steroid therapy, bed rest, poor nutritional status, and cholestasis. Osteoporosis is most common in the first 6 months following OLT and these patients require calcium and sometimes vitamin D supplementation along with serial bone density measurements.

8.2.6.9 Posttransplant Lymphoproliferative Disorder (PTLD) An in depth discussion of PTLD and other posttransplant malignancies is found in other chapters. In brief, PTLD is a life threatening complication of chronic immunosuppression. It is strongly associated with replicating Epstein-Barr virus (EBV) in B cells and has primarily been observed in patients who received antilymphocytic antibody induction therapy [407, 526]. PTLD has a low incidence in OLT patients and the prognosis depends on the histological characteristics of the tumor. Polyclonal PTLD is usually successfully treated with reduction or removal of immunosuppression and antiviral therapy. Monoclonal PTLD usually requires chemotherapy, radiation therapy and/or surgical resection [526].

8.3 Approach to the Liver Transplant Recipient: Maintenance of Allograft Function Jeffrey S. Crippin This chapter will present and discuss the management of liver transplant recipients from the perioperative period

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and beyond. Pathologic findings and how they guide clinical decision making will be the key to much of this chapter. Keep in mind that practice patterns and approaches vary from transplant center to transplant center. As in any aspect of clinical medicine, evidence based medicine is the basis of most decisions. However, due to the relatively new specialty of transplantation medicine and the evolution of new pharmaceutical agents, anecdotal experience frequently comes into play.

8.3.1 Immunosuppressive Agents Although tacrolimus based immunosuppression represents the most frequently used regimen in the United States [209], treatment decisions are often based on specific characteristics of a specific patient. The literature is full of trials of different immunosuppressive regimens. This section will address each of the agents and outline general approaches to transplant recipients and their problems. Calcineurin inhibitors remain the backbone of immunosuppression in liver transplantation. Cyclo­ sporine markedly improved survival rates when introduced in the United States in 1984. Within lymphocytes, cyclosporine binds to cyclophilin. This complex binds to calcineurin, leading to inhibition of the formation of interleukin-2 (IL-2), a potent cytokine and stimulus of lymphocyte proliferation, as well as other activated lymphocyte generated cytokines [649]. Drug levels are followed in order to maximize the therapeutic effect, while minimizing toxicity. Immediately following the transplant, trough levels of 200–300 ng/mL and peak levels (“C2” levels drawn 2 h following ingestion) of 750–1,000  ng/mL are targeted. Higher levels than those outlined can lead to neuro- and nephrotoxicity. Neurologic effects range from tremulousness to headaches to convulsions to blindness, though the latter two are rarely seen. Nephrotoxicity can result in azotemia and acute renal failure, the latter is reversible as levels decrease. Hyperlipidemia, diabetes mellitus, and gingival hyperplasia are long-term complications related to calcineurin inhibitor therapy, though other cofactors, such as body habitus and family history contribute, as well. As patients progress following their transplant, cyclosporine levels are routinely decreased, due to the increase in immunologic tolerance seen with time. Unfortunately, no single formula predicts

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long-term control of acute rejection, though trough levels usually are dropped to 100–200 ng/mL in months 6–12 following the transplant and to 50–100  ng/mL after the first anniversary of the transplant. Some patients may continue to remain free of acute rejection in spite of cyclosporine trough levels in the range of 25–50 ng/mL. Unfortunately, no assay currently exists that allows the clinician to use this as a target, except for trial and error and the risk of precipitating an episode of rejection. Tacrolimus binds to FK506 binding protein, and inhibits a number of cytokines, similar to cyclosporine. Trough levels are much lower than those targeted in patients on cyclosporine, with levels of 10–15 ng/mL following the transplant, levels of 5–10  ng/mL in months 6–12, and levels of 4–6 ng/mL long term. The side effect profile of tacrolimus mirrors that of cyclo­ sporine. Diabetes mellitus occurs more commonly in patients on tacrolimus [428], while hyperlipidemia is seen more often in patients on cyclosporine [69]. Lymphocyte inhibitors are routinely used in combination with calcineurin inhibitors following the transplant. Historically, azathioprine at a dose of 1–2 mg/ kg, was used for 2–3 months following the transplant. Mycophenolate mofetil (MMF) has largely replaced azathioprine in the treatment of liver transplant recipients. MMF inhibits inosine monophosphate dehydrogenase, a key factor in B and T lymphocyte proliferation. Common side effects include gastrointestinal symptoms, such as nausea, vomiting, crampy abdominal pain, and diarrhea. Bone marrow suppression may also complicate the care of liver transplant recipients, resulting in leucopenia, thrombocytopenia, and/or anemia. All side effects are reversible with lower dosing or drug cessation. MMF is usually tapered off in the first 2–4 months following the transplant, though it is frequently used long term in patients with episodes of ACR. Corticosteroids have been used in solid organ transplantation since its genesis. Practice patterns differ, though most centers give large doses, ranging from 500 to 1,000 mg at the time of the transplant, followed by a rapid taper over the course of the first few postoperative weeks. Patients are often tapered off corticosteroids within the first 6–12 months following the transplant, though some centers taper patients off within days to a few weeks. Other clinical situations (described later) may dictate ongoing treatment with low dose corticosteroids. Corticosteroids have a wide range of immunologic effects, including suppression

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of cytokine production, macrophage suppression, and a decrease in adhesion molecule expression. Unfor­ tunately, side effects related to corticosteroid therapy can be devastating and include diabetes mellitus, accelerated osteoporosis, cataracts, weight gain, and fluid retention. Polyclonal antibody therapy is often used as “induction” therapy at the time of the transplant. Some centers use this approach in all patients, though many reserve use of these agents for patients with pretransplant renal insufficiency, hoping to minimize the nephrotoxic effects of calcineurin inhibitors at the time of transplantation [169]. Polyclonal rabbit antithymocyte preparations bind multiple antigens on T and B cells, reduce the number of lymphocytes, and block T cell activation by cross linking T cell receptors [649]. Side effects include serum sickness, leucopenia, and thrombocytopenia. In patients with pretransplant renal insufficiency, calcineurin inhibitors will be started as the serum creatinine improves, a process that may take 2–4 days as the effects of the hepatorenal syndrome resolve. Historically, monoclonal antibodies to the CD3 T cell receptor were used for refractory rejection and induction therapy. Unfortunately, its use was complicated by the cytokine release syndrome, a syndrome of variable severity marked by fever, chills, headaches, and neuropsychiatric complications. Currently, this preparation is rarely used in liver transplant recipients. Monoclonal antibody therapies, directed at receptors for cytokines, continue to search for a place in the initial treatment of liver transplant recipients. Anti­ bodies to CD25, the alpha chain of the intereukin-2 receptor (basiliximab and daclizumab), are effective when used in combination with calcineurin inhibitors and corticosteroids [483]. Monoclonal antibodies to CD52 (alemtuzumab) markedly decrease B and T cells, an effect that may last for months. However, preliminary studies suggest this may lead to more severe recurrence of hepatitis C and an increased risk of opportunistic infection and malignancy. The prevention of activation of the kinase enzyme mTOR leads to effective immunosuppression without the nephrotoxicity seen with calcineurin inhibitors. Sirolimus was studied as initial therapy in liver transplant recipients, however, an increased incidence of HAT has made most centers wary of its use in the immediate posttransplant state [672]. Many centers switch to sirolimus from calcineurin inhibitor based immunosuppression in patients with progressive renal

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insufficiency. Preliminary studies have shown a mild improvement in creatinine clearance following the discontinuation of the calcineurin inhibitor [471]. Side effects of sirolimus include hyperlipidemia, oral ulcers, diarrhea, and nephrotic syndrome [74]. There is no one rule for the choice of immunosuppression following a liver transplant. To coin a phrase, “one size does NOT fit all.” Many of these decisions are based on a combination of physician preference, the disease leading to transplantation, and a number of other patient characteristics. For example, calcineurin inhibitors are the most commonly used agents following a transplant. However, whether a center uses this in combination with a lymphocyte inhibitor and corticosteroids, vs. corticosteroids alone, is often based on personal preference. A large retrospective study showed fewer episodes of late acute rejection and prolonged survival in patients started on “triple” therapy (MMF, corticosteroids, and tacrolimus) [715]. Some centers prefer to avoid the use of a calcineurin inhibitor initially, regardless of the severity of renal insufficiency, with the intent of improving renal function long term. Several preliminary studies support this hypothesis, though larger studies are needed to verify this claim [169]. As time evolves, specific disease and patient directed immunosuppression is likely.

8.3.2 Posttransplant Allograft Dysfunction: Causes and Evaluation Assessment of the liver allograft is not based on a single laboratory, radiologic, or patient finding. All aspects of the patient’s history, physical examination, and diagnostic testing must be used if an accurate diagnosis is to be established and an appropriate therapeutic intervention applied. As an example, abnormal liver biochemistries in a patient 4 weeks following the transplant is a much different finding than similar abnormalities 4 years after a transplant. Likewise, abnormalities in a patient with hepatitis C are frequently addressed differently than similar findings in a patient transplanted for other causes of end-stage liver disease. The remainder of this chapter will address allograft dysfunction in a number of different clinical settings. Although the evaluation of these abnormalities may not differ much in each case, the timing and urgency of the studies will vary, depending on the clinical setting.

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8.3.2.1 Allograft Dysfunction: What Does It Mean? From the standpoint of the clinician, allograft dysfunction is defined as any abnormality in the metabolic, excretory, circulatory, or synthetic function of the transplanted liver. Defining the cause, severity, treatability, and reversibility of the problem is a key at the bedside and in the outpatient clinic. These changes may occur in the immediate postoperative hospital room or at a distance far from the transplant center, long after the patient has returned home. The evaluation usually starts with relatively simple blood tests. Additional evaluation can include radiologic studies and/or a liver biopsy. Liver allograft dysfunction is usually tied to a micro- or macroscopic finding at the level of blood flow, bile flow, hepatocyte dysfunction, or some combination of any or all of the above. As one would expect, abnormalities in one of the above can lead to significant changes in either of the other two. Thus, any assessment of allograft dysfunction must address each of the three components, particularly if diagnostic studies evaluating one component fail to reveal the cause of the problem. For example, a rapid rise in transaminases within the first few days to weeks of a transplant raises a concern for ischemic hepatopathy, a consequence of impaired arterial flow to the allograft. In the early period following a transplant, HAT must be ruled out with an imaging study of the hepatic artery. Due to the noninvasive nature of the study, Doppler examination of the hepatic artery is a frequent first step, with hepatic arteriography serving as the definitive study. However, if both of these studies show normal flow to the allograft, hepatocyte or biliary tract abnormalities must be considered and pursued. Thus, a liver biopsy or cholangiogram may be the next diagnostic study to be performed. If flow abnormalities are found, ischemic biliary damage is common, with stricturing of the biliary tree due to the lack of collateral blood flow. Thus, one component affects another and a negative study of one means the others must be assessed. Liver allograft dysfunction is usually first detected by abnormal liver biochemistries. Immediately following a transplant, liver biochemistries are usually checked daily, with decreasing frequency as the patient’s condition stabilizes. Labs are then checked twice a week for the first few postoperative weeks,

then weekly, bi-weekly, and subsequently monthly for the long term. Many programs allow their stable patients to have their labs checked every 3–4 months once they have had the transplant for several years, unless there is some clinical indication to check them sooner. Although the AST, ALT, alkaline phosphatase, GGT, and total bilirubin are relatively poor indicators of liver function, they serve as easily obtainable tests for liver injury. Unfortunately, these studies are far from perfect, though it is unusual for an acute injury to occur in the absence of some biochemical abnormality. Chronic liver injury, unfortunately, may not present with significant biochemical abnormalities, thus, an index of suspicion, based on the clinical setting, may lead to the need for diagnostic studies. Patient symptoms can lead to a more directed evaluation. For example, right upper quadrant abdominal pain often leads to radiologic imaging of the allograft by sonography, computed tomography, or magnetic resonance imaging. Findings on these studies may lead to angiography and/or cholangiography. Finally, the liver biopsy is often an important piece of the evaluation, again, depending on the clinical setting.

8.3.2.2 Allograft Dysfunction in the Early Postoperative Setting The two main complications leading to allograft dysfunction and failure in the early postoperative period are primary nonfunction and HAT. Both can have devastating effects on allograft function and may lead to the need for retransplantation or complications resulting in death. Primary nonfunction is best described as irreversible delayed graft function that fails to improve, leading to retransplantation or death within 7 days of the transplant [524]. The diagnosis is based on the absence of other causes of allograft failure and occurs in less than 5% of all transplants. Patients often remain hypo­ tensive and jaundiced following the transplant and exhibit no significant improvement in postoperative allograft dysfunction. Due to the relative absence of allograft function, patients become coagulopathic and encephalopathic. Urgent retransplantation is life saving and the only known treatment. A number of studies have examined different agents to reverse this syndrome; however, none have proven effective. A liver biopsy is usually not necessary and is often done at

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significant risk, due to bleeding secondary to the coagulopathy. Explanted livers with primary nonfunction show profound necrosis. This syndrome is more severe than a milder entity, termed initial poor function (IPF). IPF affects up to 20% of transplanted livers and is marked by transaminase elevations in the range of 1,000–2,500, a bilirubin greater than 10 mg/dL, a prolonged prothrombin time, and/or hepatic encephalopathy between the second and seventh postoperative day. Though associated with a lower patient and allograft survival, retransplantation is not always necessary, as it is for the patient with primary nonfunction. Risks for both primary nonfunction and IPF include retransplants, older donors, prolonged cold ischemia time, and donor steatosis [524, 679]. HAT is associated with a rapid increase in transaminases, due to a decrease in arterial flow. Allograft function may deteriorate and retransplantation is often indicated. A liver biopsy is usually not necessary, as the diagnosis is established on the combination of clinical and radiologic findings. Extensive biliary ductal damage occurs due to the nature of blood flow in the liver transplant recipient. The biliary tree in the liver allograft recipient derives all of its blood flow from the hepatic artery. The native biliary tree receives collateral arterial blood flow from branches of the gastroduodenal artery, flow that disappears in the allograft. The lack of, or a marked decrease in blood flow can lead to ischemic injury to the biliary tree. Cholangiographi­ cally, strictures develop and can be confused with those seen in patients with PSC. These strictures are usually irreversible, even with improvement in blood flow. Efforts to improve flow are usually too little and too late, due to the irreversibility of the strictures. Long-term management is directed at maintaining bile flow, usually by repeated efforts at dilating the strictures through percutaneously or endoscopically placed drainage tubes and/or stents. Refractory biliary strictures can lead to bacterial cholangitis, liver abscesses, and secondary biliary cirrhosis.

8.3.2.3 Allograft Dysfunction in the First 3 Months Preservation injury occurs in every donor liver, though of varying degrees. It is secondary to allograft ischemia related to the time the donor liver is removed until the time of reperfusion in the recipient. This may result in

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prolonged elevations of the transaminases and cholestasis. Imaging studies show no evidence of biliary ductal obstruction. A liver biopsy is often not pursued, due to the clinical situation and the absence of other obvious precipitants of allograft dysfunction. However, if there is any question of another potential cause of allograft dysfunction, a liver biopsy is necessary. Histologically, cholestasis is present, along with hepatocellular necrosis; however, there is no evidence of rejection. Treat­ ment is supportive, as the changes of preservation injury usually resolve within a few weeks. ACR is most commonly diagnosed within the first month after the transplant. Unlike other solid organ allografts, hyperacute rejection, the result of preformed antibodies present prior to the transplant, is relatively rare in liver transplant recipients. Case reports exist, but for the most part, this is not a major consideration in the care of the liver transplant recipient. ACR is a common, though readily treatable, complication following liver transplantation. In the early days of transplantation, rejection was seen in 50–60% of transplant recipients, usually within 21 days of the transplant. This rate has dropped to 10–20% of cases [486]. ACR usually presents in the absence of symptoms, i.e., abnormal liver biochemistries are the first clue when seen as part of the normal weekly to biweekly labs drawn in follow up. These abnormalities usually lead to a liver biopsy, the definitive way to make the diagnosis. Although a number of other findings have been proposed as a sign of rejection, none have the reliability, sensitivity, or specificity of a liver biopsy. A liver biopsy, though often perceived as an invasive procedure associated with a high rate of complications, is actually a relatively straightforward procedure that can be performed easily at the bedside or in an outpatient setting. Contraindications to a percutaneous biopsy are usually limited to coagulopathy or the presence of ascites, neither of which is a common occurrence in the weeks following a transplant. However, thrombocytopenia related to the hypersplenism from pretransplant portal hypertension or ascites that has not resolved immediately following the transplant, may necessitate the need for an interventional radiologist to obtain adequate tissue via a transjugular approach. Although these specimens were often limited by their size in the early days of the technique, currently available needles allow adequate sampling [698]. If clotting parameters are within the comfort

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zone of the individual performing the procedure, the presence of ascites may lead to the radiologist obtaining a sample of hepatic parenchyma percutaneously, via sonographic guidance. Risks associated with a liver biopsy are related to pain, bleeding, or contact with another organ. Pain often leads to patient reluctance to have another biopsy, though premedication with an appropriate sedative often adequately decreases anxiety. Interestingly, liver transplant recipients tend to have less pain following a biopsy, likely related to the denervation of the allograft at the time it is retrieved. Bleeding may occur, even in the absence of coagulopathy, with bleeding severe enough to result in hospitalization in 0.35% of cases and death in 0.14% of cases, the latter consisting of biopsies of malignant lesions [466]. This data was from a series of patients, not all of whom were transplant recipients. Contact with another organ resulting in bowel perforation, hemothorax, or pneumothorax occurs relatively infrequently, though must be considered with the appropriate clinical picture. The relatively low complication rate and the potential effect on patient management make the liver biopsy one of the more common diagnostic studies in transplant recipients, even to the point of performing frequent biopsies to try to further define the cause of allograft dysfunction or as a follow up assessment of drug therapy. Careful inspection of the biopsy with the hepatopathologist is a key to delivering optimal patient care to the liver allograft recipient. Although rejection is frequently perceived as an end point in the management of a transplant recipient, the diagnosis may actually lead to a slightly different approach, depending on the clinical setting. For example, a patient who has had a transplant for cirrhosis secondary to alcoholic liver disease with elevated AST and ALT levels and Banff grade 2 or higher ACR on a liver biopsy, will almost always receive some type of medical treatment for ACR. In many centers, this will include a bolus dose of corticosteroids, followed by a rapidly tapering dose over the course of a few weeks. However, a milder grade of rejection may not necessarily lead to an urgent need for treatment. One study showed that untreated, subclinical rejection actually improved the long-term survival of liver transplant recipients [661]. This may be related, in part, to the role of rejection and its treatment in the induction of tolerance [706]. Furthermore, high dose corticosteroids are associated with a number of side effects, including emotional lability, risk of

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opportunistic infections, hyperglycemia, hypertension, weight gain, and fluid retention, making their use unattractive, unless absolutely needed. High dose corticosteroids have been associated with an increased risk of mortality in patients with hepatitis C, presumably related to accelerated destruction of the allograft by the HCV [110]. Thus, the approach to the patient with hepatitis C is distinctly different from patients transplanted for other causes of end-stage liver disease. Many of the histologic features of hepatitis C in the allograft are similar to those seen in patients with ACR. Bile duct injury, portal inflammation, and mild endotheliitis can appear in patients with hepatitis C, similar to the findings seen in patients with ACR [517]. In the early days of transplantation, many of these patients were treated for ACR with high dose corticosteroids, monoclonal T cell receptor antibodies, or a change in calcineurin inhibitor. Subsequent follow up of these patients showed higher rates of mortality and an accelerated course of the hepatitis C infection in the allograft [557]. This has led many centers to follow these patients closely, withholding treatment of ACR until the diagnosis has been definitively established. Even then, most clinicians will treat confirmed ACR with an increase in the dose of calcineurin inhibitor, particularly if the tacrolimus trough level is in the range of 4–7 ng/mL. Consideration is also given to the addition of MMF, a lymphocyte inhibitor. These therapies clearly do not have the same potency as a corticosteroid bolus; however, the rejection may still be controlled, without the effect on the progression of hepatitis C. Consider this example: an asymptomatic 52 year old male, 3 weeks out from an uncomplicated transplant for cirrhosis secondary to hepatitis C, presents asymptomatically with routine laboratory follow up, with an AST level of 130 and an ALT level of 107. These values are in contrast to previously normal levels 1 week ago. An abdominal ultrasound of the liver with Doppler study of the hepatic vasculature shows no parenchymal abnormalities and no evidence of impaired flow in the hepatic artery, portal vein, or hepatic veins. Based on the history and findings above, the clinician cannot differentiate between hepatitis C infection of the allograft and ACR without a liver biopsy. Histologically, there is evidence of portal infiltrates and bile duct injury, but no definitive endotheliitis. At many centers, due to the absence of definitive ACR, observation is usually the recommendation. The

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approach may vary from center to center; however, follow up labs within the week is the usual recommendation. If the liver biochemistries continue to increase, another biopsy may be obtained. This may lead to a clearer and more definitive diagnosis of ACR. If present, calcineurin inhibitor dosing may be increased and MMF added to the regimen, with close follow up of the liver biochemistries. If the liver biochemistries improve over the course of the next few weeks, further close follow up is recommended. If the numbers continue to rise, another biopsy may be performed. If severe findings of rejection are seen, high dose corticosteroids will usually be started, in spite of the risk outlined above. However, if the changes appear more consistent with hepatitis C, additional observation may be recommended or the possibility of antiviral therapy considered. Two key points are illustrated in this example: (1) in liver transplantation, waiting another week, particularly if the diagnosis is unclear, can usually be done safely; and (2) in the liver transplant recipient, there is no such thing as too many biopsies! Another problem must be considered in the above case: what if there is no evidence of ACR, but there is clear evidence of progressive hepatitis C in the liver transplant recipient within the first two postoperative months? This can create a similarly difficult situation for the clinician from the standpoint of treatment. Tragically, a small percentage of patients develop rapidly progressive hepatitis C in the allograft within the first few months of the transplant [655]. This may progress in the form of fibrosing cholestatic hepatitis (FCH), a rapidly accelerated form of allograft infection that may lead to allograft failure, need for another transplant, or death [159]. Unfortunately, many transplant centers will not retransplant patients with this form of allograft infection, due to the poor outcomes often seen in patients with this syndrome [91]. Other patients develop an acute hepatitis C infection of the allograft, characterized by a progressive increase in transaminases that may peak and subsequently slowly decrease, or may lead to continued deterioration of allograft function. Similar to the situation when rejection is suspected, follow up biopsies are helpful in managing these patients. Transaminase levels do not necessarily correlate with histologic changes, thus, even high levels of transaminases may be followed. Furthermore, there is no evidence that minimizing immunosuppression in these cases necessarily leads to an improved outcome, in spite of what logic would

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suggest. Finally, antiviral therapy with pegylated interferon alfa 2a or 2b, in combination with ribavirin, is complicated by significant side effects and poor efficacy [600]. It is unclear if this is due to the presence of immunosuppression or the need to decrease the dose due to intolerance to standard doses. Antiviral therapy is often started with the thought that treatment may lead to a better outcome than ongoing observation. Unfortunately, no controlled studies have been performed. Most centers rely on anecdotal experience and the experience of the clinician, based on the clinical situation.

8.3.2.4 Other Early Viral Infections Although ACR and hepatitis C are the main complications in the early posttransplant period, other viral infections may impact the course of the allograft recipient. Cytomegalovirus (CMV), an DNA virus in the Herpes family, may cause transaminase evaluations and/or cholestasis. CMV has been less of a problem to liver transplant recipients, due to the frequent use of antiviral prophylaxis following the transplant [225]. However, patients may still develop an infection, particularly if they had no preoperative evidence of CMV and received a donor organ from a CMV positive donor. Likewise, patients may reactivate a CMV infection if they have previously been infected. Patients may present asymptomatically with elevations of transaminases and/or alkaline phosphatase. Others may present with fever and malaise. CMV viremia may result in bone marrow suppression, particularly leucopenia and thrombocytopenia [383]. Histologically, viral inclusions may be seen and positive CMV stains are diagnostic. CMV viremia may be detected using the PCR assay [217]. The EBV is a less frequent cause of posttransplant allograft dysfunction and is usually related to reactivation of a previous infection, since 90% of adults have had previous exposure. Transaminase levels may be in the 200–1,000 range, with atypical lymphocytosis, leucopenia, and/or fever [538]. Histologic changes are outlined in other chapters of the textbook. Other potentially hepatotropic viruses include herpes simplex, a virus that may lead to acute liver failure. Although this syndrome has been well described in native livers in immunocompromised patients and pregnant women, it is a rare complication of liver transplant recipients.

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8.3.2.5 Allograft Dysfunction from 3–9 Months As the doses of immunosuppressive medications are decreased, the causes of allograft dysfunction evolve. Although many of the complications outlined above must always be considered in the liver allograft recipient, the incidence decreases, except in specific clinical situations. For example, the incidence of ACR decrea­ ses to a rate of 19% after the first 24 postoperative months [678]. These “late” ACR episodes are most frequently precipitated by low levels of immunosuppressive medications, usually related to non-compliance or illness. For example, a bout of ACR is common in patients with a prolonged viral gastroenteritis manifested by vomiting and diarrhea, preventing a patient from taking their medicines. Most transplant centers have also seen similar bouts in patients who fail to notify the center regarding a lack of medicines due to a misunderstanding or miscommunication. Although it may occur, ACR occurs infrequently outside of these clinical situations. Chronic allograft rejection is a relative misnomer. Yes, this may persist “chronically,” however, its presentation is distinctly different from ACR. By definition, presentation must be at least 3 months following the transplant and is usually asymptomatic and characterized by cholestasis [481]. The main risk factor for the development of chronic rejection is previous episodes and/or late onset of ACR [146]. Ductopenia is the predominant histologic feature, secondary to an ongoing attack on arteriolar endothelial cells with subsequent bile duct loss secondary to ischemia. In many ways, this is the microscopic version of HAT. Macroscopic bile duct injury must be ruled out, as in any case of cholestasis, with a cholangiogram obtained either endoscopically, percutaneously, or via an MRI. Furthermore, arterial blood flow must be assessed to rule out HAT. Treatment is directed at minimizing immunologic injury, usually by manipulation of the immunosuppressive regimen. Early studies showed a change in calcineurin inhibitor from cyclosporine to tacrolimus was effective [604]. Whether the addition of other agents such as MMF or an increase in corticosteroid dose improves outcomes is unclear. Arterial blood flow problems can occur at any time following a transplant. Though HAT is more common in the days following a transplant, hepatic arterial stenosis, with or without thrombosis, can complicate a transplant months to years after the transplant. A

patient presenting with the new onset of biliary strictures, biliary obstruction, or acute bacterial cholangitis should have a careful assessment of hepatic arterial flow with Doppler studies, MR angiography, or arteriography. Cholestasis is the characteristic laboratory finding. A liver biopsy may not add much to the diagnosis, if inadequate flow is documented. Efforts to maintain adequate bile flow may ultimately be ineffective if stricture formation is extensive enough. Recur­ rent episodes of cholangitis, jaundice, and/or secondary biliary cirrhosis may ultimately lead to allograft failure and the potential need for another transplant.

8.3.2.6 Recurrent Diseases The most frequent recurrent disease following liver transplantation is hepatitis C, however, other diseases leading to the need for a liver transplant can appear in the liver allograft. In each case, other causes of allograft dysfunction must be ruled out so appropriate treatment recommendations can be made. Primary biliary cirrhosis is a chronic cholestatic liver disease most commonly presenting in middle aged women with an elevated alkaline phosphatase. Symp­ toms are frequently not present, though many patients complain of pruritus, fatigue, or right upper quadrant abdominal pain. The disease is thought to be autoimmune in nature. Many clinicians think recurrence in the allograft is uncommon, due to the immunosuppressive medications used to prevent allograft rejection. The diagnosis of primary biliary cirrhosis in the native liver is based on cholestasis, presence of the antimitochondrial antibody, and characteristic histologic changes. Similar findings must be present to make the diagnosis of recurrent primary biliary cirrhosis. Obviously, for the disease to be “recurrent,” a firm diagnosis must have been made prior to the transplant. Cholestasis, in the absence of biliary ductal obstruction, must be verified [637]. Early studies of recurrent disease showed an increase in the titer of the antimitochondrial antibody, though this may be difficult to document if this has not been checked following the transplant. If biliary ductal abnormalities are not present, the liver biopsy becomes the diagnostic tool of choice. A lymphocytic portal infiltrate with bile duct damage is characteristic. This can be differentiated from ACR based on the absence of endothelial damage. Treatment of recurrent primary biliary cirrhosis may follow two pathways. The use of

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bile acid therapy in the non-transplant patient has been associated with prolonged survival [380]. However, similar large scale studies in transplant patients have not been performed, due to the 5–10% rate of recurrence in transplant recipients. Ursodeoxycholic acid is safely administered to transplant recipients and does not interact with other immunosuppressive medications. Others use the approach of treating an “autoimmune disease.” Anecdotal experience has shown the use of MMF improves both liver biochemistries and histologic damage; however, controlled studies have not been performed, due to the paucity of cases at most institutions. In rare cases, the disease is progressive and may lead to allograft failure and consideration of retransplantation. PSC is a progressive cholestatic liver disease characterized by fibrosis and stricturing of the bile ducts, ultimately leading to impaired bile flow, cholangitis, and biliary cirrhosis. Due to the findings seen in patients with poor hepatic arterial blood flow, disease recurrence cannot be diagnosed until adequate hepatic arterial flow is documented. If there is no impairment to blood flow, bile duct strictures can be documented percutaneously, endoscopically, or by MR cholangiography. Histologic changes are nonspecific, as the classic “onion skin” periductal fibrosis may not be present. Ductopenia and biliary ductal damage in the absence of endothelial damage are usually present. Based on these issues, this may be a diagnosis that is difficult to make. However, bile duct strictures will not be present in patients with chronic rejection. Treatment of recurrent PSC has been difficult to define. Bile acid therapy is not effective for PSC in patients with a native liver, yet many clinicians routinely use ursodeoxycholic acid due to its lack of toxicity and ability to promote bile flow. Similar to recurrent primary biliary cirrhosis, others have tried additional immunosuppressive medications with the hope of minimizing the potential autoimmune attack on the bile ducts. Again, anecdotal experience has seen successful treatment in occasional cases, however, controlled studies have not been performed due to the relative rarity of this syndrome, occurring in 15–20% of cases [252]. Autoimmune hepatitis recurs in the allograft approximately 5–10% of the time [163]. Elevated transaminases in the absence of symptoms is the usual presentation. Similar to primary biliary cirrhosis, autoimmune serologies are of minimal help in the diagnosis, due to their presence prior to the transplant and the absence of

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follow up levels following the transplant. Even if checked, however, a change in titer alone is inadequate for the diagnosis. Histologic changes are the rule, with a lymphoplasmacytic portal infiltrate in the absence of endothelial damage. A variant of these findings may be centrolobular damage in the appropriate clinical setting. An increase in immunosuppression is the usual treatment recommendation. An increase in calcineurin inhibitor dose/level is generally not preferred, due to the risk of renal insufficiency. Corticosteroids may be used with an initial high dose followed by a rapid taper, similar to the steroid recycle used following a bout of ACR. Azathioprine, a commonly used drug for autoimmune hepatitis in the native liver, may be added in doses up to 2 mg/kg/day. MMF is also commonly used at a dose of 1,000–1,500  mg twice a day. Addition of these medicines usually leads to both biochemical and histologic improvement. Corticosteroids routinely will be tapered off, while long-term use of a lymphocyte inhibitor is the usual recommendation. Hepatitis B is usually controlled following transplantation through the use of an antiviral agent with or without the use of high dose HBIG [29]. The latter effectively binds up any hepatitis B surface antigen circulating systemically, while the antiviral agent prevents hepatitis B viral replication in the hepatocyte. Though viral “breakthrough” can occur in the absence of inadequate dosing of either the antiviral agent or the immunoglobulin, this is relatively rare. Active viral replication is usually manifest as an increase in transaminases. Histologically, findings are similar to those seen in the native liver, including those seen with special stains for hepatitis B surface and core antigens. However, the histologic findings must be present in combination with positive hepatitis B serologies and a hepatitis B viral DNA level by PCR. Histologic changes in the absence of evidence of viral replication are confusing, and are difficult to attribute to hepatitis B. If hepatitis B viremia is documented, an increase in dose of HBIG and the addition of an antiviral agent or a change to another agent may be helpful, particularly if a hepatitis B viral mutation has been documented. Strains resistant to lamivudine, a previous commonly used antiviral agent, should lead to the addition of adefovir, a strategy that has served patients well with hepatitis B in the native liver. Patients on adefovir with resistance may require a change to entecavir or tenofovir. A severe form of recurrence called FCH, affects patients rarely, but is often associated with rapid

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deterioration of allograft function, allograft failure, need for another transplant, or death [137]. Other diseases leading to allograft dysfunction are less well studied but deserve mention. There is no documented case of severe iron overload in patients transplanted for hemochromatosis [124]. In light of the fact it takes decades for cirrhosis to occur in the native liver, the chance of iron overload being a problem in most patients would appear to be small. That being said, following a serum ferritin every year or two is reasonable. Similarly, copper overload following liver transplantation in patients with Wilson disease has not been reported. Recurrence of a1-antitrypsin deficiency has not been reported. The recipient assumes the phenotype of the donor following the transplant [693]. Nonalcoholic steatohepatitis is an increasingly common cause of liver dysfunction in the general population and may result in cirrhosis in up to 30% of cases. Obesity following the transplantation can lead to a posttransplant fatty liver [109]. This may be diagnosed by elevated transaminases and the characteristic findings seen on a liver biopsy. Weight loss through dietary measures and exercise remains the backbone of treatment.

8.3.2.7 Other Causes of Allograft Dysfunction Theoretically, the liver transplant recipient is subject to many potential causes of liver dysfunction, similar to the general population. Many of these have been outlined above. Due to the number of medicines frequently required to maintain allograft function and the side effects associated with them, drug toxicity is another common posttransplant complication. Liver transplant recipients are no more susceptible to drug toxicity than patients without transplants, however, the very fact that they have a transplant means they are frequently taking multiple medicines, thus, potentially increasing their chance of drug toxicity. De novo autoimmune hepatitis may occur in the allograft, even without a previous history of the disease prior to the transplant. The disease is characterized by classic histologic features, and the presence of positive autoimmune serologies. In one series, 82% of patients had a recent decrease in immunosuppression [194]. Treatment is centered on manipulation of immunosuppression. High dose corticosteroids followed by a taper over several months, in combination with a lymphocyte inhibitor or

a change in calcineurin inhibitor is commonly used. The goal is normalization of liver biochemistries and improvement in histologic changes.

8.3.3 Approach to the Care of the Liver Transplant Recipient Based on all of the potential causes of allograft dysfunction, it would appear there is a myriad of possibilities. Thus, how does the clinician make a decision regarding the cause of allograft dysfunction and the means of correcting it? This lesson in problem solving is no different from any other aspect of clinical medicine. All aspects of the history and physical examination play an important part in deciphering the many clues present in each clinical situation. The following questions may give some structure to the process: When was the transplant? As outlined, different problems arise at different points following the transplant. A patient with liver dysfunction years after the transplant does not have primary nonfunction. Furthermore, chronic rejection is not in the differential in a patient with cholestasis 2 weeks after a transplant. Likewise, specific infections are more likely immediately following a transplant than years later. What was the indication for the transplant? The prospect of recurrent disease in the liver allograft is a common case of allograft dysfunction. Has there been any change in immunosuppressive doses or drug levels? A decrease in the level of calcineurin inhibitor may precipitate an episode of rejection, regardless of the time following the transplant. Likewise, a recent decrease in dosing may lead to problems related to acute or chronic rejection. Does the patient have any complaints? Frequently, a recent nonspecific viral illness can lead to abnormal liver biochemistries that spontaneously resolve. However, high fever and right upper quadrant abdominal pain may mean major problems that are occurring in the allograft. Once these questions have been addressed, the clinician may decide to follow the patient for additional signs and symptoms and recheck lab results. However, if the clinical situation dictates, a number of diagnostic studies may be indicated. The basic approach involves

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assessment of blood flow, bile flow, and hepatocyte injury. Blood flow can be easily assessed with a Doppler study, usually performed with an ultrasound examination. This study allows a relatively quick assessment of hepatic arterial, portal venous, and hepatic venous flow, while ruling out bile duct dilation. Additional studies may be needed to further assess blood flow, including MR angiography or angiography. The latter runs the risk of contrast induced renal dysfunction, as well as the requirement for arterial or venous catheterization.

8.3.4 Conclusion The number of variables involved in the daily lives of liver transplant recipients makes their care incredibly challenging. However, a systematic approach to these problems, with particular attention to their timing, the history, and the results of diagnostic studies, will simplify a seemingly complex differential diagnosis.

8.4 Primary Nonfunction, Donor Liver Evaluation, Preservation And Reperfusion Injury Hanlin L. Wang The critical shortage of donor organs has led many transplant centers extend their criteria to accept livers from suboptimal donors. Although there is no universally accepted definition, extended-criteria or marginal donors may include those with advanced age, steatotic livers, prolonged cold ischemic time, inotropic support, nonheart beating status, hepatitis B or C viral infection, and extrahepatic malignancies [35, 94, 272, 487]. Studies have shown that using extended criteria to expand donor pool significantly reduces waiting list mortality and provides satisfactory outcomes to selected recipients [544, 577, 584]. However, marginal grafts appear to be more susceptible to ischemia–reperfusion injury compared with standard donors. In general, they carry a higher probability of primary nonfunction or IPF and thus subject the recipients to a greater risk of morbidity and mortality [8, 94]. For example, Briceño et al. demonstrated

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that each of the five marginal factors they examined had an independent influence on liver preservation injury. Severe preservation injury was noted in 32.7% of the grafts from donors without any marginal factor, in 46.8% from donors with one factor, in 66.2% from donors with two factors, and in 78.3% from donors with at least three factors [80]. Transplantation from donation after cardiac death has resulted in an overall lower graft and patient survival rates and a higher incidence of ischemia-related biliary complications compared with transplantation from donation after brain death, despite similar rates of primary nonfunction [202, 423, 443, 721]. Grafts from elderly donors may work as effectively as those from younger donors [104, 233, 736], but may show more aggressive recurrent hepatitis C viral infection with more rapid progression of fibrosis and early graft failure in recipients with hepatitis C [397, 539, 703]. Therefore, there is a clear correlation between graft quality and posttransplant outcome. A decision whether a marginal graft is suitable for a selected recipient is primarily made by transplant surgeons based on their assessment of donor and recipient data and gross inspection of the organ. These approaches can be subjective, however, which may lead to selecting a poor graft or discarding a liver that would function satisfactorily. In the absence of reliable functional, imaging or sonographic modalities [13, 287, 303, 699], liver biopsy for histopathologic evaluation remains the gold standard to assess the quality of the grafts before transplantation. In addition to serving the primary role of helping to more accurately estimate the degree of steatosis, histopathologic examination of the donor livers also provides baseline information for subsequent biopsy evaluations, discovers unexpected abnormalities, assists etiologic assessment of primary nonfunction, and aids in the understanding of potential future problems.

8.4.1 Primary Nonfunction 8.4.1.1 General Considerations Primary nonfunction is one of the most serious and life-threatening conditions in the immediate posttransplant period. It is defined as primary graft failure that results in retransplantation or patient death within 30

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days of initial transplantation in the absence of other identifiable causes of graft failure such as rejection, vascular thrombosis, recurrent disease, or graft-vs.host disease [323, 642]. Primary nonfunction is manifested by rapidly rising transaminases, absence of bile production, severe coagulopathy, high lactate levels, hypoglycemia, and hemodynamic instability. The diagnosis is usually made within the first week or the first 2 days on the clinical grounds and may be suspected at the end of transplant operation when the graft is observed to fail to produce bile following revascularization. Early retransplantation is the only therapy. In a study of UNOS data from 1988 to 2001 that included 761 patients who underwent retransplantation for primary nonfunction, a significantly lower short- and long-term patient and graft survival was detected compared with patients who received only primary transplantation. In addition, patients with retransplantation for primary nonfunction were more likely to lose their grafts again compared with those who underwent retransplantation for other reasons [731]. However, this latter finding is not supported by other studies, which showed that retransplantation for primary nonfunction had outcomes similar to or even better than those of retransplantation for other causes [39, 525, 679]. This discrepancy could be explained by the relatively smaller sample size of the studies, the heterogeneity of the patient population, and the different definition of primary nonfunction used in different studies. Nevertheless, patient survival is very poor if primary nonfunction occurs to retransplanted grafts [679, 731]. Sepsis, renal failure and multiorgan failure are thought to be the main causes of death in these patients. The incidence of primary nonfunction varies widely in different studies, ranging from 2 to 14%. An analysis of SRTR (The Scientific Registry of Transplant Recipients) database that included 10,545 primary liver transplants up to September 2004 revealed an incidence of 5.81% [311]. An analysis of UNOS database that included 58,576 transplant recipients between January 1990 and December 2004 showed an incidence of 3.52% [323]. Interestingly, the annual incidence is relatively stable or gradually decreases despite the increased use of marginal donors, suggesting that the donor factors are not solely responsible. Suggested risk donor factors associated with primary nonfunction include increased age, prolonged ischemia time, prolonged stay in intensive care unit, uncorrected hypernatremia,

female gender donor to male recipient, liver steatosis, and reduced graft size. Risk recipient factors may include renal insufficiency and prolonged life support [311, 524, 622, 642, 679]. The pathogenic mechanism that mediates the development of primary nonfunction remains largely unknown but is thought to be ischemia resulting from microcirculatory disturbance. In animal models, primary nonfunction involves sinusoidal endothelial cell injury, Kupffer cell activation, overproduction of proinflammatory cytokines, failure of certain antioxidant mechanisms, and release of free radicals following reperfusion, which eventually leads to a noflow status of the graft [214, 215, 228, 276, 457].

8.4.1.2 Pathologic Features Biopsied or explanted allografts may show massive or zone 3 coagulative necrosis with a variable degree of inflammatory cell response, similar to that seen in ischemic infarction or fulminant hepatitis (Figs.  8.3 and 8.4). However, since the cause of primary nonfunction is multifactorial, the allografts may show a wide spectrum of histopathologic findings reflecting different combinations of the underlying etiologies and the reperfusion time after transplantation. These may include steatosis, rupture of fat-filled hepatocytes with release of large fat droplets into the sinusoids and other extracellular spaces, preservation and reperfusion injury with centrilobular hepatocyte ballooning degeneration (Fig. 8.5), hemorrhage, necrosis, choles­ tasis and hepatocellular rosetting (Fig.  8.6). If the

Fig. 8.3  Primary nonfunction. The explanted graft shows necrosis and hemorrhage

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grafts are examined a few days after transplantation, regenerative changes may become evident featuring bile ductular reaction (Fig.  8.7), hepatocellular mitosis, binucleation and nuclear enlargement (Fig.  8.8). Inflammatory cell infiltrates and cholestasis may become more evident. In a clinicopathologic study of 8 liver allografts with primary nonfunction, Colina et al. observed the development of acute progressive microvesicular steatosis in every case [116]. Steatosis was graded as severe (involving >70% hepatocytes) and panlobular in four cases, moderate (40–70%) and centri-mediozonal in two, and mild (10–40%) and centrilobular Fig.  8.4  Primary nonfunction showing panacinar coagulative necrosis

Fig. 8.5  Primary nonfunction showing hepatocyte ballooning, microvesicular steatosis and cholestasis in a centrilobular region, similar to those seen in preservation and reperfusion injury

Fig.  8.7  Primary nonfunction showing marked bile ductular reaction with accompanied inflammatory cell infiltrates rich in neutrophils

Fig. 8.6  Primary nonfunction showing hepatocyte rosetting and marked cholestasis

Fig.  8.8  Primary nonfunction showing regenerative changes with mitosis. Note the presence of microvesicular steatosis and cholestasis

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in two. Mild macrovesicular steatosis was noted in only two cases. In five cases, steatosis was initially absent in donor livers as demonstrated by post-reperfusion biopsies performed within 2–4  h after implantation. In only three cases was fat already present before reperfusion. These three grafts clearly showed increased fat accumulation during the posttransplant period. Although the role of microvesicular steatosis in primary nonfunction remains to be understood, these observations may suggest a mechanism analogous to that occurring in Reye’s ­syndrome and acute fatty liver of pregnancy where the development of microvesicular steatosis leads to acute hepatic failure as a result of mitochondrial dysfunction with inhibition of b-oxidation of fatty acids [116, 360]. Other histopathologic changes associated with primary nonfunction observed in the study by Colina et al. included spotty single cell necrosis (acidophil or apoptotic bodies) seen in all eight cases. Zonal necrosis was seen in six cases, which primarily involved centrilobular or centri-mediozonal region. Only one case showed panlobular necrosis. In addition, hemorrhage, cholestasis, hepatocyte mitosis, and mild portal and lobular mixed inflammatory cell infiltrates consisting of neutrophils, eosinophils and lymphocytes were described [116].

8.4.1.3 Differential Diagnosis Differential diagnosis for primary nonfunction may include antibody-mediated humoral rejection, vascular thrombosis, and preservation and reperfusion injury. These entities share many histopathologic features and the distinction is primarily based on the clinical findings and clinicopathologic correlation.

8.4.2 Evaluation for Donor Liver Steatosis 8.4.2.1 General Consideration Fatty infiltration of the donor liver is a widely recognized histologic factor implicated in the development of primary nonfunction or dysfunction following

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revascularization [35, 94, 487]. In general, donor livers with severe steatosis (involving >60% hepatocytes) is associated with increased severity of ischemia–reperfusion injury that leads to a high risk of primary nonfunction or IPF, which places recipients at a considerable risk. Organs with mild steatosis (<30%) usually results in clinical outcomes comparable to non-steatotic livers. The outcomes of organs with moderate steatosis (30–60%) may vary, depending on the presence or absence of other donor and recipient risk factors [592]. In an early study by D’Alessandro et al. [133], primary nonfunction occurred in 7 of 8 livers with severe fatty infiltration, in contrast to only 1 of 26 livers with minimal to moderate steatosis and 3 of 89 livers with normal histology. Similar observations have been reported in a number of other studies [11, 524, 664, 696]. In animal studies, steatosis is associated with decreased ATP production, disturbance of sinusoidal flow and diminished regenerative capacity [592], which amplify the negative effects of cold and warm ischemia inherent in transplantation procedures [28, 186, 215, 300, 301]. However, the exact clinical impact of steatosis is still under debate. For example, it has been shown that even mild fatty infiltration may lead to significantly diminished early graft and patient survival resulting mainly from primary nonfunction or dysfunction [420]. On the other hand, studies have also shown that even severely steatotic grafts can be safely transplanted [27, 434]. In these latter studies, patients who received grafts with moderate or severe steatosis showed more significant graft dysfunction in the early posttransplant days, but the short- and long-term graft and patient survival rates were comparable to those of the control group. Interestingly, posttransplant histologic assessment showed a dramatic reduction in fat content in all the grafts that were followed by biopsies [434]. In fact, almost all the fat is cleared by the end of the first week following transplantation in both human and animal studies [338]. Though still controversial, these more recent studies challenged the dogma that organs with severe fatty infiltration should be discarded. In addition to the severity, histologic type of steatosis also has a significant impact on the outcome of the grafts. In fact, histologic type appears to be more important than severity in determining the outcome of the grafts. There are three histologic types of steatosis: macrovesicular, microvesicular, and mixed macrovesicular and microvesicular.

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Studies have shown that donor livers with even severe microvesicular steatosis can be safely used without significant adverse outcome. In a review of 40 donor livers with at least 30% microvesicular steatosis, Fishbein et al. reported an incidence of 5% for primary nonfunction, 10% for poor early graft function which did not affect patient survival, 80% for 1-year patient survival, and 72.5% for 1-year graft survival. These outcome data were no different from those of overall liver transplant population in authors’ institution. Microvesicular steatosis is commonly associated with donor obesity and traumatic death. It is reversible, usually disappearing from the grafts within 1 year in the majority of the cases [197]. Ureña et al. further confirm that grafts with >30% microvesicular steatosis can be safely utilized [682]. Although an initial dysfunction may develop following transplantation, the long-term graft and patient survival is not affected. On the other hand, severe macrovesicular steatosis has been shown to be more frequently associated with primary nonfunction [681, 682]. In a multivariate analysis, macrovesicular steatosis involving 25% or more of the hepatocytes was the only variable that was found to be independently associated with a shorter patient survival [735]. Again, microvesicular steatosis of any degree did not affect the patients’ outcome in this particular study. These observations have also been confirmed by animal studies showing that severe microvesicular steatosis does not affect graft survival but macrovesicular steatosis does [115].

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reject a liver with >30% macrovesicular steatosis irrespective of other variables, and a large proportion of the surgeons only accepted livers with <50% macrovesicular steatosis. None would accept a liver graft with >60% steatosis. However, 68% of the surgeons would accept a donor liver with a higher degree of macrovesicular steatosis in case of an urgent need. In addition, 27% of the surgeons also considered microvesicular steatosis to be a risk factor for primary nonfunction. It is thus recommended that when a donor liver is evaluated histologically, both the percentages of total fat (macrovesicular and microvesicular) and macrovesicular steatosis be reported.

8.4.2.3 Pathologic Features Assessment of the fat content in a donor liver is one of the least scientific aspects but one of the most difficult tasks in the practice of liver transplantation. Undoub­ tedly, severe steatosis can be recognized by gross examination by surgeons as yellow discoloration, rounded edges and a greasy firm texture (Figs.  8.9 and 8.10). However, gross examination is known to be unreliable and many livers with significant fatty change may be judged as normal from their macroscopic appearance [10, 316]. The positive predictive value of gross inspection at procurement was reported to be 71, 46, and 17% for severe, moderate and mild steatosis, respectively [10, 11]. Many surgeons prefer frozen section on needle or wedge biopsy of the donor liver for evaluating the

8.4.2.2 General Guidelines In general, most transplant surgeons would transplant donor livers with mild (<30%) macrovesicular steatosis and reject livers with severe (>60%) macrovesicular steatosis. Grafts with moderate (30–60%) macrovesicular steatosis may be used in the lack of other recipient and donor risk factors [487, 490]. Again, even severe microvesicular steatosis is not considered as a highrisk factor. Given the controversial nature of the issue, it is important for pathologists to be aware how the transplant surgeons in their institutions practice. In a survey of 78 transplant surgeons in the US published in 2002, the acceptable range for steatosis was found to vary widely [302]. Thirty percent of the surgeons would

Fig.  8.9  Fatty liver with yellow discoloration and rounded edges

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The standard stain for frozen section evaluation is hematoxylin and eosin (H&E) [302]. Although it may underestimate the extent of microvesicular steatosis [229, 338]. it is sufficient for the estimation of macrovesicular steatosis that is believed to be more critical as discussed previously (Fig. 8.11). Since the precise quantification is generally unnecessary, a rough estimation of the percentage of total fat and the percentage of macrovesicular fat can usually be achieved on a low-power scan (Fig. 8.12). Macrovesicular steatosis is defined by a single large fat vacuole or a few smaller droplets that eccentrically displace the nucleus to the edge of the hepatocyte Fig.  8.10  Fatty liver with yellow discoloration and a greasy texture

degree of steatosis [302]. There are several factors, however, that need to be considered when performing and interpreting a frozen section. First, fatty infiltration in the liver can be patchy and nonuniform, one short core biopsy may not be adequate for an accurate assessment. Second, the biopsy should be transported to the frozen section room on a piece of gauze or paper towel moisturized with preservation solution. Immer­ sion of the tissue in normal saline or preservation solution even for a few minutes can lead to marked frozen artifact that will confuse the interpretation for steatosis. Third, microscopic assessment for the severity of steatosis is also a subjective estimation based on an observer’s experience. The quantification can thus vary widely on a given biopsy by different pathologists, which may provide inaccurate or misleading information to surgeons [206]. Despite the limitations, frozen section remains the gold standard for pretransplant donor liver evaluation [13, 35]. In a study of 385 donor livers on which pretransplant frozen section examination was performed [415], 22 livers were rejected because of the presence of severe macrovesicular steatosis. Five additional livers were also rejected due to ischemic necrosis, prominent portal inflammation and prominent fibrosis identified on frozen sections. The primary nonfunction rate following transplantation was 1.4% in the remaining 358 grafts, which represented a significant decrease compared with the rate of 8.5% before using frozen section examination in authors’ institution. This study demonstrates that pretransplant frozen section examination is a useful tool to exclude potential grafts that may become dysfunctional after transplantation.

Fig.  8.11  Frozen section and H&E staining of donor liver biopsy for the evaluation of steatosis. The total fat was estimated to be ~80% and the macrovesicular fat ~50% in this biopsy

Fig.  8.12  Low-power scan of a donor liver biopsy on frozen section. The total fat was estimated to be >70% and the macrovesicular fat ~40%

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(Fig. 8.13). Among a variety of underlying etiologies, obesity, diabetes mellitus and excessive alcohol use are the main causes for this type of steatosis. Micro­ vesicular steatosis is defined as numerous small fat droplets that accumulate in the cytoplasm of the hepatocytes, leaving the nucleus centrally located (Fig. 8.14). The affected cells may exhibit a foamy appearance, which can be confused with ballooning degeneration. This type of steatosis is most commonly associated with drugs, toxins and certain types of metabolic disorders. In almost all the cases, the pattern is mixed but may be predominantly macrovesicular or predominantly microvesicular.

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8.4.2.4 Pitfalls and Special Stains Semi-quantification of fat infiltration on frozen sections is apparently observer-dependent. Grafts deemed to be severely steatotic in one transplant center may be acceptable to others [302]. Another compounding factor is freezing artifacts that may result from tissue immersion in saline or preservation solution during transportation, slowed freezing time, thick sections and knife markers. These artifactual changes usually do not interfere with the recognition of steatosis (Fig. 8.15). However, if marked frozen artifacts are created, the assessment can be very challenging. Particularly, the sinusoidal spaces may be misinterpreted as large fat droplets (Figs. 8.16 and 8.17). Fat stains, such as oil red O and Sudan III, are not recommended in general [302]. Although these stains are more sensitive in fat detection [229, 415], they are highly staining-technique-dependent and can yield a high rate of false-positive results [415]. For example, oil red O may accumulate in the sinusoidal spaces and overstain microvesicular fat droplets, leading to an overestimation of the fat content and erroneous rejection of a transplantable graft (Fig. 8.18). A more objective and reproducible assessment of fat content in donor livers is anticipated by using computer-based image analysis. However, currently available data in this area are limited and more studies are needed [195, 206, 419].

Fig. 8.13  Macrovesicular steatosis with large fat vacuoles that displace the nucleus to the periphery

Fig.  8.14  Microvesicular steatosis with numerous small fat droplets surrounding the centrally located nucleus

Fig. 8.15  Frozen artifacts which did not influence the microscopic interpretation in this biopsy. Lack of significant steatosis was confirmed by permanent section

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Fig.  8.16  Marked frozen artifacts which made microscopic interpretation very difficult. The sinusoidal spaces could be misinterpreted as large fat droplets

Fig.  8.17  Permanent section of the same biopsy illustrated in Fig. 8.16 showed ~70% total fat but <30% macrovesicular fat

8.4.3 Day 0 Biopsy Evaluation

Fig. 8.18  Oil red O stain performed on frozen section may lead to overestimation of the fat content in donor livers

with more rapid fibrosis progression if receiving a moderately or severely steatotic graft. Mild steatosis does not appear to have an impact on HCV recurrence and fibrosis progression [75, 79]. In a retrospective study of 79 donor livers that were transplanted to patients with HCV-related chronic liver disease, preexisting portal inflammation in the grafts as detected by liver biopsy prior to reperfusion was demonstrated to be significantly associated with subsequent fibrosis progression [42]. All the recipients who received a graft with histologically proven portal inflammation showed stage 3 or 4 fibrosis within 1 year following transplantation. In contrast, none of the patients who received a graft without portal inflammation showed more than stage 2 fibrosis. This study also showed that intrahepatic inflammation was significantly increased in older donors and donors with a prolonged stay in the intensive care unit [42]. These latter findings support the observations by others showing that donor age is an independent risk factor for HCV recurrence, fibrosis progression, and graft survival [75, 325, 397, 485, 539].

8.4.3.1 General Consideration Biopsies of the grafts immediately before implantation or near the end of the operation after revascularization provide baseline information for subsequent biopsy evaluation of the grafts, which may discover preexisting donor liver abnormalities that will help explain the future problems occurring in the grafts [6, 219]. For recipients with HCV infection, donor liver histology may also help predict their clinical outcome. It has been shown that HCV recurrence is more frequent and earlier

8.4.3.2 Pathologic Features Day 0 biopsies are usually routinely processed with formalin fixation and H&E stain. A routine liver staining panel including reticulin, trichrome, iron and periodic acid-Schiff with diastase (PAS-D) should be performed for a more thorough histologic assessment. The histologic findings in these biopsies range from complete normal to a variety of abnormalities including

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steatosis (as discussed previously), preservation injury, portal inflammation, lobular inflammation, fibrosis, glycogenated nuclei (particularly in older donors), iron overload, a1-antitrypsin hyaline globules, granulomas, amyloidosis, and benign small subcapsular nodular lesions such as bile duct hamartoma and focal nodular hyperplasia. Some of these findings are incidental and do not have any clinical significance, but others may have an adverse impact on graft survival. In a review of 1,546 liver transplantations performed at the Mayo Clinic [529], 14 grafts were found to have unusual pathology on day 0 biopsies. These included six cases of amyloidosis, three cases of schistosomiasis, three cases of a1-antitrypsin deficiency, and two cases of iron overload. All the donors had normal liver chemistries at the time of transplantation without a known history of liver diseases. Follow-up showed that three recipients who received grafts with amyloidosis developed vascular complications and required second transplantation within 18 weeks. The complications included HAT and arteritis, hepatic artery stenosis and PVT. All three recipients with donor schistosomiasis were successfully treated with praziquantel with a stable clinical course. No ova or granulomas were detected in the follow-up biopsies. Donor a1-antitrypsin deficiency did not appear to affect graft functions, even though diastase-resistant hyaline globules remained detectable for a long period of time after transplantation. Iron overload also tended to persist in the grafts. It has been shown that iron overload is associated with liver disease progression and resistance to interferon/ ribavirin therapy in HCV patients [212]. Another common finding on day 0 biopsy is small clusters of neutrophils scattered in the sinusoids with no associated hepatocyte necrosis, termed “surgical hepatitis” (Fig. 8.19). This is believed to be caused by prolonged handling of the liver, which can also be seen in other non-transplant hepatectomy specimens.

8.4.4 Living Donor Evaluation 8.4.4.1 General Consideration Living donor liver transplantation has been used to alleviate the significant shortage of cadaveric donors and to reduce waiting list mortality, initially developed for children but rapidly applied to adults [82, 200, 728].

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Fig. 8.19  Surgical hepatitis featuring small clusters of neutrophils scattered in the sinusoids with no associated hepatocyte necrosis

It has the advantage of availability of the graft at the optimal time because it is an elective procedure. It also has the advantage of no or minimal preservation injury [451]. However, it carries significant risks not only to the recipients but also the living donors. The reported complication rates in donors range from 0 to 67%, primarily including biliary complications such as bile leaks and biliary strictures, blood loss, intraabdominal bleeding, PVT, wound infection, bowel obstruction, pneumonia, pulmonary embolism, incisional hernia, and rarely donor death [81, 384]. Currently, the overall donor complication rate is estimated to be 10%, with a mortality rate between 0.2 and 0.4% [81, 412]. Preoperative living donor evaluation is of paramount importance in selecting a suitable donor with optimal graft quality and to ensure donor safety [112]. This is so not only because an unhealthy liver may cause problems in the recipient, but also because it may possess a compromised regenerative ability in both the recipient and the donor. The role of liver biopsy in this evaluation process is controversial because of its invasive nature. In a survey of transplant centers published in 2003 [82], the majority of the centers performing living donor transplantation either did not require or were selective in performing liver biopsy in prospective donors. Only 14% of the programs performed liver biopsy in all the donors and 26% of the programs did not perform liver biopsy in any of their living donors. Nevertheless, data have shown that liver biopsy not only provides most

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accurate assessment for the severity of steatosis, but also detects unexpected histologic abnormalities in apparently healthy potential donors.

8.4.4.2 Pathologic Features Similar to day 0 biopsy findings for cadaveric donors, the histologic findings in living donor biopsy range from complete normal to a variety of abnormalities including steatosis, steatohepatitis, portal inflammation, fibrosis, spotty necrosis, increased iron deposition, sinusoidal dilatation, idiopathic granulomas, and microabscesses [127, 177, 559, 574, 671]. With the exception of steatosis, all these histologic abnormalities are usually unexpected despite extensive clinical, serological and radiological investigations. In a study of 100 consecutive living donor biopsies, Ryan et al. found that biopsy was superior to imaging and body mass index (BMI) in determining the severity of steatosis [559]. In their series, 10 potential donors were denied based on liver biopsy findings. Seven had significant steatosis. Three had unexpected histologic findings including chronic hepatitis with fibrosis, incipient bridging fibrosis, and unclassifiable vascular abnormality. In another study of 62 liver biopsies from potential donors by Emiroglu et  al. [177], 30 (48%) donors were rejected because of abnormal histologic findings including moderate to severe steatosis, chronic hepatitis, fibrosis and hepatocellular injury. Although this study supports the notion that liver biopsy is mandatory to choose suitable living donors, the unusually high rejection rate raises the doubt whether some of the abnormal histologic findings are significant enough to disqualify the donors. In a study of 20 living-related liver transplantations performed for Alagille syndrome [318], Kasahara et al. reported that 4 of 36 potential donors were rejected because of paucity of intrahepatic bile ducts (no bile duct in at least half of the portal tracts) diagnosed by preoperative liver biopsy. Since Alagille syndrome is an inherited autosomal dominant disorder with variable penetration, presumably caused by mutations in the Jagged1 (JAG1) gene involved in the Notch signaling pathway [518], intrahepatic bile duct anomaly may also be present in patients’ parents or other close relatives who may be considered potential donors for livingrelated transplantation. Kasahara et al. also described a recipient who received a segmental graft despite

intraoperative diagnosis of unsuspected intrahepatic bile duct paucity by cholangiography and subsequent liver biopsy. Although the biliary drainage was reconstructed with portoenterostomy, the recipient developed recurrent cholestasis and died of sepsis [318]. In addition, Gurkan et al. reported two cases of living-related donors who had no liver function abnormalities or characteristic features of Alagille syndrome, but the operations for transplantation had to be aborted because of unsuspected bile duct paucity discovered intraoperatively [262].

8.4.5 Preservation and Reperfusion Injury 8.4.5.1 General Consideration Preservation and reperfusion injury is one of the major causes of initial graft dysfunction. It may also lead to a higher incidence of subsequent acute and chronic rejection [203, 348] increased severity of recurrent HCV [710], and a higher incidence of biliary complications [93, 374]. It is characterized by elevation of serum transaminases and poor bile production within the first 24–48  h after revascularization. A marked elevation in the enzyme levels in the first few days is usually an indication of severe injury, which can result in graft failure. The enzyme and bilirubin levels typically decrease progressively within several days if the graft survives the injury [111]. Clinical resolution is usually achieved within 1–4 weeks but abnormal liver tests may persist for several months if the injury evolves. Pathophysiologically, preservation and reperfusion injury is a multifactorial process but is essentially caused by ischemia. It is triggered by initial ischemia incurred during harvesting, preservation and transportation of the grafts, followed by reperfusion. Thus, ischemia and reperfusion injury appears to be a better term to describe this process. It occurs in 4 stags: (1) prepreservation injury, (2) cold preservation, (3) rewarming, and (4) reperfusion injury [94] and is generally categorized into 2 types: cold ischemia and warm ischemia [374]. Cold ischemia results from prolonged storage in preservation solutions, such as University of Wisconsin solution and Celsior solution that are designed to reduce cell swelling, prevent cellular uptake of calcium and sodium, provide substrates for ATP repletion, and

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decrease production of reactive oxygen species [374, 511, 622]. Warm ischemia results from compromised blood flow to the liver at body temperature before and during harvesting and from rewarming and resumption of blood flow after implantation (reperfusion). The degree and type of injury is related to the severity and duration of the ischemic stress. With prolonged pretransplant ischemia or hypoxia, cell death may become evident even before reperfusion and may thus be recognizable on day 0 biopsy. If the ischemic period is short, however, cell death may be precipitated by reperfusion injury [374]. It has been shown that cold ischemia preferentially targets sinusoidal endothelial cells, whereas warm ischemia primarily damages hepatocytes [581], although there is evidence suggesting that sinusoidal endothelial cells are probably damaged before hepatocytes even during warm ischemia [500]. Bile duct cells, Kupffer cells and Ito cells are also very sensitive to cold and warm ischemia. As mentioned earlier, preservation and reperfusion injury is a multifactorial and dynamic process that involves multiple cell types with the production, activation and coordinated actions of multiple cytokines, chemokines, adhesion molecules, reactive oxygen species, complements, coagulation factors, leukocyte protease inhibitors, and nitric oxide [94, 203, 220, 297, 347, 348, 374, 500, 594, 610, 651]. Among these, Kupffer cell activation appears to serve an important role in releasing proinflammatory cytokines, such as tumor necrosis factor a, and reactive oxygen species that not only act as direct cytotoxins to endothelial cells and hepatocytes but also recruit neutrophils into the grafts [348, 500]. The final consequence of these interrelated processes is microcirculatory disturbance resulting from imbalanced actions of vasoconstrictors and vasodilators, leading to decreased sinusoidal blood flow, endothelial cell necrosis and eventually hepatocyte loss [94, 348, 500, 651]. Various preexisting donor factors have shown to aggravate ischemia and reperfusion injury (prepreservation injury). These may include history of alcohol or drug abuse, hepatic steatosis, cardiovascular instability after brain death, hypotension during donor operation, prolonged stay in intensive care unit, and surgical trauma at the time of harvest [94]. As discussed previously, severe macrovesicular steatosis is probably the most important detrimental factor because steatotic liver is highly susceptible to both cold and warm ischemic injury [28, 191, 215, 300, 301].

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8.4.5.2 Pathologic Features Preservation injury can be recognized as early as in day 0 biopsy, albeit mild in general. This usually occurs in grafts with prolonged prepreservation injury and/or prolonged cold preservation. It is characterized by diffuse or centrilobular (zone 3) hepatocyte ballooning and the presence of scattered acidophil bodies (Fig.  8.20). Neutrophil accumulation (surgical hepatitis) may be evident in some cases. Rarely, large areas of confluent coagulative necrosis, either centrilobular or panacinar, occurs (Fig. 8.21), which usually results in graft failure. Because liver damage is aggravated by reperfusion, classic morphologic changes of preservation and

Fig. 8.20  Preservation injury detected on a day 0 biopsy. Note diffuse hepatocyte ballooning and scattered acidophil bodies

Fig. 8.21  Severe preservation injury detected on a day 0 biopsy. Note large areas of confluent coagulative necrosis in zones 3 and 2. The periportal region (zone 1) is relatively spared

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reperfusion injury usually become more evident 2–3 days after revascularization. The histologic hallmark is zone 3 hepatocyte ballooning [327], imparting a distinctive pale appearance at the centrilobular areas that can be easily recognized at low magnification (Fig. 8.22). The hepatocytes are swollen with expanded pale cytoplasm, which leads to the compression of the sinusoidal spaces. Other common findings at the centrilobular regions include microvesicular steatosis, scattered acidophil bodies, perivenular hepatocyte dropout, and cytoplasmic cholestasis (Figs.  8.23 and 8.24). There is usually no significant lobular or portal

Fig. 8.24  Preservation and reperfusion injury showing perivenular hepatocyte dropout and cytoplasmic cholestasis. No venulitis is evident

Fig. 8.22  Preservation and reperfusion injury showing centrilobular ballooning and microvesicular steatosis, giving rise to a characteristic pale appearance at the centrilobular area on lowpower view

Fig. 8.23  Preservation and reperfusion injury showing centrilobular ballooning, microvesicular steatosis and acidophil bodies

inflammatory cell infiltration, with the exception of lobular neutrophils as mentioned previously. Ultrastructurally, the sinusoidal endothelial cells show cytoplasmic swelling and vacuolization, membranous blebbing, nuclear pyknosis, cell detachment, and enlarged fenestrae. The hepatocytes also show vacuolization and blebbing, as well as glycogen depletion, mitochondrial swelling, fragmentation of the rough endoplasmic reticulum and chromatin clumping. Sinusoidal spaces may be obstructed by cell fragments. Leukocytes and platelets are also often found in the sinusoids [111, 468]. Preservation and reperfusion injury typically resolves within 2–4 weeks. Resolving injury features hepatocyte regenerative changes with frequent binucleation and repopulation of the perivenular areas in grafts with prominent hepatocyte dropout. Mitotic figures may be frequent in grafts with more severe injury. Mild centrilobular hepatocyte ballooning and cytoplasmic cholestasis may persist for several weeks (Fig. 8.25). In the early literature, these latter findings have been described as the characteristic histologic features associated with a posttransplant cholestatic syndrome (functional cholestasis) that typically resol­ ves spontaneously [14, 111, 716]. Ultrastructurally, ischemia and reperfusion cause changes in actin microfilaments in bile canaliculi, loss of canalicular micro­ villi and canalicular dilatation, which lead to bile stasis [122, 128]. Morphologic changes of preservation and reperfusion injury can extend to a panacinar distribution in

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Fig. 8.25  Resolving preservation and reperfusion injury showing residual mild centrilobular hepatocyte ballooning and cytoplasmic cholestasis. The centrilobular area is repopulated by hepatocytes that show frequent binucleation. No significant inflammatory cell infiltration is present

Fig. 8.27  Preservation and reperfusion injury showing bile duct degeneration, bile ductular reaction and cholangiolar cholestasis. Note the presence of neutrophils in the portal tract, mainly surrounding the proliferative ductules

severe cases. Severe injury may also manifest as centrilobular necrosis, which is a histologic sign of poor prognosis for the graft and the patient [240]. Centri­ lobular necrosis is defined as varying degrees of zonal hepatocyte necrosis and dropout around the terminal hepatic venules (central vein) with sparing of the periportal hepatocytes (Fig. 8.26). It typically occurs within 1 week post transplantation and lacks inflammatory cell response. Bile ducts are very sensitive to preservation and reperfusion injury, which causes detachment of the duct epithelium from the basement membrane. A greater number of cholangiocytes are found in the bile as

preservation time increases [97]. In addition, hydrophobic bile salts can directly damage the duct epithelium to amplify the preservation injury [56, 122, 347]. Histo­ logically, bile duct degeneration, periportal bile ductular reaction, and cholangiolar cholestasis with bile plugs may be evident, similar to that seen in sepsis (Fig. 8.27). In response to ductular reaction, a variable number of neutrophils may be present in portal tracts, usually surrounding the proliferating ductules. It has been shown that biliary tree needs a much longer time to recover from preservation and reperfusion injury compared with hepatocytes and endothelial cells [347]. Macrovesicular steatosis is generally considered to be a preexisting donor abnormality. In grafts with significant macrovesicular steatosis, preservation and reper­ fusion injury may cause fat release from damaged hepatocytes into extracellular spaces including the sinusoids, a phenomenon termed lipopeliosis or pseudopeliotic steatosis [66, 105, 191]. The released fat may coalesce to form large globules, giving rise to large clear spaces. Subsequently, macrophages and other inflammatory cells accumulate around the fat (Fig. 8.28). If the graft survives, the fat globules eventually resolve over several weeks.

8.4.5.3 Differential Diagnosis Fig. 8.26  Preservation and reperfusion injury showing centrilobular necrosis. Note the lack of inflammatory cell infiltration

The diagnosis of preservation and reperfusion injury is usually straightforward in cases that exhibit classic

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Fig. 8.28  Lipopeliosis or pseudopeliotic steatosis resulted from fat release from damaged hepatocytes secondary to preservation and reperfusion injury. Note the presence of inflammatory cells around the fat globules

centrilobular hepatocyte ballooning, microvesicular steatosis, acidophil bodies and cholestasis within the first 1–2 weeks after transplantation. However, there are a few conditions that can also occur in early posttransplant days that may show histologic abnormalities similar to those seen in preservation and reperfusion injury. In cases with large areas of confluent coagulative necrosis, antibody-mediated humoral rejection and vascular thrombosis should be included in the differential diagnosis. While the distinction is primarily based on the clinical findings and clinicopathologic correlation, new onset of hepatocyte necrosis several days after transplantation should not be attributed to preservation and reperfusion injury because it takes only several hours for hepatocytes to undergo apoptosis. In that case, other etiologies, particularly ischemia secondary to vascular complications, should be sought. The distinction between severe preservation and reperfusion injury and humoral rejection in ABOcompatible grafts can be very difficult. Clinical find­ ings of unexplained low complement levels and thrombocytopenia, angiographic findings of segmental or diffuse narrowing or sausage-like appearance of the hepatic artery, and histologic findings of arteritis with fibrinoid necrosis all support the possibility of humoral rejection. Immuno­histochemical detection of C4d deposition may serve as an additional useful marker in the diagnosis of humoral rejection, but the staining specificity needs to be further evaluated [55,

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76, 264, 484, 566]. This staining will be discussed in more detail in the Sect. 8.6.1.3. In cases with centrilobular necrosis, the differential diagnosis can be broad [240, 274, 327, 478], which will be discussed in more detail in the Sect. 8.6.2.4. In the early posttransplant period, however, the primary differential should include acute rejection if vascular complications have been clinically excluded. In the study by Khettry et  al. [327], centrilobular necrosis was broadly divided into two types: one related to preservation and reperfusion injury and the other as part of rejection. Centrilobular necrosis related to reversible or irreversible preservation and reperfusion injury was detected within the first 6 months after transplantation, as expected, and was characterized by centrilobular hepatocyte dropout, ballooning and/or cholestasis. Interestingly, cases with all the three findings in this study were associated with biliary complications and poor patient survival. On the other hand, centrilobular necrosis as part of rejection could be seen at any time and was frequently associated with central venulitis. Neil and Hubscher also demonstrated that fat, cholestasis and hepatocyte ballooning were features of preservation and reperfusion injury but centrilobular necrosis might not [478]. The authors found that centrilobular necrosis correlated with hepatic venular endothelial inflammation and centrilobular inflammation, suggesting a manifestation of rejectionrelated parenchymal injury. Therefore, centrilobular necrosis should be suspected as a rejection-related lesion, even if typical portal changes for rejection are not prominent [478]. Apparently, acute rejection can occur in grafts that are suffering from severe preservation and reperfusion injury with centrilobular necrosis. Typically, histologic features of rejection in the portal tracts are evident in those cases. In cases with ischemic biliary injury, sepsis, ascending cholangitis and large bile duct obstruction may enter the differential diagnosis. Antibody-mediated humoral rejection may cause portal neutrophil infiltration and bile ductular reaction, which can also be confused with preservation and reperfusion injury. In addition to different clinical findings, preservation and reperfusion injury lacks portal edema, which is typically seen in large bile duct obstruction. Neutrophil accumulation within the lumen of native bile ducts and neutrophil infiltration of the duct epithelium, characteristic of ascending cholangitis, should not be seen in preservation and reperfusion injury.

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8.5 Technical Complications Mathew Augustine, and Robert A. Anders Improvements in technique, critical care management, immunosuppression, and early diagnosis of complications by radiologic imaging have resulted in 1-year graft survival and patient survival rates in the United States of approximately 80 and 85%, respectively [552]. Despite the recent success of liver transplantation, graft loss and mortality remain a major concern. Two of the most significant causes of morbidity, graft failure, and mortality from liver transplantation relate to vascular and biliary complications, affecting more than 10% of all liver transplant recipients. Vascular and biliary complications compromise both the short and long-term viability of the graft and are intimately related to the technical success of the operation. Phy­ siologic and metabolic abnormalities that affect blood flow, rejection, infection, and clotting disorders may also contribute to the development of these complications. In this chapter, we describe the causes, diagnosis, consequences, and treatment of early and late vascular and biliary complications with special emphasis on the pathology and differential diagnosis pertaining to each entity.

8.5.1 Hepatic Artery Thrombosis HAT is the most common vascular complication following OLT, resulting in increased morbidity, allograft loss, and mortality. It is also the most common technical complication requiring retransplantation. The reported incidence of HAT following liver transplantation varies from 2.5 to 7% in adult recipients [164, 497, 609, 624, 700] and 1.7 to 11% in pediatric recipients [275, 543, 602]. Retransplantation rates are reported to occur in 62% of pediatric and 50% of adult patients with HAT [53]. The overall mortality rate is approximately 33% [53]. Various etiologies for HAT have been described and are broadly divided into technical and non-technical causes. Technical errors include problems with the donor-recipient anastomosis, dissection of the arterial wall, celiac stenosis or compression of the median arcuate ligament, aberrant donor or recipient arterial anatomy, kinking of the artery, and performance of

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complex back table arterial reconstruction of the allograft [77, 432, 588, 675, 676, 719]. Non-technical factors include the preoperative utilization of transarterial chemoembilization (TACE), chronic rejection, blood group type-incompatible grafts, tobacco use, CMV infection, coagulation abnormalities, and graft preservation solution [183, 275, 400, 461, 510, 571, 572, 583, 725]. The different etiologies of HAT result in the varied time to presentation and severity of the liver injury that ensues. Based on this time to clinical onset or identification, HAT is generally divided into early and late forms. Currently, there is no specifically agreed upon date with various publications utilizing different cutoff time points including 15 days, 30 days, 2 months, and 6 months. The early form of HAT, seen in approximately one third of patients, usually occurs in the early postoperative period and results in fulminant hepatic necrosis, resulting in higher rates of allograft loss and patient mortality. Elevations in liver transaminases, leukocytosis, and worsening coagulation profile are the prelude to the development of fever, sepsis, altered mental status, hypotension, and coagulopathy. While hepatic artery revision or thrombectomy may be attempted, these patients often show improved shortterm survival with retransplantation [164]. In the remaining two thirds of patients with HAT, symptoms and clinical presentation are associated with the development of bile duct necrosis or abscess formation as a consequence of bile duct ischemia. The fulminant liver necrosis commonly identified with the early form of HAT is not as extensive in late HAT due to the interval development of hepatic arterial collateralization. Late HAT patients may develop transaminitis, leukocytosis, cholangitis, hepatic abscess formation, and/ or sepsis. These patients do not require retransplantation or revascularization. Instead, late HAT patients are typically managed with percutaneous biliary and/or abscess drainage. HAT patients may also present with a mild or asymptomatic picture. The diagnosis of HAT in these patients typically occurs incidentally on routine follow-up or from work-up for another condition. While these patients have limited or no symptoms, the identification of subtle hepatic injury from HAT in this setting reveals a latent problem that must be addressed and discriminated from potential confounders including rejection or recurrence of the primary disease. The diagnosis of HAT is substantially aided by radiologic assessment of the arterial inflow to the graft

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and can be achieved by various modalities. Doppler ultrasound is commonly used during the immediate postoperative period for surveillance or primary assessment and is reported to detect 100% of early and 72.7% of late HAT [290]. Doppler examination of HAT can identify complete absence of proper hepatic or intrahepatic arterial flow as well as document a high arterial resistive index, suggestive of a restrictive lesion or kinking. However, Doppler examination is operator dependent and the development of collateralization may drop the resistive index, thereby decreasing the specificity of the exam. The documentation of HAT by Doppler examination is typically followed by diagnostic angiography, which is considered the gold standard for diagnosis of HAT. More recently, the use of computer tomographic angiogram (CTA) or magnetic resonance angiogram (MRA) has been utilized to visualize HAT by the absence of hepatic arterial enhancement. Pathologic examination soon after thrombosis reveals a recently formed clot that is obstructing the hepatic artery (Fig.  8.29). Liver biopsy findings in early HAT predominately focus upon centrilobular hepatocyte injury. The changes range from mild centrilobular hepatocyte swelling to severe coagulative necrosis which can become confluent and involve large patches of the liver (Fig. 8.30). The necrotic tissue may become secondarily infected resulting in a localized hepatic abscess or disseminated bacteremia. If the liver of a patient requiring retransplantation for early HAT is examined, necrosis of the hilar and large

Fig. 8.29  Acute hepatic artery thrombosis. Cross section of the hepatic artery with an acute thrombus tethered to the arterial wall (asterisk). This thrombosis developed soon after orthotopic liver transplant

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Fig.  8.30  Hepatic artery thrombosis-induced centrilobular necrosis. Liver with portal tract (PT) showing centrilobular coagulative necrosis (N) following acute hepatic artery thrombosis. The hepatocytes immediately surrounding the portal tract are spared from ischemic necrosis

bile ducts may be seen. The morphologic changes in late HAT are variable. As expected, asymptomatic patients may show non-specific changes on liver biopsy, while others display bile duct necrosis or bile ductular reaction. Chronic HAT is characterized by marked intimal thickening which can impede vascular flow and cause atrophy of the interlobular bile ducts (Figs. 8.31 and 8.32).

Fig. 8.31  Chronic hepatic artery thrombosis. Large intrahepatic branch of the hepatic artery shows marked intimal proliferation with a narrow central lumen (CL) and peripheral lumen (L). The intimal hyperplasia (I) is evident from the elastic interna (EI) layer to the central lumen

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Fig. 8.32  Chronic hepatic artery thrombosis with bile duct atrophy. Portal tract with hepatic artery (HA), portal vein (PV) and atrophic interlobular bile duct (BD) and paucity of inflammation. The degenerating biliary epithelium has scant cytoplasm and nuclear anisocytosis secondary to ischemia

8.5.2 Biliary Complications Biliary complications have been reported to occur in 10–40% of all adult OLTs [657, 734]. Split liver, smallfor-size, and living-related donor liver transplantation (LDLT) generally have a higher rate of biliary complications. The most common biliary complications following liver transplantation are biliary leaks and strictures; however, various other complications can arise in the postoperative setting including biliary abscess, sludge, stones, cast syndrome, mucocele, and bile duct necrosis. Biliary strictures occur in 4–9% of liver transplant patients [158, 255, 463, 694]. Biliary leaks are reported to occur in 1–25% of patients [674]. Biliary complications are typically divided into early and late complications with approximately two thirds of complications occurring in the first 3 months after OLT. Biliary complications carry significant morbidity and mortality, requiring a high level of suspicion along with adequate intervention. In the past, surgical and percutaneous interventions for biliary complications were common. Recently, advances in radiologic imaging, including magnetic resonance cholangiography (MRC) and endoscopy, have shifted the focus onto less interventional procedures. Biliary complications can be classified according to the underlying etiology. Similar to HAT, the broadest classification distinguishes technical from non-technical causes for biliary complication develop­

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ment. Poor suturing of the biliary anastomosis, excessive tension on the biliary anastomosis, T-tube dislodgement, excessive dissection of periductal tissues during organ procurement, and extensive use of electrocautery can result in biliary stricture or leak. Ischemia of the biliary structures is typically a consequence of poor hepatic artery inflow as a result of hepatic artery stenosis, HAT, splenic artery steal syndrome, or celiac axis stenosis. Ischemia and cholangitis comprise the majority of non-technical complications. Less common etiologies including immunologic injury from humoral and cellular immune rejection, preservation injury, hemobilia, biliary stones or sludge, and sphincter of Oddi dysfunction should also be considered. Strictures from recurrent PSC should also be considered in patients with elevated alkaline phosphatase and gamma-glutamyl transferase levels. Patients with biliary complications can present in a myriad of ways with none providing high sensitivity or specificity. Biliary strictures and leaks that occur soon after transplant tend to have a technical etiology, while those that occur later are more likely to be multifactorial with a more complex presentation. Right upper quadrant abdominal pain, right shoulder pain, and anorexia are symptoms associated with biliary tract disorders. Due to hepatic denervation, pain may be totally absent upon presentation. Patients with fever and the above constellation of symptoms should have biliary leak and cholangitis considered in their differential diagnosis. Jaundice and the presence of acholic stools is an indicator of biliary tract obstruction in the posttransplant period. It is important to remember that the use of corticosteroids in this patient population may mask the clinical signs of infection. Screening using total bilirubin or gamma-glutamyl transferase levels may indicate biliary system complications. In one study, gamma-glutamyl transferase was found to be the best single test during the early (days 0–30) period while total bilirubin level is more sensitive between 30 and 90 days post transplant [165]. Ele­vations of AST and ALT levels are indicative of liver injury that could be related to primary biliary problems but are clearly non-specific for this event. Various imaging modalities can be applied toward the diagnosis of biliary complications after OLT. The gold standard imaging modality for the assessment of post-OLT biliary complications is cholangiography. Cholangiography can be performed through intraoperatively placed T-tubes, percutaneous transhepatic cholangiography (PTC), or endoscopic retrograde

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cholangiography (ERC). These modalities can detect leaks, strictures, T-tube migration, or papillary dysfunction. Investigation of the arterial system should also be considered, especially in late-onset biliary complications. Asymptomatic patients with abnormal liver enzymes following OLT should have biopsy performed instead of routine cholangiography with consideration toward excluding rejection, ischemia, or recurrence of primary disease. Three-dimensional CT can identify intra-abdominal fluid collections, such as bilomas or cholangitis-induced hepatic abscesses, as well as visualize the vascular supply of the graft. Mag­ netic resonance cholangiopancreatography (MRCP) is another noninvasive imaging modality to delineate the anatomy of the biliary system and can also provide information on intraparenchymal disease or infection, fluid collections, and vascular complications. Pathologically, biliary obstruction can be divided into intrinsic and extrinsic etiologies. Intrinsic etiologies include foreign object (stent or T tube), bile casts, or stones while extrinsic etiologies include abscess, biloma, mucocele, and hematoma. Biliary strictures can be thought of as incomplete obstructions and may be the result of a technical complication (usually at the anastomosis), partially resolved complete obstruction, or healing in response to an acute insult. Liver biopsy obtained in the setting of bile outflow obstruction is classically divided into early (days to weeks) or late (months) changes. Immediately following obstruction, centrilobular canalicular cholestasis, portal edema, and inflammation develop (Fig.  8.33). Cholestatic hepatocellular rosettes (three or more hepatocytes arranged in a ring often containing bile material in a

Fig. 8.33  Canalicular bile plug. An early indicator of bile outflow obstruction is the formation of canalicular bile plug (open arrow head) between hepatocytes (H)

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central lumen) and individual apoptotic hepatocytes (acidophil body) can develop. Proliferation of bile duct-like cells (bile ductular reaction) develops in the periportal regions and may be associated with neutrophilic infiltration (Fig. 8.34). With persistent obstruction, hepatocytes become increasingly filled with bilirubin and undergo feathery degeneration in which small clusters of hepatocytes exhibit fine foamy (nongranular) cytoplasm. Another distinctive change is cholate stasis, which first appears as ballooned periportal hepatocytes and progresses with toxic intracellular bile salts leading to further hepatocyte degeneration with Mallory bodies and visible bile within swollen hepatocytes (Fig.  8.35). Bile lakes and infarcts are

Fig.  8.34  Bile ductular reaction in response to bile outflow obstruction. The portal tract, identified by the portal vein (PV), hepatic artery (HA) and bile duct (B), are encircled by duct structures lined by biliary type cuboidal epithelium. Biliary epithelium located further from the portal tract is present in small ductular structures

Fig. 8.35  Cholate stasis. Accumulation of hepatocellular bilirubin products (B) is toxic to hepatocytes and induces cell swelling and in some cases condensation of cytoplasmic structural elements into Mallory bodies (open arrow head)

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hallmarks of advanced stage bile outflow obstruction (Fig. 8.36). Portal fibrosis with abundant bile ductular proliferation and ultimately cirrhosis with the distinctive lacey or geographic pattern typically seen in biliary cirrhosis are the end-stage manifestation of chronic biliary obstruction. Impaired biliary flow is a risk factor for the development of a secondary bacterial infection in the biliary tree, resulting in ascending cholangitis. Within the liver, histologic changes of biliary obstruction often occur with periductal or intraductular collections of neutrophils (Fig. 8.37). Compromise of the bile duct epithelium secondary to the intense inflammation and rising backpressure in the duct may result in bile extravasation into the hepatic parenchyma. The extruded bile

Fig. 8.36  Bile infarct. Bile infarct or lake lies next to a portal tract with hepatic artery (HA) and bile duct (BD) and is associated with proliferating bile ducts and ductules (PD). Immediately adjacent to the bile is an intense inflammatory (I) response and hepatocyte loss (HL)

Fig. 8.37  Cholangitis. Neutrophils (open arrow head) are evident around (pericholangitis) and infiltrating into (cholangitis) bile ducts (B)

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elicits an intense neutrophilic infiltrate leading to foci of hepatocellular degeneration (Fig.  8.36). In longstanding or repeated bouts of obstruction, portal fibrosis or cirrhosis can develop. The hepatic artery exclusively perfuses the bile ducts and adequate perfusion of the peribiliary plexus is essential to maintain the integrity of the biliary tree [492, 640]. It is reported that 50% of the blood from the hepatic artery is used to perfuse the bile ducts [143]. Ischemic cholangiopathy, sometimes referred to as ischemic cholangitis [393], is a term meant to encompass the damage to the biliary system caused by reduced perfusion of the biliary system and not hepatocellular cholestasis induced by ischemia [702]. Inflammation is not a prominent component of ischemic cholangiopathy, however, CMV infection (particularly in immunosuppressed patient), can induce ischemia secondary to CMV vasculitis [103]. Ischemic cholangiopathy primarily affects the middle third of the common bile duct, a watershed region between the two main arteries feeding the large bile ducts [143]. The first pathologic change consists of bile duct epithelial necrosis, causing sloughing of the epithelium and formation of a necrotic biliary coagulum, resulting in biliary cast formation (Fig. 8.38). The casts consist of a coagulum of necrotic biliary epithelium which causes ductal obstruction. These casts may be seen as a filling defect on endoscopic retrograde cholangiopancreatography (ERCP). More severe ischemia can induce full thickness coagulative necrosis of the bile duct (Fig. 8.39). The necrotic region may weaken and result in a bile leak. If the segment does not rupture it

Fig.  8.38  Bile cast. This bile cast was recovered in the hilar biliary system of the liver. The cast consists of necrotic debris such as sloughed ductal epithelium (DE), hemorrhagic debris, and bile concretion (BC)

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Fig. 8.39  Bile duct necrosis. Cross section of extrahepatic bile duct with transmural necrosis. Bile sludge and concretions are seen in the duct lumen while coagulative necrosis effects the duct wall with bile embedded in the superficial layers

may heal with the development of a fibrotic stricture. The liver biopsy from a patient with ischemic cholangiopathy may show non-specific changes; however, biopsy usually shows features of biliary obstruction [393]. If the ischemic compromise affects the intrahepatic biliary tree, there may be epithelial atrophy or ductopenia of the interlobular bile ducts. Strictures secondary to ischemic cholangiopathy may induce chronic bile outflow obstruction leading to a biliary pattern of fibrosis or eventually cirrhosis.

8.5.3 Portal Vein Thrombosis In comparison to HAT, PVT is a less commonly seen form of vascular complication from liver transplantation, occurring in less than 5% of all transplants perfor­ med [677]. However, the consequences of posttransplant PVT can be devastating, resulting in loss of allograft or patient death [164]. The portal vein may be anastomosed primarily to the recipient portal vein or a venous conduit (typically donor iliac vein). The long-term data suggests no difference in graft and patient survival in select patients with donor iliac vein conduit to the portal vein despite the additional anastomosis [489]. Various factors are reported to be associated with the development of PVT and include poor technique, misalignment of the anastomosis, excessive vessel length resulting in kinking, hypercoaguable state, small portal vein size, use of venous conduits, or previous PVT requiring thromboendovenectomy. The presentation can vary

from the classic manifestations of PVT including gastrointestinal bleeding from gastroesophageal varices, intestinal congestion, ascites, encephalopathy, and splenomegaly to fulminant liver failure requiring retransplantation. Some centers perform color-flow Doppler ultrasonography to monitor the patency of the portal vein on the day following surgery and at regular intervals thereafter [352]. While many patients are naturally coagulopathic in the immediate postoperative period following OLT, continuous infusion of low molecular weight dextran or heparin may be used to prevent recurrent PVT [456, 677]. Aspirin has also been used in the long-term setting [456]. Treatment of PVT previously required surgical intervention in the form of surgical thrombectomy and revision of the anastomotic site. If these options failed then retransplantation was required. More recently, however, the use of percutaneous interventional procedures and thrombolysis has been applied to the management of PVT [167]. Mechanical fragmentation along with thrombolytics and stent deployment has also been used to treat patients with this condition [41]. PVT, occurring immediately after transplantation, manifests as acute graft dysfunction that must be immediately corrected. The morphologic changes in late PVT mirror what is seen in nontransplant patients. The thrombosed vessel is re-organized with formation of a partially or totally calcified intimal plaque or development of webs within the vessel lumen. Small portal vessels downstream of an obstructed portal vein branch may show minor changes while those downstream of a patent branch may become dilated secondary to increased portal pressure. In some cases, portal obstruction is sufficient to induce global liver atrophy, or in segmental obstructions, might induce lobar atrophy [709]. Interestingly, compensatory regeneration, likely due to increased arterial perfusion, can be seen in the hilum.

8.5.4 Hepatic Vein and Inferior Vena Cava Stenosis and Thrombosis Stenosis or thrombosis of the IVC and hepatic veins are uncommon vascular complications after liver transplantation. IVC thrombosis occurs in less than 1% of liver transplants [442, 719]. The rate of hepatic vein stenosis varies according to the type of liver transplant

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performed. In pediatric liver transplants it occurs in 1% of whole liver grafts and 2% of living-related grafts [87]. However, hepatic vein stenosis occurs in 4% of patients who receive reduced-size or split grafts [87]. Piggyback transplants have been reported to reveal hepatic vein obstruction in less than 1% of cases [707]. Multiple factors are associated with the development of IVC or hepatic vein occlusion. Technical problems as well as recurrence of underlying disease are the major causes for the development of hepatic vein occlusion [595]. Technical causes include anastomotic narrowing, mismatch between the donor and recipient veins, kinking or twisting of the vein, or the development of an abnormal intimal flap [136]. Anastomotic narrowing in the early postoperative period is related to surgical narrowing. When it occurs in a delayed fashion, it is usually related to intimal hyperplasia and/or fibrosis. Compression due to graft edema has been suggested as a cause of hepatic outflow obstruction. Growth of partial liver transplants may also lead to displacement in the venous outflow structures, resulting in twisting of the veins and subsequent stenosis or thrombosis. The clinical presentation of caval or hepatic vein occlusion is in many ways similar to that of the BuddChiari syndrome or PVT and portal hypertension. Patients may present with abdominal pain, increasing abdominal girth, shortness of breath, and weight gain related to ascites. Of note, the diagnosis of venous outflow obstruction may be delayed in liver transplant patients with pre-existing ascites. Variceal formation may result in gastrointestinal bleeding. Hydrothorax may be another associated finding. Unlike in portal thrombosis, the liver can be enlarged and firm during clinical examination. Laboratory studies will reveal increases in AST and ALT, alkaline phosphatase, bilirubin, and gamma-glutamyl transferase levels. Another laboratory value that may indicate hepatic venous occlusion is decreased clearance of the immunosuppressive transplant medication tacrolimus [670]. It should be mentioned that IVC occlusion might occur above or below the liver. In cases of infrahepatic occlusion, liver dysfunction will not be seen; however, bilateral lower extremity edema, hematuria, and renal failure can be seen on initial presentation. For patients suspected of having venous outflow obstruction, imaging is performed to determine both the level and extent of occlusion. Doppler ultrasound is typically performed in the initial diagnostic phase due

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to its ease of utility and portability. In addition to the ability to visualize stenosis, Doppler ultrasound can identify venous outflow occlusion by decreased mean velocities in the hepatic veins with transition from triphasic waveforms to biphasic and monophasic waveforms depending on the severity of outflow obstruction [685]. Hepatopetal flow may reverse into hepatofugal flow. The gold standard imaging study for the identification of venous outflow occlusion is venography with measurement of pressure gradient. Interventional procedures including balloon angioplasty and stenting have been described in the literature as recent modalities for resolving venous outflow obstruction. While the literature reports success rates as high as 100% for percutaneous transluminal angioplasty, restenosis is common and requires several sessions for durable results [136]. The use of anticoagulation in the peri- and postprocedural period has also been reported. The resolution of venous outflow obstruction will lead to resolution of liver enzyme elevation, hepatic enlargement, and the various manifestations of portal hypertension. The morphologic changes in a transplanted liver mirror what is seen in a native liver following hepatic outflow obstruction. Since the occlusion rarely involves all the draining hepatic veins, the pathologic changes within the liver can be patchy. A liver with marked regional injury may show no histologic changes or subtle centrilobular sinusoidal dilatation. In more severely affected regions, the changes initially are dilated vascular channels and markedly congested hepatic sinusoids with red blood cells filling the space of Disse [366] (Figs.  8.40 and 8.41). The congestion

Fig. 8.40  Marked hepatic congestion from hepatic vein obstruction (Budd-Chiari). This low magnification shows blood markedly dilating the central veins (CV). The regions around the central veins are also congested with blood, while the portal area (P) is relatively spared

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8.5.5 Small-For-Size Syndrome

Fig. 8.41  Marked hepatic congestion from hepatic vein obstruction (Budd–Chiari). This high magnification view demonstrates blood infiltrating the space of Disse resulting in hepatic plates filled with blood rather than hepatocytes. The hepatic sinuses (S) are dilated secondary to elevated central venous pressure

may induce centrilobular hepatocyte atrophy and even necrosis (Fig. 8.42). Disease progression results in loss of hepatic veins into fibrous septa leading to the development of a venocentric cirrhosis pattern that spares the portal tracts. Secondary hepatocellular regeneration can occur in lobes drained by non-occluded veins and can be particularly prominent in the caudate lobe. The occluded veins and thrombus undergo remodeling, resulting in total obliteration into a fibrous cord, partial recanalization, or development of luminal webs.

Fig. 8.42  Chronic congestion and hemorrhagic necrosis from hepatic vein obstruction (Budd–Chiari). Chronic venous congestion induces hemorrhagic necrosis (HN) in the central vein (CV) region. The pale area immediately surrounding the central vein represents regions of hepatocyte loss (asterisk)

The size of the liver transplant graft is a crucial factor in graft survival as an inadequately sized liver may fail to perform the required metabolic and synthetic functions necessary of a normal liver. This can result in cholestasis, coagulopathy, gastrointestinal variceal bleeding, portal hypertension, ascites, and in the most severe cases encephalopathy, pulmonary and renal failure, and death. The constellation of conditions from an inadequately sized graft is referred to as the small-for-size syndrome (SFSS). In addition to insufficient liver mass, elevated portal vein pressures have been implicated in the development of SFSS. This increased portal vein pressure is correlated with elevated total bilirubin levels on postoperative day 2 [241]. SFSS patients are at increased risk of developing sepsis and have increased morbidity and mortality. It should be noted that not all patients with SFSS develop allograft failure. Patients who survive the initial insult from SFSS may undergo adequate liver regeneration to a sustainable functional mass. Various studies provide evidence that the graft weight to recipient body weight ratio (GRBWR) is an important measure for assessing the adequacy of the donor graft. Kiuchi et  al. reported that transplanted grafts with less than 1% of recipient body weight have lower graft survival rates [334]. Other studies have indicated that a GRBWR of >0.8% is acceptable [57, 362, 386]. Furthermore, reports of successful grafting with GRBWR <0.8% have also been reported [321, 334, 335, 385]. While graft size is clearly a factor in the development of SFSS, other factors are also considered in the assessment of adequate liver graft including steatosis, donor and recipient age, liver disease status, donor anatomic vascular supply and outflow, the duration of warm/cold ischemia time, and the type of vascular reconstruction [335]. SFSS grafts show evidence of sinusoidal congestion, mitochondrial swelling in hepatocytes, and collapse of the space of Disse [406]. Furthermore, SFSS intergraft mRNA endothelin-1 levels have been found to be increased while the hemeoxygenase-1 and heat shock protein 70 were decreased with reduced plasma nitric oxide levels [406]. The pathologic features in SFSS can be quite diverse. The lack of synthetic function is usually accompanied by prolonged cholestasis. Prolonged cholestasis may show some of the early changes seen in bile outflow obstruction discussed above. Elevated portal and venous pressures can enhance ischemia–reperfusion

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Fig.  8.43  Mitotic hepatocytes after small-for-size liver transplant. Hepatocyte proliferation occurs in liver grafts that are small for the size of the recipient as early as 24 h after transplant. A liver biopsy of a small-for-size transplant obtained 1 week after surgery continues to shows a brisk regeneration with five mitotic hepatocytes (open arrow) evident within this field

injury, causing hepatic congestion and necrosis, but this is poorly studied in the human regenerating liver. Animal studies replicating small-for-size grafts with portal hyperperfusion have shown sinusoidal congestion and hemorrhage [322]. The liver’s innate ability to regenerate by increasing the number of hepatocytes is a consistent feature [160, 188, 446, 648]. Technically, the regeneration is a compensatory hyperplasia in which more hepatocytes form in the liver remnant. The ordinarily dormant hepatocytes re-enter the cell cycle as evidenced by frequent observation of mitotic hepatocytes (Fig.  8.43). The start of the replication process occurs within 24 h and requires many non-hepatocyte cell types such as inflammatory, vascular, and possibly even bone marrow-derived cells which produce cytokines and growth factors to stimulate hepatocyte division. Most restoration of hepatic mass occurs in the first week and interestingly does not differ in donors or recipients [413]. Physiologic factors such as warm and cold ischemia, portal flow, and pressure have been shown to affect regeneration by influencing the delicate cellular and cytokine network driving normal regeneration [172, 591, 593, 722].

8.6 Rejection Hanlin L. Wang Rejection is an immune-mediated host reaction against graft antigens, which has the potential to result in graft

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damage and, if not controlled, graft failure. In theory, all graft recipients are at risk of developing rejection, but the occurrence and severity vary among different patients. The outcome of the host immune response largely depends on the type of antigen presenting cells that initially detects the foreign antigens, the interplay between pro- and anti-inflammatory lymphocytes, the activation of tumor necrosis factor superfamily receptors for the transduction of apoptotic signals, and the engagement of hepatic parenchymal cells. These components determine the occurrence and severity of rejection and also provide targets for pharmacological intervention [12, 174]. Apparently, the development of rejection can be markedly modified by immunosuppressive drugs and inadequate immunosuppression is thus a major risk factor. However, in a small population of recipients, immunosuppression can be successfully weaned off without any long-term adverse effect [337, 431, 502, 663]. This clinical tolerance, defined as stable normal graft function without the need for maintenance of immunosuppression, has been the subject of extensive research and is the ultimate goal of organ transplantation. Ideally, this would not only reduce the cost of immunosuppression and improve the quality of life, but also alleviate the patients from complications of life-long immunosuppression such as infections, direct drug toxicities, metabolic disorders and malignancies. The mechanisms of graft tolerance are poorly understood but are believed to involve liver-specific cell populations because liver itself possesses immunomodulatory properties. As a result, liver recipients are more prone to develop effective clinical tolerance than recipients of other solid organs [502, 663]. Development of microchimerism by donor-derived stem cells, production of soluble major histocompatibility complex (MHC) of donor origin, and contribution by passenger donor leukocytes (particularly regulatory T-cells) are also suggested mechanisms [174, 337, 502]. Rejection is broadly divided into three categories: humoral (antibody-mediated), acute (cellular), and chronic [653, 654].The classification is primarily based on time of occurrence, underlying immunological mechanisms and clinicopathological presentations. It is important to note that morphologic rejection may not always be clinically relevant and whether additional immunosuppression is needed usually requires clinicopathological correlation.

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8.6.1 Humoral (Antibody-Mediated) Rejection 8.6.1.1 General Considerations Humoral rejection is mediated by preformed antibodies directed against donor antigens. It mainly occurs in the setting of ABO incompatibility. In an early study, ABOincompatible grafts showed a 46% failure rate during the first 30 days in contrast to a 11% failure rate for ABOcompatible grafts [149]. In another early study, the 2-year graft survival for ABO-incompatible emergency liver transplants was significantly lower (30%) than that for ABO-compatible emergency transplants (76%). Among the 17 patients who received ABO-incompatible grafts, humoral rejection occurred in six cases (35%) [258]. For this reason, crossing the ABO barrier is generally avoided except for emergent conditions. In recent years, ABO-incompatible grafts have been used for living donor liver transplantation in combination with vigorous immunosuppression employing novel therapeutic strategies such as perioperative plasmapheresis, anti-CD20 monoclonal antibody (rituximab) and splenectomy [320, 342, 427, 656]. Data from Japan have shown that these new modalities significantly reduced the incidence of antibody-mediated rejection and significantly improved the patient survival rate in ABO-incompatible living donor liver transplantation [170, 171, 634]. Humoral rejection in ABO-compatible liver transplants is a rare event and the incidence is estimated to be <1% [481]. Although preformed lymphocytotoxic antibodies against human leukocyte antigens (HLA) have been suggested as potential predisposing factors, the detrimental role of HLA incompatibility in liver transplants has not been firmly established [101, 424]. Earlier studies have suggested that a high antibody titer (>1:32) and a IgG class are required [409]. Currently, HLA typing is not a prerequisite for liver transplantation given the undetermined risk. The primary targets of preformed donor-specific antibodies are antigens expressed on endothelial cells. Binding of the antibodies to graft vasculature results in fixation and activation of the complement components and initiation of subsequent cascade of events, leading to impaired blood flow, thrombosis and tissue damage. Humoral rejection typically occurs immediately (hyperacute) or during the first week (acute) following

liver transplantation [653]. However, late humoral rejection occurring several years after transplantation has also been described in a case report [717]. With increasing utilization of C4d immunostaining, the definition of humoral rejection may become more broad than that originally defined (discussed below). Clinically, humoral rejection presents with severe graft dysfunction without an obvious cause in a presensitized patient, usually over a period of hours to days following revascularization. An initial short period of normal reperfusion and bile production followed by a rapid rise in serum liver enzyme and bilirubin levels, coagulopathy, thrombocytopenia, hypocomplementemia and other signs of acute liver failure is characteristic. The diagnosis is confirmed by the presence of preformed donor-reactive antibodies in the recipient either before transplantation or in tissue eluates from failed allografts [653].

8.6.1.2 Pathologic Features Grossly, the grafts with hyperacute rejection may be swollen, cyanotic and mottled, and show widespread hemorrhagic necrosis. Thrombosis in large vessels may be seen. The histopathologic findings of humoral rejection vary with the severity of rejection and the time of tissue examination after revascularization [653], and may be modified by the new perioperative therapeutic interventions as mentioned earlier [34, 320, 342, 427, 656]. Typically, fibrin thrombi in portal and central veins, sinusoidal congestion and fibrin deposition, hemorrhage into the portal tracts and the space of Disse, portal and periportal edema, neutrophil infiltration in the portal tracts and lobules, and periportal hepatocyte necrosis are earliest changes observed from 2 to 24 h after implantation (Fig. 8.44) [149, 171, 265, 462]. In severe cases (hyperacute form), massive hemorrhagic necrosis develops within 1–2 days (Fig.  8.45), often accompanied by thrombosis and circumferential fibrin deposition in portal veins (Fig. 8.46). Fibrinoid necrosis of the hepatic artery branches may also be seen. Mild cases (acute form of humoral rejection) may show portal edema, mild portal neutrophilic infiltration, mild ductular reaction, spotty hepatocyte necrosis, centrilobular hepatocyte ballooning and cholestasis. Lymphocytic infiltration and other classical features of ACR are typically lacking. These cases are difficult to

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Fig.  8.44  Early signs of humoral rejection characterized by portal edema, mild portal inflammatory cell infiltrates including neutrophils, and hemorrhage

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Fig. 8.46  A thrombus noted in a portal vein in a case of humoral rejection. Note the presence of circumferential fibrin deposition (arrow). The adjacent liver parenchyma shows hemorrhagic necrosis (right)

8.6.1.3 C4d Immunohistochemical Staining

Fig. 8.45  Massive hemorrhagic necrosis seen in severe humoral rejection. Note the absence of inflammatory response

diagnose on biopsy because they may resemble biliary obstruction and preservation/reperfusion injury histologically. Even with immunofluorescent staining on frozen tissue for immunoglobulins (IgG and IgM) and complements (C1q, C3 and C4), the diagnosis may not be easy because the deposits tend to be transient and patchy, and can be difficult to distinguish from background staining. These allografts usually show a higher incidence of acute and chronic rejection and a higher incidence of biliary and hepatic artery complications during follow-up [171, 265, 572, 573].

Immunohistochemical detection of C4d, a marker of activation of the complement pathway, is now widely accepted as a useful diagnostic tool of antibodymediated rejection in renal and cardiac allografts [465]. Much less is known about its role in liver transplantation, however. In a study of 34 patients with ABO-incompatible living donor liver transplantation, Haga et al. reported that 50% of their patients showed strong C4d immunostaining in portal stroma in their liver biopsies performed within the first 3 postoperative weeks [264]. Positive staining was associated with a high postoperative anti-donor antibody titer and a poorer overall survival rate. Ten of 11 cases showing histologic evidence of acute humoral rejection, such as portal and periportal edema, neutrophilic infiltration, hemorrhage and necrosis, were positive for C4d, all of which had high antibody titers. These observations suggest that C4d deposition in the portal stroma is an indication of acute humoral rejection and the need for plasmapheresis in the setting of ABO-incompatible liver transplantation [484]. In ABO-compatible liver transplants, positive C4d immunostaining has been reported in 8–80% of postoperative biopsies with ACR and 23–100% cases of chronic rejection [55, 85, 135, 307, 388, 566, 575, 580, 673]. There is no consensus as to the staining patterns

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among these studies and positive staining has been variably detected in endothelial cells lining the portal veins, arteries, capillaries and sinusoids, and portal stroma. There appears to be a tendency that cases with higher grade of acute rejection are more likely to show C4d positivity and to show a sinusoidal staining pattern (Fig. 8.47). Interestingly, positive C4d staining is frequently associated with accumulation of macrophages in the portal tracts [135, 575], similar to the finding in renal allografts where macrophage is thought to be an important cellular player of humoral rejection [402]. It is thus suggested that in cases with unexplained macrophage-rich infiltrates, humoral rejection should be suspected if C4d is also positive [55]. Although the currently available data suggest a role of humoral mechanism in conventional ACR and chronic rejection [55, 85, 135, 307, 388, 566, 575, 580, 673], whether there is a circulating anti-donor antibody in the recipients is unknown because this is not examined in most of the studies. There have been only two studies that have demonstrated a possible correlation of C4d immunostaining with lymphocytotoxic antibody status [55, 566]. In the study by Sakashita et al. [566], positive C4d staining was seen in 82% of biopsies from lymphocyte crossmatch-positive patients in contrast to 33% from crossmatch-negative recipients. In the 9 C4d-positive cases in the crossmatch-positive group, graft biopsies showed moderate and severe acute rejection in two recipients, cholangitis in four, cholestasis in

one, lobular inflammation in one, and portal hemorrhage in one. Four patients died. This study thus suggests that positive lymphocyte crossmatch is associated with a higher frequency of C4d deposition and reduced patient survival, although only 11 crossmatch-positive cases were examined in this study. Positive C4d immunostaining has also been reported in allografts with other complications such as recurrent hepatitis C, recurrent hepatitis B, recurrent primary biliary cirrhosis, cholangitis and biliary obstruction, albeit with lower frequencies [85, 264, 566, 580]. Furthermore, positive C4d staining is detected in a high frequency in autoimmune hepatitis and chronic viral hepatitis B and C in a pediatric population without previous liver transplantation [76]. In addition to endothelial staining, a peculiar staining pattern of the portal lymphoid aggregates and periductal accentuation is observed in some of the cases [76]. Although these perplexing observations may suggest a role of humoral immune response in inflammatory liver diseases, it raises the doubt regarding the specificity of C4d immunostaining as a reliable diagnostic tool of humoral rejection in liver transplants. It is known that in addition to the classical antibody-mediated pathway, C4d can be activated by antibody-independent mechanisms such as the lectin pathway and C-reactive protein [119, 465]. It is also unclear at present whether detection of C4d deposition in an allograft biopsy that shows typical histologic features of ACR, chronic rejection or other posttransplant complications would offer any management benefit.

8.6.1.4 Differential Diagnosis

Fig.  8.47  A case of severe acute rejection with central perivenulitis shows a sinusoidal pattern of C4d deposition by immunohistochemistry, suggesting an involvement of humoral mechanism

The minimal diagnostic criteria for hyperacute humoral rejection were established by the International Working Party published in 1995. It includes rapid onset of graft dysfunction, histologic features of ischemic necrosis, predominantly neutrophilic infiltrates, and absence of other clearly defined causes of ischemia or infarction. The diagnosis is strengthened if neutrophilic or necrotizing arteritis is present, if immunoglobulin deposits can be demonstrated in the liver, and if preformed antidonor antibodies are found [653]. In addition to hyperacute rejection, other causes of acute graft failure during the early posttransplant period may include primary nonfunction, vascular thrombosis,

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severe hypotension and sepsis. The distinction is primarily based on clinical findings and clinicopathologic correlation. For example, hyperacute rejection may be associated with an initial short period of graft function, in contrast to primary nonfunction, and the finding of fibrinoid arteritis is helpful. As discussed earlier, C4d immunostaining has shown promise in confirming the diagnosis of humoral rejection [264]. In ABO-compatible grafts, antibody-mediated rejection can be difficult to distinguish from preservation/ reperfusion injury and biliary obstruction. Again, clinical findings and clinicopathologic correlation are imperative. Prominent infiltration with neutrophils and/or macrophages in the portal tracts with minimal ductular reaction should raise the suspicion of humoral rejection. The diagnostic role of C4d immunostaining in this setting needs to be further defined and the staining does not seem to be useful to help distinguish acute rejection from recurrent liver diseases [76, 85, 388, 566, 580].

8.6.2 Acute Rejection 8.6.2.1 General Consideration Acute rejection is an inflammatory process primarily mediated by cellular immunity. Hence, ACR and cellular rejection are alternative terms. The presumed target is the MHC antigen system [337]. It mainly involves T lymphocytes (both CD4+ and CD8+ T cells) and macrophages. Natural killer cells may also contribute. The role of humoral immunity and participation of B lymphocytes have been recently suggested, as evidenced by positive C4d immunostaining in histologically confirmed acute rejection [55, 85, 135, 307, 388, 566, 575, 580, 673]. Acute rejection is the most common type of rejection and the most common complication in the early posttransplant period. The incidence varies between 20 and 80% in different studies and is influenced by different regimens of immunosuppression [49, 150, 199, 245, 382, 481, 599, 712, 741]. In most of the cases, however, the rejection is mild, which does not lead to significant long-term sequelae if it is not associated with biochemical graft dysfunction and thus does not require additional immunosuppression. Clinically significant rejection is estimated to occur in 20–40% of patients and this rate appears to decline in recent years due to improved immunosuppressive therapies.

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Acute rejection may occur as early as 2 days or as late as many months after transplantation, but the majority of the cases are seen between 5 and 30 days. Patients with clinically relevant acute rejection may present with fever, enlargement and tenderness of the graft, and reduced bile flow. Laboratory findings generally lack specificity. Liver tests may show a preferentially cholestatic pattern with high serum levels of gamma-glutamyl transferase and alkaline phosphatase, but serum alanine aminotransferase and aspartate aminotransferase levels can also be significantly elevated. Leukocytosis and eosinophilia in the peripheral blood are common [46, 653]. Apparently, the occurrence of acute rejection is usually associated with suboptimal immunosuppression. It has been shown, however, that an increased risk of acute rejection is also associated with a number of recipient and donor parameters such as young recipient age, older donor age, prolonged cold ischemic time, higher pretransplant aspartate aminotransferase level, and fewer HLA-DR matches [150, 481, 599, 712]. Several earlier studies have suggested that the original recipient liver diseases have an impact on the incidence. For example, patients with autoimmune liver diseases and hepatitis C may have a higher risk to develop acute rejection than those with alcoholic hepatitis and hepatitis B [63, 184, 277, 438, 481, 590, 712].

8.6.2.2 Pathologic Features The pathologic features of acute rejection have been well defined as a triad of portal inflammation, bile duct damage, and endotheliitis (or endothelialitis). It is generally accepted that at least 2 of these 3 features are required for the diagnosis [46, 51, 295, 365, 653]. The diagnosis is strengthened if unequivocal endotheliitis is identified or >50% of the ducts are damaged [46, 653]. However, like many other liver diseases, the diagnostic features of acute rejection can vary considerably in different areas in the grafts. It is thus recommended that a minimum of five portal tracts and at least two sections at different levels need to be examined when evaluating a needle core biopsy for acute rejection [46]. The inflammatory infiltrates in the portal tracts are typically mixed (Fig. 8.48), but consist predominantly of small and large lymphocytes (Figs. 8.49). The large lymphocytes are activated immunoblastic forms that exhibit large nuclei, prominent nucleoli and abundant

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Fig.  8.48  Acute rejection featuring mixed inflammatory cell infiltrates in the portal tracts. Note the presence of mild bile duct damage (white arrow) and focal endotheliitis (black arrow) at this power

Fig. 8.49  The portal inflammatory infiltrates in acute rejection consist predominantly of small and large lymphocytes. Note the presence of mild bile duct damage (white arrow) and focal endotheliitis (black arrow)

basophilic cytoplasm. Most of the lymphocytes are CD8+ T lymphocytes, but immunophenotyping is unnecessary for the diagnosis of rejection unless PTLD is in the differential which is a B cell process. Other cell components include eosinophils, neutrophils, macrophages and plasma cells. The number of portal mast cells is also variably increased [31, 175], but this does not appear to be rejection-specific because similar findings have also been observed in other liver diseases including recurrent hepatitis C [161, 426]. Eosinophils are generally conspicuous, though still representing a minor component of the infiltrates (Fig. 8.50). Studies have suggested that the presence of

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Fig. 8.50  A case of acute rejection showing prominent eosinophils in portal inflammatory infiltrates. Note the presence of bile duct damage

prominent portal eosinophils is a useful feature of acute rejection [154, 333, 469], but its diagnostic value should not be overemphasized because increased portal eosinophils can be seen in many other liver diseases including drug hepatotoxicity and viral hepatitis [647]. It should be noted, however, that the commonly used immunosuppressive agents in liver transplants, such as cyclosporine, tacrolimus (FK506) and corticosteroids, do not cause histologic eosinophilia in allografts. In most cases, the inflammatory infiltrates are limited to the portal tracts. The presence of prominent interface hepatitis signifies a more severe form of acute rejection. Bile duct damage is characterized by inflammatory cell infiltration of the duct epithelium, mainly by lymphocytes and less frequently by neutrophils. Lympho­ cytes are typically found in the spaces between duct epithelial cells and between the epithelial cells and basement membrane (Figs.  8.48–8.50). Neutrophils may also be found within the lumens, similar to that seen in ascending cholangitis (Fig. 8.51). The demonstration of endotheliitis, presence of portal eosinophils, presence of centrilobular necroinflammation, lack of portal edema, lack of ductular reaction, and lack of cholestasis favor the diagnosis of acute rejection and help distinguish it from acute cholangitis [65]. Typically, the bile ducts are cuffed by mixed inflammatory cell infiltrates, and may become obscured in cases with heavy portal infiltrates (Fig. 8.52). In those cases, immunostaining for cytokeratins, usually cytokeratin 7 or 19, may be necessary to help identify their presence. The duct epithelium may show degenerative changes characterized by nuclear

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Fig. 8.51  Acute rejection with inflammatory cell infiltration of a bile duct, mainly by neutrophils. Neutrophils are also found in the lumen, reminiscent of ascending cholangitis

Fig. 8.52  Damaged bile duct (arrow) in acute rejection becomes obscured in a case with heavy portal infiltrates

enlargement, overlapping, pleomorphism and apoptosis, and cytoplasmic vacuolation and eosinophilia (Figs.  8.50–8.53). As mentioned above, bile ductular reaction is usually insignificant [65]. When prominent, it may signify another cause of bile duct damage such as preservation/reperfusion injury, cholangitis or large bile duct obstruction. Endotheliitis is the most specific diagnostic feature of acute rejection, most commonly seen in the portal veins but can also involve the terminal hepatic venules (central veins). It is characterized by subendothelial lymphocytic infiltration, often lifting up and disrupting

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Fig. 8.53  Bile duct damage in acute rejection characterized by lymphocytic infiltration of the duct epithelium. Note that the duct epithelium exhibits nuclear enlargement and overlapping, cytoplasmic eosinophilia, and perinuclear vacuolation (arrow)

Fig. 8.54  Portal vein endotheliitis in acute rejection characterized by subendothelial lymphocytic infiltrates that lift up and disrupt the overlying endothelium

the overlying endothelium (Fig.  8.54). Endotheliitis may also manifest as direct attachment of lymphocytes to the endothelial cells through cytoplasmic processes from the lumenal side (Fig. 8.55). The endothelial cells may be swollen and detached from the basement membrane. Endotheliitis is usually focal (Fig. 8.54) but can be circumferential in more severe cases (Fig.  8.56). Arterial changes including necrotizing arteritis have also been reported as a sign of severe acute rejection, but these findings are rarely seen in needle biopsies because the large vessels are typically not sampled.

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Fig. 8.55  Portal vein endotheliitis in acute rejection featuring direct attachment of lymphocytes to the endothelial cells from the lumenal side (arrow). Note the presence of dense subendothelial inflammatory infiltrates and focal disruption of the endothelial lining

posttransplant days as detailed above, but may show fewer blastic lymphocytes, less conspicuous bile duct damage, less intense subendothelial lymphocytic infiltration, slightly greater interface activity, and slightly more lobular activity [147]. In some cases, isolated central perivenulitis may be the only histologic finding (discussed below). Patients with late-onset acute rejection may show a poorer response to increased immunosuppression. An increased risk to evolve into chronic rejection and graft loss has been observed in some studies [22, 536, 678]. Histologically, late-onset acute rejection can be more difficult to diagnose because it may share more features with chronic hepatitis as described above. Mult­ iple biopsies and exclusion of other potential etiologies may be necessary to establish the diagnosis.

8.6.2.4 Central Perivenulitis

Fig. 8.56  Endotheliitis in acute rejection that circumferentially involves a portal vein

8.6.2.3 Late-Onset Acute Rejection Late-onset acute rejection occurs more than several months after transplantation, variably defined in the literature as >3 months, >6 months, or >1 year [18, 134, 201, 314, 460, 536, 636, 678, 733]. The reported incidence ranges from 7 to 32% in different studies, and it usually develops as a consequence of inadequate immunosuppression [22, 134, 150, 460, 692, 733]. The histologic features of late-onset acute rejection are essentially the same as those occurring in the early

Central perivenulitis is characterized by a spectrum of necroinflammatory changes involving the central veins and centrilobular hepatocytes. It has been described under various terms in the literature including centrilobular necrosis, central venulitis, centrilobular injury, centrilobular alterations, centrilobular changes, centrilobular inflammation, centrilobular necroinflammation, and hepatitic phase of rejection [294]. Histologically, central perivenulitis displays endotheliitis of the central veins with subendothelial mononuclear cell infiltration (Fig. 8.57). Frequently, there is perivenular hepatocyte necrosis and dropout, with extension of the inflammatory infiltrates into perivenular damaged hepatic parenchyma (Fig. 8.58). In some cases, centrilobular necrosis is prominent but the histologic features of endotheliitis are not apparent (Fig.  8.59). The inflammatory cell infiltrates may not be significant in damaged centrilobular regions in these cases. In the presence of typical portal changes of rejection, central perivenulitis is generally regarded as a sign of severe acute rejection and usually does not cause much diagnostic dilemma [144, 147, 294, 473]. However, isolated central perivenulitis, which is not accompanied by concomitant portal changes of rejection, frequently pose diagnostic challenges. In most of the cases, it may still represent a manifestation of rejection (either acute or chronic rejection), but other causes of centrilobular damage need to be investigated

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Fig. 8.57  Central perivenulitis in acute rejection characterized by subendothelial inflammatory cell infiltrates surrounding a central vein. Focal disruption of the endothelial lining is evident. There is only minimal damage to the surrounding hepatic parenchyma in this case

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Fig.  8.59  Central perivenulitis showing centrilobular necrosis but endotheliitis and perivenular inflammatory infiltrates are not prominent

was also implicated but this drug is no longer routinely used in most U.S. transplant centers as part of immunosuppressive therapies. The incidence of isolated central perivenulitis varies between 9 and 31% in different studies. It may occur in early posttransplant days but is most commonly seen as a late event after 3 months [294]. This late occurrence makes preservation/reperfusion injury an unlikely differential diagnosis. A number of studies have demonstrated that compared with portal-based rejection, cases with central perivenulitis are less likely to respond to increased immunosuppression, more likely to develop subsequent episodes of acute rejection, chronic rejection, perivenular (zone 3) fibrosis and graft failure [5, 20, 154, 274, 343, 389]. Fig. 8.58  Central perivenulitis showing perivenular hepatocyte necrosis/dropout accompanied by inflammatory cell infiltrates

[295, 327, 345, 478]. These mainly include recurrent or de novo autoimmune hepatitis, preservation/reperfusion injury, vascular thrombosis, and drug toxicity. Fortunately, both rejection and autoimmune hepatitis can be effectively treated with increased immunosuppression, and thus a clear-cut distinction may have minimal clinical significance. Centrilobular necrosis secondary to preservation/reperfusion injury or vascular thrombosis typically lacks significant inflammatory cell infiltration. The toxic effect of FK506 has been debated but it is generally believed to be an unlikely cause of centrilobular necrosis [144, 345]. Azathioprine

8.6.2.5 Grading of Acute Rejection The Banff schema is a widely used grading system for acute rejection, which was developed as a consensus by an international panel of pathologists, hepatologists and transplant surgeons [46]. It is a measure of the severity of necroinflammatory process associated with acute rejection, i.e., portal inflammation, bile duct damage and endotheliitis. It works in two ways: a global assessment of overall rejection (Table 8.6) and a semiquantitative measurement of rejection activity (Table 8.7). The global assessment grades acute rejection as indeterminate, mild (or grade I), moderate (or grade II),

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8  Liver Table  8.6  Banff schema for grading liver allograft acute rejection – global assessment [46] Global Criteria assessment Indeterminate

Portal inflammatory infiltrate that fails to meet the criteria for the diagnosis of acute rejection

Mild

Rejection infiltrate in a minority of the triads, that is generally mild, and confined within the portal spaces

Moderate

Rejection infiltrate, expanding most or all the triads

Severe

As above for moderate, with spillover into periportal areas and moderate to severe perivenular inflammation that extends into the hepatic parenchyma and is associated with perivenular hepatocyte necrosis

or severe (or grade III) after the histologic diagnosis of acute rejection is established. The diagnosis of indeterminate rejection should be restricted to cases that only have mild portal inflammatory infiltrates but lack convincing histologic evidence of bile duct damage or endotheliitis (Fig.  8.60). The infiltrates should not be explainable by other conditions such as recurrent liver diseases, and thus could represent minimal or early acute rejection. This grading schema is commonly used by practicing pathologists on a daily basis.

Fig. 8.60  Allograft biopsy on day 12 after transplantation for end stage primary sclerosing cholangitis showing mild portal mixed inflammatory cell infiltrates. Bile duct damage and endotheliitis are not evident. The findings are interpreted as indeterminate acute rejection

The semiquantitative measurement provides numerical scores for each of the three histologic features of acute rejection on a scale of 0 (absent) to 3 (severe). The scores are then added up to produce an overall rejection activity index (RAI) of 0–9. Roughly, a RAI score of 1–2 is considered indeterminate for acute rejection, 3–4 for mild rejection, 5–6 for moderate, and 7–9 for severe. Although RAI appears to offer a greater degree of precision, there have been no data to support

Table 8.7  Banff schema for grading liver allograft acute rejection – rejection activity index [46] Category Criteria Portal inflammation

Bile duct inflammation and damage

Venous endothelial inflammation

Score

Mostly lymphocytic inflammation involving, but not noticeably expending, a minority of the triads

1

Expansion of most or all of the triads, by a mixed infiltrate containing lymphocytes with occasional blasts, neutrophils and eosinophils

2

Marked expansion of most or all of the triads by a mixed infiltrate containing numerous blasts and eosinophils with inflammatory spillover into the periportal parenchyma

3

A minority of the ducts are cuffed and infiltrated by inflammatory cells and show only mild reactive changes such as increased nuclear: cytoplasmic ratio of the epithelial cells

1

Most or all of the ducts infiltrated by inflammatory cells. More than an occasional duct shows degenerative changes such as nuclear pleomorphism, disordered polarity and cytoplasmic vacuolization of the epithelium

2

As above for 2, with most or all of the ducts showing degenerative changes or focal lumenal disruption

3

Subendothelial lymphocytic infiltration involving some, but not a majority of the portal and/or hepatic venules

1

Subendothelial infiltration involving most or all of the portal and/or hepatic venules

2

As above for 2, with moderate or severe perivenular inflammation that extends into the perivenular parenchyma and is associated with perivenular hepatocyte necrosis

3

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that it is a better approach than global assessment [46]. In a study of 231 cases, acute rejection was classified as indeterminate, mild, moderate and severe in 4 (1.7%), 41 (17.8%), 83 (35.9%) and 103 (44.6%) cases by RAI, and 7 (3.0%), 52 (22.5%), 94 (40.7%) and 78 (33.8%) cases by global assessment, respectively, which demonstrated a good agreement between the two approaches (k-value: 0.70) [289]. However, the RAI schema may force pathologists to look for the critical histologic features when evaluating allograft biopsies and may be more useful for research and clinical trials. Studies have shown that the Banff schema is simple to use, reasonably reproducible and clinically useful [150, 152, 480, 503]. Patients with moderate or severe rejection are more likely to have abnormal liver function tests and more likely to develop perivenular fibrosis, chronic rejection and graft failure [70, 150, 152]. There are also studies, however, that failed to show correlation between the histologic grade or RAI score of acute rejection and graft outcomes [162, 289]. These observations may reflect the effects of successful early treatment of cases with more severe changes [295].

8.6.2.6 Response to Treatment As mentioned earlier, mild rejection without associated biochemical graft dysfunction usually does not require specific management. The vast majority of the cases will recover spontaneously without long-term sequelae. On the other hand, additional immunosuppression is necessary for mild rejection with associated abnormal liver function tests and for moderate or severe acute rejection. Most cases of moderate or severe acute rejection can be successfully treated and in only a small subset of the patients, more severe complications, such as steroid-resistant rejection, chronic rejection, perivenular fibrosis and graft loss, may result [49, 150]. Effective treatment of acute rejection with increased immunosuppression is evidenced by subsidence of the inflammatory infiltrates, usually occurring within 24 h. Complete recovery normally takes 7–10 days, and endotheliitis usually resolves before bile duct damage. In cases with complete resolution, follow-up biopsy may show regenerative changes such as mild ductular reaction and hepatocyte mitosis and binucleation. Mild cholestasis may be present. However, these cases are seldom rebiopsied if they also show a satisfactory biochemical response to additional immunosuppression.

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Fig. 8.61  Resolving acute rejection showing partial response to increased immunosuppression. Note the presence of residual inflammatory infiltrates in the portal tract and residual bile duct damage. Endotheliitis has completely resolved

In cases with partial response, some of the features of acute rejection are still present but greatly reduced comparing with the original biopsy (Fig. 8.61). In nonresponders, repeat biopsy shows ongoing or worsening rejection, indicating a need for more vigorous immunosuppression. It is always a good practice to compare with previous biopsies and to discuss the histologic findings with transplant team for a more accurate assessment and better clinicopathologic correlation.

8.6.2.7 Differential Diagnosis Acute rejection typically occurs within 30 days after transplantation and its diagnosis is usually straightforward if the histologic triad of mixed portal inflammation, bile duct damage and endotheliitis is present. Differential diagnosis may include preservation/reperfusion injury, acute cholangitis and large bile duct obstruction, all of which may show bile duct damage with degenerative changes of the duct epithelium. Prominent ductular reaction, predominantly neutrophilic portal infiltration, portal edema and lack of endotheliitis help the distinction from acute rejection. Late-onset acute rejection poses more diagnostic challenges because it needs to be distinguished from a variety of conditions, particularly recurrent liver diseases. The detailed histopathologic features of these diseases are described in Sect. 8.7. Only the distinction between recurrent viral hepatitis C (HCV) infection and

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acute rejection is discussed here because this is a major diagnostic problem in posttransplant liver biopsies. HCV reinfection occurs immediately after transplantation and HCV replication in the graft begins as soon as a few hours in most patients [231]. Histologic features of recurrent HCV may be recognizable as early as 7 days, but typically become evident within 3–8 weeks. The early or acute phase of recurrent HCV is dominated by lobular changes featuring lobular disarray, hepatocyte ballooning, frequent acidophil bodies, sinusoidal dilatation, spotty sinusoidal lymphocytic infiltration, Kupffer cell prominence, and mild fatty change (Fig. 8.62) [43, 254, 494, 516, 517, 576]. Portal inflammation may be absent, minimal or mild at this phase, and consists primarily of lymphocytes if present. Steatosis is predominantly macrovesicular, typically without a particular zonal distribution. In patients with HCV genotype 3 infection, steatosis can be severe and may serve as the initial histologic sign of recurrent disease (Fig.  8.63) [244]. Although these “hepatitic” lobular changes have also been described in cases with rejection [532, 575], their presence is generally regarded as signs of early recurrent HCV. With time, recurrent HCV evolves into chronic hepatitis, histologically similar to that seen in non-transplanted livers. This usually occurs 4–12 months after transplantation. The lobules may still show the presence of acidophil bodies, foci of lymphocytic lobulitis and steatosis, the dominant feature is portal mononuclear cell infiltration, often associated with lymphoid

Fig. 8.63  Fat change is common in recurrent hepatitis C

Fig.  8.64  Recurrent hepatitis C with portal mononuclear cell infiltrates and a lymphoid aggregate. The inflammatory infiltrates appear to stay away from bile duct and vasculature

Fig. 8.62  Early recurrent hepatitis C featuring lobular changes characterized by lobular disarray, hepatocyte ballooning, frequent acidophil bodies, and spotty lymphocytic infiltration in the lobules

aggregates (Fig. 8.64) and interface activity (Fig. 8.65). The portal infiltrates consist predominantly of lymphocytes with occasional plasma cells. Eosinophils and neutrophils are not prominent. Typically, the infiltrates stay away from bile ducts and veins (Figs. 8.64 and 8.65), but in some cases, particularly those with dense portal infiltrates, bile duct damage and even endotheliitis can be seen, which makes the distinction from acute rejection very difficult (Figs.  8.66 and 8.67). However, bile duct damage in recurrent HCV is usually mild and focal and does not cause bile duct loss. Endotheliitis in recurrent HCV typically does not involve the central veins [370, 494, 617, 727]. These latter findings are helpful in difficult cases. The presence of significant portal and/or periportal fibrosis,

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Fig. 8.65  Recurrent hepatitis C with prominent interface activity (piecemeal necrosis). The bile duct is spared from the inflammatory infiltrates

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Fig. 8.67  Focal endotheliitis (arrow) noted in a case of recurrent hepatitis C

Table  8.8  Distinction between acute rejection and recurrent hepatitis C Features Acute rejection Recurrent hepatitis C

Fig. 8.66  Mild bile duct damage noted in a case of recurrent hepatitis C

bridging fibrosis or cirrhosis is characteristic of recurrent HCV, whereas centrilobular fibrosis can be seen as a result of centrilobular rejection (central perivenulitis). Prominent bile ductular reaction is also a feature of recurrent HCV, usually associated with the progression of fibrosis. Table  8.8 lists a number of clinicopathologic features that can be used to help distinguish acute rejection from recurrent HCV, but there is a significant overlap between these two entities and none of the features is absolutely specific [92, 295, 516, 739]. An integrated assessment is thus imperative. It should also be noted that even for experienced liver transplant pathologists, the interobserver and intraobserver agreement

Timing

Usually <1 month

Usually >1 month

Blood viral load

Low

High

Serum levels of immunosuppressants

Low

Adequate

Portal inflammation

Mixed

Mononuclear

Lymphoid aggregates No

Common

Portal eosinophils

Conspicuous

Inconspicuous

Interface activity

Mild

Variable

Bile duct damage

Prominent

Mild and focal

Bile duct loss

Seen in cases progressing to chronic rejection

No

Ductular reaction

Uncommon

Common

Portal endotheliitis

Prominent

Mild

Fibrosis

No

Common

Acidophil bodies

Occasional

Frequent

Fatty change

No

Common

Central perivenulitis

Yes

No

of the histopathologic diagnosis of acute rejection vs. recurrent HCV can be low [542]. It is thus not surprising that there are cases where a clear-cut distinction is

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very difficult or impossible. These cases are usually those with convincing histologic evidence of bile duct damage and/or endotheliitis, and may be regarded as concurrent recurrent HCV and acute rejection. In the majority of these cases, however, the rejection component is mild at most in severity and should be considered as a secondary process as a general rule. Thus, these patients should not be treated for rejection. Instead, they should be closely followed and may be rebiopsied if liver function tests continue to rise [148]. Increased immunosuppression is indicated only when rejection is felt to be the primary diagnosis, and such cases are usually graded as moderate or severe. The rationale is that recurrent HCV carries a much worse prognosis than acute rejection and that overtreating rejection with increased immunosuppression in those patients have shown significant deleterious effects [108, 354]. On the other hand, the detrimental effects of antiviral therapies on rejection are much less well established [92, 569]. Although some studies have shown that antiviral therapies increase the risk of acute and chronic rejection [561, 623, 631, 705], the prevalence and severity remain controversial [92, 569].

Early recognition is crucial, which allows earlier effective therapeutic interventions. Studies have shown that chronic rejection can be reversed and grafts can be salvaged if diagnosed early enough in the course of the disease [70, 71, 585, 604, 711]. Advanced chronic rejection is generally considered irreversible, which does not respond to additional immunosuppressive therapies and leads to graft loss. A number of risk factors have been reported to be associated with the development of chronic rejection [70, 71, 146, 230, 260, 481, 711]. Among these, the number and severity of prior episodes of acute rejection, inadequate immunosuppression and transplantation for autoimmune liver diseases appear to be most reproducible in various studies. Patients who had graft failure due to chronic rejection are also more likely to develop chronic rejection again in their new grafts [481]. The role of other factors, such as CMV infection, interferon therapy for recurrent HCV, sex mismatch, donor and recipient age, ethnic origins, and HLA incompatibility remain controversial [146, 230, 481, 569, 711].

8.6.3.2 Pathologic Features

8.6.3 Chronic Rejection 8.6.3.1 General Consideration Chronic rejection usually develops as the end result of persistent or severe acute rejection and thus involves similar immunological mechanisms that mediate the acute rejection. It is a dynamic process that may begin within the first month as acute rejection, become evident within several months (usually after 2 months), and lead to graft failure within the first year after transplantation [146, 653, 711]. Chronic rejection can also develop insidiously without documented episodes of prior acute rejection. This scenario may occur as a late event (after 1 year) and typically result from inadequate immunosuppression [146]. Clinically, patients with chronic rejection show progressive jaundice and a cholestatic pattern of abnormal liver function tests with a preferential elevation of serum gamma-glutamyl transferase and alkaline phosphatase levels. Chronic rejection affects only 2–5% of liver allografts [146, 481, 711], but remains to be an important cause of late graft dysfunction and failure [147].

Chronic rejection features two major histopathologic abnormalities: progressive bile duct loss (ductopenia) and obliterative arteriopathy. In the majority of the cases, both features are present, but cases with pure ductopenia or pure arteriopathy have been well documented [146, 653]. Obliterative arteriopathy primarily affects large and medium-sized arterial branches, usually located in or near the hilar region. In explanted failed allografts, obliterative arteriopathy can be readily appreciated, characterized by intimal thickening and intimal accumulation of lipid-laden foamy macrophages, which cause lumenal narrowing and occlusion (Fig.  8.68). The vascular lesion mainly affects the intima but the entire arterial wall can be involved in severe cases. With progression, proliferation of myofibroblasts and fibrosis ensue, which replace foamy cells and may lead to complete obliteration of the arteries and secondary ischemic damage to the hepatocytes and bile ducts. Large bile ducts may show necrosis, detachment of the lining epithelium, mural fibrosis and inflammatory cell infiltration (Fig. 8.69). However, the diagnostic value of the pathognomonic obliterative arteriopathy is limited because large vessels

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Fig. 8.68  Obliterative arteriopathy in chronic rejection characterized by intimal thickening with lipid-laden foamy macrophages. The lumen is near completely occluded and the lesion focally involves the entire arterial wall

Fig. 8.70  Pigmented foamy macrophages within the sinusoids are sometimes seen in chronic rejection

Fig. 8.69  A large bile duct in an explanted failed allograft due to chronic rejection showing inflammatory cell infiltrates in the duct wall

Fig.  8.71  Chronic rejection featuring centrilobular fibrosis, resembling veno-occlusive disease

are almost never sampled by a needle biopsy. Histologic clues in a biopsy that may suggest its presence are centrilobular ischemic changes manifested by hepatocyte ballooning, necrosis/dropout, and cholestasis in the centrilobular regions. Clusters of foamy macrophages, usually pigmented, within the sinusoids are also a common finding, which are believed to represent a nonspecific response to hepatocyte damage and cholestasis (Fig. 8.70). A variable degree of centrilobular/perivenular fibrosis may develop. In advanced cases, this may lead to fibrous obliteration of the central veins resembling veno-occlusive disease [147, 474], and central-tocentral or central-to-portal bridging fibrosis (Fig. 8.71).

These centrilobular ischemic changes secondary to obliterative arteriopathy overlap with central perivenulitis related to acute rejection, but the associated inflammatory infiltrates are usually inconspicuous in chronic rejection. Correlation with the findings in the portal tracts should be helpful. Although obliterative arteriopathy typically does not affect small arteries, loss of small arterial branches in the portal tracts has been observed in chronic rejection [70, 479]. This occurs early in chronic rejection before bile duct loss [425, 479] and appears to be a predictor of graft failure [70]. The currently available data have failed to show a direct association of small

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arterial loss with large vessel arteriopathy, suggesting that it is probably mediated by other mechanisms, such as an inflammatory process [479]. Progressive bile duct loss is a hallmark of chronic rejection, primarily mediated by immunological and ischemic mechanisms [373, 472, 711]. It mainly affects small interlobular bile ducts present in the portal tracts and thus can be readily recognized on a needle biopsy (Fig. 8.72). It has been recommended that 20 or more portal tracts are needed in order to make a confident determination whether ductopenia is present [653], but chronic rejection can be reliably diagnosed by experienced pathologists with considerably fewer portal tracts. In an early study, there was a near unanimous diagnosis of chronic rejection by 5 participating patho­ logists in 18 biopsies that contained only 4–15 portal tracts (mean: 8.4) [151]. In cases where both bile ducts and small arterial branches are vanished, identification of portal tracts can be difficult, which may require a subjective interpretation based on the shape, location and internal structure of the connective tissue that is presumed to represent a portal tract (Fig. 8.73) [146]. A firm diagnosis of chronic rejection may sometimes require examination of several subsequent biopsies. Complete bile duct loss is preceded by a phase of dystrophic or degenerative changes of the duct epithelium. These early changes manifest as eosinophilic transformation of the cytoplasm, unevenly spaced nuclei, nuclear enlargement, hyperchromasia and pleomorphism (Figs.  8.74 and 8.75). The ducts may be

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Fig.  8.73  Chronic rejection with both bile duct and hepatic artery vanished in the portal tract. Only portal veins are present. Note the absence of ductular reaction. There is only a minimal inflammatory cell infiltrate

Fig.  8.74  Early chronic rejection featuring dystrophic/degenerative changes of the duct epithelium, characterized by cytoplasmic eosinophilia, unevenly spaced nuclei, and nuclear pleomorphism. The portal tract is infiltrated by inflammatory cells at this stage

Fig.  8.72  Chronic rejection featuring bile duct loss. Note the presence of a hepatic artery (arrow) and portal veins. There is only a minimal inflammatory cell infiltrate in the portal tract. No ductular reaction is evident

only partially lined by epithelial cells [146, 653]. It has been shown that the expression level of a senescencerelated protein, p21WAF1/Cip1, is increased in degenerating duct epithelium and is decreased with successful treatment, suggesting that replicative senescence accounts for the characteristic cytological changes in the early phase of chronic rejection [395]. There is usually a mild inflammatory cell infiltrate in the portal tracts at this stage, consisting predominantly of lymphocytes. Compared with acute rejection, however, the portal inflammation is usually less intense, eosinophils

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Fig. 8.75  Early chronic rejection featuring dystrophic/degenerative changes of the duct epithelium, characterized by cytoplasmic eosinophilia, unevenly spaced nuclei, and nuclear pleomorphism. The duct is partially lined by epithelial cells. The inflammatory cell infiltration of the duct epithelium is not evident

are less frequent, lymphocytic infiltration of the duct epithelium is less prominent, and endotheliitis is not evident. By the time the bile ducts are completely gone, there may be only minimal or no inflammatory cells present in the portal tracts, giving rise to a “burntout” appearance (Figs. 8.72 and 8.73). A salient feature of chronic rejection is the lack of ductular reaction despite bile duct degeneration and loss (Figs. 8.72 and 8.73). This is in marked contrast to other liver diseases that cause bile duct destruction, such as primary biliary cirrhosis and PSC, which is almost always accompanied by proliferating bile ductules. Thus, the presence of ductular reaction is an unusual finding for chronic rejection. In cases with established diagnosis of chronic rejection, finding ductular reaction usually signals a recovering process resulting from successful treatment [71]. The underlying mechanisms for the lack of ductular reaction in chronic rejection remain to be elucidated, but may involve increased apoptotic activity [340], impaired regenerative potential of the hepatic progenitor cells [688], and denervation [99]. Chronic rejection usually does not cause significant portal or periportal fibrosis, and thus cirrhosis secondary to chronic rejection is an unusual finding. However, ischemic damage secondary to obliterative arteriopathy can lead to centrilobular fibrosis, central vein obliteration, and central-to-central or central-to-portal bridging fibrosis, as described previously. In advanced cases with extensive liver parenchymal extinction, this

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may generate a cirrhosis-like appearance [474]. True regenerative nodules are not formed, however. Several studies have observed a “hepatitic” phase in chronic rejection, usually prior to bile duct loss. This is characterized by spotty inflammatory cell infiltrates in the lobules, apoptotic hepatocytes and interface activity, similar to those seen in chronic hepatitis [286, 479, 532, 585]. Although this is generally regarded as a transitional phase from acute rejection to chronic rejection (so-called “transition” hepatitis), some studies have suggested that this may actually represent a unique chronic hepatitis-like pattern of chronic rejection that primarily target hepatocytes probably by both humoral and cellular mechanisms. Cases with interface hepatitis have been shown to be more likely to develop portal-based fibrosis and cirrhosis. In the study by Herzog et al. [286], interface hepatitis was observed in 29 of 119 (24.4%) allografts at a mean interval of 23.9 months after transplantation, of which 23 (79.3%) cases were noted after 1 year. Sixteen allografts had other concurrent features of chronic rejection, such as foamy cell arteriopathy, bile duct loss, or centrilobular fibrosis. During a mean follow-up of 12 years, 23 (79.3%) cases showed at least periportal fibrosis, in contrast to only 11% in cases without interface hepatitis. Seven patients developed bridging fibrosis and four had definitive cirrhosis. Five patients died and seven required retransplantation. In the absence of classical features of chronic rejection, the diagnosis of this purported hepatitic form of chronic rejection appears difficult and needs to be differentiated from other hepatitic diseases such as recurrent viral hepatitis and de novo autoimmune hepatitis.

8.6.3.3 Staging of Chronic Rejection Chronic rejection is staged as early and late based on the recommendation by an international panel (Table 8.9) [146]. This staging schema emphasizes the recognition of histologic features that characterize the early stage of chronic rejection, and has greatly improved the sensitivity for the diagnosis [585]. It is potentially important in guiding patient management because early chronic rejection is potentially reversible with increased immunosuppression, whereas late chronic rejection is generally considered irreversible and usually requires retransplantation. It is recommended, however, that the clinical decision to either increase immunosuppression or retransplant should be

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8  Liver Table 8.9  Histopathologic staging of liver allograft chronic rejection [146] Structure

Early chronic rejection

Late chronic rejection

Small bile ducts (<60 mm)

Degenerative changes involving a majority of ducts: eosinophilic transformation of the cytoplasm; increased nuclear: cytoplasmic ratio; nuclear hyperchromasia; uneven nuclear spacing; ducts only partially lined by biliary epithelial cells Bile duct loss in <50% of portal tracts

Degenerative changes in remaining bile ducts

Terminal hepatic venules and zone 3 hepatocytes

Intimal/lumenal inflammation Lytic zone 3 necrosis and inflammation Mild perivenular fibrosis

Focal obliteration Variable inflammation Severe (bridging) fibrosis

Portal tract hepatic arterioles

Occasional loss involving <25% of portal tracts

Loss involving >25% of portal tracts

Other

So-called “transition” hepatitis with spotty necrosis of hepatocytes

Sinusoidal foam cell accumulation; marked cholestasis

Large perihilar hepatic artery branches

Intimal inflammation, focal foam cell deposition without lumenal compromise

Lumenal narrowing by subintimal foam cells Fibrointimal proliferation

Large perihilar bile ducts

Inflammation damage and focal foam cell deposition

Mural fibrosis

based on a complete clinicopathologic evaluation of the patients, rather than histologic findings alone [146]. A small fraction of the patients with advanced bile duct loss may still recover spontaneously or with additional immunosuppression. In needle biopsy specimens, early chronic rejection features bile duct dystrophic/degenerative changes, seen in >50% of the portal tracts. Bile duct loss may be appreciated but should be seen in <50% of the portal tracts. It is important to remember, however, that up to 20% of the portal tracts may physiologically lack bile ducts. Thus, bile duct loss is defined as <80% of the portal tracts contain bile ducts [146]. A tedious counting of the portal tracts is necessary in equivocal cases. In late chronic rejection, bile duct loss is more readily recognizable, seen in >50% of the portal tracts. Severe perivenular fibrosis with at least focal central-to-central or central-toportal bridging and/or veno-occlusive-like fibrous obliteration of the central veins is also a sign of late chronic rejection. Inflammatory activity typically subsides at the late stage. The liver parenchyma may show marked canalicular and intracytoplasmic cholestasis.

8.6.3.4 Differential Diagnosis In needle biopsy specimens, problems in differential diagnosis center on bile duct degeneration and loss,

Loss in ³50% of portal tracts

which can be seen in a number of conditions unrelated to rejection. Examples may include recurrent PSC, recurrent primary biliary cirrhosis, ischemic cholangiopathy secondary to hepatic artery stricture or thrombosis, biliary stricture or obstruction at anastomosis, adverse drug effects, and CMV infection. In general, the characteristic clinicopathologic features of these conditions, detailed in other chapters, can be readily distinguished from ductopenic chronic rejection [145, 147, 711]. As mentioned earlier, a variable degree of ductular reaction is always present in these conditions, in contrast to chronic rejection. Centrilobular hepatocyte injury and perivenular fibrosis also cause diagnostic dilemma, which has been discussed previously in Sect.8.6.2.4. When isolated perivenular fibrosis is present, various etiologies of venous outflow obstruction including those that cause veno-occlusive disease and Budd–Chiari syndrome should be ruled out. When ductopenia is evident, chronic rejection usually does not cause confusion with recurrent viral hepatitis such as recurrent HCV. However, chronic rejection and recurrent HCV can coexist. This features an unusual mononuclear cell infiltrate in the portal tracts in the absence of bile ducts and ductular reaction (Fig. 8.76). Interface activity, lymphoid aggregates and lobular inflammation may be prominent as seen in a typical case of HCV. In difficult cases, immunostaining

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Fig. 8.76  Concurrent chronic rejection and recurrent hepatitis C showing inflammatory cell infiltrates in the portal tract with foci of interface activity. There is a lack of bile duct

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management of liver transplant recipients. In a study that evaluated the causes of death in 299 adult liver transplant recipients who survived >3 years, 9 (56%) of the 16 patients who died of hepatic diseases had recurrent diseases, accounting for 24% of all 38 late deaths [528]. In a more recent study of 679 patients, 29% of all the late deaths were attributed to recurrent liver diseases [394]. With the exception of inherited metabolic liver diseases, most conditions for which liver transplantation is performed can recur in allografts. Like native livers, allografts can also acquire disorders that are not the causes for transplantation, for instance, new infection by hepatitis viruses and de novo autoimmune hepatitis. The histological diagnosis of recurrent or newly acquired liver diseases can be challenging because of compounding transplant-related factors. For example, recurrent hepatitis C viral infection can be difficult to distinguish from ACR. Recurrent primary biliary cirrhosis or PSC may be difficult to separate from chronic rejection or ischemic cholangiopathy. In addition, rejection and recurrent disease can occur simultaneously, which further perplexes the biopsy interpretation.

8.7.1 Hepatitis C Virus Infection

Fig. 8.77  Immunostaining for cytokeratin 7 confirms the absence of bile duct and ductular reaction in a case of concurrent chronic rejection and recurrent hepatitis C. Note the presence of inflammatory cells in the portal tract caused by recurrent hepatitis C

for cytokeratins, usually cytokeratin 7 or 19, may be necessary to confirm the absence of bile ducts (Fig. 8.77). The clinical management can be problematic in these cases.

8.7 Recurrent and De Novo Liver Diseases Andres Roma, and Hanlin L. Wang With improved graft and patient survival, disease recurrence has become a common problem in the

HCV infection is the most common recurrent disease after liver transplantation, which is virtually universal in recipients with positive HCV RNA at the time of transplantation [61, 222]. The viral particles are present in the circulation and infect the allografts immediately following reperfusion. Viral replication in the grafts begins as early as a few hours in most patients [231, 535]. Similar to primary HCV infection in nontransplant patients, recurrent HCV may be clinically insidious at the beginning in many cases but will eventually evolve into chronic hepatitis with fibrosis over time. Comparing with that in non-transplant patients, the natural history of HCV reinfection is accelerated in transplant recipients, with 10–30% of the patients progressing to cirrhosis within 5 years after transplantation and >40% after 10 years. The median interval from transplantation to cirrhosis is 9.5 years in these patients with a fibrosis progression rate of 0.3–0.6 stage per year (using a 0–4 scale for fibrosis staging), in contrast to 30 years in non-transplant patients with a progression rate of 0.1–0.2 stage per year [222]. A number of viral, recipient, donor and posttransplant factors have been shown to be associated with severe

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recurrence, such as high pretransplant viral load, infection by HCV genotype 1b or 4, and HIV coinfection. However, accelerated viral replication in the setting of immunosuppression appears to be the most important risk factor for the aggressive clinical course [535]. The histological features of recurrent HCV are essentially similar to those seen in native livers but vary along the posttransplant course [254, 257, 508]. As described in Sect. 8.6.2.7 the histological features of recurrent HCV may be recognizable as early as 7 days after transplantation, and typically become evident within 3–8 weeks. During this early acute phase, lobular disarray, hepatocyte ballooning, acidophil bodies, Kupffer cell hyperplasia, increased sinusoidal lymphocytes, sinusoidal dilatation and steatosis are the most common findings in allograft biopsy (Figs.  8.62 and 8.78). Portal infiltrates are usually insignificant at this phase. Steatosis, predominantly macrovesicular, can be severe in cases with HCV genotype 3 infection and may be the initial histological sign of recurrent disease [244]. Later in the course of the disease, more typical features of chronic hepatitis develop, characterized by a variable degree of mononuclear cell infiltrates in the portal tracts while some of the lobular changes, such as spotty foci of lymphocytic lobulitis, acidophil bodies and steatosis, persist. The portal infiltrates are composed mainly of lymphocytes, but may also include occasional plasma cells and eosinophils. Lymphoid aggregates, often with fully developed germinal centers, are characteristic, though not pathognomonic (Fig.  8.64). A variable degree of interface hepatitis (piecemeal necrosis) may also be present (Fig. 8.65). Fibrosis is commonly seen in biopsies obtained after

Fig. 8.78  Early phase of recurrent hepatitis C featuring lobular inflammation with increased sinusoidal lymphocytes

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Fig. 8.79  Recurrent hepatitis C with periportal fibrosis and septal formation highlighted by trichrome stain. Note the presence of mild steatosis that is commonly seen in recurrent hepatitis C

the first year of transplantation (Fig.  8.79), but can develop much earlier in some cases [60, 196]. Recurrent HCV in allografts is graded and staged in the same fashion as for primary infection in native livers. It should be noted, however, that there have been a number of grading and staging systems available for chronic hepatitis, which all share the assessment of portal and lobular necroinflammation for grade and portalbased fibrosis for stage [84]. A consistent application of a selected grading and staging system to allograft biopsies is important to the assessment of disease progression and effectiveness of therapeutic intervention. In the absence of significant portal inflammation, the lobular changes in early recurrent HCV may need to be distinguished from preservation/reperfusion injury. Characteristically, lobular changes secondary to preservation/reperfusion injury involve centrilobular (zone 3) hepatocytes whereas those associated with recurrent HCV do not have a particular zonal distribution. Centri­ lobular hepatocyte dropout or necrosis may be prominent in preservation/reperfusion injury but confluent necrosis is exceedingly rare in recurrent HCV. One of the main differential diagnoses to consider is ACR. This is particularly problematic because the portal infiltrates in HCV can involve bile ducts to cause bile duct damage (the Poulsen lesion) and can show subendothelial inflammation in portal veins, identical to “endotheliitis.” The histological distinction between recurrent HCV and acute rejection is detailed under Sect.8.6 (Table 8.8). Suffice it to say here that the bile duct damage in HCV is usually mild and focal, and does not cause bile duct loss (Fig. 8.66). HCV-associated

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endotheliitis is also mild and focal (Fig. 8.67), and typically does not involve the central veins. The findings of significant steatosis, predominantly lobular inflammation, portal lymphoid aggregates, ductular reaction and fibrosis all favor recurrent HCV. There have been a number of studies to suggest that various ancillary tools can be utilized to help distinguish recurrent HCV from acute rejection [516]. Immunohistochemical studies performed on allograft biopsies have shown that there is a correlation of hepatocyte expression of HCV antigen with serum viral load and histological changes of recurrent HCV [190, 250, 494, 515, 697]. In a study of 215 allograft biopsies from 152 HCV-positive recipients [250] , HCV antigen was detected in the cytoplasm of the hepatocytes in 78% of the biopsies. Among these, 57% of the biopsies performed within 30 days after transplantation, 92% of the biopsies performed between 31 and 180 days and 74% of the biopsies performed after 180 days showed positive staining. Overall, cases with histological features of recurrent hepatitis were more frequently positive for HCV antigen comparing to those with rejection (89 vs. 59%) and showed a higher median percentage of positive hepatocytes (40 vs. 1%). Interestingly, in 16 cases where a high number of antigen-positive hepatocytes were detected, a diagnosis of recurrent HCV was rendered clinically despite inconclusive histological findings. These findings are quite promising but the currently available antibodies for immunohistochemical detection of HCV antigens do not appear to be reliable for paraffin sections. In addition, the staining specificity needs further investigation as positive staining was also observed in cases with definitive clinical and histological evidence of rejection and in the absence of serological markers of HCV infection [250, 697]. In situ PCR has been shown to be a more sensitive method for HCV RNA detection in allograft biopsies [2, 205, 494]. Differential gene expression profiling has also shown promise in the distinction between recurrent HCV and rejection [267, 619, 646]. These techniques are not readily available in surgical pathology laboratories for the purpose of diagnosis, however.

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recurrence rate was 1% at 1 year and 4% at 5 years in a recent study [223] , while the patient survival rate after transplantation improved from 50% in the late 1980s and early 1990s to almost 80% in 2002 [331]. Several antiviral drugs (nucleoside or nucleotide analogues) have been approved by the Food and Drug Administration for the treatment of chronic hepatitis B, with the objective to achieve viral suppression to undetectable HBV DNA levels prior to transplantation [25, 218, 355]. After transplantation, combined use of HBIG and antiviral drugs has emerged as the current prophylaxis to prevent reinfection [123, 268, 723]. In a recent review, it was concluded that pretransplant complete viral suppression and posttransplant combined HBIG/antiviral drug therapy lead to successful prevention of HBV reinfection in ~95% of the patients [52]. The spectrum of histological findings of recurrent HBV is similar to that seen in the non-transplant setting [660]. In the acute phase of reinfection, usually occurring between 1 and 6 months post transplantation, the histological changes related to HBV reinfection can be subtle and may resemble other early posttransplant complications such as ACR or preservation/reperfusion injury. Allograft biopsy may show varying degrees of lobular disarray, hepatocyte ballooning, spotty foci of lobular inflammation, acidophilic bodies, Kupffer cell hypertrophy, and portal mononuclear cell infiltrates. Rarely, confluent or multiacinar bridging necrosis with collapse of the lobular framework may be seen in patients with severe acute hepatitis (Figs.  8.80 and

8.7.2 Hepatitis B Virus Infection In contrast to recurrent HCV, recurrent HBV infection is an uncommon event nowadays [58]. The reported

Fig.  8.80  Recurrent hepatitis B showing bridging necrosis. Note the presence of inflammatory cell infiltrates at the interface. The remaining hepatocytes exhibit a nodular appearance, resembling cirrhosis

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Fig. 8.81  Recurrent hepatitis B with bridging necrosis and collapse of the lobular framework. It differs from cirrhosis in that there is minimal, if any, collagen deposition at collapsed areas as shown by trichrome stain

8.81). Viral antigens can be demonstrated by immunohistochemistry, usually with nuclear and cytoplasmic expression of HBV core antigen (HBcAg). Stainable HBV surface antigen (HBsAg) may also be detected, but ground-glass hepatocytes are usually not discernable in the acute phase. Bile duct damage and endothelialitis are not features of recurrent HBV, and if present, should raise the concern for acute rejection. In the chronic phase, usually occurring 6 months post transplantation, lobular changes of acute hepatitis may persist, but portal and periportal inflammation becomes more pronounced. The inflammatory infiltrates consist mainly of lymphocytes with frequent plasma cells. Interface and lobular activity is evident. Portalbased fibrosis may develop and rapid progression to cirrhosis has been reported [570, 665]. A relatively specific finding in chronic HBV hepatitis is the presence of ground-glass hepatocytes (Fig. 8.82), which exhibit pale eosinophilic and homogeneous cytoplasm containing enriched smooth endoplasmic reticulum filled with HBsAg. Occasionally, hepatocytes show eosinophilic, sanded-appearing nuclei due to excess HBcAg accumulation in the nuclei (Fig.  8.83). A definitive diagnosis can be established by immunohistochemical detection of HBsAg (Fig. 8.84) in the cytoplasm and HBcAg in the nucleus (Fig. 8.85). There is no correlation between the number of antigen-expressing hepatocytes and the severity of necroinflammation; biopsies showing minimal necroinflammatory activity may contain numerous ground-glass hepatocytes. However, completely nega-

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Fig. 8.82  Ground-glass hepatocytes (arrows) seen in recurrent hepatitis B showing pale eosinophilic and homogeneous cytoplasm

Fig. 8.83  Sanded nuclei (arrows) occasionally seen in recurrent hepatitis B

tive stains for viral antigens should suggest an alternative explanation for allograft dysfunction. A number of studies have shown that coinfection with HCV or hepatitis D virus (HDV) after transplantation may inhibit HBV replication, which in turn diminishes the risk of HBV recurrence, reduces the severity of necroinflammatory activity, and improves the survival of the patients [292, 367, 392, 548, 629, 645]. In patients with HBV/HCV coinfection, the histological findings are usually attributed to HCV, given the successful prophylaxis for HBV reinfection. In HBV/ HDV coinfection, immunohistochemical detection of

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solid organ transplantation, and in patients with HBV/ HIV or HCV/HIV coinfection [720]. Clinically, FCH is characterized by rapid and progressive deterioration in graft functions, evidenced by severe jaundice, coagulopathy, encephalopathy and possible death within 4–6 weeks of onset. The first reported cases were all rapidly progressive and fatal, and were associated with high levels of viral antigen expression [496]. Histologically, FCH features severe hepatocyte damage and a rapid progression of fibrosis [137, 392, 496]. Allograft biopsy shows marked hepatocyte ballooning (Fig. 8.86), intracellular and canalicular cholestasis, marked ductular reaction, and periportal and pericellular/sinusoidal fibrosis (Fig.  8.87). Portal Fig. 8.84  Cytoplasmic accumulation of HBsAg in hepatocytes in recurrent hepatitis B demonstrated by immunohistochemistry

Fig.  8.86  Fibrosing cholestatic hepatitis showing hepatocyte ballooning and cholestasis Fig.  8.85  Nuclear accumulation of HBcAg in hepatocytes in recurrent hepatitis B demonstrated by immunohistochemistry

HDV intranuclear antigen is helpful in establishing the diagnosis.

8.7.3 Fibrosing Cholestatic Hepatitis FCH is an aggressive form of viral hepatitis occurring in patients with severe immunosuppression. The term FCH was first introduced in 1991 to describe a severe, fulminant form of recurrent HBV in liver transplant recipients [137, 496], and FCH has now also been reported in recurrent HCV following liver transplantation, HBV or HCV infection in patients with other

Fig.  8.87  Fibrosing cholestatic hepatitis showing marked ductular reaction and periportal fibrosis as highlighted by trichrome stain. There is only mild lymphocytic infiltrates in the portal tract

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and lobular infiltration by inflammatory cells is characteristically inconspicuous. However, inflammation in HCV-associated FCH can be more severe, which is primarily periportal and consists of mixed neutrophils and lymphocytes. Confluent or bridging necrosis with parenchymal collapse may be seen. It is not uncommon for these allografts to progress to fibrosis, nodular regeneration, and early or full-blown cirrhosis in a short period of time [579]. Histological recognition of FCH is important because it carries a dismal clinical outcome. Reduction or complete stop of immunosuppressive therapies combined with aggressive antiviral treatment is necessary. Most importantly, FCH should not be confused with rejection because erroneous diagnosis may lead to stronger immunosuppression, which will further precipitate the progression of FCH. Helpful histological features to distinguish from rejection include lack of significant portal inflammation, lack of bile duct damage or loss, lack of endotheliitis, presence of ductular reaction, and presence of periportal and sinusoidal fibrosis. Immuno­ histochemical detection of HBsAg and HBcAg is also very helpful in HBV-related cases. Other differentials may include large bile duct obstruction, ischemic injury, steatohepatitis and drug toxicity. The constellation of parenchymal changes and the unique pattern of fibrosis of FCH should help the distinction.

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8.7.4 Autoimmune Hepatitis

a history of liver transplantation for AIH, sustained rise in serum aminotransferase activity, autoantibodies (antinuclear, anti-smooth muscle or anti-liver-kidney micro­ some type 1 antibodies) in a significant titer (³1:160), hypergammaglobulinemia, diagnostic or compatible liver histology (see below), corticosteroid dependency, and exclusion of other causes of graft dysfunction such as rejection, HCV infection or drug-induced hepatitis [147, 582]. The criteria and scoring system proposed by the International Autoimmune Hepatitis Group for the diagnosis of AIH in native livers provide useful guidelines but their diagnostic utility has not been tested in allografts [21]. The reported outcome of recurrent AIH varies considerably in different studies. This is probably because different diagnostic criteria, different protocols for monitoring graft function and different immunosuppressive therapies were used by different authors. In general, recurrent AIH responds well to increased immunosuppression or addition of corticosteroids [293, 464]. There is increasing recognition, however, that recurrent AIH may be associated with a more aggressive behavior, including progression to cirrhosis, graft failure and need for retransplantation. A high re-recurrence rate of AIH has also been reported following retransplantation [293]. As seen in native liver, the typical histological features of recurrent AIH include a predominantly portalbased, plasma-cell-rich mononuclear cell infiltrate with a variable degree of interface and lobular activity (Fig. 8.88) [163, 293, 449]. Hepatocyte rosetting may

The reported recurrence rate of autoimmune hepatitis (AIH) after liver transplantation is calculated to be 22% based on a number of studies [234]. Recurrence usually occurs between 1 and 5 years post transplantation, but cases with features of recurrent AIH have been reported within the first year [37]. The risk factors associated with AIH recurrence may include suboptimal immunosuppression, recipient HLA-DR3 haplotype with a HLA-DR3-negative graft, type 1 AIH as the original liver disease, and the presence of severe necroinflammatory activity in the explant [293], but these observations have been controversial [234]. Diagnosis of recurrent AIH can be problematic because it shares clinicopathological features with rejection or other late-onset posttransplant complications, and thus requires clinical, serological and histological correlation. Suggested diagnostic criteria include

Fig.  8.88  Autoimmune hepatitis showing dense mononuclear cell infiltrates in the portal tract with prominent interface activity. Numerous plasma cells are present

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be evident (Fig.  8.89). It should be noted, however, that though characteristic, portal plasmacytic infiltrate is not an absolute diagnostic requisite. Some cases may present with lobular hepatitis as the initial manifestation of recurrent disease before typical portal inflammatory changes appear. Others may show zone 3 necroinflammatory lesions (central perivenulitis) and confluent or bridging necrosis (Fig. 8.90) [37, 452]. It is important to know that the histological features of disease recurrence can be seen in patients with normal liver function tests and can precede clinical and biochemical recurrence by several years [163]. On the other hand, the histological observation by itself should not be regarded as diagnostic of recurrent AIH because

Fig. 8.89  Autoimmune hepatitis showing hepatocyte rosetting

Fig. 8.90  Centrilobular necrosis seen in autoimmune hepatitis. Note the presence of prominent plasma cells. The histological findings may also be regarded as evidence of rejection

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it lacks specificity. As mentioned earlier, the diagnosis relies on a constellation of clinical, laboratory and histological findings. Numerous studies have shown that classical biochemical, serological and histological features of AIH can be seen in patients transplanted for liver diseases other than AIH [26, 59, 261, 282, 284, 312, 324, 454, 554]. This condition has been described under various names, such as graft dysfunction mimicking AIH or posttransplant immune hepatitis, but de  novo AIH is the currently preferred term [447]. It is more commonly seen in pediatric liver transplant recipients where a 5–10% incidence has been reported. In adults, the incidence is 1–2% and primary biliary cirrhosis is a frequently documented underlying liver disease [282, 312, 554]. Occasionally, de novo autoimmune hepatitis and recurrent primary biliary cirrhosis may occur concurrently [641]. Similar to recurrent AIH, most cases of de novo AIH respond well to additional immunosuppression [324, 568, 691]. The pathogenesis of de novo AIH is poorly understood. It remains debated whether the word “autoimmune” is appropriate even for recurrent AIH because both recurrent and de novo conditions are not necessarily autoimmune, but rather alloimmune. It has been shown that in addition to the presence of classical autoantibodies, some patients with de novo AIH have circulating, donor-specific antibodies to glutathione S-transferase T1 (GSTT1), a cytosolic enzyme that is expressed abundantly in liver and kidney [15, 16, 555]. In a recent study [567] , anti-GSTT1 antibodies were detected in 29 of 419 (6.9%) adult liver transplant recipients with donor/recipient GSTT1 genotype mismatch. Twenty of 27 assessable patients (74%) developed clinically evident de  novo AIH after a median follow-up of 26 months. The probability of developing de novo AIH was 11, 44, and 60% at 12, 24, and 36 months, respectively. These novel findings suggest that de novo AIH represent an anti-graft reaction in recipients who lack the GSTT1 phenotype. As a result, the host immune system recognizes the donor GSTT1 protein as a foreign antigen. In essence, de  novo AIH appears to be a special form of graft rejection that is not directed against HLA antigens. Fiel et  al. recently proposed an alternative name, posttransplant plasma cell hepatitis (PCH), to describe a group of patients who underwent liver transplantation for HCV and subsequently developed a dense

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plasma cell infiltrate in allograft biopsies [194]. The authors concluded that PCH represents a variant of rejection rather than true de novo AIH. They observed that PCH frequently developed in association with recent lowering of immunosuppression or subtherapeutic calcineurin inhibitor levels, was frequently seen in patients with prior episodes of acute rejection, and responded to treatment rapidly with resolution of plasma cell infiltrate. In addition, 58% of their PCH cases were diagnosed within 2 years post transplantation and 34% within 12 months. Almost all the patients in this study had low titer autoantibodies but only 13 of 23 patients (61%) had autoantibody titers of ³1:40. These features did not seem to be consistent with those seen in AIH to the authors. On the other hand, Khettry et  al. found 9 of 92 (10%) patients transplanted for HCV to have a plasmacell-rich AIH-like histology in posttransplant biopsies. In comparison with cases showing typical histological features of recurrent HCV, those with an AIH-like histology tended to show more rapid progression to fibrosis and a higher incidence of central perivenulitis. Although autoantibodies and/or increa­ sed serum immunoglobulins were detected in six patients (67%), these cases were regarded as an AIHlike variant of recurrent HCV, rather than de  novo AIH or rejection, by the authors [328]. Therefore, it remains to be investigated whether an AIH-like histology (plasma-cell-rich infiltrates with or without centrilobular lesions) detected in posttransplant biopsies from HCV patients, represents de  novo AIH, rejection or recurrent HCV. While the separation between AIH and rejection may have little clinical significance because both conditions can be effectively managed with increased immunosuppression, the distinction between rejection/AIH and recurrent HCV is clinically relevant. In general, the finding of central perivenulitis that involves the majority of the central veins should suggest the diagnosis of rejection or AIH, regardless of plasma-cell-rich or not (Fig. 8.90). These cases may show an adequate antiHCV immunity with low or negative HCV RNA levels, and thus treatment by optimization of immunosuppression (without the use of corticosteroids) appears justified and effective [59, 153, 194]. Nevertheless, the development of PCH in the setting of recurrent HCV is a negative prognostic factor for graft and patient outcomes [194, 328].

8.7.5 Primary Biliary Cirrhosis In a recent review of multiple studies that included >1,200 patients with primary biliary cirrhosis (PBC), the recurrence rate after liver transplantation was 18% after a median posttransplant follow-up of 69 months (range: 36–114 months). The median time for PBC to recur was 46.5 months (range: 25–78 months). The majority (90%) of the patients with recurrent PBC were women [234]. Although rare cases with histological findings suggestive of recurrent PBC may be seen during the first few months post transplantation, most cases occur after 1 year and the recurrence rate increases with time, approaching 50% at 10 years [482]. Despite the high incidence, recurrent PBC appears to have little impact on long-term graft or patient survival. Only rare cases progress to cirrhosis or graft failure that requires retransplantation [304, 375, 464, 582]. Diagnosis of recurrent PBC can be difficult because most patients may be asymptomatic with normal or only mild elevation in serum alkaline phosphatase and gamma-glutamyl transpeptidase levels. Serum antimitochondrial antibody (AMA) titer offers no diagnostic value because it usually remains elevated after transplantation. The diagnosis is thus almost entirely based on histological findings, frequently detected by protocol biopsies. As seen in native livers, early stage recurrent PBC features mixed portal inflammatory cell infiltrates consisting of lymphocytes, plasma cells and eosinophils. Ductular reaction, lymphoid aggregates with or without germinal centers and interface hepatitis may be seen. In classical cases, the pathognomonic florid duct lesion may be detected, which is characterized by granulomatous bile duct destruction or lymphocytic bile duct infiltration (Fig. 8.91). The affected duct may become ruptured and the duct epithelium may exhibit degenerative changes. Ductopenia, collection of foamy macrophages in the portal tracts and periportal fibrosis may become evident in more advanced cases. Cholate stasis, characterized by ballooning degeneration, Mallory hyaline, copper deposition and copper-binding protein accumulation in periportal hepatocytes, may also be seen. The lobular changes in recurrent PBC are usually mild and nonspecific. Recurrent PBC needs to be differentiated from lateonset acute rejection, chronic rejection, drug toxicity, large bile duct obstruction and ischemic cholangiopathy. The finding of destructive granulomatous cholangitis is

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Fig.  8.91  Recurrent primary biliary cirrhosis showing florid duct lesion. The portal tract is involved by granulomatous inflammation. The bile duct is damaged and difficult to appreciate (arrow)

highly specific for recurrent PBC in the appropriate clinical setting. However, this diagnostic feature may be seen in only a subset of the patients [298]. Other useful features in the distinction from acute rejection include ductular reaction, cholate stasis, lymphoid aggregate and lack of endotheliitis. In addition, the portal involvement is usually patchy in recurrent PBC but usually diffuse in rejection. On the other hand, portal granulomas can be seen in other conditions such as HCV, fungal or mycobacterial infections and sarcoid, but these conditions usually lack the distinctive granulomatous cholangitis. Histological features of AIH have been observed in allograft biopsies from patients with PBC [298, 326]. It is unclear however, whether this represents an alternative form of recurrent PBC, conversion of PBC to AIH, overlap syndrome, de novo AIH or an alternative form of rejection (posttransplant PCH).

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higher proportion of the patients with recurrent PSC developed graft failure and loss comparing with those with recurrent PBC [558]. The diagnosis of recurrent PSC is challenging because the characteristic cholangiographic findings of biliary stricture in native livers can be caused by a variety of additional conditions in allografts, such as technical biliary complications, ischemia, biliary sepsis and chronic rejection [251]. The proposed diagnostic criteria for recurrent PSC require a confirmed pretransplant history of PSC; cholangiographic findings of intrahepatic and/or extrahepatic biliary stricturing, beading or irregularities at least >90 days after transplantation; histological findings of fibrous cholangitis and/or fibroobliterative lesions with or without ductopenia, biliary fibrosis or biliary cirrhosis; and the exclusion of other causes of biliary strictures [234, 251]. As seen in native livers, histological examination of an allograft biopsy may or may not reveal diagnostic features because of the patchy nature of the disease. Mild portal lymphocytic infiltration and mild bile ductular reaction may be the only findings (Fig. 8.92). Mild portal edema and mild portal infiltration by neutrophils may also be noted. The relatively specific finding, concentric periductal fibrosis with an “onion-skin” appearance, is only seen in a subset of the biopsies (Fig. 8.93). When present, however, the involved ducts usually exhibit atrophic and degenerative changes in

8.7.6 Primary Sclerosing Cholangitis PSC recurs in 11% of the patients [234]. It usually first manifests after 1 year post transplantation and the incidence increases with time thereafter. Although the overall patient and graft survival in patients with recurrent PSC was thought to be similar to those without recurrence [251], a recent study showed that a significantly

Fig.  8.92  Recurrent primary sclerosing cholangitis featuring mild portal lymphocytic infiltration and mild bile ductular reaction. Classic periductal “onion-skin” fibrosis is not evident in this biopsy. Note the lack of endotheliitis and rejection-type duct damage

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Fig. 8.93  Classic periductal “onion-skin” fibrosis seen in recurrent primary sclerosing cholangitis

Fig.  8.95  Large bile obstruction featuring portal edema and ductular reaction with associated neutrophilic infiltration. These findings are more pronounced in comparison with recurrent primary sclerosing cholangitis

intrahepatic bile ducts are affected, bile duct loss, fibro-obliterative lesion and biliary cirrhosis may develop, histologically resembling PSC. The distinction from recurrent PSC relies primarily on clinical history. Large bile duct obstruction/stricture secondary to other causes may cause more pronounced portal edema, ductular reaction, neutrophilic infiltration with neutrophils surrounding the proliferating ductules, and perivenular cholestasis (Fig. 8.95), in comparison with recurrent PSC. Fig. 8.94  Characteristic fibro-obliterative scar (arrow) seen at the end stage of primary sclerosing cholangitis. Note the presence of ductular reaction and mild inflammatory cell infiltrates. Similar findings can also be seen in ischemic cholangiopathy

epithelium, and will eventually be replaced by characteristic fibro-obliterative scar (Fig. 8.94) [253, 329]. Recurrent PSC should be differentiated from chro­ nic rejection, ischemic cholangiopathy and large bile duct obstruction. In chronic rejection, bile duct loss is typically not accompanied by fibro-obliterative scar, ductular reaction, or copper/copper-binding protein accumulation. Centrilobular/perivenular fibrosis and loss of small arterial branches in the portal tracts may be seen in chronic rejection [145]. Ischemic cholangiopathy is defined as focal or extensive damage to bile ducts due to impaired blood supply [143]. When

8.7.7 Alcoholic Liver Disease Alcoholic liver disease is a common indication for liver transplantation [437]. Patient survival following transplantation is similar to that for other indications but resumption of alcohol consumption appears to be associated with decreased survival, though controversial [90, 126, 390, 398, 667]. The incidence of recurrent alcohol use ranges from 10 to 50% based on different definitions for recidivism, which vary from any consumption to heavy intake [398, 666]. The histological features of alcohol-related liver injury after liver transplantation are similar to those seen in general population, which encompass a clinicopathological spectrum including fatty liver, alcoholic hepatitis and alcoholic cirrhosis [90, 505, 512, 643].

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Steatosis and pericellular/sinusoidal fibrosis are the most common findings in allograft biopsy from heavy drinkers. Steatosis may be predominantly macrovesicular and primarily involves the centrilobular hepatocytes. Microvesicular steatosis can also be seen, however. Hepatocyte ballooning and Mallory hyaline may be prominent. Inflammatory infiltrate is typically rich in neutrophils and frequently distributed in the lobules adjacent to hepatocytes containing Mallory hyaline (Fig. 8.96). In addition to the distinctive “chicken-wire” pericellular/sinusoidal distribution, fibrosis may also involve the central veins, which leads to the thickening of the wall and luminal occlusion in association with

Fig.  8.96  Alcoholic hepatitis showing steatosis, hepatocyte ballooning, Mallory hyaline (arrow), and neutrophilic lobular inflammation

Fig.  8.97  Central hyaline necrosis seen in alcoholic hepatitis (trichrome stain), resembling veno-occlusive disease

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necrosis of adjacent hepatocytes, a lesion referred to as central hyaline necrosis (Fig. 8.97).

8.7.8 Nonalcoholic Fatty Liver Disease Nonalcoholic fatty liver disease (NAFLD) resembles alcohol-induced liver injury, but by definition, occurs in the absence of alcohol consumption. It is considered the hepatic manifestation of the metabolic syndrome, a constellation of obesity, hypertension, diabetes mellitus and hyperlipidemia. These conditions usually persist after liver transplantation and recurrent fatty liver disease is thus a common finding in allograft biopsy. De novo NAFLD has also been observed, usually in patients with a prior history of cryptogenic cirrhosis, suggesting that the actual etiology for cirrhosis be NAFLD [38, 89, 98, 102, 121, 308, 332, 455]. Recurrent NAFLD usually occurs between 3 weeks and 2 years post transplantation. Approximately 10–40% of the patients may develop nonalcoholic steatohepatitis (NASH) and up to 12.5% may progress to cirrhosis [109]. In a recent study of 98 patients who underwent liver transplantation for NASH cirrhosis, the 5-year survival was comparable to patients with PBC, PSC, alcoholic cirrhosis, HCV and cryptogenic cirrhosis when matched for age, sex, MELD score and years of transplantation. However, the early mortality, usually due to posttransplant infections, was higher among patients transplanted for NASH as a result of combinational effects of older age, higher BMI, diabetes and hypertension [404]. Most but not all the histological features described for alcoholic hepatitis can be found in NASH [726]. These mainly include predominantly macrovesicular steatosis that primarily involves centrilobular regions, hepatocyte ballooning, lobular inflammation rich in neutrophils, acidophil bodies, small lipogranulomas, glycogenated nuclei, megamitochondria and patchy pericellular/sinusoidal fibrosis (Figs.  8.98–8.100). Portal inflammation is typically insignificant. The presence of heavy portal inflammation should raise the suspicion of overlapping disease such as viral hepatitis, autoimmune hepatitis or acute rejection. In contrast to alcoholic hepatitis, central hyaline necrosis is not a feature of NASH. Mallory hyaline may or may not be seen, and is typically poorly formed if present.

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Fig.  8.98  Nonalcoholic steatohepatitis showing diffuse, predominantly macrovesicular steatosis

Fig. 8.99  Nonalcoholic steatohepatitis showing steatosis, hepatocyte ballooning, glycogenated nuclei (arrow), and neutrophilic lobular inflammation (insert)

8.7.9 Hemochromatosis and Iron Overload Hereditary hemochromatosis (HH) is an autosomal recessive iron-overload disorder, primarily associated with mutation of the HFE gene. Increased intestinal iron absorption leads to excessive iron deposition in various organs, resulting in cirrhosis, diabetes, cardiomyopathy, arthropathy, skin hyperpigmentation and hypogonadism [124, 738]. HH is an uncommon indication for liver transplantation. In a study of 3,600 adult primary liver transplants, only 22 patients were transplanted for cirrhosis related

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Fig.  8.100  Nonalcoholic steatohepatitis showing pericellular/ sinusoidal fibrosis. This pattern of fibrosis is also seen in alcoholic hepatitis

to HH [124]. Among them, 11 also consumed excessive alcohol, 2 had additional causes of liver disease (a1antitrypsin deficiency and HCV), and 8 had HCC. Thus, in only 9 patients (0.26%) was transplantation performed solely for HH. In that study, the 1-, 3- and 5-year posttransplant survival rates were 72, 62 and 55%, respectively; and recurrent HCC was the cause of death in 5 patients. Of the 11 surviving patients, 10 had neither biochemical nor histological evidence of iron reaccumulation after a median follow-up of 4 years. One patient showed a progressive increase in serum ferritin levels with grade 1–2 siderosis in allograft biopsy. The donor HFE status was unknown in that case, but abnormal iron store was not noted at the time of transplantation [124]. These observations suggest that reaccumulation of iron in donor livers in HH patients is an uncommon event, but a longer follow-up period is needed to determine if a slow reaccumulation, if occurring, has any potential to compromise the late graft function. In another study participated by 12 liver transplantation centers in the United States that involved 14 patients with homozygous HH and 11 patients with compound heterozygous HH, the 1-, 3- and 5-year posttransplant survival rates were 64, 48 and 34%, respectively, which were significantly lower than those for recipients who carried a wildtype or simple heterozygous HFE gene [341]. The causes of death included infections, cardiovascular disorders and malignancies. No death was apparently attributed to iron reaccumulation. Histologically, iron deposition initially occurs in periportal hepatocytes and the iron granules are

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Fig. 8.101  Iron reaccumulation in hepatocytes after transplantation for hemochromatosis (Perls’ Prussian blue stain). Note that the characteristic pericanalicular pattern may be seen in some cases (insert)

concentrated along the border of the canaliculi (Fig.  8.101). Uncontrolled HH may show iron accumulation throughout the entire lobule, and iron granules may also be seen in Kupffer cells, bile duct epithelial cells and portal macrophages. There is usually little to no inflammatory infiltrate. Iron deposition is best evaluated by Perls’ Prussian blue stain and positive hepatocyte staining can be semiquantitatively scored using a 1–4 scale. If a higher score is appreciated (such as 3+ or 4+), chemical quantitation of iron concentration in dry hepatic tissue to determine the hepatic iron index should be performed. A hepatic iron index of >1.9 is strongly suggestive of HH. Secondary iron overload is a common finding in patients undergoing liver transplantation for other causes of cirrhosis unrelated to HH, such as HCV, HBV, alcoholic hepatitis and NASH. Iron deposition in these conditions primarily involves Kupffer cells (Fig.  8.102). Accumulation in hepatocytes is usually mild and rarely exceeds 2+.

8.7.10 Budd-Chiari Syndrome Budd-Chiari syndrome is characterized by obstruction of the hepatic venous outflow in the absence of right heart failure or constrictive pericarditis [686]. By convention, hepatic veno-occlusive disease (recently renamed as sinusoidal obstruction syndrome), commonly occurring in the setting of bone marrow or hematopoietic stem cell

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Fig. 8.102  Secondary iron overload with iron deposition predominantly in Kupffer cells (Perls’ Prussian blue stain). There is only minimal iron noted in hepatocytes

transplantation, hepatic radiation, and exposure to chemotherapeutic agents, toxic plants or herbal medicines, is also excluded. Therefore, Budd–Chiari syndrome results from obstruction at any level of the hepatic venous system between the liver and the IVC or the right atrium, which involves a variety of thrombotic and nonthrombotic causes including hypercoagulable states secondary to hematologic disorders [440]. In a study of 39 patients who received liver transplants for Budd–Chiari syndrome in an 18 year span, Ulrich et al. reported that the 1-, 5-, and 10-year survival rates were 92.3, 89.4 and 83.5%, respectively, which were comparable to those for patients transplanted for other indications. Retransplantation was necessary in 3 (7.7%) patients who developed PVT or recurrent Budd–Chiari syndrome [680]. A prior study reported the posttransplant survival rates at 1-, 5- and 10-years to be 81, 65 and 65%, respectively [125]. Three of the 11 patients in this study developed recurrent Budd–Chiari syndrome, including two who died due to rapid graft failure within days after transplantation. Similar results have also been reported by Srinivasan et al. in an earlier study [621]. The histological features of Budd-Chiari syndrome are similar to those seen in congestive heart failure, characterized by centrilobular sinusoidal dilatation and congestion. Acute onset may also give rise to a hemorrhagic appearance at centrilobular regions, with extravasation of red cells under the space of Disse to replace hepatocytes within the cords and to cause hepatocyte necrosis (Fig. 8.103). The portal tracts are

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Fig.  8.103  Acute onset of Budd–Chiari syndrome showing centrilobular hemorrhage

typically uninvolved. If unrelieved, Budd–Chiari syndrome will result in substantial perivenular and bridging fibrosis, and eventually cirrhosis.

8.7.11 Idiopathic Posttransplantation Hepatitis Idiopathic posttransplantation hepatitis (IPTH) refers to a form of chronic hepatitis that cannot be ascribed to a specific cause. It is characterized by mononuclear cell infiltrates in the portal tracts with varying degrees of interface and lobular activity (Fig. 8.104). By definition, bile duct damage, ductopenia and endotheliitis are not present. The patients with IPTH are typically asymptomatic clinically with normal or near-normal liver tests. The diagnosis is established by protocol biopsy of the allografts after exclusion of other potential etiologies [147, 598]. Some studies have shown a 10–50% incidence of IPTH after 1 year and up to 60% at 10 years post transplantation. The true incidence is difficult to determine, however, because of the lack of standardized definition and variable application of protocol biopsies. A significant proportion of the patients followed up for a minimum of 10 years have been shown to develop progressive fibrosis and ultimately cirrhosis [155, 181, 596, 638]. It remains controversial whether some of the IPTH cases may actually represent a hepatitic form of rejection or a modified type of de novo AIH. In cases that

Fig.  8.104  Idiopathic posttransplantation hepatitis featuring mononuclear cell infiltrates in the portal tract, resembling hepatitis C or indeterminate for acute rejection. Note the absence of bile duct damage and endotheliitis

show confluent or bridging necrosis resembling central perivenulitis, these two possibilities should certainly be considered. Other potential causes for IPTH may include infections attributable to uncommon viruses, such as human herpesvirus 6 (HHV-6) and hepatitis G, as well as undiscovered transmissible agents [598]. In a recent study of 944 Japanese patients who survived for at least 6 months following living donor liver transplantation, 42 (4.4%) showed histological features of IPTH in at least one biopsy at a mean of 5.2 years (range: 0.7–10.8 years) after the possibility of recurrent diseases, viral hepatitis or drug-induced hepatitis was excluded [453]. Progression to fibrosis was observed in 8 (19%) patients and 5 (12%) required retransplantation. Interestingly, autoantibodies were detectable in 29 of 40 patients (73%) at the time IPTH was diagnosed, with antibody titers ranging from 1:40 to 1:640. However, no differences were observed between the high-titer (³1:160) and low-titer (<1:160) autoantibody groups in terms of clinicopathological features, response to treatment or outcome. The authors concluded that the distinction between IPTH and de novo AIH did not seem to be necessary [453].

8.7.12 Malignancies Liver transplantation is a treatment option for selected patients with HCC, and the use of specific selection

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criteria has improved tumor-free survival. The Milan criteria are the currently most widely used system for recipient selection, which allows patients with a solitary tumor of £5 cm or up to 3 tumors (each £3 cm) to be transplanted [433]. These criteria were latter expanded to allow patients with a solitary tumor of £6.5  cm or up to 3 tumors (each £4.5  cm, £8  cm in sum) to be transplanted (the expanded San Francisco criteria) [724]. With the universal application of the MELD system for organ allocation in the United States, in which patients with HCC have the priority on the waiting list for cadaveric organs, the number of patients who undergo transplantation for HCC is rising [714]. The incidence of recurrent HCC after transplantation varies from 6.4 to 56.5% in different studies [740]. The 5-year survival is 22–48% in patients with recurrence in comparison to 64–75% for patients without. The most important risk factor for recurrence while using the Milan or the expanded San Francisco criteria is vascular invasion. Other documented adverse factors include poorly differentiated histology and the presence of satellite nodules [281, 371, 414, 418, 541, 550, 578, 607, 732, 737]. Tumors incidentally identified in the explants are usually solitary and small, and thus unlikely to recur. In most cases, recurrence is detected by elevated serum a-fetoprotein levels and imaging studies. Allograft biopsy may be occasionally needed to confirm the diagnosis. Rarely, HCC occurs de  novo in cirrhotic allografts, usually secondary to recurrent HCV. Transplantation for cholangiocarcinoma has been controversial. Early experiences with this neoplasm, particularly hilar cholangiocarcinoma, showed high rates of recurrence after transplantation and poor patient survival even in combination with adjuvant chemotherapy and radiation [704]. Recent studies using stringent criteria to select patient population in combination with neoadjuvant treatment have shown promise. The reported 5-year survival rates were as high as 82% [140, 553, 606, 611, 633]. Transplantation has also been performed for other uncommon hepatic neoplasms such as epithelioid hemangioendothelioma, angiosarcoma and hepatoblastoma, and even for metastatic tumors [100, 291]. In a recent study of 59 patients who underwent liver transplantation for epithelioid hemangioendothelioma, 14 (23.7%) developed recurrent disease after a median follow-up of 49 months (range: 6–98 months), among whom 9 died of recurrence. The 1-, 5- and 10- year

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patient survival rates were 93, 83 and 72%, respectively. The disease-free survival was not significantly affected by pretransplant treatment, nodal status, extrahepatic disease localization or vascular invasion [368]. The use of allografts from older donors or donors with a history of neoplasms carries an increased risk of transmitting malignancies to recipients. The estimated incidence of donor-derived malignancies is between 0.02 and 0.2%, and the reported cases include a variety of tumors such as malignant melanoma, choriocarcinoma, neuroendocrine tumors, and adenocarcinomas of the lung, prostate and pancreas [67, 86, 467]. Determination of donor origin of the tumors, which may have little impact on the clinical management of the recipients, may be facilitated by cytogenetic, fluorescent in situ hybridization and other molecular methods [221]. Kaposi sarcoma is a slow-growing endothelial tumor caused by human herpesvirus 8 (HHV-8). It is 400–500 times more common in transplant recipients comparing with the general population [439]. It is generally believed that HHV-8 reactivation in the recipient secondary to immunosuppression or rarely direct transmission of the virus from donor is responsible. In liver transplant recipients, the incidence is approximately 1% [33, 523], and involvement of the allografts has been observed [33, 232]. Histologically, Kaposi sarcoma is characterized by proliferation of plump spindle cells, sometimes with intracytoplasmic eosinophilic hyaline globules, red cell extravasation and hemosiderin deposition. The early lesions are usually portal based with tumor cells forming slit- or sieve-like thin-walled vascular channels (Fig.  8.105). When invading the adjacent liver parenchyma, the tumor cells may follow the sinusoidal pattern, similar to angiosarcoma (Fig.  8.106). It differs from angiosarcoma by lacking nuclear pleomorphism and lacking significant mitotic activity. The tumor cells are immunohistochemically positive for endothelial markers such as Fli-1 and CD34, and characteristically show nuclear staining for HHV-8 (Fig. 8.107).

8.7.13 Hepatic Architectural Alterations A varying degree of alterations in the hepatic architecture can be seen in allograft biopsy, probably secondary to altered blood supply, vascular problems, drug effects and/or rejection-related damage. The common

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Fig. 8.105  Kaposi sarcoma showing plump spindle cells involving the portal tract. Note the presence of a bile duct (arrow). Hyaline globules and red cell extravasation are not evident in this case

Fig. 8.106  Kaposi sarcoma showing tumor cells growing along the sinusoidal spaces

histological findings include sinusoidal dilatation, thickening of the hepatocyte plates, and nodular regenerative hyperplasia (NRH). Abnormal portal vasculature is also a frequent observation, which may show aberrant vascular structures adjacent to portal tracts or aberrant extension of portal veins into adjacent hepatic lobules. The portal veins may be fragmented, diminished, sclerosed or absent [344]. Although these abnormal histological findings tend to correlate with HCV infection, increased portal fibrosis, episodes of rejection, and increased posttransplantation time, their clinical significance needs to be interpreted in the appropriate clinical setting. Occasionally, these

Fig.  8.107  Tumor cells in Kaposi sarcoma showing nuclear staining for human herpesvirus 8 by immunohistochemistry

findings may explain portal hypertension in the absence of cirrhosis. Patients with NRH may be asymptomatic or present with portal hypertension. In an early study of 9 liver transplant recipients who were diagnosed NRH between 6 and 144 months (median: 64 months) post transplantation, 6 developed portal hypertension. Interestingly, all 9 patients were found to take azathioprine [224]. In a more recent study of 14 patients who developed NRH between 3 months and 11 years after transplantation, however, the development of NRH was found to be unrelated to azathioprine in most cases. In this study, 7 patients showed signs of portal hypertension [156]. The histological features of NRH can be subtle and are best appreciated with reticulin stain, which highlights alternating regenerative hepatocytes with thickened plates within the nodules and atrophic or compressed hepatocytes at the periphery (Fig. 8.108). It differs from cirrhosis in that there is no or only minimal fibrosis and the portal structure is usually unaltered.

8.7.14 Others Rarely, other diseases affecting the liver have been reported to recur or develop de novo in allografts. These include sarcoidosis [5, 193, 381], hepatitis A virus infection [173], hepatitis G virus infection [315], hepatic venoocclusive disease [422], hepatic lymphangiomatosis

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Fig. 8.108  Nodular regenerative hyperplasia characterized by regenerative hepatocytes that form small nodules and compressed hepatocytes at the periphery of the nodules (reticulin stain)

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ballooning and necrosis, cholestasis, Mallory hyaline, and sinusoidal collagen deposition. As seen in native liver, the histological hallmark of recurrent sarcoidosis is the presence of multiple granulomas. If giant cells are present, they may contain Schaumann and asteroid bodies. Small areas of central fibrinoid necrosis may be occasionally present but tuberculosis-like caseating necrosis should never be seen. It should be noted that non-caseating granulomas can be seen in a wide variety of conditions and the underlying etiologies may not always be evident [235]. Examples include HCV hepatitis, mycobacterial or fungal infection, PBC, drug reaction, schistosomiasis, Hodgkin lymphoma, and idiopathic granulomatous hepatitis.

8.8 Hepatic Complications of Immunosuppression Hanlin L. Wang

Fig.  8.109  Giant cell hepatitis featuring syncytial giant cell transformation of hepatocytes. Ballooning change and Mallory hyaline are noted in this case

[533], erythropoietic protoporphyria [142], echinococcosis [78], and Ito cell hyperplasia [5]. Recurrent and de novo giant cell hepatitis can also occur in both pediatric and adult patients [273, 470, 507, 597]. The potential etiologies may include autoimmune hepatitis, drug toxicity or unusual viral infections such as HHV-6A [527] , but may remain elusive despite extensive work-up. Histologically, giant ell hepatitis is characterized by syncytial transformation of hepatocytes with individual hepatocytes containing multiple nuclei (Fig. 8.109). Other findings may include varying degrees of mixed inflammatory cell infiltrates including neutrophils, hepatocyte

Immunosuppression serves an important role in the success of liver transplantation, but is associated with a variety of adverse effects [58, 230]. Many of the adverse effects are not unique to liver transplant recipients but rather common to all patients who receive long-term immunosuppression for various organ transplantations. Examples include diabetes mellitus, obesity, hypertension, dyslipidemia, osteoporosis, renal dysfunction, bone marrow suppression, gastrointestinal toxicity, neurotoxicity and infections. These general adverse effects are discussed in detail in Part I, Chap. 2 and other relevant chapters. Tumor surveillance by the immune system is also impaired by long-term immunosuppression. Liver transplant recipients thus carry an increased risk for the development of malignancies than the general population. The reported incidence of posttransplant malignancies ranges from 4.7 to 15.7% in various studies depending on the length of follow-up [4, 58, 310]. Nonmelanoma skin cancers are by far the most common malignancies observed in these patients followed by PTLD. The incidence of Kaposi sarcoma is also increased. Whether or not other extrahepatic malignancies, such as colorectal, lung, prostatic, breast, cervical, oropharyngeal, thyroid, brain and kidney cancers, that are commonly seen in the general population also occur

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more frequently in liver transplant recipients is controversial and remains to be further investigated [40, 687]. In this chapter, only the hepatic complications of immunosuppression that occur after liver transplantation are discussed. These include drug hepatotoxicities and hepatic infections. Recurrent and de novo malignancies that occur in the allograft are discussed in Sect. 8.7.12.

8.8.1 Drug Hepatotoxicity The potential hepatotoxicity of immunosuppressive drugs is difficult or impossible to assess in an allograft biopsy because of lack of morphologic specificity and the presence of many other potential contributing factors in the transplant setting such as ischemia, rejection and recurrent diseases. Combined use of multiple immunosuppressive drugs and other hepatotoxic medications such as prophylactic antibiotics, antiviral and antifungal agents further makes the histologic assessment challenging. Accurate diagnosis requires a high index of suspicion and discontinuation or dose reduction of the suspected drugs.

8.8.1.1 Cyclosporine Unlike kidney, liver appears much less sensitive to the toxic effects of cyclosporine. In animal models, cyclosporine has been shown to cause cholestasis by interfering with bile formation via inhibiting ATPdependent export of bile salts and secretion of glutathione [56, 122]. Thus, it should be considered a potential cause of unexplained cholestasis in transplant patients [72, 95]. Usually, cyclosporine-induced cholestasis is mild and asymptomatic [319] , occurs shortly after the initiation of therapy, and can be easily reversed by dose reduction or switch to tacrolimus. However, continued administration of cyclosporine does not appear to lead to long-term sequelae [56, 122]. The histopathologic features of cyclosporine hepatotoxicity are poorly defined and lack specificity. These may include cholestasis, centrilobular hepatocyte ballooning, fatty change and acidophil bodies. In a follow-up of 87 pediatric patients who had received cyclosporine for at least 5 years for liver transplantation, hepatotoxicity was noted in 2 (2.3%) patients

[271]. In this study, hepatotoxicity was defined as elevated transaminases and histologic evidence of centrilobular damage with cholestasis and necrosis.

8.8.1.2 Tacrolimus (FK506, Prograf) Similar to cyclosporine, tacrolimus has also been shown to cause cholestasis [56, 122]. In a study of 112 pediatric liver transplant recipients, the incidence of tacrolimus-induced cholestatic complications was reported to be 5.4% [226]. Cholestasis in these cases developed within 2 weeks after the start of tacrolimus therapy and all the patients had trough levels within the therapeutic range. Liver biopsies showed spotty hepatocyte necrosis and centrilobular cholestasis (Fig. 8.110). After the immunosuppression was switched to cyclosporine and prednisone, the clinical signs and laboratory findings of cholestasis were completely resolved. Similar findings have been occasionally reported in adult patients [644]. Centrilobular necrosis or central perivenulitis has been attributed to the hepatotoxic effect of tacrolimus in earlier studies [198, 299], but this has been controversial. As discussed in Sect. 8.6.2.4, there is increasing evidence to suggest that even isolated central perivenulitis (in the absence of portal pathology) may represent a histologic variant of rejection if other potential etiologies, such as ischemic damage and autoimmune hepatitis, have been excluded [144, 294, 343, 345].

Fig.  8.110  Cholestatic hepatitis characterized by cytoplasmic and canalicular cholestasis. Occasional hepatocyte dropout is noted. The inflammatory infiltrate is insignificant. The histologic findings are commonly seen in drug-induced hepatitis

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8.8.1.3 Corticosteroids

8.8.1.6 Sirolimus (Rapamycin)

Long-term use of corticosteroids may be associated with steatosis and the development of NRH. There have been only rare case reports suggesting that methylprednisolone may be the cause of toxic hepatitis with mixed hepatocellular and cholestatic injury [668]. In general, corticosteroids are not considered hepatotoxic agents. Instead, they may be the treatment of choice for severe hepatitis induced by other medications [192, 237, 618].

In a study of 210 patients who received sirolimus after liver transplantation, 10 (4.8%) showed evidence of hepatotoxicity [477]. The mean time between the initiation of sirolimus therapy and the increase in liver enzymes was 21 days (ranging from 7 to 40 days) and the mean time of resolution was 27 days (ranging 14–56 days) following discontinuation. Interestingly, the sirolimus dose was further increased in these patients because the elevation in liver enzyme levels was initially thought to be the result of rejection, which led to a further increase in serum transaminase levels. Although all the patients were infected with HCV, there was no significant change in their blood viral load during sirolimus therapy. Their antiviral therapies were not modified. Some patients were also concomitantly using tacrolimus, but the tacrolimus dose was not changed during the administration of sirolimus. Six patients had liver biopsies which showed sinusoidal dilation in two, eosinophilia in one, minimal inflammation in one, and moderate inflammation in one. None of the biopsies showed evidence to suggest moderate or severe rejection. Mild portal infiltration by lymphocytes and macrophages is also observed in a liver biopsy from a renal transplant patient with presumed sirolimus-induced hepatotoxicity [488]. Sirolimus has been implicated as a risk factor for HAT, but this has been controversial [120, 166, 458, 672]. Most of these patients might also have other predisposing factors that increased the risk of thrombosis.

8.8.1.4 Azathioprine (Imuran) The hepatotoxic effects of azathioprine are believed to center on endothelial cells, causing sinusoidal dilatation, peliosis hepatis and veno-occlusive disease [630]. NRH has also been reported [224]. In addition, azathioprine may have direct toxic effects on hepatocytes, which cause mitochondrial dysfunction and activation of stress-activated protein kinase pathways, leading to hepatocyte necrosis and cholestasis [361, 441]. In some cases, discontinuation of azathioprine has led to improved liver function tests and improved histology on follow-up biopsies [224, 630]. However, permanent hepatic damage leading to progressive fibrosis, persistent portal hypertension and progressive graft failure is also reported [139, 224], which requires retransplantation. Fortunately, azathioprine has largely been replaced by MMF in the treatment of liver transplant patients in recent years.

8.8.1.5 Mycophenolate Mofetil (CellCept) MMF has not been reported to be hepatotoxic in liver transplant recipients. In an early study of 75 renal transplant recipients who received MMF, elevated liver enzymes were observed in 5 (7%) patients [616]. In a more recent study of 79 renal transplant recipients treated with MMF, 11 (14%) showed a progressive increase in liver enzymes [44]. The median time between the initiation of MMF and the increase in liver enzymes was 28 days (ranging from 4 to 70 days). The enzymes were normalized in 16 days (ranging from 4 to 210 days) after withdrawal or dose reduction of MMF in all 11 patients. No liver biopsies were performed in these studies.

8.8.2 Infections Despite the advance in antimicrobial prophylaxis, infection remains a significant problem in liver transplantation. In a long-term survival study on 4,000 liver transplant recipients, infection was found to be the most common cause of death at all time points, accounting for 28.4% of all deaths [306]. Eighty percent of infection-related deaths occurred in the first year following transplantation. Autopsy data showed that the infections were bacterial in origin in 48% of the cases, fungal in 22%, and viral in 12%. Two thirds of the infections occurred during the first 100 days [669]. In general, infections occurring in the early

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posttransplant days (up to 6 weeks) are similar to those commonly seen in immunocompetent patients with major abdominal surgeries [141]. Bacterial infections predominate in this period. Opportunistic infections secondary to the cumulative effect of immunosuppression occur most frequently between 1 and 6 months and are primarily caused by viral and fungal pathogens. Opportunistic infections are relatively uncommon after 6 months and these late-onset infections may involve a diverse spectrum of common and uncommon bacterial pathogens [141]. The clinical aspects of posttransplant infections are discussed in detail elsewhere. Only those infectious complications that directly involve liver allografts are addressed here. Under immunosuppression, liver allografts are also vulnerable to recurrent or new infections by hepatotropic viruses, such as hepatitis C and hepatitis B viruses, which is covered in Sect. 8.7.1 and Sect. 8.7.2.

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Staphylococcus aureus and Klebsiella pneumoniae are frequent isolates [330, 563, 565, 639]. The histologic hallmark of ascending cholangitis is the presence of neutrophils in the lumens of the bile ducts (Fig. 8.112). Infiltration of bile duct wall by neutrophils with epithelial damage or necrosis may be seen. The portal tracts may also show neutrophilic infiltration, accompanied by edema and ductular reaction. Abscess may develop in severe cases (Fig. 8.113). Histologically, ascending cholangitis is distinguished from acute rejection that may also show neutrophilic infiltration of the bile ducts by the lack of lymphocyte-predominant mixed portal infiltrates and the lack of endotheliitis.

8.8.2.1 Bacterial Infections Bacterial infections in liver transplant recipients often involve other organ systems and thus usually do not need allograft biopsy for diagnosis. Liver manifestations may include ascending cholangitis and sepsisrelated changes. Intrahepatic abscess and infected bilioma can also be seen, often resulting from HAT that leads to ischemic bile duct necrosis with secondary bacterial colonization (Fig.  8.111). Enterococcus species, Escherichia coli, coagulase-negative Staphylococci,

Fig.  8.112  Ascending cholangitis with neutrophils present within the lumen of the bile duct. The portal tract is edematous and infiltrated by neutrophils. The duct epithelium is also infiltrated by neutrophils and exhibits reactive changes

Fig. 8.111  Gram-positive cocci colonized in an infected biloma in a failed liver graft secondary to hepatic artery thrombosis

Fig.  8.113  Cholangitis with destruction of the bile duct and abscess formation

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organisms are typically associated with necrotic tissue or bilioma [563, 565], and special stains may be necessary for their detection.

8.8.2.3 Cytomegalovirus Hepatitis

Fig. 8.114  Ductular cholestasis seen in a patient with sepsis. It is characterized by ductular reaction with inspissated bile plugs in dilated ductules. Portal edema and mild neutrophil infiltration are also noted

Sepsis is associated with a unique histologic pattern of cholestasis characterized by ductular reaction with inspissated bile plugs in dilated ductules at the periphery of portal tracts (Fig. 8.114). The native bile ducts are unaffected and portal inflammation is typically insignificant unless ascending cholangitis is also present. This characteristic lesion, variably termed cholangitis lenta, subacute nonsuppurative cholangitis or ductular cholestasis in the literature [364, 379], is seen in only a fraction of patients with sepsis, however. In a study of 15 patients who died of sepsis, this lesion was demonstrated in only two patients [339]. In the majority of the cases, the histologic findings in the liver are nonspecific, including portal and lobular inflammation, centrilobular necrosis, acidophil bodies, steatosis and cholestasis.

8.8.2.2 Fungal Infections Fungal infections in liver transplant recipients are frequently caused by Candida or Aspergillus species. Other uncommon and unusual fungal infections may include Cryptococcus neoformans, Histoplasma cap­ sulatum, Zygomycosis (Mucor), Trichosporon, Fusa­ rium, Coccidioides and Aureobasidium. Most of the infections involve other organ systems or manifest as disseminated diseases, and are only rarely detected in allograft biopsy [216, 534]. In liver allografts, fungal

With the frequent use of antiviral prophylaxis in recent years, the incidence of CMV infection has dramatically decreased. Yet, it remains to be the most common opportunistic viral pathogen that contributes significantly to the morbidity and mortality in liver transplant recipients. The currently estimated incidence is 4.8% overall but 12–30% for high-risk patients who are seronegative for CMV and receive livers from seropositive donors [358, 540, 587]. Most clinically apparent CMV infections occur 3–12 weeks after trans­plantation, but delayed presentation after 3 months or even several years has been increasingly reported mainly due to discontinuation of antiviral prophylaxis [32, 377, 587, 605]. A high percentage of CMV infections are subclinical. In a study of 254 CMV infections among 1,146 liver transplants, 50.4% of the patients were found to be asymptomatic. A viral syndrome occurred in 28.0% and tissue invasive disease in 21.7%. Only 24 (9.5%) patients were diagnosed to have CMV hepatitis, accounting for 2.1% among all transplant recipients [587]. In 19 patients who developed CMV infection after cessation of antiviral prophylaxis, only 2 (11%) had CMV hepatitis [32]. Persistent CMV infection of the allografts has been shown to be associated with chronic ductopenic rejection, other biliary complications and HAT, but these associations remain to be open questions [53, 180, 266, 540, 587]. Histologically, CMV hepatitis features lobular inflammation with small neutrophil aggregates known as microabscesses (Fig. 8.115). The number of microabscesses varies widely from biopsy to biopsy and ranges from 1 to 15 in one study [358]. Spotty lobular lymphocytic infiltration, hepatocyte ballooning, lobular disarray and acidophil bodies can be seen. Kupffer cells may become prominent and may aggregate to form microgranulomas. The portal tracts are usually only mildly infiltrated by lymphocytes and plasma cells. Large area of hepatocytes necrosis is not typical of CMV hepatitis. Histologic diagnosis of CMV hepatitis relies on the identification of CMV-infected cells, which are usually

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Fig.  8.115  CMV hepatitis with a microabscess. An enlarged cell with viral inclusions is present within the microabscess

hepatocytes with large eosinophilic nuclear inclusions, typically surrounded by a clear halo. Smaller cytoplasmic viral inclusions may be also present. The infected hepatocytes are typically found in the vicinity of microabscesses, and thus the finding of microabscesses in an allograft biopsy warrants immunohistochemical staining for CMV if viral inclusions are not apparent on routine examination (Fig. 8.116). Occa­sionally, viral inclusions can be found in bile ducts, endothelial cells and inflammatory cells [358]. In general, however, viral inclusions

Fig. 8.116  CMV hepatitis confirmed by immunohistochemical staining. The infected cell exhibits large nuclear viral inclusions with a clear halo, and is associated with a microabscess

are sparse and there may be only 1–2 infected cells present in a biopsy core even on immunohistochemistry. Though characteristic, microabscess is not entirely specific for CMV hepatitis. In a study of 97 liver transplant recipients [356], 62 of 372 (17%) biopsies from 43 patients showed microabscesses. However, only 19% of the biopsies had CMV infection. The remaining biopsies with microabscesses were associated with a variety of conditions including bacterial, fungal or other viral infections (27%), biliary obstruction and/or cholangitis (15%), graft ischemia (10%), and combined sepsis and ischemia (3%). The underlying etiologies could not be determined in the remaining 26% biopsies. In a different study [396], CMV-negative microabscesses were found to be smaller and more numerous than those associated with CMV infection, but these findings were not confirmed by others [356].

8.8.2.4 Adenovirus Hepatitis Adenovirus hepatitis is an uncommon but potentially fatal complication in liver transplant recipients. The reported cases in the literature are mainly pediatric and there have been only a few adult cases. The clinically apparent infection usually occurs between 15 and 130 days after transplantation and is primarily caused by serotypes 1, 2 and 5 [179, 435, 436, 513, 562]. Typical histologic findings in adenovirus hepatitis include small or large areas of coagulative hepatocyte necrosis with no particular zonal distribution. Inflam­ matory response is typically insignificant, but mild lobular and portal inflammatory cell infiltrates consisting of lymphocytes, neutrophils and eosinophils may be present. Poorly formed granulomas and microabscesses can be seen [513, 562, 708]. The diagnosis is established by identification of nuclear viral inclusions with characteristic smudgy appearance and marginal condensation (Fig.  8.117). These virus-containing hepatocytes are most commonly seen at the periphery of necrotic foci and do not exhibit cytomegaly. Immunohistochemical stain is useful to confirm the diagnosis and to help distinguish from other viral infections particularly herpes simplex virus (Fig.  8.118). Adenovirus has also been shown to infect biliary tree with viral inclusions noted in the biliary epithelium. This causes cholangiohepatitis with necrotizing cholangitis and progressive bile duct loss [83].

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HSV hepatitis is characterized by focal, confluent or diffuse coagulative necrosis of the hepatic parenchyma with no particular zonal distribution [351]. Hemorrhage and mild neutrophil infiltration may be seen in the areas of necrosis (Fig. 8.119). The hepatocytes at the periphery of necrotic foci may be slightly enlarged and multinucleated, and contain nuclear viral inclusions with a smudgy and ground-glass appearance. Immunostains for HSV-1 and HSV-2 antigens are useful in the distinction from adenovirus hepatitis.

8.8.2.6 Varicella-Zoster Virus Hepatitis Fig.  8.117  Adenovirus hepatitis characterized by coagulative necrosis. The infected hepatocytes exhibit smudgy chromatin with marginal condensation

Reactivation of latent varicella-zoster virus (VZV) infection or primary infection is not an infrequent complication in liver transplant recipients, reported in 1.2–18% of the patients in various studies. The time of onset varies from several days to 12.9 years after transplantation and has been shown to be associated with immunosuppression [19, 248, 285, 372]. Most infections cause painful cutaneous eruptions (herpes zoster or shingles) with a dermatomal distribution. Disse­ minated infection with VZV hepatitis has only rarely been documented [350]. Histologically, VZV hepatitis is essentially indistinguishable from HSV hepatitis, characterized by foci of coagulative necrosis with minimal or mild inflammatory cell response. Nuclear viral inclusions and multinucleated cells can be seen [350]. Antibodies

Fig.  8.118  Adenovirus hepatitis confirmed by immunohistochemical staining. Note the presence of necrosis at the lower portion

8.8.2.5 Herpes Simplex Virus Hepatitis Herpes simplex virus (HSV) hepatitis is a rare complication after liver transplantation, which carries a high mortality rate if left unrecognized and untreated. It most commonly results from reactivation of latent infection by HSV types 1 and 2 in recipients who are HSV seropositive before transplantation, but primary infection or direct transmission of HSV from allografts has also been described. Allograft involvement can occur as early as 5 days or as late as several years after liver transplantation [50, 68, 96, 256, 351, 476].

Fig.  8.119  HSV hepatitis characterized by confluent hemorrhagic necrosis. A few residual cells contain nuclear viral inclusions with smudgy chromatin and marginal condensation

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against VZV antigens for immunohistochemistry are available, which should aid in the differential diagnosis. Immunostains should be negative for HSV-1 and HSV-2. In addition, patients with VZV hepatitis typically have skin lesions whereas patients with HSV hepatitis usually do not even with disseminated disease that involves multiple organs [350, 351].

8.8.2.7 Human Herpesvirus 6 Hepatitis Human herpesvirus 6 (HHV-6) is a member of the bherpesvirus family. Two variants have been identified, HHV-6A and HHV-6B, which share similar biologic properties. The incidence of HHV-6 infection in liver transplant recipients has been reported to range from 14 to 82%. Most infections are believed to represent reactivation because HHV-6 is a ubiquitous virus that infects >90% of the general population by the age of 2 years. Primary HHV-6 infection occurs only in a minority of the cases, primarily in pediatric transplant patients who are younger than 2 years. The infection typically occurs between 2 and 8 weeks after transplantation but can be seen as late as 5 years. Most of the infections are subclinical or result in a febrile illness with or without skin rash. Other documented complications of HHV-6 infection include hepatitis, myelosuppression, pneumonitis and neurological diseases. HHV-6 infection has also shown to exacerbate CMV infection, recurrent hepatitis C, fungal and other opportunistic infections, and ACR [3, 498]. In a study of eight posttransplant patients who were thought to have HHV-6 hepatitis based on serologic findings and immunohistochemical demonstration of HHV-6 specific antigens in liver biopsies, only two cases were found to have pure HHV-6 infection without coexisting CMV infection or acute rejection [359]. Histologically, HHV-6 hepatitis features mild to moderate predominantly lymphocytic infiltrates in the portal tracts, without evidence of bile duct damage or endotheliitis. Scattered acidophil bodies and foci of lobular inflammation consisting of lymphocytes and neutrophils can also be seen. Usually, there is no significant portal or lobular fibrosis. Since the virus primarily infects CD4+ T-lymphocytes, and to a lesser extent, CD8+ T-lymphocytes and natural killer cells, HHV-6 antigen-positive cells are predominantly seen in portal lymphocytes on immunohistochemistry, with cytoplasmic positivity [106, 270, 359]. However,

cytoplasmic immunoreactivity in hepatocytes has also demonstrated in some reports [349, 527]. The diagnosis of HHV-6 hepatitis requires a high index of clinical suspicion and can be established by serology, PCR and immunohistochemistry [3]. The histologic findings in liver biopsy are nonspecific and can be easily interpreted as indeterminate or mild acute rejection, or recurrent liver diseases. Rarely, HHV-6 infection causes giant cell hepatitis [527] or fulminant hepatitis [349], which can be confused with autoimmune hepatitis, drug toxicity or infections by other unusual pathogens such as paramyxoviruses. A case of fatal hemophagocytic syndrome associated with HHV-6 reactivation 2 weeks after liver transplantation has also been reported [157].

8.8.2.8 Epstein–Barr Virus Infection EBV is a member of the herpesvirus family that infects >90% of the adult population. Like other herpes viruses, EBV infection persists for the life of the patient. The virus stays in memory B lymphocytes at a concentration of ~1 in 1 × 105 to 1 × 106 cells [658]. In liver transplant recipients, reactivation or primary infection of the virus causes a broad spectrum of liver diseases ranging from EBV hepatitis to malignant PTLD, reported in up to 5% of adult patients and in up 15% of pediatric patients [47]. EBV hepatitis usually occurs within the first 6 months after transplantation [47, 357]. Histologically, it features mild, moderate or marked mixed inflammatory cell infiltrates in the portal tracts. The infiltrates consist predominantly of lymphocytes with occasional admixed plasma cells, eosinophils and neutrophils. Foci of interface activity, mild bile duct damage and mild endotheliitis can be seen (Fig. 8.120). Characteristically, there is beaded sinusoidal lymphocytic infiltration, giving rise to an “Indian file” pattern (Fig. 8.121). Most of the portal and sinusoidal lymphocytes are small in size, but some are large and irregular, consistent with atypical lymphocytes. Other histologic findings in EBV hepatitis may include focal lobular disarray, focal hepatocyte ballooning, scattered acidophil bodies, mild steatosis, microgranulomas and canalicular cholestasis [47, 635]. EBV hepatitis causes allograft dysfunction and histologically simulates ACR and recurrent hepatitis C. The diagnosis thus requires a high index of suspicion. The presence of peripheral lymphocytosis with

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Fig.  8.120  EBV hepatitis showing lymphocyte-predominant portal infiltrates with foci of interface activity. Mild bile duct damage is noted

Fig. 8.121  EBV hepatitis showing characteristic “beaded” lymphocytic infiltration in the sinusoids. Microgranulomas are noted at the right

atypical lymphocytes and the histologic finding of prominent sinusoidal lymphocytic infiltration should prompt further investigation including serologic tests, in situ hybridization (ISH) and PCR. It has been shown that ISH for EBV-encoded RNA (EBER) is equally sensitive to PCR in detecting EBV in liver biopsy specimens (Fig. 8.122), and thus is a very useful ancillary tool in confirming the diagnosis [47, 635]. It should be mentioned, however, that EBER-positive cells detec­ ted by ISH can be sparse in number in EBV hepatitis because EBV infects only B lymphocytes whereas the infiltrating lymphocytes are predominantly T cells. This may raise the doubt about the significance of detecting rare positive cells in a liver biopsy from a

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Fig.  8.122  EBV hepatitis confirmed by in situ hybridization for EBV encoded RNA (EBER)

patient with no clinical suspicion of EBV hepatitis considering the fact that the virus may be present in memory B lymphocytes at a very low concentration [658]. Thus, ISH results should be interpreted with caution and should be correlated with clinicopathologic findings. PTLD develops as a consequence of uncontrolled EBV replication. It accounts for >50% of all tumors in children and ~15% of all tumors in adults following liver transplantation. About 80% of the cases occur in the first 2 years. Heavy immunosuppression for the treatment of rejection has been shown to be the major risk factor for the development of PTLD. Other reported risk factors include lack of pretransplant immunity to EBV, CMV infection, and transplantations for fulminant hepatitis, hepatitis C and alcoholic cirrhosis [168, 227, 263, 283, 346, 408]. The majority of PTLD cases are of host origin, but occasionally they can be of donor origin, indicating that the donor lymphocytes carried in an allograft can survive and undergo malignant transformation [387, 493]. Constant awareness of PTLD is crucially important because its clinical presentation is often nonspecific and the prognosis relies on early diagnosis. Fortunately, PTLD-related mortality has markedly decreased in recent years due to increased awareness and better management of the disease [305, 613]. Histopathologically, PTLD encompasses a wide spectrum of diseases ranging from benign lymphoproliferation to aggressive lymphoma. It is divided into three major categories according to the World Health Organization (WHO) classification: early lesions,

8  Liver

polymorphic PTLD, and monomorphic PTLD [247, 353]. Cases diagnosed as monomorphic PTLD are essentially the same as lymphomas occurring in immunocompetent hosts, which are classified according to standard lymphoma classification. The majority of the cases (>80%) derive from uncontrolled B cell proliferation and the most common subtype is diffuse large B cell lymphoma. Burkitt or Burkitt-like lymphoma, plasma cell myeloma, peripheral T cell lymphoma, g/d T cell lymphoma, T/NK cell lymphoma, Hodgkin lymphoma and Hodgkin lymphoma-like PTLD can also be seen but these variants are rare. Hepatic involvement by lymphoma usually presents as a solitary mass or multiple masses with destruction of the hepatic architecture (Fig.  8.123). A diffuse pattern with extensive infiltration of the portal tracts and/or sinusoids by neoplastic cells is less common. The diffuse pattern can be seen in both T and B cell lymphomas, but predominantly sinusoidal infiltration appears to be more likely caused by T cell lymphoma, particularly g/d T cell lymphoma (Fig. 8.124). It is not as destructive as the nodular pattern and the hepatic architecture is usually relatively well preserved [176, 491]. Early lesions are characterized by reactive plasmacytic hyperplasia or infectious mononucleosis-like lesions. This often involves the portal tracts with mixed mononuclear cell infiltrates including small and medium-sized lymphocytes, atypical lymphocytes, immunoblasts and plasma cells (Fig.  8.125). There may be sinusoidal lymphocytic infiltration, similar to

287

Fig. 8.124  A case of g/d T cell lymphoma diffusely involving the sinusoids. The hepatic architecture is relatively well preserved. The infiltrative neoplastic cells in the sinusoids exhibit cytologic atypia and frequent mitoses

Fig.  8.125  Early PTLD characterized by mixed mononuclear cell infiltrates in the portal tracts

Fig.  8.123  Monomorphic PTLD (diffuse large B cell lymphoma) involving a liver allograft detected on a needle biopsy. The tumor presents as a mass lesion with destruction of the hepatic architecture

that seen in EBV hepatitis. Polymorphic PTLD is also characterized by mixed lymphoplasmacytic proliferation, which can be either polyclonal or monoclonal. When PTLD is suspected, workup should include B and T cell markers, k and l light chains for clonality, in situ hybridization for EBV encoded RNA, and molecular and cytogenetic studies including gene arrangement for immunoglobulins and T cell receptors. Immunohistochemical stain for EBV latent membrane proteins (LMP) can also be used but is less sensitive than in situ hybridization for detecting the virus (Fig. 8.126). Immunohistochemical studies for B

288

Fig.  8.126  Immunohistochemical detection of EBV latent membrane proteins (LMP) in a case of monomorphic PTLD

and T cell markers can be very helpful in distinguishing early PTLD lesions from other conditions that also feature portal lymphocytic infiltration such as ACR and recurrent hepatitis C. In PTLD, the portal infiltrates consist predominantly of B cells (Fig.  8.127) whereas in rejection and recurrent hepatitis C, the infiltrates are predominantly T cells. Other subtle histologic features favoring PTLD may include nodular infiltrates, frequent immunoblasts, frequent plasmacytoid cells, absence of neutrophils, inconspicuous eosinophils, presence of cytologic atypia, and frequent mitoses [475, 549]. It should be borne in mind, however, that early PTLD and acute rejection can coexist

Fig. 8.127  Early PTLD (the same case presented in Fig. 8.125) showing B lymphocyte proliferation in the portal tracts as demonstrated by immunohistochemical stain for CD20

H.L. Wang et al.

and the histologic distinction can be very difficult. In those cases, bile duct damage and endotheliitis are typically more prominent. It has been shown that a cautious increase in immunosuppression is safe and may improve graft function if a rejection component is present [537]. On the other hand, the long-term allograft function in patients with PTLD can be suboptimal because maintenance of a lower than needed level of immunosuppression may lead to the progression to chronic rejection [260, 537]. In ~20% of the cases, the presence of EBV cannot be demonstrated. The diagnosis of PTLD in these cases may be more difficult, particularly at the early stage. These EBV-negative PTLD cases usually occur later after transplantation and are more likely to be of T cell origin [247, 475]. The etiopathogenesis of EBV-negative PTLD is poorly defined but is still believed to result from decreased immune competence. However, some cases may simply represent sporadic lymphomas arising in patients who happen to be immunosuppressed.

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314 MELD allocation policy. Gastroenterology 127, S261–S267 (2004) 715. Wiesner, R.H., Steffen, B.J., David, K.M., Chu, A.H., Gordon, R.D., Lake, J.R.: Mycophenolate mofetil use is associated with decreased risk of late acute rejection in adult liver transplant recipients. Am. J. Transplant. 6, 1609–1616 (2006) 716. Williams, J.W., Vera, S., Peters, T.G., Van Voorst, S., Britt, L.G., Dean, P.J., Haggitt, R., Massie, J.D.: Cholestatic jaundice after hepatic transplantation. A nonimmunologically mediated event. Am. J. Surg. 151, 65–70 (1986) 717. Wilson, C.H., Agarwal, K., Carter, V., Burt, A.D., Hubscher, S., Talbot, D., Jaques, B.C., Manas, D.M.: Late humoral rejection in a compliant ABO-compatible liver transplant recipient. Transplantation 82, 988–989 (2006) 718. Winston, D.J., Emmanouilides, C., Busuttil, R.W.: Infections in liver transplant recipients. Clin. Infect. Dis. 21, 1077–1089 (1995). quiz 1090–1071 719. Wozney, P., Zajko, A.B., Bron, K.M., Point, S., Starzl, T.E.: Vascular complications after liver transplantation: a 5-year experience. AJR Am. J. Roentgenol. 147, 657–663 (1986) 720. Xiao, S.Y., Lu, L., Wang, H.L.: Fibrosing cholestatic hepatitis: clinicopathologic spectrum, diagnosis and pathogenesis. Int J Clin Exp Pathol 1, 396–402 (2008) 721. Yagci, G., Fernandez, L.A., Knechtle, S.J., D’Alessandro, A.M., Chin, L.T., Musat, A.I., Lucey, M.R., Said, A., Pirsch, J.D., Leverson, G., Kalayoglu, M.: The impact of donor variables on the outcome of orthotopic liver transplantation for hepatitis C. Transplant. Proc. 40, 219–223 (2008) 722. Yagi, S., Iida, T., Taniguchi, K., Hori, T., Hamada, T., Fujii, K., Mizuno, S., Uemoto, S.: Impact of portal venous pressure on regeneration and graft damage after living-donor liver transplantation. Liver Transpl. 11, 68–75 (2005) 723. Yan, M.L., Yan, L.N., Li, B., Zeng, Y., Wen, T.F., Wang, W.T., Yang, J.Y., Xu, M.Q., Li, Z.H., Chen, Y.B.: Intramuscular hepatitis B immune globulin combined with lamivudine in prevention of hepatitis B recurrence after liver transplantation. Hepatobiliary Pancreat. Dis. Int. 5, 360–363 (2006) 724. Yao, F.Y., Ferrell, L., Bass, N.M., Watson, J.J., Bacchetti, P., Venook, A., Ascher, N.L., Roberts, J.P.: Liver transplantation for hepatocellular carcinoma: expansion of the tumor size limits does not adversely impact survival. Hepatology 33, 1394–1403 (2001) 725. Yao, F.Y., Kinkhabwala, M., LaBerge, J.M., Bass, N.M., Brown Jr., R., Kerlan, R., Venook, A., Ascher, N.L., Emond, J.C., Roberts, J.P.: The impact of pre-operative loco-regional therapy on outcome after liver transplantation for hepatocellular carcinoma. Am. J. Transplant. 5, 795–804 (2005) 726. Yeh, M.M., Brunt, E.M.: Pathology of nonalcoholic fatty liver disease. Am. J. Clin. Pathol. 128, 837–847 (2007) 727. Yeh, M.M., Larson, A.M., Tung, B.Y., Swanson, P.E., Upton, M.P.: Endotheliitis in chronic viral hepatitis: a comparison with acute cellular rejection and non-alcoholic steatohepatitis. Am. J. Surg. Pathol. 30, 727–733 (2006) 728. Yeh, H., Olthoff, K.M.: Live donor adult liver transplantation. Curr. Opin. Organ Transplant. 13, 241–246 (2008) 729. Yersiz, H., Renz, J.F., Farmer, D.G., Hisatake, G.M., McDiarmid, S.V., Busuttil, R.W.: One hundred in situ

H.L. Wang et al. split-liver transplantations: a single-center experience. Ann. Surg. 238, 496–505 (2003). discussion 506–497 730. Yersiz, H., Shaked, A., Olthoff, K., Imagawa, D., Shackleton, C., Martin, P., Busuttil, R.W.: Correlation between donor age and the pattern of liver graft recovery after transplantation. Transplantation 60, 790–794 (1995) 731. Yoo, H.Y., Maheshwari, A., Thuluvath, P.J.: Retrans­ plantation of liver: primary graft nonfunction and hepatitis C virus are associated with worse outcome. Liver Transpl. 9, 897–904 (2003) 732. Yoo, H.Y., Patt, C.H., Geschwind, J.F., Thuluvath, P.J.: The outcome of liver transplantation in patients with hepatocellular carcinoma in the United States between 1988 and 2001: 5-year survival has improved significantly with time. J. Clin. Oncol. 21, 4329–4335 (2003) 733. Yoshida, E.M., Shackleton, C.R., Erb, S.R., Scudamore, C.H., Mori, L.M., Ford, J.A., Eggen, H., Wynn, V., Partovi, N., Keown, P.A.: Late acute rejection occurring in liver allograft recipients. Can. J. Gastroenterol. 10, 376–380 (1996) 734. Zajko, A.B., Campbell, W.L., Bron, K.M., Lecky, J.W., Iwatsuki, S., Shaw Jr., B.W., Starzl, T.E.: Cholangiography and interventional biliary radiology in adult liver transplantation. AJR Am. J. Roentgenol. 144, 127–133 (1985) 735. Zamboni, F., Franchello, A., David, E., Rocca, G., Ricchiuti, A., Lavezzo, B., Rizzetto, M., Salizzoni, M.: Effect of macrovescicular steatosis and other donor and recipient characteristics on the outcome of liver transplantation. Clin. Transpl. 15, 53–57 (2001) 736. Zapletal, C., Faust, D., Wullstein, C., Woeste, G., Caspary, W.F., Golling, M., Bechstein, W.O.: Does the liver ever age? Results of liver transplantation with donors above 80 years of age. Transplant. Proc. 37, 1182–1185 (2005) 737. Zavaglia, C., De Carlis, L., Alberti, A.B., Minola, E., Belli, L.S., Slim, A.O., Airoldi, A., Giacomoni, A., Rondinara, G., Tinelli, C., Forti, D., Pinzello, G.: Predictors of longterm survival after liver transplantation for hepatocellular carcinoma. Am. J. Gastroenterol. 100, 2708–2716 (2005) 738. Zhang, K.Y., Tung, B.Y., Kowdley, K.V.: Liver transplantation for metabolic liver diseases. Clin. Liver Dis. 11, 265– 281 (2007) 739. Ziarkiewicz-Wroblewska, B., Wroblewski, T., Wasiutynski, A.: Morphological features and differential diagnosis of hepatitis C recurrence after liver transplantation – literature review and results of single transplantation center. Ann. Transplant. 13, 12–20 (2008) 740. Zimmerman, M.A., Ghobrial, R.M., Tong, M.J., Hiatt, J.R., Cameron, A.M., Hong, J., Busuttil, R.W.: Recurrence of hepatocellular carcinoma following liver transplantation: a review of preoperative and postoperative prognostic indicators. Arch. Surg. 143, 182–188 (2008). discussion 188 741. Ziolkowski, J., Paczek, L., Niewczas, M., Senatorski, G., Oldakowska-Jedynak, U., Wyzgal, J., Foroncewicz, B., Mucha, K., Zegarska, J., Nyckowski, P., Zieniewicz, K., Patkowski, W., Krawczyk, M., Ziarkiewicz-Wroblewska, B., Gornicka, B.: Acute liver transplant rejection: incidence and the role of high-doses steroids. Transplant. Proc. 35, 2289–2291 (2003)

9

Heart Dylan V. Miller, Hannah Krigman, and Charles Canter

9.1 Introduction 9.1.1 Historic Perspective The first human heart transplantation was performed in South Africa in 1967 [5]. Results of this and subsequent early heart transplants were disappointing until the advent of calcineurin inhibitor-based immunosuppression regimens in the early 1980s. Since then, heart transplantation has become a widely available option for end-stage heart disease in patients from infancy to the seventh decade around the world. The International Society for Heart and Lung Transplantation (ISHLT) has maintained a heart transplant database since 1982 with annual published reports and updates [129]. These data show the number of heart transplants performed worldwide peaked in the mid-1990s at approximately 4,000 per year. More recently (1995–2004), this number has been approximately 3,000 procedures annually. About two thirds of heart transplants are performed in the United States [97]. Pediatric (<18 years of age at time of transplantation) recipients comprise 10% of all transplantations performed annually with 40% being age 1–10 years and 25% in adolescents [129].

D.V. Miller (*) Intermountain Central Lab, 5252 South Intermountain Drive, Salt Lake City, UT 84157, USA e-mail: [email protected] H. Krigman Department of Pathology and Immunology, Washington University School of Medicine, Saint Louis, MO, USA C. Canter Saint Louis Children’s Hospital, Washington University School of Medicine, Saint Louis, MO, USA

9.1.2 Outcomes Overall survival after heart transplantation has improved substantially, ever since the 1980s [17, 129]. Progress in monitoring and perioperative care has improved postoperative survival and advances in immune modulation have reduced rejection and graft loss. Current 1-year survival is approximately 85–90% for all age groups. In the first 3 years after transplantation, most deaths result from primary graft failure and rejection, though the causes vary according to age (Table 9.1); the primary difference being that acute cellular rejection is more common in pediatric patients [17, 95, 129]. Obviously failure of cardiac allografts has catastrophic consequences given the vital function of the heart and limited alternatives for long-term artificial circulatory support. Still, analysis of graft survival has been separated from recipient survival in the ISHLT database. Infant recipients have an overall graft half-life (interval from transplantation at which 50% of heart transplant recipients have died or required retran­splantation) of 15.8 years; adolescent and young adults about 11 years; and adult recipients approximately 9 years.

9.1.3 Immunosuppressive Therapy For the first heart transplant recipients, as with other early solid organ transplants, steroids were the only medications available for suppression of rejection. The development of cyclosporine in the 1980s revolutionized transplantation and led to an explosion of solid organ transplants in the ensuing decades. Commonly used agents in the current era are summarized in Table 9.2. Cyclosporine and tacrolimus are calcineurin

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Table 9.1  Causes of cardiac allograft failure in pediatric and adult recipients in the first 3 years post-transplant Adults Patients £18 years old Transplant coronary artery disease

Transplant coronary artery disease

Graft failure “not otherwise specified”

Graft failure “not otherwise specified”

Acute cellular rejection

Malignancy

Malignancy

Infection

Infection

Table  9.2  Common immunosuppression agents in cardiac transplantation Anti-cellular immunity Calcineurin inhibitors

Cyclosporine Tacrolimus (FK506)

Cell cycle inhibitors

Mycophenolic acid Azathioprine Cyclophosphamide

MTOR inhibitors

Sirolimus Everolimus

Corticosteroids

Methylprednisolone Prednisone

Induction agents

Muronumab-CD3 Anti-thymocyte globulin Daclizumab Basiliximab

Anti-humoral immunity Therapeutic antibodies 26s proteosome inhibitors

Rituximab IVIg Bortezomib

inhibitors; agents that interfere with lymphocyte production of interleukin 2 (IL-2). Currently, tacrolimus use is more prevalent than cyclosporine-based regimens [129]. The further introduction of other agents has led to a “triple therapy” approach that has become the cornerstone of current immunosuppression after heart transplantation. This includes the combined use of calcineurin inhibitors, cell cycle inhibitors (such as mycophenolic acid, azathioprine, and cyclophosphamide), and steroids [97]. In addition to triple therapy, approximately 50% of heart recipients are initially treated with pharmaceutical antibodies directed against lymphocytes/thymocytes or against the IL-2 receptor, though there is some evidence that prolonged use of induction agents may cause endothelial damage and

complement activation, similar to antibody mediated rejection (discussed in Sect. 9.5.4) [58, 88]. Despite great interest and investigation, there is no consensus on optimal immunosuppressive therapy and regimens vary from center to center. Each immunosuppressive agent has nuanced differences in efficacy and side effects. Mycophenolic acid may be more effective in preventing rejection and subsequent graft coronary disease than azathioprine when used in combination with calcineurin inhibitors [42]. Some centers substitute calcineurin inhibitors with sirolimus, an agent inhibiting MTOR (molecular target of rapamycin) mediated signaling pathways like those activated by IL-2. Sirolimus is thought to be less nephrotoxic than calcineurin inhibitors [109] and initial studies suggest it may decrease the progression of transplant coronary disease [107]. Many centers routinely attempt to discontinue maintenance steroids 6–12 months after transplantation to avert Cushingoid side effects. The toxicities of commonly used immunosuppressants are discussed in Sect. 9.8.4.

9.2 Native Disease in Explanted Hearts Cardiomyopathy, mostly the idiopathic dilated form (45%), and ischemic heart disease (41%) are the most common indications for heart transplantation in adults [129]. In infants, congenital heart disease accounts for 65% of the transplants [35] and cardiomyopathy is the indication for another 30% of pediatric patients [17]. Retransplantation comprises <10% of heart transplants performed in children and adults [105].

9.2.1 Cardiomyopathy Non-ischemic dilated cardiomyopathy can be classified as acquired, secondary to systemic disease, or idiopathic. Idiopathic disease accounts for about 50% of dilated cardiomyopathy. Toxins (mostly alcohol) and viral or post-myocarditis etiologies constitute the majority of acquired cardiomyopathies. Ischemic heart disease can result in a form of dilated cardiomyopathy, so there is some overlap in the reporting of cardiomyopathy and coronary artery disease. The presence of transmural infracts and critical coronary obstructions

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advances in plaque detection, myocardial infarction recognition, and revascularization therapies, post infarction heart failure due to massive myocardial infarction or progressive remodeling (wall thinning and dilatation) of the ventricle following smaller infarctions and chronic ischemia is still a leading reason for transplant in both men and women.

9.2.3 Myocarditis

Fig. 9.1  Idiopathic vs. ischemic dilated cardiomyopathy. Gross photographs showing short-axis sections of explanted hearts. The top section shows changes typical of idiopathic dilated cardiomyopathy with increased left ventricle internal diameter, wall thinning and circumferential fibrosis with accompanying fat, but not in a subendocardial distribution. There are no obvious infarctions. The lower section shows ischemic dilated cardiomyopathy with a large antero-septal infarction and extensive ventricular remodeling

The incidence of myocarditis varies with geography. Myocarditis remains a significant cause of endstage cardiomyopathy. Up to 8% of cardiac transplants are performed for myocarditis, both inflammatory and infectious [129]. Initial reports suggested poorer outcome for patients transplanted with myocarditis; however, recent studies find the outcome for patients transplanted with myocarditis equals that of the general population [94]. While not technically a form of myocarditis, up to 25% of patients with systemic sarcoidosis have cardiac involvement. This is rarely symptomatic but can be a cause of heart failure in up to 5% of cases [94]. Giant cell myocarditis (Fig. 9.2) is a highly aggressive form of myocarditis, often requiring transplantation, and showing good outcomes with infrequent recurrence [28].

provide a basis for differentiating these (Fig.  9.1). Secondary causes include hemochromatosis, autoimmune, familial, and peripartum forms. Non-dilated cardiomyopathies (such hypertrophic, tachycardia induced and primary restrictive) can also progress to become indications for transplant [132].

9.2.2 Ischemic Heart Disease Coronary artery atherosclerosis is the most common form of heart disease in developed countries. Despite

Fig.  9.2  Giant cell myocarditis. Photomicrograph from an endomyocardial biopsy (EMB) showing widespread myocyte destruction, mixed inflammation and prominent multinucleated giant cells without granuloma formation (H&E, × 200)

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9.2.4 Metabolic Disorders Storage disorders, hemochromatosis, familial hyperlipidemia, and amyloidoses can all be causes of heart failure. Because of the systemic nature of some diseases and the resultant risk of recurrence after transplantation, many are not generally considered eligible for transplant. There have been reports of combined liver heart transplants for familial hypercholesterolemia and familial amyloidosis [3, 103] with some success. Heart transplantation following bone marrow transplantation for primary amyloidosis has also been successful [80]. Cardiac involvement by certain systemic storage diseases (such as Fabry) can be successfully treated with heart transplant alone, since the allograft myocytes contain functional enzymes [102].

9.2.5 Evaluation of the Explanted Native Heart Examination of the native heart is important in confirming the clinical diagnosis and may provide significant findings that may require altering therapy for the allograft recipient or that may alter the prognosis after transplantation. Still, adequate fixation is important for proper examination and outweighs the need for immediate diagnosis in most cases. Photographs, gross descriptions and histologic sections should adequately document the native disease. The external configuration, weight, ventricular wall thicknesses, valve circumferences and chamber diameters (in short axis) are important components of the gross examination. A template for recording gross examination findings is included after this chapter as Appendix 1. Prior to dissection, the overall shape, symmetry, presence of adhesions, evidence of prior interventions (coronary bypass or ventricular assist devices), and position of any internal or external wire leads, should be documented. In order to obtain an accurate weight of the explant, the aortic and pulmonary vessels should be trimmed to within approximately 2  cm or less of the valves, and the pericardium removed, as completely as possible. In cases of ischemic heart disease the coronary arteries may be stripped from the specimen prior to dissection and then decalcified prior to cross sectioning.

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Bypass graft anastomoses should be carefully identified and examined histologically for obstruction. If stents are encountered, vessel segments can be sent for special microtomy using diamond or tungsten carbide blades [111] or the stents can be dissolved by reverse electroplating [18]. There are different approaches to the actual sectioning of the heart, including the flow-of-blood approach, combined short-axis and inflow-outflow, unroofing of structures to create “windows,” and cuts resulting in views approximating the standard echocardiographic planes. The combination of short-axis sectioning of the apical and mid ventricles with flow-of-blood technique for the remaining base and atria is recommended as it allows for examination of infarcts as well as ventricular chamber size, and preserves the majority of the coronary vasculature and valves. It also allows for optimal sampling of the myocardium across vascular territories for histology. In this combined technique, the fixed heart is laid with the left posterior-inferior ventricle side down and sectioned along the short axis (perpendicular to the long axis) of the left ventricle up to the level of the papillary muscles (Fig.  9.3). The remaining intact heart base is then dissected using the flow-of blood approach, as follows. The vena cavae and atria are typically absent, as these structures are needed for anastomosis to the allograft. Any remnants of atrial appendage should be evaluated for thrombus

Fig. 9.3  Dissection using the combined short axis and inflowoutflow method. Gross image of a whole heart specimen viewed anteriorly, showing the approximate planes for sectioning the ventricles in short axis (1), the right ventricular outflow tract (2), and left ventricular outflow tract (3)

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and the oval fossa should be inspected for competence if present. The right ventricle is opened by an incision through the tricuspid valve along the posterior aspect about 1  cm from the septum. A second cut is made from below the right ventricle following the outflow tract and through the pulmonary valve. The open circumference of the valves should be measured and the leaflets should be carefully inspected. An incision is then made through the mitral valve along the left lateral aspect of the ventricle, between the papillary muscles. The last incision begins from below the left ventricle on the anterior aspect following the ventricular septum and out the outflow tract through the aortic valve. Care should be taken not to damage the anterior mitral leaflet by deviating to the left or the pulmonary valve by deviating to the right. The left anterior des­ cending artery is transected by this incision (if it wasn’t removed previously). The coronary artery ostia should be inspected and, if not stripped for decalcification, the coronary arteries should be serially sectioned at 0.3  cm intervals along their epicardial course to evaluate for thrombi and luminal stenosis. Significant lesions should be submitted for microscopy to evaluate plaque composition and the nature of any thrombotic/embolic material. Histologic assessment of the coronaries is particularly important when examining allografts removed at retransplantation, to document allograft vasculopathy (discussed in Sect. 9.6). Describing the other dissection approaches is beyond the scope of this chapter, but several helpful references are available in the literature [1, 41, 137, 138].

9.2.6 Recurrence of Native Disease in Allografts Though cardiac transplantation for diseases with potential for recurrence is infrequent, and often curative (at least for the years of survival post transplant), there are a number of well-documented reports of primary diseases recurring the cardiac allografts (Table  9.3). These include giant cell myocarditis [94]. The histologic appearance is described as identical to that in the native hearts. Sarcoidosis, which occasionally recurs in pulmonary allografts, has been reported to recur in a transplanted heart allograft [147]. Recurrence of lymphocytic myocarditis can be difficult to distinguish from cellular

Table  9.3  Native diseases with potential for recurrence in cardiac allografts Inflammatory

Lymphocytic myocarditis Giant cell myocarditis Sarcoidosis Eosinophilic endomyocardial disease

Infiltrative

Amyloidosis Light chain deposition disease

Infectious

Chagas’ disease Toxoplasma Viral (CMV, EBV, parvovirus, enterovirus)

Neoplasms

Sarcomas Metastases

rejection (discussed in Sect. 9.5.3) since it is characterized by interstitial T-cell infiltration and myocyte damage. Myocarditis in general is also difficult to understand conceptually since transplant patients already have substantial suppression of cellular immunity. Infection with Trypanosoma cruzii (Chagas’ disease), while uncommon in the United States, is a significant cause for cardiomyopathy in Brazil and other parts of South America. Transplantation is an accepted treatment for chagasic cardiomyopathy, though there is a recurrence rate of 30–50% [20, 39]. There have also been reports of recurrent neoplasms following transplantation for tumor, most notably with primary cardiac sarcomas [2]. Patient transplanted for systemic amyloidosis are also at risk for recurrence if the source of amyloidogenic proteins (with the bone marrow for primary AL amyloidosis and the liver for hereditary amyloidosis) is not adequately addressed [40, 84].

9.3 Allograft Selection and Procurement 9.3.1 Donor Criteria Evaluation of a potential donor heart includes review of history, echocardiography, and electrocardiography. The ideal donor is under the age of 30, of similar body size, with no evidence of coronary artery disease, chest trauma, or infection. Implantation of the allograft ideally transpires within 4 h of harvesting. As the recipient list has grown in recent years, an expansion of the donor criteria has allowed for

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transplantation of organs not meeting all of these ideal criteria in certain donors. Approximately one tenth of donors in recent years are over 50 years of age [17, 53]. While these expanded criteria provide increased availability of organs, there is some decrement in the outcome [129]. Furthermore, increased donor age is associated with increased coronary artery disease and ventricular hypertrophy. Myocardial hypertrophy can be assessed directly with echocardiography and indirectly by electrocardiogram. While myocardial hypertrophy is associated with increased risk of graft failure, specific acceptance criteria vary from center to center [53]. It has been suggested that screening angiography be utilized in donors over 40 years of age or when indicated for the presence of ischemic heart disease [53].

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Fig. 9.4  Acute myocyte ischemic injury. Photomicrograph from an EMB showing contraction band necrosis, karyorrhexis, loss of myocyte nuclei and sarcoplasmic cross striations, and prominent neutrophilic infiltrate (H&E, × 400)

9.3.2 ABO Compatibility ABO incompatible (ABOi) cardiac allografts for adults have led to poor outcomes, presumably due to preformed anti-ABO antibodies (isoagglutinins) inciting rejection. By contrast, ABO incompatible transplants in infants under 1 year of age have been successful, presumably due to immature humoral immunity. Serum isoagglutinins do not develop until 12–14 months of age. Interestingly, many of these recipients eventually develop anti A or anti B antibodies. The mechanism appears to be one of induced tolerance of B-lymphocytes to A and B blood group antigens [44]. Three year survival for ABOi heart transplants in this age group is comparable to that of ABO compatible transplants [141] and there appears to be no overall increase in risk of mortality or retransplantation in comparison to infants receiving ABO compatible organs [141]. Infants receiving transplants before the development of serum isoagglutinins have an overall 80% survival rate [142].

(“harvesting” injury). The type of preservation solution used during transportation may have an effect on outcomes [34, 52]. Hearts from donors who have required inotropic agents prior to transplantation or organs with lengthy ischemic time may have myocardial ischemic damage and early graft dysfunction [117]. This may be evident when these hearts are sampled in the perioperative period, and may manifest as contraction band necrosis, or myocyte necrosis – karyorrhexis and loss of nuclei with homogenization of sarcoplasm and loss of cross striations (Fig. 9.4). Signs of reperfusion injury such as interstitial hemorrhage and abundant neutrophils are also common since blood flow resumes after implantation. Similar to ischemia caused by coronary obstruction, these changes are more prominent in the subendocardial myocardium and, as such, may been seen on endomyocardial biopsy (EMB). Myocyte ischemic injury is discussed further in Sect. 9.7.

9.4 Endomyocardial Biopsy 9.3.3 Preservation Injury Graft failure in the perioperative period may result from hyperacute rejection (discussed in Sect.  9.5.1), ischemia/reperfusion (due to intraoperative complications), inadequate cardioplegia, or suboptimal graft preservation during procurement or transportation

9.4.1 Background The refinement of EMB techniques and interpretation [13, 22] were critical to recognition of acute rejection and response to immunosuppressive therapy. Prior to this, rejection was observed only in animal heart

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transplant models [13]. The advent of endomyocardial surveillance immediately resulted in substantial improvement in 1-year survival after transplantation [10]. Over the years, a number of noninvasive alternatives to the EMB have been proposed [30] including MRI for examination of microvasculature [87] and the development of a genetic profile of acute rejection in heart transplant recipients [36] (discussed in Sect. 9.9). To date, none of these techniques has replaced pathologic examination of EMB material as the gold standard for the diagnosis of rejection. The biopsy procedure has minimal morbidity with minor complication rates of 1–6% [27]. Major complications such as myocardial perforation are unusual, especially in transplant recipients; however, the risk of disrupting the chordal apparatus of the tricuspid valve, leading to progressive tricuspid regurgitation increases with each biopsy performed [68, 123]. Rarely, small coronary arterial-venous fistulae may also develop as a consequence of endomyocardial biopsies [136].

9.4.2 Timing of Biopsies and Surveillance Strategies EMB may be routinely performed at specified intervals (“protocol” or “surveillance” biopsies) or when graft dysfunction is evident clinically, to assess for rejection. Some institutions also include a baseline biopsy immediately after transplantation, often with frozen section evaluation to assess for hypertrophy, fibrosis, and severe ischemic damage. EMB is also routinely used to assess response to changes in immunosuppressive therapy. Approximately 60% of patients experience an episode of rejection within the first year [21, 44], most occurring in the first 6 months. Accor­dingly, surveillance biopsies are most frequent in that time period and late routine surveillance biopsies may have less clinical utility [124], though clearly some patients seem to spontaneously develop late, severe rejection [78].

9.4.3 Specimen Adequacy and Handling The 2004 revised ISHLT working formulation (discussed in Sect. 9.5) requires three fragments of tissue

in order for a biopsy to be considered adequate for diagnosis. This is largely because acute rejection can be a focal process [126]. Prior to this, 4–6 undivided pieces of myocardium had been considered the minimum, reportedly reducing sampling error to approximately 2% [12]. The 2004 ISHLT criteria also specify that evaluable fragments should contain more than 50% myocardium, to avoid over interpreting findings in a scar or previous biopsy site. Tissue fragments should not be cut into pieces after being obtained by bioptome, as this may introduce further crush artifact. The sampling of multiple sites is more important than the quantity of myocardium obtained from a given site. Tissue should not be allowed to air-dry, and should not be crushed by forceps. Room temperature fixative (10% formalin) is preferred since cold fixative can increase contraction band artifact [127]. Additional tissue specimens may be frozen in embedding medium or placed in transport medium (such as Michels or Zeus) in preparation for freezing. Frozen tissue may be used for immunofluorescence studies to detect complement deposition along with other antigens (discussed in Sect. 9.5.4.4) or saved for future studies. Immunoperoxidase studies can also be performed on formalin-fixed paraffinembedded tissue sections for many of antigens detected by immunofluorescence as well. Submission of tissue in glutaraldehyde for electron microscopy could also be considered, but is rarely indicated. For permanent sections, a minimum of three 4–5 mm sections on at least 3 slides each should be examined histologically. Slides are routinely stained for hematoxylin and eosin. A connective tissue stain, such as the trichrome stain, may be included to help assess fibrosis and myofilament loss, but is not necessary.

9.4.4 Biopsy Site Changes and Incidental Findings The defect left in the myocardium by a bioptome heals through platelet and fibrin clotting, granulation tissue formation, and scarring just as any other wound. Subsequent sampling of the endomyocardium may include areas of scarring from previous biopsies. Depending on the timing and frequency of biopsies, up to two thirds of biopsy samples can contain areas of resolved or healing biopsy site [127]. The histologic

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appearance varies with the interval from the prior biopsy. Biopsy beds from the same procedure can even be sampled, showing fibrin deposition and neutrophils in direct apposition to frayed or crushed myocardium (without intervening endocardium) (Fig.  9.5), and should not be mistaken for ischemic injury of mural thrombus. Recently (within days to weeks) biopsied

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areas may show hemorrhage and hemosiderin-laden macrophages, loose connective tissue, with plump, fibroblasts and granulation tissue. Older biopsy sites can contain myxoid to collagenized stroma (Fig. 9.6). Adipose tissue is a normal finding in the right ventricle; the presence of adipocytes adjacent to myocytes tissue does not indicate epicardial fat from perforation of the ventricle. However, perforation can be assumed if biopsy fragments contain strips of mesothelial lining (Fig.  9.7). Telescoped fragments of arterioles or venules are occasionally interpreted as reorganized thrombi, but are actually another stretch artifact from the bioptome (Fig. 9.8).

a

Fig. 9.5  Sampling of biopsy site from the same biopsy procedure.Photomicrograph from an EMB showing a fibrin aggregate with entrapped leukocytes (right) directly apposed to myocytes showing some crush artifact (left), without an intervening endocardial layer (H&E, × 400)

b

Fig. 9.6  Sampling of old biopsy site. Photomicrograph from an EMB showing a central area of replacement fibrosis and puckering of the endocardial lining, suggestive of prior biopsy site scar (H&E, × 40)

Fig. 9.7  Epicardial mesothelial lining indicating right ventricle perforation. Photomicrograph from an EMB showing adipose tissue with the epicardial layer of mesothelium (a), indicating perforation of the ventricle (H&E, ×200) and a large nerve fiber (b), also suggestive of epicardial sampling (H&E, × 100)

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the graft implantation. Nonetheless, the risk of acute rejection diminishes with time from transplantation, so that the relative risk of the biopsy may exceed the risk of missing rejection for normally functioning allografts many years after transplantation [124]. Though less well understood, chronic rejection in the heart seems to manifest as microvascular and epicardial coronary changes as well as alterations in the endocardium and myocytes [56, 134].

9.5.2 Hyperacute Rejection Fig.  9.8  “Telescoping” artery artifact. Photomicrograph from an EMB showing this common artifact that should not be mistaken for vascular pathology (H&E, × 200). This artifact is caused by retraction of elastic vessel tissue as a consequence of tugging by the bioptome

9.5 Allograft Rejection 9.5.1 Overview Rejection is a leading cause of death in the first 3 years after heart transplantation, followed closely by primary graft failure [17, 129]; therefore, accurate diagnosis of acute rejection is crucial in the management of heart transplant recipients. Both humoral and cellular arms of the immune system can be involved in rejection. Each has a distinct histologic appearance. Cellular rejection is a T-cell mediated process that can also involve monocytes and granulocytes. Antibody mediated rejection occurs when antibodies bind to endothelial antigens in the cardiac allograft microvasculature. Antibody binding leads to activation of the complement cascade and direct endothelial cell damage with recruitment of macrophages and other inflammatory cells. Mixed antibody mediated and cellular rejection has been described in some cases. While cellular and antibody mediated rejection have traditionally been considered “acute” forms of rejection, both can occur late (>3 years) into the posttransplant course. In other words, “acute” refers to the rapidity of episode onset and not timing in relation to

Hyperacute rejection occurs immediately after implantation and reperfusion of cardiac allografts. This form of rejection can be thought of as the most severe form of antibody mediated rejection and occurs in patients with preformed antibodies. Hyperacute rejection develops within minutes to hours of transplantation in the presence of pre-formed circulating antibodies in the recipient [114] targeted against donor HLA antigens. The term hyperacute rejection originated in renal transplantation to describe graft destruction occurring literally before the eyes of the surgeon in the operating room. In the heart, hyperacute rejection after transplantation has been primarily observed with cardiac xenotransplantation [116]. Fortunately, it is rare in human transplantation and is usually seen in the setting of ABO incompatibility, but not HLA mismatch [26]. Hyperacute rejection is not increased in cardiac retransplantation, but retransplantation has been associated with antibody mediated rejection [112]. In the experimental setting of cardiac xenotransplantation, the onset of hyperacute rejection may be delayed beyond 24  h after transplantation [114]. The histopathology of hyperacute rejection reflects its pathogenesis. The cellular and tissue level changes in hyperacute rejection result from antibody mediated activation of the complement system, causing capillary endothelial damage and thrombi in the venules of the myocardium. Congestion at this level results in edema and hemorrhage. A histopathologic grading system has been proposed [115], but is not included in the current ISHLT working formulation. In that system, initial (mild) findings include observation of thrombi within venules and swollen capillary endothelium. An intermediate stage

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These difficulties finally led to the development of a standardized consensus formulation for the grading of rejection in endomyocardial biopsies through the auspices of the ISHLT [12].

9.5.3.2 1990 ISHLT Classification

Fig.  9.9  Hyperacute rejection. Photomicrograph from an autopsy specimen from a patient dying 24 h after transplantation due to hyperacute rejection. There is intense lymphocytic and granulocytic inflammation, karyorrhexis, fading loss of myocyte nuclei (center) and sarcoplasmic smudging (H&E, × 100)

includes these findings as well as interstitial edema, congestion and sludging of capillary erythrocytes with scattered capillary thrombi. A late, severe stage will exhibit all of the above findings plus disruption of capillaries draining into thrombosed venules and interstitial hemorrhage, areas of parenchymal necrosis, and/or arterial thrombi (Fig. 9.9). Immune studies demonstrate vascular deposits of immunoglobulins IgM and IgG, along with complement fractions and fibrin [127].

The 1990 working formulation was based on histologic changes seen in patients on standard immunosuppressive therapy of the era (azathioprine, cyclosporine, and steroids). Its application was inten­ded for biopsy specimens and full thickness heart sections (from autopsies or explants). The severity of rejection was graded on the concentration and distribution of inflammatory cell infiltrates and the presence of myocyte damage, edema, hemorrhage, and (in severe forms) vasculitis. Six categories were developed, ranging from 0 (no evidence of rejection) to 4 (severe rejection). Myocyte “necrosis” separated grade 1 (mild rejection) from higher grades, which were then stratified according to the intensity/ composition of inflammation and extent of myocyte necrosis. Grades 1 and 3 (multifocal moderate rejection) were further subdivided into A and B categories based on focal (A) or diffuse (B) distribution of inflammation. Grade 2 was by definition focal, and grade 4 was diffuse, so these were not further divided.

9.5.3.3 2004 ISHLT Classification

9.5.3 Acute Cellular Rejection 9.5.3.1 Background Billingham first proposed an initial, unifying system for classification of cellular rejection in 1982 [7]. During the decade to follow several other grading schemes were proposed [75, 89, 113]. These shared a number of similarities including: (1) varying grades and degrees of acute rejection; (2) the use of descriptive terms in characterizing a specific biopsy grade; and (3) a tendency to grade the entire biopsy by the grade of the worst area observed in the entire specimen. These parallel grading systems confounded efforts to compare outcomes from different centers or to develop multicenter clinical research efforts [10].

With widespread application of the 1990 ISHLT Working Formulation, some key points of confusion became apparent. Interobserver variability was substantial, outcomes for grades 1A, 1B, and 2 were not significantly divergent, and some non-rejection findings were also occasionally confused with rejection and vice versa. Central review by experts in multicenter studies identified widespread inconsistencies in the application of the grading system among individual pathologists, particularly for grade 2 (single focus of myocyte necrosis) and 3A rejection (moderate multifocal inflammation and myocyte necrosis) which can be difficult to distinguish from single and multiple Quilty lesions (discussed in Sect.  9.5.3.4) [86, 145]. Several studies also demonstrated that neither grade 1A/1B nor grade 2 was significantly associated with progression to higher

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grade rejection on subsequent biopsy in the absence of therapeutic intervention. Hence, these grades were not felt to warrant increased immunosuppression [83, 92, 144, 145, 148]. Late (>3 year) biopsies demonstrating multiple foci of lymphocytic infiltration and myocardial necrosis (Grade 3A) were also reported to resolved spontaneously in some cases [78]. These difficulties with the working formulation eventually led to the reconvening of a consensus group and revision of the working formulation in 2004 [126]. Adequacy criteria were revised, defined as at least three fragments of tissue with myocardium constituting at least 50% of the sample. It was recognized that other elements (chordae tendineae, valve leaflets) should be described as appropriate. Grades greater than 0 in the new system all end with “R” to indicate that they represent interpretation under the 2004 revision of the working formulation (Table 9.4). These are described in detail below. The new formulation also addressed the composition of inflammation. For cellular rejection this was defined as primarily lymphocytic (T-cell) inflammation with occasional macrophages and/or eosinophils (especially in early and/or severe rejection). Granulocytes could be seen in severe rejection. Plasma cells are not usually present and their presence could suggest healing ischemic injury (see Sect. 9.7), Quilty lesion (see Sect.  9.5.3.4), or rarely lymphoproliferative disease (see Sect. 9.8.2). The designation myocyte “necrosis” in the 1990 formulation was also modified to myocyte “damage” or “injury,” acknowledging that sublethal myocyte injury is the predominant mode of cell injury, even in the severe forms of rejection [59]. Manifestations of myocyte injury had been described as vacuolization, perinuclear

halo formation, irregular myocyte border (ruffled sarcoplasmic membrane), increased branching of myocytes, and myocyte encroachment by lymphocytes with partial disruption of the myocytes [75, 150]. Coagulative necrosis, hypereosinophilia and nuclear pyknosis, indicators of irreversible myocyte necrosis, were felt to be rare in rejection. Contraction bands and hemorrhage can be seen as artifacts of compression by the bioptome and should not be mistaken for a pathologic process. In the revised working formulation, myocyte damage is described as “clearing of the sarcoplasm and nuclei with nuclear enlargement and occasionally prominent nucleoli” [126] (Fig. 9.10). Lymphocyte encroachment into the sarcoplasm and irregular myocyte borders are also commonly seen. Myocyte dropout can also be signaled

Fig.  9.10  Myocyte damage in cellular rejection. Photo­ micrograph from an EMB showing cellular rejection and myocyte damage. There is loss of nuclei, scalloping of the myocyte border, and homogenization of the cytoplasm (H&E, × 400). Myocytolysis (sarcoplasmic clearing) is not shown in this example

Table 9.4  Summary of the 1990 and 2004 (revised) ISHLT working formulations for acute cellular rejection in cardiac allografts 2004 Revision 1990 Working formulation Grade 0 – no rejection

Grade 0 – no rejection

Explanatory notes

Grade 1R – mild

Grade 1A – focal mild Grade 1B – diffuse mild Grade 2 – focal moderate

Any mononuclear infiltrate with up to a single focus of myocyte damage

Grade 2R – moderate

Grade 3A – multifocal moderate

Mononuclear infiltrate with multifocal areas of myocardial damage

Grade 3R – severe

Grade 3B – borderline severe Grade 4 – severe

Diffuse mononuclear or polymorphous infiltrate with widespread myocardial damage, edema, interstitial hemorrhage and/or vasculitis

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by the presence of “space occupying” aggregates of inflammation that significantly expand the interstitium and push away the surrounding myocytes.

Grade 1R Rejection (Includes 1990 ISHLT Working Formulation Grades 1A, 1B, 2) Two separate patterns are included in this designation, reflecting in part its fusion of two groups of biopsies from the 1990 working formulation. The first pattern is that of predominantly lymphocytic inflammation either in a perivascular distribution or among myocardial cells without myocyte damage (Fig. 9.11). The second pattern is a single focus of mononuclear inflammation associated with myocyte damage. Because rejection can be a focal process, sampling error likely explains for similar clinical behavior of rejection assuming any of these 3 patterns.

 rade 2R Rejection (Formerly 1990 ISHLT G Working Formulation Grade 3A) Multiple (two or more) foci of mononuclear cells with myocyte damage constitute Grade 2R or moderate

Fig.  9.12  Moderate cellular rejection (ISHLT 2004 2R). Photomicrograph from an EMB showing more widespread, but not diffuse inflammation with foci of myocyte injury (H&E, × 200)

cellular rejection (Fig.  9.12). The inflammation is less likely to reflect selective sampling given its multifocality, and may include eosinophils (especially in early biopsies), but neutrophils are generally absent. Criteria do not require that the two foci be on the same fragment, or that the intervening fragments contain significant rejection.

Grade 3R (Includes 1990 ISHLT Working Formulation Grades 3B and 4)

Fig.  9.11  Mild cellular rejection (ISHLT 2004 1R). Photo­ micrograph from an EMB showing perivascular lymphocytic inflammation without myocyte damage. This could be a focal or diffuse finding and up to a single focus of myocyte damage may be seen (H&E, × 200)

In severe cellular rejection, the biopsy fragments contain a diffuse inflammatory infiltrate. The infiltrate can be polymorphous. Myocyte damage and necrosis is seen in numerous foci. Most fragments should be involved by at least some degree of rejection, but this is not required (Fig.  9.13a). Interstitial edema and hemorrhage may be present (Fig.  9.13b). If larger arteries are sampled, arteritis can be present. The significance of vasculitis (Fig. 9.14) in cardiac allografts is controversial, but does not seem to be a manifestation of severe cellular rejection (as is the case for renal allografts). In addition to rejection (cellular or antibody mediated), it can also be associated with cardiac allograft vasculopathy (CAV) (see Sect. 9.6), infection, and infarction [65, 70, 133].

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Fig.  9.13  Severe cellular rejection (ISHLT 2004 3R). Photo­ micrograph from an EMB showing diffuse lymphocytic inflammation with extensive myocyte damage (H&E, × 100)

9.5.3.4 Nodular Endocardial Lymphocytic Infiltrates (Quilty Effect) In 1981, Billingham et  al described the presence of large endocardial aggregates of mononuclear cells (Fig. 9.15) in an EMB from a transplant patient with normal allograft function. These large mononuclear cell aggregates were characterized as “Quilty effect,” after the surname of the index patient though similar lesions were subsequently recognized in several other patients without compromised graft function [69]. The a

Fig. 9.15  Quilty effect. Photomicrograph from an EMB showing a well-demarcated (nodular) endocardial inflammatory collection, or Quilty lesion (H&E, × 40) (courtesy Joseph Maleszewski, Mayo Clinic)

non-eponymous term “endocardial lymphocytic infiltrates” is also used to describe this change [127]. Associations have been drawn between these lesions and the use of cyclosporine [47], but this has not been a consistently reproducible correlation. Immuno­phenot­ yping the constituent inflammatory cells typically demonstrates B-cells, macrophages, plasma cells, and CD4 T-cells (as opposed to CD8 positive T-cells in >1R rejection). Though Quilty effect consistently does not correlate with graft dysfunction across several studies, there are a

b

Fig. 9.14  Vasculitis in small vessels and arteries of cardiac allografts. Photomicrographs from endomyocardial biopsies showing transmural inflammation of small vessels (a) (H&E, × 200) and intramyocardial muscular arteries (b) (H&E, × 200)

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small number showing a loose association with cellular rejection [126] and possible CAV [24]. Staining for C4d has also been recognized in Quilty lesions, though no relation to antibody mediated rejection (discussed in Sect. 9.5.4.4) has been demonstrated [127]. A large series of patients with Quilty effect found no association with cytomegalovirus or Epstein–Barr virus, or any propensity for progression to lymphoproliferative disorder [71]. These remain enigmatic lesions. The revised 2004 ISHLT classification retains the “Quilty effect” terminology although the term nodular endocardial infiltrates may be also used. The presence of Quilty effect should be recorded separately from the presence or absence of rejection. Quilty effect can be confined to the endocardium (formerly “Quilty A” in the 1990 ISHLT working formulation) or can infiltrate into the myocardium (“Quilty B” in the 1990 ISHLT working formulation). The revised ISHLT classification does not make a distinction between these two subtypes. The histologic appearance of Quilty effect is that of a nodular aggregate of inflammatory cells, predominantly lymphocytes, with rare plasma cells, dendritic cells and histiocytes. They are well demarcated from the surrounding myocardium, well circumscribed, and symmetric. Dendritic components of lymphoid follicles may be seen, especially when stained for CD21 [118]. Small vascular lumina may be present as well (Fig.  9.16). The aggregates may be large, and can extend into myocardium. Myocyte damage can be

Fig. 9.16  Microvasculature in Quilty lesions. Photomicrograph from an EMB at high power showing small capillaries that are often seen within Quilty lesions and can be helpful in distinguishing deep Quilty lesions from rejection (H&E, × 400)

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seen, particularly in intramyocardial lesions, but the hallmark feature of symmetric circumscription allows for distinction from rejection. Even for deep-seated lesions, examination of serial sections almost always demonstrates continuity with the endocardium, a finding helpful confirming the nature of these foci.

9.5.4 Antibody-Mediated Rejection (AMR) 9.5.4.1 Background In the late 1980s and early 1990s, a distinct clinical syndrome of left ventricular dysfunction soon after transplantation was observed in some patients. In these cases, endomyocardial biopsies lacked both the lymphocytic infiltration of acute rejection and the associated pattern of myocyte damage [61, 93]. Routine light microscopic examination of endomyocardial biopsies demonstrated endothelial cell swelling, interstitial edema, and acute “vasculitis” or inflammatory cells within the lumen and walls of arterioles and small arteries. Immunofluorescence studies detected immunoglobulin heavy chains (IgG, IgM, or IgA) as well as complement deposition [46]. Serum assays of the affected recipients found evidence of antibodies to HLA class I and class II markers usually with donorspecificity [23, 32, 82, 130]. Patients were more likely to have prior exposure (pre-sensitization) to HLA antigens through blood transfusions (often with prior surgery such as ventricular assist device placement), pregnancy, and previous solid organ allografts though de novo antibody formation after transplantation also seemed to occur [110]. Interestingly, the interval to first rejection did not seem to differ between initial transplantation and retransplantation [105]. This constellation of serum antibodies, capillary inflammation and damage, and absent cellular rejection mirrored the changes of so-called antibody-mediated (or humoral or vascular) rejection as it was defined in other organ transplants, such as kidney [25]. AMR is most often diagnosed early after transplantation. Two thirds of patients with AMR seen in the first month post transplant exhibit graft dysfunction [110]. However, acute onset AMR may occur years after transplantation (late AMR) [91, 124]. In one series of patients for whom late AMR was documented, AMR occurred 60–163 months after transplantation [112].

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AMR and cellular rejection are not mutually exclusive and concomitant AMR has been reported in up to one fourth of biopsies with acute cellular rejection [110].

9.5.4.2 2004 ISHLT Criteria The 2004 revision of the working formulation included a separate immunopathology task force to evaluate AMR. Based on consensus that patients with episodes of antibody mediated rejection have diminished longterm graft survival as compared to those who do not [110], the revised working formulation included criteria for histologic features indicative of antibody mediate rejection including endothelial cell activation, endovascular macrophages, myocyte damage without cellular infiltration, thrombosis, edema, and pericapillary neutrophils [57, 110, 126]. Immunopathologic criteria were also set forth and included positive immunofluorescence staining of frozen tissue sections for immunoglobulin heavy chains and complement (C4d and/or C3d). The 2004 classification included a score for AMR; graded as present (AMR1) or absent (AMR0). In 2006, further clarification from the task force was provided in a follow-up document [110]. Their recommendation was that “the combination of clinical, histologic, and immunopathologic findings as well as demonstration of circulating donor specific antibodies, in the absence of cellular rejection, are recommended to diagnose acute antibody-mediated rejection” [110]. This was a significant expansion beyond the previously published histologic and immunopathologic criteria to include clinical (including imaging studies), and laboratory data in the pathologic diagnosis scheme (Table  9.5). The histopathologic and immunopathologic parameters for diagnosing AMR remained unchanged, but it was recognized that they may not be entirely specific and the appropriate clinical context was needed in order to diagnose AMR with certainty. This was a departure from the grading scheme for cellular rejection, which can be applied without respect to the clinical and laboratory findings.

9.5.4.3 Histopathology of AMR The interstitial capillaries are the primary locus of activity in AMR. In the acute phase of AMR, they

Table 9.5  2004 ISHLT criteria for AMR Clinical findings Allograft dysfunction, as defined by hemodynamic compromise Histology Capillary endothelial damage (swelling, activation of nuclei) Interstitial hemorrhage and perivascular neutrophils Myocyte damage (coagulative necrosis, cytoplasmic clearing) Fibrinoid necrosis or fibrin thrombi within vessels Immunopathology Capillary deposition of immunoglobulins (IgG, IgM, or IgA) Diffuse activation of complement with demonstration of C4d fraction in vessels (frozen or paraffin embedded tissue) Endovascular macrophage deposition as demonstrated by CD68 immunostain, in concert with vascular markers CD31 or CD34 Serology Donor specific antibodies (anti HLA I or anti HLA II) Adapted from Reed et al. [110]

appear congested with evidence of endothelial injury including cytoplasmic swelling and nuclear enlargement (Fig. 9.17a). Interstitial hemorrhage and edema in addition to neutrophilia in and around the capillaries can be observed in severe cases (Fig.  9.17b). This is most often a diffuse change, seen throughout all biopsy pieces (Fig. 9.17c). When the cellularity within vessels is pronounced, occasionally even spilling into the perivascular space, this pattern can resemble the 1990 1B classification of mild multifocal cellular rejection. For biopsies with this pattern, a diagnosis of mixed cellular and antibody mediated rejection is often entertained (see mixed rejection in Sect.  9.5.4.6), though this diagnosis requires demonstrating perivascular accumulation of CD3 positive T-cells in significant numbers (with or without myocyte damage) in addition to AMR by histology and immunopathology. The most severe manifestation of AMR may resemble hyperacute rejection (see Sect. 9.5.2) with microangiopathic changes, thrombi, and widespread edema and myocyte injury with prominent neutrophils. The histologic appearance (or even existence) of chronic antibody mediated rejection is a controversial topic. Biopsies from patients with repeated episodes of AMR, but who do not succumb to acute graft failure have been reported to show a characteristic and conserved pattern of changes on biopsies [54, 64]. This subject is discussed further in Sect. 9.5.4.5.

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a

Fig.  9.17  Antibody mediated rejection (ISHLT 2004 AMR1). Photomicrograph from an EMB showing diffuse interstitial edema and endothelial swelling as well as macrophage accumulation

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b

within the capillaries (a) (H&E, × 200). In the acute phase, eosinophils may also be seen in the interstitium (b)

9.5.4.4 Immunopathology of AMR Several studies have shown that histological parameters associated with the diagnosis of AMR alone do not have a sufficiently high sensitivity to serve as a screening tool for AMR and further justify the need for immunostaining by showing independent prognostic significance for complement and fibrin deposition [57, 66]. Immunopathologic features in frozen biopsy specimens subjected to immunofluorescence include diffuse capillary staining for complement components (C3d, C4d), variable demonstration of immunoglobulins (most often IgG or IgM), and in severe forms, fibrin (but not fibrinogen) (Fig. 9.18). There are commonly observed staining artifacts with immunofluorescence that should be noted, including: autofluorescence of fine endocardial elastic fibers and perinuclear lipochrome pigment, bright homogenous sarcoplasmic staining of non-viable myocytes (which may indicate ischemia), and diffuse staining of the sarcolemmal membrane without specific capillary staining (the significance of which is unclear) (Fig. 9.19). Immunoperoxidase staining of paraffin sections can also be used to aid in AMR diagnosis. The recommended target antigens include a macrophage marker (CD68) in combination with a vascular marker (CD31 or CD34) and C4d (Fig. 9.20) [126]. Accumulation of macrophages within capillaries (identified by CD31 or CD34) together with diffuse capillary staining for C4d is seen in AMR. There is substantial variability in the application of immunopathology for AMR throughout the world.

Fig.  9.18  Immunofluorescence staining patterns in AMR. Immunofluorescence photomicrograph from an EMB showing strong diffuse capillary staining for C4d (×400). Staining for other antigens will have a similar appearance in AMR

The panel of antibodies and methods of staining vary considerably from center to center, with C4d being the most constant target antigen. A brief summary of other antigens thought to have a role in AMR diagnosis follows (Table 9.6).

C3d There is evidence that the additional diagnostic and prognostic contribution of C3d staining warrants inclusion in AMR immunopathology panels. In pediatric patients, C3d deposition has been shown in patients

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a

b

Fig.  9.19  Immunofluorescence staining artifacts. Immuno­ fluorescence photomicrographs showing common non-specific staining artifacts including peri-myocyte or sarcolemmal

a

staining (a) (× 400) and homogenous staining of necrotic myocytes, mostly in the subendocardial region (b) (× 100)

b

Fig.  9.20  Immunoperoxidase staining patterns in AMR. Photomicrograph from an EMB stained for C4d (a) (× 100) and CD68 (b) (× 200) using immunoperoxidase methods. Diffuse

Table 9.6  Immunofluorescence studies for AMR C4d C3d

staining of capillaries for C4d (a) and accumulation of CD68 positive macrophages within vessels (b) is suggestive of AMR

HLA-DR vascular

HLA-DR extravascular

Fibrin or platelets

CD59, etc.

ACR

–

–

+

–

± *Severity

–

Active AMR

+

+

+

–

± *Severity

± *Accomodation

Late AMR

+

+

–

+

± *Severity

± *Accomodation

Ischemia

+ *Myocyte

+ *Myocyte

–

–

*Myocyte

–

Quilty

+

+

–

+ *Lymphs

–

–

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receiving positive crossmatch allografts even in the absence of findings of cellular rejection [66]. In adults, staining for C4d and C3d has been associated with greater allograft dysfunction and mortality than staining for C4d alone. C3 is further downstream in the complement cascade than C4 and the presence of C3d may indicate a failure of complement regulators to halt the steps after cleavage of C4 [128]. C3d is available commercially as a primarily fluorochrome labeled antibody and many centers use this in direct immunofluorescence rather than indirect techniques (used most often for C4d). As a result, the fluorescent signal may be weaker for C3d and sensitivity may be affected.

Immunoglobulin Heavy Chains The significance of staining for immunoglobulins is obvious in AMR, but this technique appears may be less specific for AMR than expected and may not contribute additional value to staining for complement alone, though co-localization of immunoglobulin and complement was compelling evidence in the early establishment of AMR as a viable entity [61]. Staining for immunoglobulins has been observed in perioperative ischemic injury [4, 14] and in patients receiving prolonged anti-lymphocyte antibody (so-called induction) therapy [60].

Fig.  9.21  Immunofluorescence staining for HLA-DR in late AMR. Immunofluorescence photomicrograph from an EMB showing moderately intense staining for HLA-DR with a frayed or lace-like appearance. The punched-out round dark areas represent mononuclear inflammatory cell nuclei in negative relief (× 400)

membrane attack complex. These may play a role in preventing cell injury from occurring in the setting of antibody bound to capillary endothelium. This has led to speculation about these factors participating in the process of graft “accommodation” [128]. There are few commercially available antibodies for these and those available require extended incubation times, making them impractical for real-time diagnostic utility.

HLA-DR HLA-DR (an MHC class II antigen) plays a role in antigen presentation and its expression is largely confined to leukocytes. Expression on capillary endothelium can also be induced by endothelial injury and “activation” [55]. Thus, staining for HLA-DR can be seen in both AMR and cellular rejection and expression can persist long after resolution of these. A paradoxic loss of staining or a “ragged” staining pattern (Fig. 9.21) may signal loss of capillary integrity in late or chronic AMR (see Sect. 9.5.4.5) [76, 77].

Fibrin

Complement Regulators (CD55, CD59)

Platelet Antigens (CD61, CD63)

A number of proteins (including CD35, CD46, CD55, CD59 and others) serve to inhibit progression of the complement cascade and prevent formation of the

Like the coagulation cascade, interactions between platelets and endothelium may also play a critical role in rejection pathogenesis. Several recent studies have

Activation or dysregulation of the coagulation cascade (evidenced by fibrin staining) may play a role in the myocardial injury that occurs as a result of AMR. Thrombi can be seen in hyperacute rejection, the most extreme manifestation of AMR. Several studies have shown deposition of fibrin to be an adverse prognostic sign, and an indicator of greater severity in AMR [9, 76, 79].

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demonstrated the involvement of platelets in the early events of AMR as well as in allograft vasculopathy [96, 121, 151].

9.5.4.5 Serum Antibody Studies Demonstrating the presence of circulating anti-donor antibodies is of obvious importance in the clinical diagnosis of AMR. Identification of anti-HLA antibodies using solid phase (ELISA plate or latex bead flow cytometry) methods have markedly improved sensitivity and specificity compared to previous generation cytotoxicity-based assays [19]. These techniques also provide quantitative measurements that can be followed over time. Newer generation assays can also determine the immunoglobulin class, subclass and complement fixing capacity [122]. Such monitoring of donor-specific antibodies (DSA) has provided critical insights into the development, temporal progression, and response to therapy in AMR. Their use has almost completely ameliorated hyperacute rejection in the current era. Still, these assays are not perfect and convincing cases of AMR (by histology and immunopathology) have been reported in the absence of DSA [128]. Flow cytometric strategies include B-cell and T-cell crossmatching and single antigen bead testing. In flow crossmatching, the donor’s lymphocytes are incubated with the recipient’s serum and then interrogated for CD20 or CD3 expression and surface bound antibody. Significant populations of antibody coated T-cells correlate with anti-MHC class II antibody and antibody coated B-cells generally indicate anti-MHC class I antibody. These methods are powerful as in  vitro approximations of the donor-host interactions in vivo, but do not provide information about the specific antigen target of the recipient antibody. Single antigen bead testing involves incubating serum with a panel of fluorescent-labeled latex beads that have been coated with different known MHC class I and II antigens. Typically a screening step is employed followed by specific testing for sera testing positive in screening. Using this technique, the exact specificity of the antibodies in a recipient can be identified. This information together with HLA typing (usually by molecular methods) of donors can be combined to perform a “virtual crossmatch” to accurately predict the likelihood of compatibility without actually mixing cells and sera.

9.5.4.6 Controversies in AMR Mixed Rejection Since AMR and cellular rejection proceed through different cellular pathways with separate regulatory controls, they are not mutually exclusive. Cardiac allograft biopsies from patients with hemodynamic compromise can show histologic and immunopathologic evidence of combined cellular and antibody mediated rejection (mixed rejection) [15, 104]. Most reports have occurred within the first 4 weeks of transplantation, but this phenomenon can occur at any time after transplant. It may be more commonly seen in patients with prior episodes of AMR and subtherapeutic immunosuppression levels. In cases of subtle perivascular infiltrates, differentiating macrophages from lymphocytes by histology alone can be difficult. Staining for macrophage and T cell antigens (e.g., CD68 and CD3) can be helpful in distinguishing mixed rejection from AMR with focal extravascular macrophages.

Severity of AMR Inclusion of AMR scoring in the 2004 revised ISHLT classification was a major step forward in recognizing, treating, and understanding AMR. However, as a binary (present or absent) variable, it does not capture gradations across a spectrum of changes that seem to exist in AMR (analogous to cellular rejection). Graded severity schemes for AMR have been proposed and shown to be reproducible and to correlate with increased risk of mortality or graft loss [76], but involve complicated algorithms and employ immunostains not routinely used at many institutions. Using the current classification scheme, pathologists interpret the histologic and immunopathologic findings in the context of a subjective threshold for “positive,” and then incorporate clinical and laboratory data (if available) in assigning the AMR score. Given the many opportunities for variation at each of those junctures, the inter- and intra-observer agreement for this score is likely to be poor. Providing a graduated scheme for AMR reporting has potential to address some of the subjectivity as a source of variation (some pathologist may ignore minor or mild features of AMR and only consider the most severe forms as “positive”), but may also add further confusion to an already complex endeavor.

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“Chronic” AMR and CAV The histologic appearance of AMR in biopsies evolves over time and in response to therapy [54, 76, 128]. Even when there is resolution of endothelial swelling and endovascular macrophage accumulation, subtle indicators of so-called chronic AMR (increased interstitial collagen, capillary wall damage (evident on CD34 staining), and capillary depletion) may persist [76, 77]. It has been hypothesized that ongoing or repeated episodes of AMR lead to destruction and loss of capillaries [38]. There are speculations about findings in cardiac transplants that could be analogous to late manifestations of AMR in renal allografts (capillary basement membrane remodeling, transplant glomerulopathy, and peritubular capillary depletion) [120]. These changes are poorly understood and at the moment lack sufficient evidence, but the added dimension of chronicity is important in considering the morphologic manifestations of AMR and these factors have likely confounded attempts to achieve consensus about the histopathology of AMR. Since endothelial cells are injured in AMR, the effects on larger vessels have also been considered extensively. CAV is a common complication of heart transplantation, affecting more than half of all patients at 10 years posttransplant and is a major cause of eventual graft loss [63]. Because CAV is characterized by concentric intimal (subendothelial) collagen matrix accumulation (see Sect. 9.6), implicating AMR in this process seems intuitive. There are several reports linking the two [6, 51, 73, 146] but the evidence is far from

a one-to-one correlation. In studies showing poor outcomes following episodes of AMR, the incidence of CAV not 100% (ref), suggesting this is not the only mechanism for graft failure in AMR. CAV has also been associated with many factors besides AMR including cellular rejection [108], calcineurin inhibitors [107], and CMV infection [85, 135].

Incorporating Clinical and Laboratory Data in AMR Diagnosis The contributions of imaging, invasive hemodynamics, and laboratory data, in addition to biopsy, are critical in the clinical recognition of AMR. However, they can also introduce diagnostic dilemma when there is not complete agreement between the modalities (clinical graft dysfunction, circulating DSA, biopsy histology, and immunopathology) (Table 9.7). For example, acute graft dysfunction can be seen in the setting of a biopsy with findings of AMR but no evidence of donor-specific anti-HLA antibodies [110]. One hypothesis in this setting is that grafts may absorb antibody so that it can no longer be detected in circulation, but there is little precedence for such a phenomenon. Antibodies to antigens other than HLA have also been implicated, including antibodies to major histocompatibility class I-related chain A (MICA), vimentin, heat shock proteins, skeletal muscle, and cardiac myosin [72, 81, 140], as well as yet-to-be characterized “anti-endothelial antigens” identified by indirect immunofluorescence using recipient serum applied to

Table 9.7  Possible explanations for discordant histology, immunopathology, and/or donor-specific antibody studies Pattern

Histology + Complement + DSA –

Histology + Complement – DSA +

Histology – Complement + DSA +

Possible Explanation

Non-HLA antibodies Non-DSA “3rd party” Ig Nadir of Ig titer oscillation

Non complement fixing Ig Chronic AMR

Complement regulators

Pattern

Histology + Complement – DSA –

Histology – Complement + DSA –

Histology – Complement – DSA +

Possible Explanation

Non complement fixing Ig Non-HLA antibodies Non-DSA “3rd party” Ig Nadir of Ig titer oscillation Chronic AMR

Mannin/lectin binding pathways of complement activation

Complement regulators

Non-HLA antibodies Non-DSA “3rd party” Ig Complement regulators

Non complement fixing Ig

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cultured endothelial cells [29]. Assays for DSA are not perfect and even the solid phase assays are prone to overlooking a clinically important low titer DSA when another non-donor specific but very high titer antiHLA antibody is present in the serum (the so-called third party antibody phenomenon). Another common dilemma is the absence of complement staining when histology, DSA, and clinical findings are all suggestive of AMR. This could be explained by the presence of non-complement fixing antibody. Normal histology when histologic findings and DSA suggest AMR could be explained by the successful intervention of complement regulators, halting progression of the complement cascade and preventing endothelial damage. This may be a key event in the so-called induction of tolerance or “accommodation” of a cardiac allograft [143].

9.6 Cardiac Allograft Vasculopathy A peculiar form of intimal disease affecting the epicardial coronary arteries is a well-recognized and common complication of heart transplantation. Also known as transplant coronary artery disease, transplant coronary arteriopathy, allograft coronary vasculopathy, CAV is the most common cause of graft loss after 5 years from transplantation [129]. Both alloantigen dependent and independent risk factors have been identified for the development of transplant coronary artery disease [106]. These include HLA mismatch, previous episodes of rejection, immunosuppression regimen, ischemia/reperfusion injury at time of transplantation, mode of donor death, and CMV infection [85, 107, 108, 135]. Risk factors for the development of native vessel atherosclerosis, particularly hyperlipidemia, are also associated with the development of CAV [139]. Because of transplant denervation, typical anginal symptoms are absent and clinical detection of CAV is challenging [43]. Noninvasive diagnostic techniques are relatively insensitive, especially in the early phase of disease [74]. For these reasons, coronary angiography is the mainstay and gold standard for CAV surveillance and diagnosis in heart transplant recipients [49]. Because CAV is often a diffuse process without abrupt changes in lumen diameter, luminography techniques such as coronary angiography can underestimate the severity of CAV. Because smaller caliber (intramyocardial) vessels

not visualized, attention must be paid to the rate of contrast dissipation as an indicator of flow through these branches. Despite these limitations, angiography remains the most useful means of estimating CAV severity and several studies have demonstrated the value of the prognostic information it provides [31, 98]. Intra­ vascular ultrasound in conjunction with coronary angiography is more sensitive and specific than angiography, but also considerably more expensive and also unable to image smaller caliber vessels. The sensitivity of this technique is highlighted by studies finding that early CAV may be detected in up to 75% of transplant recipients at 1 year after transplant [149]. The histopathologic appearance of allograft vasculopathy in the coronary arteries, compared to native artery atherosclerosis, is quite distinct (Table 9.8) [13, 106]. Allograft vasculopathy is a diffuse, concentric process that is observed in proximal and distal epicardial vessels (Fig. 9.22), as well as intramural (penetrating) arteries [11, 74]. Venous changes have also been described. The focal, eccentric lesions of naturally occurring atherosclerosis may also be present from donor-transmitted disease, but the presence of native disease does not necessarily affect the progression of transplant coronary artery disease [16]. The severity of luminal narrowing is generally related to the magnitude of intimal thickening [45]. CAV progresses over months to years, whereas for native atherosclerosis the timeframe is years to decades. This allows less time for remodeling and enlargement of the overall arterial diameter to occur. Intimal lesions in longstanding CAV can undergo calcification and typical atheromatous changes, but this is not the typical appearance and is not seen in early disease. Endomyocardial biopsies from patients with CAV can demonstrate chronic subendocardial ischemic changes (diffuse and distinct sarcoplasmic vacuoles and interstitial fibrosis) or even acute ischemia, similar to what occurs in the first week after transplant. If such changes are seen, testing for serum troponin and coronary angiography can be suggested to assess for clinically significant CAV progression.

9.7 Myocardial Ischemia Myocardial ischemic changes are frequently seen in the immediate post transplantation period as a result myocardial injury in the donor, preservation damage,

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Table 9.8  Histopathological features of transplant coronary artery disease compared to native atherosclerosis Features CAV Atherosclerosis Vessel involvement

Epicardial and intramural arteries are involved Diffuse and very extensive vessel involvement Veins can also be involved Affects the proximal and distal epicardial vessels, as well as their branches A disease of the intima, media, and adventitia

Epicardial arteries involved only Predominant involvement in proximal epicardial coronary arteries in patchy discrete plaques Veins are never involved Three layers, intima, media and adventitia, are involved Primarily an intimal process without substantial wall remodeling

Lesion pattern

Diffuse concentric fibroblast and smooth muscle proliferation in the intima. Lipid and foam cells less prominent Surface endothelial erosion is rare, but intimal inflammation may be seen

Focal eccentric proliferative, and degenerative lesions with lipid accumulation Surface endothelial erosion with adherent fibrin is common

Temporal evolution

Fatty streaks are seen initially Slow progression of lesion development (decades) Complicated plaques (soft necrotic core with thin fibrous cap) lead to rupture and “atherothrombosis”

Intimal inflammation may be seen initially Accelerated development (months to years) Fibrotic plaques tend to be more stable as they remodel, though necrotic plaque may be seen

Adapted from Rahmani et al. [106]

the allograft. For these reasons, biopsies are typically avoided during the first week post transplant. Ischemic changes may also be seen later in the post transplant period and may signal significant CAV. These changes can include typical acute ischemic injury, but also chronic subendocardial ischemia with prominent myocyte vacuolization (Fig. 9.23). Serum troponin studies and/or angiography may be recommended when this finding is seen in later biopsies.

Fig.  9.22  Cardiac allograft vasculopathy. Photomicrograph from an epicardial coronary artery involved by CAV. There is concentric intimal fibrosis without typical atheroma formation (H&E, × 40)

inadequate cardioplegia, or reperfusion injury [8, 131]. This typically manifests as myocyte injury, homogenization and hypereosinophilia of the sarcoplasm, fraying of the myocyte borders, interstitial edema and occasionally neutrophils and eosinophils (as discussed in Sect. 9.3.3). Nuclear pyknosis and karyorrhexis can rarely be seen, and coagulative necrosis is distinctly unusual and would indicate significant compromise of

Fig. 9.23  Chronic subendocardial ischemia. Photomicrograph showing subendocardial vacuolization and myocytolysis in a patient with chronic myocardial ischemia due to coronary obstruction (H&E, × 200)

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9.8 Complications of Immunosuppression 9.8.1 Infection Though fortunately uncommon, donor hearts can be a source of infection transmitted during the transplant procedure with significant consequences in the immunosuppressed host. Such infections have been reported for Toxoplasmosis [37] (Fig. 9.24), methicillin resistant

Staphylococcus aureus and Listeria monocytogenes (both causing pericarditis), and Parvovirus [90]. Infection is one of the primary consequences of immunosuppression and a common cause of death in the first 10 years after transplant [129]. Bacterial infections are the most common, followed by viruses (primarily CMV and EBV) [67, 90, 95]. Fungal infections, Pneumocystis jirovecii, and protozoan (primarily Toxo­ plasmosis) infections also occur [95]. Pneumonia and sepsis are the most common presentation. Immuno­ suppression also potentiates latent viral infections such as HPV Patients should be monitored for both urogenital and oropharyngeal precancerous lesions.

9.8.2 Lymphoproliferative Disorder

Fig. 9.24  Recurrent Toxoplasmosis. Photomicrograph from an EMB showing a parasitophorous vacuole containing Toxoplasma tachyzoites within an infect cardiomyocyte (arrow) (H&E, × 400). Myocyte necrosis is seen in adjacent cells (courtesy Helen Liapis, Washington University)

a

Fig. 9.25  Post transplant lymphoproliferative disorder. Photo­ micrograph from an EMB showing a polymorphous infiltrate resembling cellular rejection, but including eosinophils and

Post transplant lymphoproliferative disorder (PTLD) encompasses a spectrum from reactive and frankly neoplastic proliferation of hematolymphoid cells, with up to 80% being associated with Epstein–Barr virus infection. The majority is B-cell type, but T-cell and Hodgkin phenotypes also occur [62]. PLTD is the third leading cause of death for heart transplant recipients [129] and occurs in 5–15% of patients [48, 50, 99]. PTLD may involve lymph nodes, but 50% of cases arise in extranodal sites, with the lung and gastrointestinal tract being the most common. Primary involvement of the cardiac allograft is distinctly uncommon [99]. This can present a particular challenge as a mimic of cellular rejection (Fig.  9.25). The presence of plasma cells, large lymphocytes, and

b

plasma cells (a) (H&E, ×400). In-situ hybridization staining for EBV is positive (b) (×400)

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pleomorphic cells can be important clues and the detection of EBV (by immunohistochemistry or in-situ hybridization) in these cells is confirmatory.

9.8.3 Solid Organ Neoplasms Squamous cell carcinomas of skin and mucosa, as a consequence of sun damage or HPV infection, are the most common malignant neoplasms in the transplant setting [99] but malignancies occur with increased frequency in other organs as well, most notably the lungs where carcinomas often present at an advanced stage. [33, 101]. Whether this reflects impaired immune surveillance or significant smoking history in transplant patients with ischemic heart disease is unclear. Breast cancer does not appear to occur with increased frequency [125], though there is a reported link between cyclosporine and multiple bilateral fibroadenomas [119].

9.8.4 Toxicity of Immunosuppressants Though the specific cardiac toxicities of these agents are obviously outweighed by the benefits of avoiding rejection, nearly all transplant patients have untoward effects of these medications within 10 years of transplantation including hyperlipidemia (93%), hypertension, osteoporosis, diabetes (37%), and renal insufficiency (14%) [129]. Cyclosporine and its derivatives can induce

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hypertension, which can lead to left ventricular hypertrophy and stiffening. Cyclosporine also has significant microvascular effects leading to renal toxicity, further detailed in Chap. 6. Sirolimus is thought to be less nephrotoxic, but is associated with development of proteinuria [109]. Mycophenolate mofetil is associated with significant gastrointestinal side effects [100] though newer enteric-coated preparations may reduce these.

9.9 Molecular Assessment of Rejection Extensive research efforts have been expended to devise and validate a noninvasive means of monitoring for rejection. In the CARGO (Cardiac Allograft Rejection Gene expression Observational) study, gene expression microarrays were employed to identify a molecular signature (or specific combination of upregulated and downregulated mRNA transcripts) detectable in peripheral blood from patients with rejection. A select set of 11 transcripts was identified including a number of cytokines, T-cell signaling molecules, and corticosteroid responsive proteins that were most informative in distinguishing patients with rejection from those without. This test received FDA approval as a “rule out” test with a high negative predictive value, but biopsies are still needed to assess for rejection if the gene expression signature is not “negative.” It is important to note that only cellular rejection was studied in determining the gene signature and this test has not been validated for antibody mediated rejection.

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Appendix 1 Gross Pathology Record for Cardiac Explants NATIVE DISEASE:

Case #

INTERVENTIONS: (VAD – type, AICD, ablation, bypass grafts, stenting, prior surgeries) GENERAL Heart weight (g): (before / after fixation) Expected (mean): Expected (range): Measurements (cm) LV anterior: LV septum: RV anterior:

LV inferior: LV lateral: RV inferior:

Pericardium: Foramen ovale:

to

Aortic: Mitral: Pulmonary: Tricuspid:

fused / patent (potential diameter:

cm)

VALVE ABNORMALITIES: Aortic: Mitral: Pulmonary: Tricuspid:

(vegetations, fibrosis, calcification, prostheses, etc.)

CHAMBERS: Hypertrophy

Dilatation

Fibrosis

Other

Left Ventricle Right Ventricle Left Atrium Right Atrium INFARCTIONS: 1. Location: from: 2.

Location:

3.

Location:

from: from:

transmural / subendocardial base / midventricle to: mid / apex transmural / subendocardial base / midventricle to: mid / apex transmural / subendocardial base / midventricle to: mid / apex

Chamber remodeling (dilatation): Papillary muscles: CORONARY ARTERIES: Calcification: Dominance:

mild / mod. / severe right / left / shared

focal / multifocal / diffuse coronary ostia: _________________

LMA:

LCX:

LAD:

RCA:

Stents: Bypass Grafting: target vessel 1. 2. 3.

graft type

graft stenosis

anastamosis

distal stenosis

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343   97. Orens, J.B., Shearon, T.H., Freudenberger, R.S., Conte, J.V., Bhorade, S.M., Ardehali, A.: Thoracic organ transplantation in the United States, 1995–2004. Am. J. Transplant. 6(5 Pt 2), 1188–1197 (2006)   98. Pahl, E., Naftel, D.C., Canter, C.E., Frazier, E.A., Kirklin, J.K., Morrow, W.R.: Death after rejection with severe hemodynamic compromise in pediatric heart transplant recipients: a multi-institutional study. J. Heart Lung Transplant. 20(3), 279–287 (2001)   99. Palma, J.H., Guilhen, J.C., Gaia, D.F., Teles, A., Teles, C.A., Branco, J.N., Buffolo, E.: Post-transplant lymphoproliferative disease presenting as a mass in the left ventricle in a heart transplant recipient at long-term follow-up. J. Heart Lung Transplant. 28(2), 206–208 (2009) 100. Parfitt, J.R., Jayakumar, S., Driman, D.K.: Mycophenolate mofetil-related gastrointestinal mucosal injury: variable injury patterns, including graft-versus-host disease-like changes. Am. J. Surg. Pathol. 32(9), 1367–1372 (2008) 101. Pham, S.M., Kormos, R.L., Landreneau, R.J., Kawai, A., Gonzalez-Cancel, I., Hardesty, R.L., Hattler, B.G., Griffith, B.P.: Solid tumors after heart transplantation: lethality of lung cancer. Ann. Thorac. Surg. 60(6), 1623–1626 (1995) 102. Pierre-Louis, B., Kumar, A., Frishman, W.H.: Fabry disease: cardiac manifestations and therapeutic options. Cardiol. Rev. 17(1), 31–35 (2009) 103. Pilato, E., Dell’Amore, A., Botta, L., Arpesella, G.: Combined heart and liver transplantation for familial amyloidotic neuropathy. Eur. J. Cardiothorac. Surg. 32(1), 180– 182 (2007) 104. Platt, J.L., Fischel, R.J., Matas, A.J., Reif, S.A., Bolman, R.M., Bach, F.H.: Immunopathology of hyperacute xenograft rejection in a swine-to-primate model. Transplantation 52(2), 214–220 (1991) 105. Radovancevic, B., McGiffin, D.C., Kobashigawa, J.A., Cintron, G.B., Mullen, G.M., Pitts, D.E., O’Donnell, J., Thomas, C., Bourge, R.C., Naftel, D.C.: Retransplantation in 7,290 primary transplant patients: a 10-year multi-institutional study. J. Heart Lung Transplant. 22(8), 862–868 (2003) 106. Rahmani, M., Cruz, R.P., Granville, D.J., McManus, B.M.: Allograft vasculopathy versus atherosclerosis. Circ. Res. 99(8), 801–815 (2006) 107. Raichlin, E., Bae, J.H., Khalpey, Z., Edwards, B.S., Kremers, W.K., Clavell, A.L., Rodeheffer, R.J., Frantz, R.P., Rihal, C., Lerman, A., Kushwaha, S.S.: Conversion to sirolimus as primary immunosuppression attenuates the progression of allograft vasculopathy after cardiac transplantation. Circulation 116(23), 2726–2733 (2007) 108. Raichlin, E., Edwards, B.S., Kremers, W.K., Clavell, A.L., Rodeheffer, R.J., Frantz, R.P., Pereira, N.L., Daly, R.C., McGregor, C.G., Lerman, A., Kushwaha, S.S.: Acute cellular rejection and the subsequent development of allograft vasculopathy after cardiac transplantation. J. Heart Lung Transplant. 28(4), 320–327 (2009) 109. Raichlin, E., Khalpey, Z., Kremers, W., Frantz, R.P., Rodeheffer, R.J., Clavell, A.L., Edwards, B.S., Kushwaha, S.S.: Replacement of calcineurin-inhibitors with sirolimus as primary immunosuppression in stable cardiac transplant recipients. Transplantation 84(4), 467–474 (2007) 110. Reed, E.F., Demetris, A.J., Hammond, E., Itescu, S., Kobashigawa, J.A., Reinsmoen, N.L., Rodriguez, E.R.,

344 Rose, M., Stewart, S., Suciu-Foca, N., Zeevi, A., Fishbein, M.C.: Acute antibody-mediated rejection of cardiac transplants. J. Heart Lung Transplant. 25(2), 153–159 (2006) 111. Rippstein, P., Black, M.K., Boivin, M., Veinot, J.P., Ma, X., Chen, Y.X., Human, P., Zilla, P., O’Brien, E.R.: Comparison of processing and sectioning methodologies for arteries containing metallic stents. J. Histochem. Cytochem. 54(6), 673–681 (2006) 112. Rodriguez, E.R., Skojec, D.V., Tan, C.D., Zachary, A.A., Kasper, E.K., Conte, J.V., Baldwin 3rd, W.M.: Antibodymediated rejection in human cardiac allografts: evaluation of immunoglobulins and complement activation products C4d and C3d as markers. Am. J. Transplant. 5(11), 2778– 2785 (2005) 113. Rose, A.U.: Pathology of acute rejection. In: Cooper, D.L. (ed.) Heart Transplant, pp. 157–176. MTP, Lancaster (1984) 114. Rose, A.G.: Understanding the pathogenesis and the pathology of hyperacute cardiac rejection. Cardiovasc. Pathol. 11(3), 171–176 (2002) 115. Rose, A.G., Cooper, D.K.: A histopathologic grading system of hyperacute (humoral, antibody-mediated) cardiac xenograft and allograft rejection. J. Heart Lung Transplant. 15(8), 804–817 (1996) 116. Rose, A.G., Cooper, D.K., Human, P.A., Reichenspurner, H., Reichart, B.: Histopathology of hyperacute rejection of the heart: experimental and clinical observations in allografts and xenografts. J. Heart Lung Transplant. 10(2), 223–234 (1991) 117. Santise, G., D’Ancona, G., Falletta, C., Pirone, F., Sciacca, S., Turrisi, M., Biondo, D., Pilato, M.: Donor pharmacological hemodynamic support is associated with primary graft failure in human heart transplantation. Interact. Cardiovasc. Thorac. Surg. 9(3), 476–479 (2009) 118. Sattar, H.A., Husain, A.N., Kim, A.Y., Krausz, T.: The presence of a CD21+ follicular dendritic cell network distinguishes invasive Quilty lesions from cardiac acute cellular rejection. Am. J. Surg. Pathol. 30(8), 1008–1013 (2006) 119. Seo, Y.L., Choi, C.S., Yoon, D.Y., Yun, E.J., Lee, Y.J., Park, S.J., Moon, J.H., Cho, S.J., Lee, S., Han, H., Kim, S.S., Lee, J.Y.: Benign breast diseases associated with cyclosporine therapy in renal transplant recipients. Transplant. Proc. 37(10), 4315–4319 (2005) 120. Shimizu, A., Colvin, R.B.: Pathological features of antibody-mediated rejection. Curr. Drug Targets Cardiovasc. Haematol. Disord. 5(3), 199–214 (2005) 121. Shimizu, A., Hisashi, Y., Kuwaki, K., Tseng, Y.L., Dor, F.J., Houser, S.L., Robson, S.C., Schuurman, H.J., Cooper, D.K., Sachs, D.H., Yamada, K., Colvin, R.B.: Thrombotic microangiopathy associated with humoral rejection of cardiac xenografts from alpha1, 3-galactosyltransferase geneknockout pigs in baboons. Am. J. Pathol. 172(6), 1471–1481 (2008) 122. Smith, J.D., Hamour, I.M., Banner, N.R., Rose, M.L.: C4d fixing, luminex binding antibodies - a new tool for prediction of graft failure after heart transplantation. Am. J. Transplant. 7(12), 2809–2815 (2007) 123. Stahl, R.D., Karwande, S.V., Olsen, S.L., Taylor, D.O., Hawkins, J.A., Renlund, D.G.: Tricuspid valve dysfunction in the transplanted heart. Ann. Thorac. Surg. 59(2), 477– 480 (1995)

D.V. Miller et al. 124. Stehlik, J., Starling, R.C., Movsesian, M.A., Fang, J.C., Brown, R.N., Hess, M.L., Lewis, N.P., Kirklin, J.K.: Utility of long-term surveillance endomyocardial biopsy: a multiinstitutional analysis. J. Heart Lung Transplant. 25(12), 1402–1409 (2006) 125. Stewart, T., Tsai, S.C., Grayson, H., Henderson, R., Opelz, G.: Incidence of de-novo breast cancer in women chronically immunosuppressed after organ transplantation. Lancet 346(8978), 796–798 (1995) 126. Stewart, S., Winters, G.L., Fishbein, M.C., Tazelaar, H.D., Kobashigawa, J., Abrams, J., Andersen, C.B., Angelini, A., Berry, G.J., Burke, M.M., Demetris, A.J., Hammond, E., Itescu, S., Marboe, C.C., McManus, B., Reed, E.F., Reinsmoen, N.L., Rodriguez, E.R., Rose, A.G., Rose, M., Suciu-Focia, N., Zeevi, A., Billingham, M.E.: Revision of the 1990 working formulation for the standardization of nomenclature in the diagnosis of heart rejection. J. Heart Lung Transplant. 24(11), 1710–1720 (2005) 127. Tan, C.D., Baldwin 3rd, W.M., Rodriguez, E.R.: Update on cardiac transplantation pathology. Arch. Pathol. Lab. Med. 131(8), 1169–1191 (2007) 128. Tan, C.D., Sokos, G.G., Pidwell, D.J., Smedira, N.G., Gonzalez-Stawinski, G.V., Taylor, D.O., Starling, R.C., Rodriguez, E.R.: Correlation of donor-specific antibodies, complement and its regulators with graft dysfunction in cardiac antibody-mediated rejection. Am. J. Transplant. 9(9), 2075–2084 (2009) 129. Taylor, D.O., Edwards, L.B., Boucek, M.M., Trulock, E.P., Aurora, P., Christie, J., Dobbels, F., Rahmel, A.O., Keck, B.M., Hertz, M.I.: Registry of the International Society for Heart and Lung Transplantation: twenty-fourth official adult heart transplant report–2007. J. Heart Lung Transplant. 26(8), 769–781 (2007) 130. Taylor, D.O., Yowell, R.L., Kfoury, A.G., Hammond, E.H., Renlund, D.G.: Allograft coronary artery disease: clinical correlations with circulating anti-HLA antibodies and the immunohistopathologic pattern of vascular rejection. J. Heart Lung Transplant. 19(6), 518–521 (2000) 131. Tazelaar, H.D.: Spectrum and diagnosis of myocardial rejection. Cardiol. Clin. 8(1), 119–139 (1990) 132. Tazelaar, H.D., Edwards, W.D.: Pathology of cardiac transplantation: recipient hearts (chronic heart failure) and donor hearts (acute and chronic rejection). Mayo Clin. Proc. 67(7), 685–696 (1992) 133. Uber, W.E., Self, S.E., Van Bakel, A.B., Pereira, N.L.: Acute antibody-mediated rejection following heart transplantation. Am. J. Transplant. 7(9), 2064–2074 (2007) 134. Uehara, S., Chase, C.M., Cornell, L.D., Madsen, J.C., Russell, P.S., Colvin, R.B.: Chronic cardiac transplant arteriopathy in mice: relationship of alloantibody, C4d deposition and neointimal fibrosis. Am. J. Transplant. 7(1), 57–65 (2007) 135. Valantine, H.A.: The role of viruses in cardiac allograft vasculopathy. Am. J. Transplant. 4(2), 169–177 (2004) 136. Veinot, J.P., Ascah, K., Davies, R.A., Smith, S.: Coronary artery-posterior interventricular vein fistula after endomyocardial biopsy in a heart transplant patient. Can. J. Cardiol. 18(2), 193–194 (2002) 137. Waller, B.F., Taliercio, C.P., Slack, J.D., Orr, C.M., Howard, J., Smith, M.L.: Tomographic views of normal and abnormal hearts: the anatomic basis for various cardiac imaging techniques. Part I. Clin. Cardiol. 13(11), 804–812 (1990)

9  Heart 138. Waller, B.F., Taliercio, C.P., Slack, J.D., Orr, C.M., Howard, J., Smith, M.L.: Tomographic views of normal and abnormal hearts: the anatomic basis for various cardiac imaging techniques. Part II. Clin. Cardiol. 13(12), 877–884 (1990) 139. Wang, S.S.: Treatment and prophylaxis of cardiac allograft vasculopathy. Transplant. Proc. 40(8), 2609–2610 (2008) 140. Warraich, R.S., Pomerance, A., Stanley, A., Banner, N.R., Dunn, M.J., Yacoub, M.H.: Cardiac myosin autoantibodies and acute rejection after heart transplantation in patients with dilated cardiomyopathy. Transplantation 69(8), 1609– 1617 (2000) 141. West, L.J., Karamlou, T., Dipchand, A.I., Pollock-BarZiv, S.M., Coles, J.G., McCrindle, B.W.: Impact on outcomes after listing and transplantation, of a strategy to accept ABO blood group-incompatible donor hearts for neonates and infants. J. Thorac. Cardiovasc. Surg. 131(2), 455–461 (2006) 142. West, L.J., Pollock-Barziv, S.M., Lee, K.J., Dipchand, A.I., Coles, J.G., Ruiz, P.: Graft accommodation in infant recipients of ABO-incompatible heart transplants: donor ABH antigen expression in graft biopsies. J. Heart Lung Transplant. 20(2), 222 (2001) 143. Williams, J.M., Holzknecht, Z.E., Plummer, T.B., Lin, S.S., Brunn, G.J., Platt, J.L.: Acute vascular rejection and accommodation: divergent outcomes of the humoral response to organ transplantation. Transplantation 78(10), 1471–1478 (2004) 144. Winters, G.L., Loh, E., Schoen, F.J.: Natural history of focal moderate cardiac allograft rejection. Is treatment warranted? Circulation 91(7), 1975–1980 (1995) 145. Winters, G.L., McManus, B.M.: Consistencies and controversies in the application of the International Society for

345 Heart and Lung Transplantation working formulation for heart transplant biopsy specimens. Rapamycin Cardiac Rejection Treatment Trial Pathologists. J. Heart Lung Transplant. 15(7), 728–735 (1996) 146. Wu, Y.W., Yen, R.F., Lee, C.M., Ho, Y.L., Wang, S.S., Hsu, R.B., Chou, N.K., Huang, P.J.: Usefulness of progressive inhomogeneity of myocardial perfusion and chronotropic incompetence in detecting cardiac allograft vasculopathy: evaluation with dobutamine thallium-201 myocardial SPECT. Cardiology 104(3), 156–161 (2005) 147. Yager, J.E., Hernandez, A.F., Steenbergen, C., Persing, B., Russell, S.D., Milano, C., Felker, G.M.: Recurrence of cardiac sarcoidosis in a heart transplant recipient. J. Heart Lung Transplant. 24(11), 1988–1990 (2005) 148. Yeoh, T.K., Frist, W.H., Eastburn, T.E., Atkinson, J.: Clinical significance of mild rejection of the cardiac allograft. Circulation 86(5 Suppl), II267–II271 (1992) 149. Yeung, A.C., Davis, S.F., Hauptman, P.J., Kobashigawa, J.A., Miller, L.W., Valantine, H.A., Ventura, H.O., Wiedermann, J., Wilensky, R.: Incidence and progression of transplant coronary artery disease over 1 year: results of a multicenter trial with use of intravascular ultrasound. Multicenter Intravascular Ultrasound Transplant Study Group. J. Heart Lung Transplant. 14(6 Pt 2), S215–S220 (1995) 150. Zerbe, T.R., Arena, V.: Diagnostic reliability of endomyocardial biopsy for assessment of cardiac allograft rejection. Hum. Pathol. 19(11), 1307–1314 (1988) 151. Zhang, Y., Zhang, X.D., Ma, L.L., Guan, D.L.: Relationship between platelet activation and acute rejection after renal transplantation. Transplant. Proc. 41(5), 1547–1551 (2009)

10

Small Intestine Frances V. White and Sarangarajan Ranganathan

Abbreviations ACR Acute cellular rejection AMR Antibody mediated rejection CR Chronic rejection CMV Cytomegalovirus DSA Donor specific antibodies EBV Epstein–Barr virus EBER Epstein–Barr virus-encoded RNA HPF High power field ITx Isolated intestinal transplant SGS Short gut syndrome LD Living donor L-ITx Liver-intestinal transplant MVTx Multivisceral transplant PRA Panel reactive antibodies PN Parenteral nutrition PTLD Posttransplant lymphoproliferative disease SCR Subclinical rejection

10.1 Overview Among all solid organ allografts, the small intestine stands out for its abundant lymphoid tissue, mucosal innate and adaptive immunity, and commensal bacteria. These factors have challenged transplant clinicians and

F.V. White (*) Department of Pathology and Immunology, Washington University School of Medicine, 660 S. Euclid, Campus Box 8118, St. Louis, MO 63110, USA e-mail: [email protected] S. Ranganathan Department of Pathology, Children’s Hospital of Pittsburgh of UPMC, Children’s Hospital Drive, 45th and Penn, Pittsburgh, PA 15201, USA

are reflected in a historical lag of progress in intestinal transplantation compared to other organ transplants [71, 112]. Small intestinal transplantation was first attempted in humans in the 1960s; however, success was limited until the use of cyclosporine for immunosuppression in the early 1980s. With the introduction of tacrolimus in 1990, intestinal transplantation became a reasonable treatment option for patients with irreversible short gut syndrome (SGS) and severe complications of parenteral nutrition. Immunosuppression with tacrolimus and steroids resulted in acceptable short-term patient and graft survival, but with high mortality and graft loss due to infection, acute rejection and posttransplant lymphoproliferative disease (PTLD). Only in the past decade, with the introduction of induction protocols based on antilymphocyte antibodies, have clinicians been able to achieve 1 year graft and patient survival rates comparable to other organ allografts. With an increased understanding of immune pathology of the intestine, there is now an emphasis on protocols to increase long-term graft survival, including recipient pretreatment and minimization of maintenance immunosuppression [1, 5, 59, 73]. Improved graft and patient survival also reflects increasing experience among large transplant centers. New surgical techniques include variations on vascular anastomoses, modifications in multivisceral transplantation, and allograft abdominal wall closure. Improved infectious disease protocols with viral monitoring by polymerase chain reaction (PCR), prophylaxis and new antimicrobials have reduced viral and fungal infections and PTLD. Early referral of SGS patients to intestinal rehabilitation centers, close monitoring, and a multidisciplinary team approach are essential. Medical, surgical and nutritional rehabilitation may facilitate intestinal adaption and reduce the need for parenteral nutrition (PN) and transplantation. The timing of referral for transplantation has

H. Liapis and H.L. Wang (eds.), Pathology of Solid Organ Transplantation, DOI: 10.1007/978-3-540-79343-4_10, © Springer-Verlag Berlin Heidelberg 2011

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been controversial; however studies indicate that a more timely consideration for intestinal transplantation results in better posttransplant outcomes. Referral of patients for intestinal transplantation before there is irreversible PN-associated liver damage decreases the need for a combined liver-intestine allograft and results in better patient survival. Patient survival is also higher in nonhospitalized patients, compared to hospitalized patients [9, 37, 74]. The Intestinal Transplant Registry (ITR) and the Organ Procurement and Transplantation Network (OPTN) maintain data on worldwide and United States intestinal transplants, respectively [42, 67]. Data from the ITR, presented at the tenth annual meeting of the International Small Bowel Transplant Association, was recently reviewed [112]. Between April 1985 and May 2007, there were 1,720 documented transplants, including 746 isolated small intestine transplants (ITx), 594 combined liver and small intestine transplants (L-ITx), and 380 multivisceral/modified multivisceral transplants (MVTx). As of May 2007, there were 909 survivors, including one patient 18 years posttransplant. Age at transplantation has ranged from less than 2 months to over 65 years of age, with approximately 60% of patients under 18 years of age. In the United States, more than 10% of transplants have been performed in infants [67]. Worldwide, more than 80% of survivors have been able to stop PN [37, 112]. Short-term graft and patient survival has increased significantly over the past decade. In the period from 2005 to 2007, patient and graft 1 year survivals for ITx were 90 and 80%, respectively. One year survival rates for L-ITx and MVTx were lower (patient and graft survival both at approximately 70%), thought to reflect the poorer health status of these patients and increased surgical procedure complexity. Decreased patient survival is seen in patients hospitalized at the time of transplantation and in infants [42, 50, 57, 112]. With the implementation of new induction protocols and immunomodulatory procedures in selected populations, even better short-term survival rates have been reported. At one pediatric center using perioperative lymphoid depletion and tacrolimus monotherapy, 2 year patient and graft survivals were 100 and 94%, respectively [79]. Long term patient and graft survival, however, is still low [67, 112]. In the United States, based on data from 1997 to 2006, 5 and 10 year patient survival for ITx was only 54 and 43%, respectively. In addition, graft survival at 5 and 10 years posttransplant was only 37 and 23%, respectively. Similar survival rates were

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reported for combined L-ITx [67]. Newer protocols to improve long term survival include preconditioning regimens with the goal of minimizing maintenance immunosuppression, in order to decrease episodes of infection and lower drug complications such as nephrotoxicity. Using these strategies, one center has recently reported 5 year patient and graft survival rates of 75 and 61%, respectively [1].

10.2 Indications for Small Intestinal Transplantation Intestinal transplantation should be considered for patients with irreversible SGS or functional intestinal failure and severe complications of PN. Short gut syndrome is defined by an inability of the intestine to provide adequate nutrition, secondary to previous extensive resection of small intestine. Underlying etiologies include primary intestinal disease, ischemia, trauma or mesenteric disease [20]. The primary standard of care for patients with SGS is PN and intestinal rehabilitation, with the goal of eventually weaning the patient from PN. Rehabilitation utilizes both nutritional and medical treatments, and may also involve nontransplant surgical procedures to increase intestinal absorptive area, such as repeat serial transverse enteroplasty [7]. However, in patients that fail intestinal adaption and remain on PN, severe complications can occur. These complications, which are considered indications for intestinal transplantation, include liver failure, recurrent line sepsis, loss of vascular access, and fluid/electrolyte imbalances. Neurologic complications and failure to thrive may also occur. In the past, many pediatric short gut patients were considered for intestinal transplantation only after irreversible liver failure, necessitating liver transplantation along with intestine. Studies indicate, however, increased survival in patients considered for intestinal transplant before the occurrence of irreversible liver failure [9, 37, 74]. The etiologies of SGS and functional intestinal failure in intestinal transplant candidates are diverse (Table 10.1). In children, the most frequent indications for transplantation include gastroschisis, volvulus, necrotizing enterocolitis, and aganglionosis. Other indications include pseudo-obstruction, intestinal atresia, tufting enteropathy and microvillous inclusion disease. In adults, the most common indications are ischemia,

10  Small Intestine Table 10.1  Indications for intestinal transplantation Short gut syndrome Volvulus Mesenteric thrombosis Gastroschisis Necrotizing enterocolitis Crohn disease Intestinal atresia Trauma Familial adenomatous polyposis Dysmotility Aganglionosis Chronic intestinal pseudo-obstruction Poor absorption Microvillous inclusion disease Tufting enteropathy Tumor Desmoid Low grade neuroendocrine tumor Hemangioma Allograft failure

Crohn’s disease, dysmotility, trauma and volvulus [112]. Intestinal transplantation has been used successfully for patients with mesenteric desmoid tumors. It has also been used for localized malignant tumors, but with limited success due to tumor recurrence and metastasis [61, 106]. Unusual indications for transplantation include radiation enteritis, necrotizing enterovasculitis (Churg Strauss), cystic fibrosis with meconium ileus, and multiple intestinal atresia with immunodeficiency [27, 31, 88]. Retransplantation following severe acute rejection and chronic rejection (CR) is another indication in both children and adults [37, 59].

10.3 Contraindications for Transplantation and Donor Selection Contraindications for intestinal transplantation are similar to those for other solid organ transplants. Contraindications include severe cardiopulmonary disease, multiorgan system failure, neurologic impairment and systemic infection. Noncompliance and substance abuse are also contraindications [13, 112]. Donors should be hemodynamically stable and without evidence of intestinal ischemia. In the presence of ischemia, resulting inflammatory mediators may

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potentiate intestinal dysmotility, reperfusion injury, bacterial translocation and acute rejection [34, 86, 87]. Donor size is important for intestinal transplantation, as recipients often have a relatively small abdominal cavity and wall for closure, due to previous surgeries and scarring. Optimal donor size is 50–75% of recipient size [34, 112]. Large grafts can be reduced on the backbench during transplantation, however, manipulation increases inflammatory mediators that potentiate intestinal injury [107]. The selection of an ABO identical donor is preferred, however, ABO compatible grafts have been used when identical donors were not available [34, 112]. The role of HLA cross-matching is controversial. A negative cross-match is preferred for isolated intestinal transplants (ITx), while it is considered less essential for combined organ transplants [34]. At many institutes, pretransplant sera are obtained for cross-match tests, but results are often not available until after transplantation. Following transplantation, patients are monitored for panel reactive antibodies (PRA). Plasmapheresis and desensitization protocols with immunoglobulins are available for patients with preformed lymphocytotoxic antibodies [33]. In general, CMV-negative recipients should receive CMV-negative intestinal grafts [13, 34, 112]. However, recent advances in viral monitoring, prophylaxis and treatment have significantly decreased morbidity from CMV infection. Although donors with negative CMV serologies are preferred, grafts from CMV positive donors are used when negative donors are not available [34, 112].

10.4 Types of Intestinal Transplantation The small intestine can be transplanted in isolation or with other organs (Table 10.2). The type of organ transplant depends upon multiple factors, including underlying disease, previous surgeries and residual abdominal domain, donor size, and center expertise and preference. The three major types of intestinal transplantation include ITx, combined liver-intestinal transplant (L-ITx) and MVTx. Based on recent data from the ITR, ITx, L-ITx and MVTx represented 36, 35, and 29% of pediatric intestinal transplants, respectively. In adults, ITx, L-ITx and MVTx represented 50, 11, and 39% of intestinal transplants, respectively [112].

350 Table 10.2  Types of small intestinal transplant Isolated small intestine (with or without colon) Small intestine – liver (with or without colon) Multivisceral: stomach, pancreaticoduodenal complex and small intestine (with or without liver, kidneys, colon, abdominal wall)

Cadaveric ITx involves transplantation of the entire jejunum through terminal ileum, with or without the ileocecal valve and colon. The rationale for including colon is to increase transit time through graft and to increase water absorption resulting in well-formed stools. Studies vary as to whether there is increased survival with the ileocecal valve and colon. Early reports suggested that colon transplantation increased bacterial translocation and increased risk of infection and graft loss [16, 103]. More recent studies, however, indicate that the colon does not decrease patient or graft survival and may actually have a protective effect [36, 49, 85]. At one large center, comparison of intestinal allografts with and without colon showed no significant impact on overall or specific cause graft survival, or on hazard rates of death from infection. In addition, there were better formed stools. In this study, acute rejection in the colon occurred in 23% of patients, but was always associated with simultaneous small intestinal acute rejection [49]. Combined L-ITx is performed in patients who also have irreversible liver failure, usually from long-term PN. In the United States, approximately 65% of pediatric patients on the intestinal transplant wait list also require a liver [28, 112]. In simultaneous transplantation, the liver and small intestine are transplanted en bloc or separately during the same procedure. The pancreas is also included with en bloc transplantation of intestine and liver. In sequential organ transplantation, the liver is transplanted first. For young children, sequential transplantation provides more time for potential adaption of the short gut [63]. Although controversial, some studies indicate an immunoprotective effect of the liver for intestinal transplantation [1, 44]. MVTx typically includes small intestine, liver, pancreas, and stomach. In modified MVTx, the liver is excluded. Other organs may also be transplanted, including spleen, colon, and kidney. Indications for MVTx include catastrophic abdominal trauma, extensive dysmotility of GI tract including long segment Hirschsprung’s disease involving the stomach, and extensive mesenteric thrombosis or tumor [56, 61]. At

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some centers, MVTx is preferred in small patients for technical reasons [28]. MVTx has an immunological advantage compared to ITx and L-ITx [50, 71, 89, 112]. Patients with MVTx have improved patient and graft survival, with decreased incidence of overall rejection and severe rejection compared to ITx and L-ITx patients [109]. In a study of MVTx examined at resection and autopsy, acute rejection was most severe in the small intestine, and acute rejection did not occur in the other organs without concurrent small intestinal rejection. This study suggests that the intestine can be used as a surrogate marker for acute rejection in the other transplanted organs [97]. New surgical techniques include the use of donor abdominal wall for better closure in MVTx patients and retention of native spleen, pancreas and duodenum. The retention of native spleen and pancreas may decrease episodes of infection, PTLD, and pancreatic insufficiency [2]. Living donor (LD) ITx and L-ITX have been performed in a small number of patients [8, 11, 30, 55, 67, 75, 88, 99–101, 112]. The major indication for LD intestinal transplantation is an available identical twin or an HLA-identical sibling, in order to avoid or minimize immunosuppressive treatment and decrease risk of rejection and infection. However, living related donors with partial HLA match and even full mismatch have been used [11]. In addition to optimal HLA matching, advantages include elimination of a waiting list, optimal timing as an elective procedure, and decreasing allograft cold ischemic time to minutes. LDs are carefully screened and significant donor morbidity has not been reported. In adult recipients, a 200-cm segment of intestine is transplanted, excluding the last 20  cm of terminal ileum. A shorter segment (150–180  cm) is used for children [11, 101]. Patient and graft survival are comparable to those of cadaveric transplants at the best transplant centers [30, 37]. In addition to isolated intestinal allografts, pediatric patients have undergone successful LD L-ITx, using both simultaneous and sequential organ transplantation [100].

10.5 Complications of Intestinal Transplantation Complications of intestinal transplantation are similar to that of other solid organ transplants, but with increased rates of infection, acute rejection, PTLD, graft loss and

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death. Increased morbidity and mortality reflect unique characteristics of the intestinal allograft, surgical complexity of transplantation including abdominal wall closure, and health of the patient with SGS. The large amount of intestinal associated lymphoid tissue, epithelial antigen-presenting cells, innate immune defense system and bacterial flora puts the patient at increased risk for acute rejection and infection. Requirements for immunosuppression are higher, with increased risk for infection and PTLD. The major early and late complication is sepsis [29, 38]. Chronic renal disease is common in intestinal transplantation candidates and nephrotoxicity is a major complication of tacrolimus, the maintenance immunosuppressant drug, especially in children [40, 92, 113]. Also, the patient with SGS often has advanced liver disease and poor nutrition, leading to increased risk for infection and delayed wound healing. Compared to other organ transplants, the wait list is longest for intestinal transplantation, reflecting a scarcity of appropriately sized allografts, especially for very young transplant candidates [26, 37, 60]. Death while on the waiting list is higher for intestinal transplant candidates than for other organ transplant candidates, with highest mortality in infants [9].

10.5.1 Surgical Complications The candidate for intestinal transplantation often has a reduced abdominal domain and insufficient abdominal wall for surgical closure, due to previous surgeries, dense adhesions, abdominal wall injury, and retention of native organs [34, 119]. Dense adhesions may result in technically difficult surgery and hemodynamic instability from blood loss. Abdominal closure with insufficient abdominal domain may be complicated by abdominal compartment syndrome, vascular thrombosis, wound dehiscence, intestinal wall necrosis, and respiratory complications [119]. Methods to improve abdominal closure include the use of nonbiological mesh, acellular dermal matrix, nonvascularized rectal fascia, and vascularized composite tissue allograft of abdominal wall [34]. Poor nutritional status delays wound and anastomotic healing, and predisposes to infection. Other surgical complications include gastrointestinal and vascular anastomotic leaks, mesh infection, abdominal

abscesses, intestinal fistula, intestinal dysmotility, and volvulus [65].

10.5.2 Preoperative, Implantation and Reperfusion Injury Injury to the intestine may occur before and during harvesting, during implantation, and with reperfusion, leading to graft motor dysfunction and mucosal damage, with increased risk for sepsis and acute rejection. Surgical manipulation of the intestine elicits an inflammatory response in the muscularis propria, with activation of resident macrophages, followed by recruitment of other inflammatory cell mediators, resulting in decreased muscle contractility [46, 107]. These recruited leukocytes remain in the muscularis for up to a week after transplant [107]. Resident macrophages are also activated by ischemia-reperfusion injury and bacterial products. Ischemia-reperfusion injury results in intestinal dysmotility, mucosal damage with impaired barrier function, and bacterial translocation, leading to peritonitis and sepsis. Mucosal injury can also potentiate acute rejection. Perioperative donor preconditioning and treatment of the recipient with glycine have been used to inhibit these inflammatory processes [86, 87, 107]. Mucosal changes during intestinal transplantation in humans were studied by Lee and colleagues [54] (Fig. 10.1). Histology of preimplantation mucosal biopsies ranged from focal separation of surface epithelium to extensive epithelial denudation, with underlying edematous lamina propria. An active inflammatory exudate was not noted. In postperfusion biopsies (0.5–6  h posttransplant), findings included capillary congestion, focal epithelial denudation, crypt epithelial regeneration with increased mitoses, and shortened villi with contracted smooth muscle bundles. In some cases, there was a mild neutrophilic infiltrate in lamina propria and surface epithelium and/or luminal exudate. Crypt apoptosis was only slightly increased. In uncomplicated cases, repeat mucosal biopsies were normal by 6–9 days. Animal models of intestinal ischemia-reperfusion injury have similar findings, but also show neutrophilic infiltrates in muscularis and mesentery [87]. In addition, in more severe cases, there can be hemorrhage and extensive epithelial denudation. Reperfusion injury results in loss of absorptive surface which correlates with increased enteral fluid loss. Following reperfusion

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Fig. 10.1  Reperfusion injury. An immediate day 0 biopsy has mucosal congestion, hemorrhage, loss of villi, and surface epithelial damage and denudation. There are regenerative changes and rare apoptotic cells in crypts

injury, there is a gradual return to normal villous height, which may take up to 10 weeks [10].

10.5.3 Antibody-Mediated Rejection In kidney and heart allografts, antibody mediated rejection (AMR) is well-documented and associated with a poor outcome. Diagnostic criteria for AMR in kidney and heart transplants include positive donor specific antibodies (DSA), characteristic histologic findings, and C4d deposition in allograft tissue [77]. In contrast, there are only limited studies on humoral rejection in human intestinal allografts, and the frequency and clinical significance of AMR is uncertain. A few published reports describe hyperacute and acute humoral rejection in patients with preformed antibodies, most frequently immunoglobulin G (IgG) lymphocytotoxic antibodies [54, 95, 117]. Protocols for patients with preformed DSA also vary among institutes, although known strong positive cross-matches are avoided. At some centers, results of pretransplant DSA serologies are routinely not available until after transplantation, and the patient is not treated unless symptomatic. High pretransplant PRA are found in up to 18–30% of patients and pretransplant plasmapheresis and desensitization protocols with IgG are available for highly sensitized recipients at some centers [33].

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The significance of the development of posttransplant DSA is also controversial. Some studies have shown an association of the development of HLA antibody with acute rejection episodes. In one study, the posttransplant development of strongly positive PRA, which occurred in the first 2 weeks after transplant, was associated with significant episodes of acute rejection at the time of positive PRA and refractory rejection with graft loss [48]. Animal models of hyperacute and acute humoral rejection demonstrate severe intestinal injury, with vascular thrombosis, hemorrhage, necrosis, acute inflammation and graft loss [104, 118]. Wu and colleagues [115] studied intestinal allograft pathology in a series of patients with preformed IgG lymphocytotoxic antibodies. Patients with a positive crossmatch had a characteristic clinicopathologic syndrome. Immediately postperfusion, there was intestinal allograft spasm, cyanotic discoloration and serosal petechial hemorrhages, which resolved within an hour. Patients with a strong positive cross-match developed severe mucosal injury, usually within 10 days. Endoscopy showed severe mucosal congestion and diffuse hemorrhage for up to 2 weeks posttransplant. Mucosal biopsies showed severe congestion, neutrophilic margination and fibrin platelet thrombi in microvasculature of lamina propria, and focal hemorrhage. Neutrophilic and necrotizing arteritis was not noted, however, only one biopsy (fullthickness) contained an artery. In this biopsy, there were intravascular thrombi and hemorrhagic infarction, without inflammation or necrosis of the sampled arterial wall. In the biopsies, there was no significant lamina propria inflammation, epithelial injury, or apoptosis. Immunofluorescence studies showed no specific capillary wall staining. The patients were successfully treated with OKT3, with resolution within 2 weeks posttransplant. At follow-up, there was no significant difference in overall graft survival or loss to CR compared to other allograft patients. In current practice, the diagnosis of AMR in intestinal allografts is based on criteria borrowed from kidney and heart allograft studies, although these criteria have not yet been published by consensus groups or completely validated. AMR is usually diagnosed during the first 2 weeks posttransplant, in the presence of circulating immunoglobulins (IgG). Clinical and endoscopic features strongly suggesting AMR include unexplained severe ischemic injury shortly after reperfusion, mucosal persistent, diffuse congestion and

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hemorrhage, and a strong positive cross-match for T or B cell lymphocytotoxic antibodies. On microscopic examination, the mucosa is characterized by dilated capillaries with marginating neutrophils and fibrin thrombi (Fig.  10.2). Immunohistochemical studies demonstrate the deposition of C4d in the endothelial cells of small capillaries and venules in a diffuse manner. Crypt damage with apoptosis may also be seen. The significance of mild mucosal vascular changes to AMR is not known. Ruiz and colleagues developed a semi-quantitative scoring system for mild vascular changes in the mucosa during the first 3 months posttransplant and correlated findings with peak reactive antibodies and graft loss [81]. Vascular changes were looked for in capillaries, small venules and arterial branches of the lamina propria and submucosa; these changes included vascular dilatation, congestion, and extravasated red blood cells. Scoring was based on the percentage of involved mucosa. The presence of a vasculitis was separately scored as: mild (rare vessels with adherent inflammatory cells), moderate (involvement of >50% of vessels) and severe (transmural inflammation, necrosis and fibrin deposition). Significant vasculitis was not seen in the biopsies, however, transient vascular changes were seen in the majority of patients within the first month, with a peak 10 days posttransplant. The vascular changes correlated with peak PRA, higher incidence of positive T-cell and B-cell cross-match, and shorter graft survival. There was no association of the vascular changes with ACR. The authors hypothesized that the vascular alterations may be a form of AMR. The

vascular changes, however, can also be seen in ischemiareperfusion injury, ACR, infection and drug reaction. In a study from another institute using the above scoring system, all patients had at least one biopsy with vascular change, none of which were severe. The vascular changes were associated with a higher incidence of ACR, without definite clinical impact [15]. Although C4d is used as a marker for AMR, studies have not yet validated its use in intestinal allografts. In one study, C4d immunostaining on paraffin embedded tissue resulted in nonspecific staining of small vessels in native intestine, normal allografts and allografts with ACR [105]. In this study, however, the number of biopsies was small, staining of native intestine was variable, and differences in intensity of staining were not described. In another small study, vascular lesions (using the above scoring system), C4d deposition (using indirect immunofluorescence on frozen tissue) and DSA were evaluated in 12 intestinal transplants [19]. C4d deposition of capillaries and venules was positive in 37% of biopsies with or without ACR, and in 50% of biopsies with severe vascular changes. In normal intestine, C4d was positive in small arterial branches, but was negative in capillaries and venules. Vascular lesions were always associated with acute rejection and poor outcome, and grade 3 lesions were associated with bowel loss. A humoral component, however, was not conclusively found and there was no clinical significance of C4d as far as clinical outcome was concerned.

Fig. 10.2  Antibody mediated rejection. (a) A mucosal biopsy contains edema, dilated capillary channels with endothelial activation and lamina propria mild neutrophilic infiltrate. (b) There

is C4d immunostaining of small vessels in lamina propria (patient with acute rejection with elevated donor specific antibodies)

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10.5.4 Acute Cellular Rejection Acute cellular rejection (ACR) is the leading cause of intestinal graft loss in the first 2 months posttransplant [37, 65, 111]. The high incidence and more frequent severity of ACR in intestinal allografts, compared to other solid organ transplants, reflects the large amount of donor associated lymphoid tissue [71]. In a study of MVTx allografts at time of retransplantation or autopsy, acute rejection was more frequent and more severe in the intestine than in other organ allografts, and severe rejection was present only in intestine [97]. ACR occurs in most intestinal allografts. At one large intestinal transplant center, 68% of patients had at least one episode of rejection [89]. The majority of episodes were in the first month posttransplant, with the first episode occurring at a median of 2.5 weeks, with a range from 3 days to over 6 years post transplant [90]. Rejection episodes doubled in length with increase in grade. Mild rejection episodes lasted 1 week, moderate 2 weeks, and severe 4 weeks. Severe acute rejection or an episode of acute rejection lasting more than 3 weeks had a negative impact on graft survival, with rapid graft loss. In 36 cases of graft failure due to rejection, graft failure resulted from severe rejection in 28, refractory moderate rejection in two, vascular rejection in one and CR in five [90]. Although ACR occurs in the majority of intestinal allografts, recent immunomodulatory preconditioning protocols, using small select patient groups, have lead to a significant decrease in acute rejection episodes [73]. The diagnosis of ACR is based on the correlation of clinical, endoscopic and histologic findings. Clinical findings include fever, nausea, vomiting, increased stomal output, abdominal distension, and abdominal pain. Endoscopic findings include edema, granularity, erythema, loss of fine mucosal vascular pattern, friability, hemorrhage and ulcer. Mucosal biopsies, obtained through an ostomy, are essential for patient monitoring and for the diagnosis of acute rejection. Surveillance biopsies are performed frequently during the first few months posttransplant. Schedules vary among centers, but in general, during the first month, biopsies are performed 2–3 times per week, followed by once a week over the next 2 months, and then on a monthly basis. Ostomy closure is performed at around 6 months posttransplant. Biopsies are obtained with clinical signs of rejection and during

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weaning of immunosuppressive drugs [89, 116]. Following closure of the original ostomy, jejunal biopsies can be obtained. An excluded blind enterostomy can be placed for long term surveillance, including deep biopsies to evaluate for CR [52]. ACR is often variable along the length of intestine. In the first month, ACR tends to be most severe in the terminal ileum at the site of Peyer’s patches [54, 114]. If ACR is present, the ileum is almost always involved, while other sites may be unremarkable [54]. In addition, mucosal lesions can be patchy, with only one of multiple pieces from a site showing ACR. Magnification endoscopy allows for closer examination of villous morphology and crypts, with guided biopsies of mucosal lesions [48]. More than one site should be biopsied, at least two pieces should be obtained from each location, and multiple levels should be examined microscopically. Stomal biopsies are inadequate due to nonspecific inflammation and gland damage [114]. At present, there are no specific surrogate markers for acute rejection in the intestine. Serial measurements of certain biological markers, however, may be useful as a prescreening tool for acute rejection. Fecal calprotectin is a stable neutrophilic cytosolic protein, which increases with inflammation, including during ACR [4]. Granzyme B and perforin, molecules released by T-lymphocytes, are increased in ACR, infection and PTLD and can be measured in blood samples [6]. Serum citrulline levels decrease during intestinal dysfunction and can be used as a nonspecific marker for ACR [17]. The histologic features of ACR in human intestinal allografts are described in studies correlating mucosal histology with clinical acute rejection and response to treatment [54, 114] (Fig. 10.3). ACR is characterized by three main histologic features: a mixed, predominantly mononuclear infiltrate with activated lymphocytes, crypt epithelial injury, and increased crypt epithelial cell apoptosis. The inflammatory infiltrate includes activated lymphocytes, usually in association with damaged crypt epithelium. Other inflammatory cells can be present, including small lymphocytes, plasma cells, eosinophils and neutrophils. Occasionally, there is prominent mucosal eosinophilia. Inflammation may be present around small venules and beneath crypts. Peyer’s patches are enlarged and contain activated lymphocytes. In allografts more than 100 days posttransplant, there is often less inflammation. Apoptotic crypt epithelial cells, which are normally

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present at less than one per ten crypts in native or normal intestinal allograft, are increased in number and are present as single bodies or clusters in glands. In mild ACR, multiple tissue levels may be needed to identify apoptotic cells. Epithelial crypt injury includes cytoplasmic basophilia and mucin depletion, loss of Paneth cells, nuclear enlargement and increased mitoses. Other findings that may be present include edema, lymphatic dilation and activated endothelium. In mild cases, crypt and villous distortion is minimal and surface epithelium is intact. With increasing severity, the inflammation increases and is more dispersed, apoptosis increases with confluent forms, crypt injury increases including focal gland drop-out, and there is increased villous blunting. Focal surface epithelial erosion may be present. In severe cases, there is ulceration and extensive gland drop-out and granulation tissue. The residual mucosa has variable epithelial regenerative change, inflammation and apoptosis. In persistent severe ACR, there may be dense inflammation with neutrophilic exudate and mucosal exfoliation (also called severe exfoliative rejection). An interesting finding during recovery from severe rejection episodes is surface reepithelialization prior to formation of crypts.

10.5.4.1 Grading System for ACR

Fig.  10.3  Acute cellular rejection (ACR). (a) Mild ACR. Mucosal architecture is preserved. There is a mild increase in lamina propria cellularity, composed of lymphocytes, plasma cells and eosinophils, along with an increase in crypt apoptosis. (b) Moderate ACR. Some architectural distortion is present and there is an increase in lamina propria mixed cellular infiltrate. Multiple crypts have apoptotic activity, including confluent apoptotic cells. Epithelial cell dropout, crypt disruption and regenerative activity are present. (c) Severe ACR. There is mucosal ulceration, inflammation of lamina propria and crypt loss. Scattered apoptosis is in residual crypts

Two similar histologic grading systems for ACR have been published [80, 83, 116]. The grading systems reflect the histologic features of ACR described above, including predominantly mononuclear infiltrate, crypt epithelial injury, and increased crypt epithelial cell apoptosis (Table 10.3). Biopsies which are indeterminate for rejection have some, but not all of the features for mild rejection. If histologic features of ACR are not present, the biopsy is graded as negative for rejection, rather than indeterminate for rejection. In mild ACR, there is a mild to moderate, mixed, primarily mononuclear infiltrate including activated lymphocytes, with crypt epithelial cell injury and six or more apoptotic epithelial cells per ten crypts. Architectural distortion, villous blunting, edema and congestion are often present, but the surface epithelium is intact. The amount of inflammation varies and can be less in biopsies greater than 100 days posttransplant. In moderate ACR, there is widely dispersed, moderate to severe mixed inflammation, predominantly mononuclear, including activated

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Table 10.3  Histologic grading of acute cellular rejection (ACR) in small intestine allograftsa No evidence of ACR

Histologic features of ACR not present (see below)

Indeterminate for ACR

Minimal, usually focal, crypt epithelial cell injury with increased epithelial cell apoptosis (less than six per ten crypts) Minimal, usually localized, mixed inflammation, primarily mononuclear, ± activated lymphocytes ± Minimal architectural distortion, villous blunting, edema, congestion Intact surface epithelium

Mild ACR

Increased crypt epithelial cell injury with epithelial cell apoptosis (six or more per ten crypts)b Mild to moderate mixed inflammation, primarily mononuclear, with activated lymphocytes, eosinophils and neutrophilsc Architectural distortion, villous blunting, edema and congestion often present Intact surface epithelium

Moderate ACR

Extensive crypt injury with increased epithelial cell apoptosis including confluent apoptosis and focal crypt drop-out Extensive, moderate-to-severe mixed inflammation, predominantly mononuclear, including activated lymphocytes, eosinophils and neutrophils Prominent architectural distortion, villous blunting, edema and congestion ± Focal erosions ± Mild to moderate arteritis

Severe ACR

Extensive, severe crypt damage with drop-out Residual mucosa with variable crypt injury, apoptosis, and reparative change Extensive mucosal erosion and/or ulceration Moderate to severe mixed inflammatory infiltrate (similar to moderate ACR)

Modified from references (Wu et al. [116], Ruiz et al. [80]) At certain institutes, multiple apoptotic cells in a single crypt is also considered sufficient, even with less than six per ten crypts c Inflammation may be less in allografts with longer posttransplant interval a

b

lymphocytes, with increased epithelial cell apoptosis including confluent apoptosis, focal gland drop-out, and architectural distortion with villous blunting. Focal erosions may be present and deep biopsies may show arteritis. In severe ACR, there is extensive crypt damage with drop-out, mucosal erosion and ulceration with moderate to severe inflammatory infiltrate. The residual mucosa has reparative change with variable inflammation and crypt apoptosis. Although both grading systems require at least six apoptotic cells per ten crypts for a diagnosis of mild ACR, at certain pediatric institutes, the presence of multiple apoptotic cells in a single crypt is considered sufficient for the diagnosis of mild rejection (SR, personal communication). The cut-off of six apoptotic cells per ten crypts is based in part on studies by Lee and colleagues [54]. Apoptotic body counts (ABC, number of apoptotic epithelial cells per ten crypts) were determined for native and allograft small intestine, with the following means (ranges): native small bowel = 0.2 (0–2); normal allograft 0.7 (0–5); acute rejection 4.1 (0–19);

preservation injury 0.5 (0–3); cytomegalovirus (CMV) 2.0 (0–6); nonspecific acute inflammation 1.4 (0–5); and PTLD 0.6 (0–2). The grading systems have been used and validated in prospective studies at intestinal transplant centers [83]. The grading systems, however, do not address variant histology in acute rejection, in the setting of new immunomodulatory protocols [116] (see below). The differential diagnosis of ACR includes ischemia-reperfusion injury, AMR, nonspecific enteritis, viral infection and PTLD. Reperfusion injury is easily distinguished from ACR by distinct histologic findings and lack of significant apoptosis. In bacterial enteritis, there is usually a neutrophilic infiltrate, often with cryptitis and infiltration of surface epithelium, but without significant activated lymphocytes or apoptosis. Nonspecific viral enteritis may be characterized by mild architectural distortion with villous blunting and intraepithelial inflammation. Usually, activated lymphocytes are not prominent [116]. Apoptosis, if present, is usually less than seen in ACR. In aden­oviral and CMV enteritis, viral

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inclusions are usually easy to identify on routine stains or with immunohistochemical markers. In adenoviral, rotaviral and calciviral enteritis, there are characteristic epithelial findings, including surface epithelial damage and disarray. Severe adenoviral and CMV enteritis can result in mucosal loss and granulation tissue similar to severe acute rejection, however, viral inclusions can be identified with routine histology, immunohistochemical stains or electron microscopy of lifted tissue sections [68]. EBV enteritis and PTLD are characterized by an atypical lymphoid infiltrate and nuclear Epstein–Barr virusencoded RNA (EBER) staining, without significant apoptosis. Infection and EBV-related processes, however, can occur in conjunction with ACR [65]. Takahasi and colleagues studied the impact of subclinical rejection (SCR), in the first 3 months posttransplant, on graft and patient survival [98]. SCR was defined as histopathologically compatible ACR without concurrent functional deterioration, clinical signs of rejection or endoscopic findings. The diagnosis was made on surveillance protocol biopsies and was not treated. Biopsies with SCR represented 6% of all biopsies and almost a fourth of all biopsies with histologic findings of ACR (most mild in grade). Only 14% of SCR biopsies were associated with the development of clinically significant rejection within 2 weeks. However, SCR was associated with a clinically significant unfavorable graft survival at 3 and 5 years and a higher hazard rate due to infection. Recent use of new immunomodulatory drugs has resulted in variant histopathologic features in intestinal biopsies [1, 117]. In one study, 48 patients were treated with thymoglobulin or alemtuzumab preconditioning protocol. In this set of patients, there were several nonclassic histologic patterns. A scattered lamina propria neutrophilic infiltrate, mixed with fewer numbers of lymphocytes and eosinophils, and accompanied by edema, was seen in a significant number of patients, and in 60% preceded the onset of acute rejection with classic histologic findings by up to 14 days. In addition, acute rejection was often associated with prominent eosinophilia with cryptitis. These findings were considered to represent impending acute rejection which should be closely monitored, if not treated. In some cases of acute rejection, there was a striking loss of crypts, but with intact surface villous epithelium. In addition, mucosal damage with granulation tissue, associated with moderate to severe acute rejection,

was able to recover completely with additional immunosuppression.

10.5.5 Chronic Rejection CR is the main cause of late intestinal graft dysfunction and loss. CR is difficult to diagnose, due t