Cell Separation Methods and Applications

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Cell Separation Methods and Applications edited by Diether Recktenwald AmCell Corporation Sunnyvale, California Andreas Radbruch Deutsches Rheuma-Forschungszentrum Berlin Berlin, Germany

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Library of Congress Cataloging-in-Publication Data Cell separation methods and applications / edited by Diether Recktenwald, Andreas Radbruch. p. cm. Includes bibliographical references and index. ISBN 0-8247-9864-3 1. Cell separation. I. Recktenwald, Diether. II. Radbruch, A. (Andreas) QH585.5.C44C435 1997 571.6—dc21 97 -33113 CIP The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright © 1998 by MARCEL DEKKER, INC. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. MARCEL DEKKER, INC. 270 Madison Avenue, New York, New York 10016 http://www.dekker.com Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

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FOREWORD It should not come as a surprise that the fields of developmental biology as an intellectual discipline and cell separation as a technological discipline have grown in an interdependent fashion. Just as the cell is the unit of biological organization, cells are organized into groupings from the most mature effector cells back to the most primitive progenitors-the stem cells. Stem cells and progenitor cells have the capacity of being clonogenic precursors for large numbers of progeny; stem cells are the most primitive subset and are defined as the cells that can both self-renew and at the single-cell level give rise to progeny of several different mature cells. Progenitors may be multipotent or oligopotent and can be distinguished from stem cells by their lack of selfrenewal capacity. The generation and regeneration of all tissues in the organs in the body depend on the actions of stem and progenitor cells. When we think of generation, we think of those events that occur in embryonic and fetal life to give rise to the organ systems; and when we think of regeneration, we think largely of repair systems, which, in the end, can or should be exploited clinically to replace, wholly or in part, tissue and organ systems that are damaged or are defective. Identification of the stem and progenitors cells involved in generation or regeneration of a particular organ system or tissue concerns fairly rare populations. When one is dealing with regeneration of tissue or organ systems in a clinical circumstance where diseased or malignant cells may contaminate that organ system, practical regeneration can occur only if the stem/progenitor cells

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are purified nearly to homogeneity and do not contain contaminating diseased cells. To do this not only in animal models but in humans, one must be thinking of separation technologies that not only are high fidelity in terms of identifying and isolating rare populations, but also are on a scale that is large enough to deliver adequate numbers of stem/progenitor cells for clinically effective and rapid regeneration. As an immunologist, I must also add that large-scale identification and separation of subsets of cells of the immune system for experiment and clinical treatment involve clonogenic cells of another sort-lymphocytes that respond to antigen by clonal expansion and differentiation into memory or effector cells. Because most methods that allow the culture and expansion of these important potential effector cells also alter their life-span and homing properties to the extent that normal regeneration of the immune system cannot occur, again largescale high-fidelity separations are required. Within this exciting volume two masters of cell separation technologies, Diether Recktenwald and Andreas Radbruch, have gathered together the most accomplished researchers at the leading edge of several cell separation technologies. It is fitting that there is input from the commercial arena, as the development of these technologies as research tools and as clinical scale separation devices can only come through entrepreneurial and commercial efforts. These articles collectively provide the technological and scientific basis for advancing cell separation and identification technologies, and I commend them to you as the state of the art just prior to a new era when this marriage of cell, developmental, and cell separation technologies is about to transform medicine and, at the same time, reveal new insights into the cell and molecular biology that allows stem and progenitor cells to be just that. IRVING WEISSMAN, PH.D. DEPARTMENT OF PATHOLOGY AND DEVELOPMENTAL BIOLOGY STANFORD UNIVERSITY STANFORD, CALIFORNIA

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PREFACE The need for ever more powerful methods in cell separation has grown tremendously with the identification of many specialized cell subsets because of rapid progress in cell biology, immunology, and molecular biology. This book on Cell Separation Methods and Applications describes all of the important methods for the analytical and preparative isolation of specific populations of biological cells. In Chapter 1, Esser includes basic methods such as lytic removal of cell subsets. These methods were developed several decades ago and are still used, primarily as a prepurification step for the preparation of certain common mammalian hematopoietic cell populations for studies in biochemistry, cell biology, immunology, molecular biology, and clinical research. The newest developments in methods using endogenous physical cell properties, such as cell size and cell density, are described in two chapters by leaders in the field of centrifugal elutriation (Van Vlasselaer and coworkers) and density gradient separations (Figdor and colleagues). All of the more specific cell separation methods, based on monoclonal antibodies against cell surface markers, are described by academic and industrial pioneers working on the perfection of the techniques. The methods include polystyrene immunoaffinity devices (Lebkowski and co-workers), biotin avidin immunochromatography (Heimfeld and co-workers), high-density particles (Kenyon and co-workers), complement-based cell lysis (Gee), various immunomagnetic methods for cell sorting (Kantor and co-workers),

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magnetophoresis (Hausmann and co-workers), and fluorescence-activated cell sorting based on flow cytometry (Hoffman and Houck) The book also describes the basic technical principles of the respective cell separation methods in detail. Typical examples with performance data are discussed, and the limitations of the methods are outlined. Applications in basic research and medicine and an outlook on future applications are reviewed. For ease of understanding, flowcharts, figures, and tables support the text in all chapters. Finally, the book includes several chapters on important applications of combinations of cell separation techniques, including research in molecular and cellular biology and genetics (Siebenkotten and co-workers) and the clinical isolation of stem and progenitor cells for cell therapy of cancer and other diseases (Hassan and co-workers). To aid the researcher with the design of cell separation projects, there is an appendix with cell properties for cell separations and one with CD antigens updated with results from the February 1996 leukocyte antigen workshop in Osaka. This book will help those working with cellular preparations to select an optimal cell separation approach, and it will provide a detailed understanding of the possibilities and limitations of cell purification for those involved in clinical cellular therapy for transplantation, cancer, and AIDS, or with gene therapy for a variety of genetic defects. We hope that the methods explained in this book will contribute to major advances in cellular therapy and diagnostics, which, in turn, will lead to refinements in methodology. We would like to acknowledge those who provided valuable support in the preparation of the book, including Larry Transue, technical typist; and Kathryn Rubenstein and her co-workers, graphics. Without MaryAnn Foote, Ph.D., this book would not exist. Without her energy and skill in organizing the book and keeping it on schedule, the project would not have succeeded. DIETHER RECKTENWALD, PH.D. ANDREAS RADBRUCH, PH.D.

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CONTENTS Foreword (Irving Weissman) Preface Contributors Part 1: Background 1. Historical and Useful Methods of Preselection and Preparative Scale Sorting Charlotte Esser Part II: Physical Methods of Separation 2. New Approaches in Density Gradient Separation Using Colloidal Silica Gradients in the Processing of Human Hematopoietic Progenitor Cells Peter Van Vlasselaer, Varghese C. Palathumpat, George Strang, and Michael H. Shapero 3. Centrifugal Elutriation: A Powerful Separation Technique in Cell Biology, Immunology, and Hematology Carl G. Figdor, Frank Preijers, Richard Huijbens, Paul Ruijs, Theo J. M. de Witte, and Willy S. Bont

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Part III: Antibody-Based Methods 4. Isolation, Activation, Expansion, and Gene Transduction of Cell-Based Therapeutics Using Polystyrene Immunoaffinity Devices Jane S. Lebkowski, Dewey J. Moody, Ramila Philip, Lisa Schain, Sohel Talib, Rukmini Pennathur Das, David A. Okrongly, and Thomas B. Okarma 5. The CEPRATE® SC System: Technology, Clinical Development, and Future Directions Shelly Heimfeld, Karen Auditore-Hargreaves, Mark Benyuenes, Michael Emde, Cindy Jacobs, Mark Jones, Nicole Provost, Grant Risdon, and Joe Tarnowski 6. High-Density Particles: A Novel, Highly Efficient Cell Separation Technology Norma S. Kenyon, Robert K. Zwerner John G. Gribben, Lee M. Nadler, Camillo Ricordi, and Thomas R. Russell 7. Antibody- and Complement-Mediated Cell Separation Adrian P. Gee Part IV: Magnetic Methods 8. Magnetic Cell Sorting with Colloidal Superparamagnetic Particles Aaron B. Kantor, Ian Gibbons, Stefan Miltenyi, and Jürgen Schmitz 9. Immunomagnetic Cell Separation Using Antibodies and Superparamagnetic Microspheres Adrian P. Gee 10. Free-Flow Magnetophoresis: Continuous Immunomagnetic Sorting of Cells and Organelles by Magnetic Deviation and Focusing Michael Hausmann, Roland Hartig, Hans-Georg Liebich, Georg H. Lüers, Armin Saalmüller, Reinhard Teichmann, and Christoph Cremer

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Part V: Flow Cytometry 11. Cell Separation Using Flow Cytometric Cell Sorting Robert A. Hoffman and David W. Houck Part VI: Special Applications 12. Employing Surface Markers for the Selection of Transfected Cells Gregor Siebenkotten, Katja Petry, Ute Behrens-Jung, Stefan Miltenyi, and Andreas Radbruch 13. CD34+ Cell Sorting and Enrichment: Applications in Blood Banking and Transplantation Hassan T Hassan, K. Gutensohn, A. R. Zander, and P. Kuehnl Appendixes A. Cellular Properties for Cell Separation B. CD Antigen Designations Glossary Index

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INDEX A N-acetyl galactosamine, 63 Acquired immune deficiency syndrome (AIDS), 62, 81 ADA, see Adenosine deaminase Adeno-associated virus (AAV), 77 (figure) plasmids, 75 Adenosine deaminase (ADA), 81, 98 Adhesion molecules, 73 (table), 285 AIDS, see Acquired immune deficiency syndrome Albumin, 190 Analysis, residual cell, 188, 198 Anemia, aplastic, 290 refractory, 290 Antibody, fluorescein-conjugated, 67, 126 fluorochrome-conjugated, 66, 79 (figure) OKT3, 138 phycoerythrin-conjugated, 66, 122 (figure), 126 use in cell sorting, 1, 5, 61, 62, 63, 67, 80, 88, 90, 96, 103, 104, 106, 115, 133–146, 154, 160, 161, 175– 176, 177, 179, 184, 186, 188, 198, 210, 225, 230, 238, 243, 258, 261, 278 Antigen, use in cell sorting, 1, 65, 68, 82, 88, 155, 186, 188, 243, 273, 274 Apoptosis, 249, 259–260

Avidin, columns, 11 (table), 88–89, 89 (figure) B B cells, 2, 7, 8, 10, 28 (figure), 34, 51 (figure), 163 (figure), 164, 272, 273, 274 depletion of, 34 Bags, gas-permeable, 69 Beads, anti-immunoglobulin, 183, 188, 191 avidin, 91, 92 (figure), 156, 161, 179, 210 coating protocols, 189 Dynal, 176, 177 (figure), 181, 210, 232 fluorochrome, 156 hapten, 156, 164 incubating protocols, 191 latex, 220, 221, 221 (table) magnetic, 154, 155, 220, 221, 229 polyacrylamide, 88, 89, 90, 91 streptavidin, 188, 228 Biohazard, 248, 265 Biotin, 228 columns, 88–89, 89 (figure), 156, 162 Blood, cord, 88, 98, 117, 284, 284 (table) murine, 2 Bone marrow cells, 19, 52, 69, 70 (figure), 75, 90, 246, 258, 261, 284

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mononuclear cells of, 63, 65, 66 (table), 67, 68 (figure), 74 (figure) transplantation of, 52–53, 62, 80–81, 94, 95, 104, 108, 111, 114, 121, 146, 166, 261, 285 vertebral, use of, 114–117, 118 (figure), 119 (figure) Borohydride reduction, 156 C Calcium, 239 Cancer, 82, 289 breast, 94, 95, 96, 98, 170, 182, 186, 289 lung, 289 neuroblastoma, 182 ovarian, 72, 73 small-cell lung, 182 Catcher tube sorting, 248–249, 248 (figure) Cell cycle, separation of cells according to, 51, 258 Cell selection, positive, 133, 158, 159–160, 162–166, 181, 182 (figure), 200, 201 negative, 133, 158, 159–160, 162, 181, 182 (figure), 200 two-step, 200–201 Cell selectors, Ceprate SC, 87–101 MacroCELLector, 62–63, 78 RPR CELLector, 62–66, 68–70, 72–76 Cell separation, direct, 191

direct versus indirect, 183–186, 184 (figure), 185 (figure) indirect, 187 (figure), 191 using flow cytometry for, 237–265 Cell sorting, efficacy of, 252–254 protocols, 2–10, 11 (table), 20, 63 purity of, 252–254 Cell types, also see specific names CD2, 96 CD3, 51 (figure), 64, 161, 165 (figure), 169, 187 CD4, 64, 66, 81, 99, 112 (figure), 126, 162, 273 (figure), 275, 276, 278 (figure) CD5, 64, 66, 71, 80, 161, 164, 170 (figure) CD8, 64, 66, 67, 72, 74 (figure), 76 (figure), 78, 80 (figure), 81, 126, 272 CD15, 115, 117 CD19, 99, 164, 165 (figure), 222, 224, 284 CD20, 27 CD25, 272 CD34, 16, 19, 20–24, 25 (figure), 28 (figure), 29, 30 (figure), 34, 48, 50, 52, 53, 63, 64, 66, 67, 68 (figure), 70, 75, 77 (figure), 91, 94, 98–99, 103, 115, 117, 169, 170 (figure), 171 (table), 183, 190, 258, 279, 283– 290 CD38, 64, 67, 68 (figure) CD45, 25 (figure), 30 (figure), 162 CD56, 99 gene-modified, 77–78, 261 H-2Kk, 273, 274, 274 (figure), 276, 277 (figure) MOLT-4, 107

Cell volume, electronic measurement of, 245 Centrifugal elutriation, 43–54 basic principles of, 44–46 chambers, types used in, 47 closed systems, 48 computer-assisted (CACE), 50 reducing sample size by, 48 use of flow systems in, 47

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Centrifugation, 24, 25, 27, 28 (figure), 52 CFU, see Colony-forming units Chemotherapy, 290 Chimerism, 113 Chromatography, 159 Chromium-release assay, 26, 74 (figure), 142 Chromosomes, 226–229, 228 (figure), 262 Chymopapain, 195 Clinical use, 48, 52, 53, 78, 80–82, 87, 104, 210, 233, 263, 283–290 Cobalt, 155 COBE 2991, 27 Colony-forming units/cells (CFU/C), 19, 24, 34, 283 Complement, 9, 10 (table), 11 (table), 176, 201 Cotton wool, 5, 6 (figure and table), 11 (table) Cyanogen bromide, 157 Cytokeratin, 166, 167 (figure) Cytokines, see specific names Cryopreservation, 289, 290 Cytostatic drugs, 52 D Dendritic cells, 51, 53, 82, 103, 104, 126, 162, 163 (figure) Density-adjusted cell sorting (DACS), 29–34 Density gradient material, characteristics of, 17

DETACHaBEAD, 195 Diaminohexane, 157 Dimethylsulfoxide (DSMO), 29, 93 (figure), 114 Diphtheria toxin, 134 Dispase, 195 DNA, analysis, 126, 228, 239, 249 transfection, 271–279 DSMO, see Dimethylsulfoxide Dyspnea, 29 E Electromagnets, 216–217 Electronic gate dissemination, 2 Elutriation, 106 Endocrine cells, 225 Eosin, 145 Eosinophils, 126 Epithelial cells, 51, 225, 226 (figure) Erythrocytes, 2, 3, 50, 115, 117, 162, 164 (figure), 223, 224 (table), 264 lysis of, 2 Erythroid burst-forming cells (E-BFC), 70 Erythropoietin (EPO), 290 Ex vivo expansion, 98, 289, 290 F FACS, see Fluorescence-activated cell sorting

Fc receptor, 157, 160, 181 Ficoll, 1, 2, 3, 4, 4 (figure and table), 11 (table), 15, 24, 25, 107, 109, 111, 117, 142, 259 Flow cytometry, 67, 145, 153, 222, 225, 237–265, 241 (figure), 272, 274 (figure) one-parameter, 50, 286 multiparameter, 239–245, 244 (figure) two-parameter, 50, 67, 116 (figure) Fluidic-switching sorting, 249–250, 249 (figure) Fluorescence cell sorting (FACS), 1, 2, 15, 33 (figure), 158, 159, 163 (figure), 165 (figure), 167 (figure), 170 (figure) Fluorochromes, 237, 238–239 Forward scatter, 240, 242, 245 (figure) G Gastrin cells, 224–225 Gaucher's disease, 98 Gene, CAT, 75, 273

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env, 72 gag/pol, 72 mdr, 273 nef, 72 rev, 81 therapy, 81, 98, 104, 166, 277, 279, 289 tk, 278 transduction, 63 transfer, 75 Glutaraldehyde, 225 Glycophorin, 109, 117, 120 (figure), 162 Graft-versus-host disease (GVHD), 26, 27, 28, 52, 81, 96, 99, 103, 288–289 Graft-versus-leukemia (GVL), 26, 288–289 Granulocytes, 3, 109, 223 Granulocyte-macrophage colony-forming cell (GM-CFC), 70, 95, 95 (table), 96 (table) GVHD, see Graft-versus-host disease GVL, see Graft-versus-leukemia H Hematopoietic progenitor cells, 18, 19, 20–23, 25, 27, 29, 50 Hemoglobinuria, 29 Heparin, 143 Hepatocytes, 51 High-density particles, separation using, 103–129 HIV, see Human immunodeficiency virus

Human immunodeficiency virus, 98 gene, 72, 74, 75 infection, 248 N-hydroxy-succinimide, 157 I IFN, see interferon IL, see interleukin Immunoabsorption, 258 Immunoaffinity, 134 Immunoglobulin, presence of, 67 Immunomagnetic techniques, 134, 135, 139, 153–171, 175–201, 209–233, 258, 273, 275 continuous, 211–233 Immunotherapy, 53, 81, 88, 101 Immunotoxin, 176, 201 Integrins, 285 Interferon (IFN), α, 61 γ, 53, 61 Interleukin (IL), -2, 61, 73, 76, 81, 272 -3, 75, 99, 285 -6, 75, 99 Internet, 238 Investigational device exemption (IDE), 140 Investigational new drug (IND), 140 Iron, 155

dextran, 155–156, 156 (table), 159 oxide, 210 Isothiocyanate, 157, 165 (figure), 170 (figure), 273, 277 (figure) K Keratinocytes, 51 Kupffer cells, 51 L Laser, 239–240, 240 (figure), 242, 242 (figure), 247 tweezers, 246 Lectin, 62, 179 L-leucine methyl ester, 1 Leukemia, 285–286 acute lymphocytic (ALL), 81, 286 acute myelogenous (AML), 81, 286 chronic myelogenous (CML), 81, 286 Leukocytes, 2, 53, 162, 190, 285 Lipofection, 81 Lipopolysaccharide, 270 Liposomes, 75 Liver transplantation, 113

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Long-term culture-initiating cell (LTCIC), 283 Lymphocytes, 1, 18, 47, 50, 51 (figure), 100 (figure), 103, 109, 126, 139, 222, 224, 225, 243, 245, 258, 279 Lymphoma, 124–125, 135, 182 M Macrophages, 2, 8, 190 MACS, see Magnetic cell sorting Magnetic cell separation, 192–195 Magnetic cell sorting (MACS), 1, 2, 153–171, 272, 274, 274 (figure), 275, 276 Magnetic filter, 211, 224 Major histocompatibility class (MHC) I, 273 (figure) II, 10, 276, 278 (figure) Markers, activation, 73 (table) other, 73 (table) surface, 271–279 Medium, Dulbecco's Modified Eagle (DMEM), 63, 75 Melanoma-associated antigens, 53 Methacrylate, 155 Methylcellulose, 15 Metrizamide, 15 MHC, see Major histocompatiblity class Monocytes, 1, 18, 43, 50, 51 (figure), 53, 162, 163 (figures), 223 mpl ligand, 285, 290

Myelocytes, 70 N Natural killer (NK) cells, 1, 18, 26, 27 (figure), 29, 162, 163 (figure) Neodymium-iron-boran magnets, 180 Nerve growth factor receptor, 78 Neutrophils, 70 recovery of, after transplantation, 34, 94, 290 NHL, see Non-Hodgkin's lymphoma Nickel, 104, 105 (figure), 016, 107, 107 (table), 108 Nitrocellulose filters, 259 Non-Hodgkin's lymphoma, 19 NK cells, see natural killer cells Nuclei, separation of, 250 Nylon wool, 2, 8, 11 (table), 190 O Optical scanning, 212, 220, 229 (figure), 237, 242 (figure) O-sialoglycoprotease, 195 Osteoblasts, 18 P Pancreatic islets, 249, 262–263 Panning, 1, 5, 7 (figure), 8 (table), 11 (table), 134, 272, 275 Parasites, 249 PBMC, see Peripheral blood mononuclear cells PBPC, see Peripheral blood progenitor cells PEG, see Polyethylene glycol

Percoll, 3, 11 (table), 17–18, 19, 24, 45 Perfusion system, 212 Peripheral blood mononuclear cells (PBMC), 3, 5 (table), 50, 62, 64, 66 (table), 72, 76, 161 (figure), 162, 163 (figure), 165 (figure), 167 (figure) Peripheral blood progenitor cells (PBPC), 63–64, 87, 90, 284 transplantation of, 94, 286–290 Peroxisomes, 230, 231 (figure) Phagocytosis, 190, 191 Phycoerythrin, 222, 273 (figure) Phytohemagglutinin, 65, 74, 76, 81 Plasma membrane, 239, 245, 250 Platelets, 28 (figure), 64, 111, 115 (figure), 223 recovery of, 34, 94 Poisson statistics, 252–253 Polyacrylamide, 88 Polyethylene glycol (PEG), 17

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Polymerase chain reaction (PCR), 106, 124, 125, 135, 145, 198, 249, 259 Polysaccharide, 155 Polystyrene, immunoaffinity devices, 61–82, 176–201, 210 Polyvinylpyrrolidone (PVP), 17 Prion, 279 Promyelocytes, 70 Pulmonary edema, 29 PVP, see Polyvinylpyrrolidone Q QBEND10, 283 R Rare cells, 210, 213, 224 (table), 249, 264, 271, 275 Recombinant human granulocyte colony-stimulating factor (rHuG-CSF), 19, 20, 23, 27, 28 (figure), 30 (figure), 34, 50, 52, 94, 94 (table), 97, 97 (table), 117, 120 (figure), 284 (table), 289 Recombinant human granulocyte-macrophage colony-stimulating factor (rHuGM-CSF), 51, 52, 53, 289 Recombinant human interleukin (rHuIL) -1, 53, 69 -2, 65, 72, 81 -3, 69, 289, 290 -7, 72 -11, 290 Recombinant human stem cell factor (rHuSCF), 69, 290 Recovery of cells, 257 Red blood cell, see Erythrocytes Renal cell carcinoma, 81

cell line, 69 rHuG-CSF, see Recombinant human granulocyte colony-stimulating factor rHuGM-CSF, see Recombinant human granulocyte-macrophage colonystimulating factor rHuIL, see Recombinant human interleukin rHuSCF, see Recombinant human stem cell factor Ricin, 134 RNA, 239 Rosette, 1, 11 (table), 179–180, 181, 192, 193, 195, 211, 275 S Samarium-cobalt magnets, 180 SBA, see Soybean agglutinin Sephadex G-10, 11 (table) Side scatter, 240, 242, 245 (figure), 286 Silica, 11 (table) separation with, 15–36 Signal transduction, 71 SK-BR3 breast cancer cell line, 34, 35 (figure) Sorters, cell killing, 250 droplet, 243 (figure), 246–247 enclosed, 247–250 Southern blot analysis, 276 Soybean agglutinin, 63 Sperm, 18, 51, 264–265 Spleen cells, murine, 2, 3 (table) Steel wool, 157–158, 162, 211

Stem cell factor (SCF), 75, 99 Stokes Law, 16 SU-DHK4 lymphoma cell line, 34, 35 (figure) Sulfhydryl, 157 T T47D epithelial tumor cell line, 166, 167 (figure) T cells, 1, 7, 8, 9 (table), 27, 32 (figure), 51 (figure), 52, 53, 75, 160, 161, 162, 163 (figure), 245, 272, 288 depletion of, 34, 35, 63, 64, 65, 67,

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80–81, 88, 95, 96, 97 (figure), 98, 104, 109–110, 169, 177, 183, 190, 199, 222 (table) expansion of, 81 receptor, 72, 73 (table) Thalassemia, 290 Thrombocytopenia, 290 Thymocytes, 51 Trypan blue, 10 (table), 145, 225 Trypsin, 195 Tumor cells, 166, 258 (table) depletion of, 34, 35 (figure), 53, 88, 121, 169, 177 Tumor-infiltrating lymphocytes (TIL), 64, 69 (figure), 75, 81, 103 V Vaccinia virus constructs, 73, 74 (figure) W White blood cells, see specific names X Xanthan gum, 263 Start of Citation[PU]Marcel Dekker, Inc.[/PU][DP]1998[/DP]End of Citation