Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline- Fourth Edition C3-A4 Vol 26 #22 (26)

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Clinical and Laboratory Standards Institute Advancing Quality in Healthcare Testing The Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS) is an international, interdisciplinary, nonprofit, standards-developing, and educational organization that promotes the development and use of voluntary consensus standards and guidelines within the healthcare community. It is recognized worldwide for the application of its unique consensus process in the development of standards and guidelines for patient testing and related healthcare issues. Our process is based on the principle that consensus is an effective and cost-effective way to improve patient testing and healthcare services. In addition to developing and promoting the use of voluntary consensus standards and guidelines, we provide an open and unbiased forum to address critical issues affecting the quality of patient testing and health care. PUBLICATIONS A document is published as a standard, guideline, or committee report. Standard A document developed through the consensus process that clearly identifies specific, essential requirements for materials, methods, or practices for use in an unmodified form. A standard may, in addition, contain discretionary elements, which are clearly identified. Guideline A document developed through the consensus process describing criteria for a general operating practice, procedure, or material for voluntary use. A guideline may be used as written or modified by the user to fit specific needs.

Most documents are subject to two levels of consensus— “proposed” and “approved.” Depending on the need for field evaluation or data collection, documents may also be made available for review at an intermediate consensus level. Proposed A consensus document undergoes the first stage of review by the healthcare community as a proposed standard or guideline. The document should receive a wide and thorough technical review, including an overall review of its scope, approach, and utility, and a line-by-line review of its technical and editorial content. Approved An approved standard or guideline has achieved consensus within the healthcare community. It should be reviewed to assess the utility of the final document, to ensure attainment of consensus (i.e., that comments on earlier versions have been satisfactorily addressed), and to identify the need for additional consensus documents. Our standards and guidelines represent a consensus opinion on good practices and reflect the substantial agreement by materially affected, competent, and interested parties obtained by following CLSI’s established consensus procedures. Provisions in CLSI standards and guidelines may be more or less stringent than applicable regulations. Consequently, conformance to this voluntary consensus document does not relieve the user of responsibility for compliance with applicable regulations. COMMENTS

The CLSI voluntary consensus process is a protocol establishing formal criteria for:

The comments of users are essential to the consensus process. Anyone may submit a comment, and all comments are addressed, according to the consensus process, by the committee that wrote the document. All comments, including those that result in a change to the document when published at the next consensus level and those that do not result in a change, are responded to by the committee in an appendix to the document. Readers are strongly encouraged to comment in any form and at any time on any document. Address comments to Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, PA 19087, USA.

•

the authorization of a project

VOLUNTEER PARTICIPATION

•

the development and open review of documents

•

the revision of documents in response to comments by users

•

the acceptance of a document as a consensus standard or guideline.

Healthcare professionals in all specialties are urged to volunteer for participation in CLSI projects. Please contact us at [email protected] or +610.688.0100 for additional information on committee participation.

Report A document that has not been subjected to consensus review and is released by the Board of Directors. CONSENSUS PROCESS

Volume 26 Number 22

C3-A4 ISBN 1-56238-610-7 ISSN 0273-3099

Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition W. Gregory Miller, PhD, DABCC, FACB Erich L. Gibbs, PhD Dennis W. Jay, PhD, DABCC, FACB Kenneth W. Pratt, PhD Bruno Rossi, MS Christine M. Vojt, MT(ASCP), MS Paul Whitehead, PhD, CChem, FRSC

Abstract CLSI document C3-A4—Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition provides information on water purity requirements for clinical laboratory testing, validation of specifications, technology available for water purification, and test procedures to monitor and trend water purity parameters. Clinical and Laboratory Standards Institute (CLSI). Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition. CLSI document C3-A4 (ISBN 1-56238-610-7). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006.

The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through two or more levels of review by the healthcare community, is an ongoing process. Users should expect revised editions of any given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or guideline, users should replace outdated editions with the current editions of CLSI/NCCLS documents. Current editions are listed in the CLSI catalog, which is distributed to member organizations, and to nonmembers on request. If your organization is not a member and would like to become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail: [email protected]; Website: www.clsi.org

(Formerly NCCLS)

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This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system, transmitted, or made available in any form or by any means (electronic, mechanical, photocopying, recording, or otherwise) without prior written permission from Clinical and Laboratory Standards Institute, except as stated below. Clinical and Laboratory Standards Institute hereby grants permission to reproduce limited portions of this publication for use in laboratory procedure manuals at a single site, for interlibrary loan, or for use in educational programs provided that multiple copies of such reproduction shall include the following notice, be distributed without charge, and, in no event, contain more than 20% of the document’s text. Reproduced with permission, from CLSI publication C3-A4—Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition (ISBN 1-56238-610-7). Copies of the current edition may be obtained from Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Permission to reproduce or otherwise use the text of this document to an extent that exceeds the exemptions granted here or under the Copyright Law must be obtained from Clinical and Laboratory Standards Institute by written request. To request such permission, address inquiries to the Executive Vice President, Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898, USA. Copyright ©2006. Clinical and Laboratory Standards Institute.

Suggested Citation (Clinical and Laboratory Standards Institute. Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition. CLSI document C3-A4 [ISBN 1-56238-610-7]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 2006.)

Proposed Standard—First Edition

Approved Guideline—Second Edition

January 1976

August 1991

Tentative Standard—First Edition

Approved Guideline—Third Edition

January 1978

October 1997

Approved Standard—First Edition

Proposed Guideline—Fourth Edition

February 1980

June 2005

Proposed Guideline—Second Edition

Approved Guideline—Fourth Edition

June 1985

June 2006

Tentative Guideline—Second Edition December 1988 ISBN 1-56238-610-7 ISSN 0273-3099 ii

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Committee Membership Area Committee on Clinical Chemistry and Toxicology David A. Armbruster, PhD, DABCC, FACB Chairholder Abbott Laboratories Abbott Park, Illinois W. Gregory Miller, PhD Vice-Chairholder Virginia Commonwealth University Richmond, Virginia John Rex Astles, PhD, FACB Centers for Disease Control and Prevention Atlanta, Georgia David M. Bunk, PhD National Institute of Standards and Technology Gaithersburg, Maryland Neil Greenberg, PhD Ortho-Clinical Diagnostics, Inc. Rochester, New York Christopher M. Lehman, MD Univ. of Utah Health Sciences Center Salt Lake City, Utah

Richard R. Miller, Jr. Dade Behring Inc. Newark, Delaware

Harvey W. Kaufman, MD Quest Diagnostics, Incorporated Lyndhurst, New Jersey

Linda Thienpont, PhD University of Ghent Gent, Belgium

Gary L. Myers, PhD Centers for Disease Control and Prevention Atlanta, Georgia

Hubert Vesper, PhD Centers for Disease Control and Prevention Atlanta, Georgia

David Sacks, MD Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts

Advisors Mary F. Burritt, PhD Mayo Clinic Rochester, Minnesota Paul D’Orazio, PhD Instrumentation Laboratory Lexington, Massachusetts Carl C. Garber, PhD, FACB Quest Diagnostics, Incorporated Teterboro, New Jersey Uttam Garg, PhD, DABCC Children’s Mercy Hospital Kansas City, Missouri

Bette Seamonds, PhD Mercy Health Laboratory Swarthmore, Pennsylvania Dietmar Stöckl, PhD University of Ghent Gent, Belgium Thomas L. Williams, MD Nebraska Methodist Hospital Omaha, Nebraska Jack Zakowski, PhD, FACB Beckman Coulter, Inc. Brea, California

Working Group on Reagent Water W. Gregory Miller, PhD, Chairholder Virginia Commonwealth University Richmond, Virginia Erich L. Gibbs, PhD High-Q, Inc. Wilmette, Illinois Dennis W. Jay, PhD, DABCC, FACB St. Jude Children’s Research Hospital Memphis, Tennessee Kenneth W. Pratt, PhD National Institute of Standards and Technology Gaithersburg, Maryland Bruno Rossi, MS Millipore SAS Guyancourt, France

Christine M. Vojt, MT(ASCP), MS Ortho-Clinical Diagnostics, Inc. Rochester, New York

Stephane Mabic Millipore SAS Guyancourt, France

Paul Whitehead, PhD, CChem, FRSC ELGA LabWater, Lane End, Bucks, United Kingdom

Alan Mortimer, CChem, FRSC ELGA LabWater, Lane End, Bucks, United Kingdom

Advisors

Keith W. Richardson Associates of Cape Cod, Inc. Woods Hole, Massachusetts

Kelli Buckingham-Meyer Montana State University Bozeman, Montana

Staff

Darla M. Goeres, MS Montana State University Bozeman, Montana Marilyn J. Gould, PhD Falmouth, Massachusetts Zenaida Maicas, PharmD Cape Neddick, Maine

Clinical and Laboratory Standards Institute Wayne, Pennsylvania John J. Zlockie, MBA Vice President, Standards Tracy A. Dooley, BS, MLT(ASCP) Staff Liaison

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Staff (Continued) Donna M. Wilhelm Editor Melissa A. Lewis Assistant Editor

Acknowledgement CLSI acknowledges the experts and their institutions listed below for their “special review,” advice, and help in preparing the approved-level, fourth edition of this guideline: Ellen Jo Baron, PhD, Stanford University Hospital and Medical School Anita Highsmith, Highsmith Environmental Consultants, Inc. Gary A. O'Neill, PhD, Selective Micro Technologies Bette Seamonds, PhD, DABCC, Mercy Health Laboratories

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Contents Abstract ....................................................................................................................................................i Committee Membership........................................................................................................................ iii Foreword .............................................................................................................................................. vii 1

Scope..........................................................................................................................................1

2

Introduction................................................................................................................................1

3

Definitions .................................................................................................................................2

4

Specifications.............................................................................................................................5 4.1 4.2 4.3 4.4 4.5 4.6 4.7

5

Validation and Trend Monitoring ............................................................................................10 5.1 5.2 5.3

6

Validation of Purified Water as Fit for Its Intended Purpose in Laboratory Procedures...................................................................................................................10 Trend Monitoring of Water Purity Specifications ......................................................11 Water Purification System Validation ........................................................................12

Design Considerations .............................................................................................................13 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8

7

Organization of Water Purity Specifications ................................................................6 Clinical Laboratory Reagent Water (CLRW) ...............................................................7 Special Reagent Water (SRW)......................................................................................8 Instrument Feed Water..................................................................................................9 Water Supplied by a Method Manufacturer for Use as a Diluent or Reagent ..............9 Commercially Bottled, Purified Water .........................................................................9 Autoclave and Wash Water Applications ...................................................................10

Filters ..........................................................................................................................14 Reverse Osmosis (RO) Membranes............................................................................14 Contactor Membranes.................................................................................................16 Ion-Exchange Resins ..................................................................................................16 Activated Carbon ........................................................................................................18 Distillation ..................................................................................................................19 Ultraviolet Light .........................................................................................................21 Storage and Distribution .............................................................................................21

Testing .....................................................................................................................................24 7.1 7.2 7.3 7.4 7.5

Resistivity ...................................................................................................................24 Microbial Content by Colony Count...........................................................................29 Microbial Content by Epifluorescence Microscopy ...................................................31 Endotoxins ..................................................................................................................34 Determination of Oxidizable Organic Substances, Expressed as Total Organic Carbon (TOC) .............................................................................................................36

References.............................................................................................................................................41 Additional References...........................................................................................................................43 Appendix A. Resistivity Measurement in a Sparged Water Sample....................................................44 v

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Contents (Continued) Appendix B. Methods for Correction or Compensation of Resistivity Measurements .......................46 Summary of Consensus and Delegate Comments and Working Group Responses .............................48 The Quality System Approach..............................................................................................................62 Related CLSI/NCCLS Publications ......................................................................................................63

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Foreword This edition of the guideline includes updated information regarding the preparation and testing of reagent water in clinical laboratories. Specifications are based on measuring parameters that serve as indicators for the total ionic, organic, and microbial contamination. Emphasis is placed on the value of trending these parameters as an effective way to control the quality and consistency of purified laboratory water, as well as the importance of validating that a given type of laboratory water is fit for its intended purpose. A new section provides guidelines for water purification system validation, ongoing maintenance, and revalidation on a recurring schedule. The Type I, II, III designations for types of purified laboratory water, used in the previous edition, have been replaced with purity types that provide more meaningful specifications for clinical laboratory testing. Clinical laboratory reagent water (CLRW) can be used in place of Type I and Type II water for most applications. Autoclave and wash water will generally be a satisfactory replacement for Type III water. The definitions of the new types of water include parameters that were not used in previous editions and some of the parameters that were used in previous editions. Resistivity measurement has been retained to monitor inorganic impurities. The previous edition recommended that water purification systems include a stage to reduce organic contamination, but required no control. This edition recognizes that organic contamination can be difficult to remove from feed water, can be introduced by components of water purification systems or biofilms, and must be controlled. Therefore, a total organic carbon (TOC) parameter has been added. Note that on-line or inhouse measurements of TOC are not required. It is acceptable to send CLRW samples to a referral laboratory for TOC measurement. (See Section 7.5 for additional information on contamination risks when TOC is at low levels.) Plate counting of colonies is a widely used method for monitoring the level of microorganisms in purified laboratory water, and this edition continues to specify this approach. However, epifluorescence and endotoxin testing have been added as optional tests, because they provide additional information and results can be determined quickly. Specifications and methods for measuring pH and silicates, as SiO2, have not been carried forward from the previous edition. Resistivity is more sensitive than pH to H+ and OH- contamination. Resistivity is not a sensitive indicator of weakly ionized contaminants, which may elute as concentrated pulses from ionexchange beds when they approach depletion. However, the release of weakly ionized contaminants (silica being but one example) can be avoided by appropriate design and regular maintenance of ionexchange components. A parameter for sterility of general-purpose purified laboratory water has not been included in this edition of the guideline, because most clinical laboratory applications do not require sterile water. Water can be sterilized as necessary for some applications; however, the method of sterilization may degrade the purity of the water. Key Words Autoclave and wash water, bottled water, clinical laboratory reagent water, high-purity water, instrument feed water, purified water, reagent water, special reagent water, water purification

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Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline—Fourth Edition 1

Scope

A number of types of purified water for use in clinical laboratory testing procedures are specified: • • • • • •

clinical laboratory reagent water (CLRW); special reagent water (SRW); instrument feed water; water supplied by a method manufacturer; autoclave and wash water; and commercially bottled, purified water.

Procedures are provided for measuring parameters that monitor ionic, organic, and microbial contamination in purified laboratory water. These parameters should be monitored over time to identify trends in performance so corrective action can be taken before a parameter exceeds specified limits. Recommendations are provided to control particulate and colloidal contamination. The guideline includes validation by the laboratory that a selected type of water is fit for its intended purpose. Suggested approaches for validation of water purification systems are included. It is beyond the scope of this guideline to recommend specific types of water purification systems. Instead, the guideline provides information about discrete purification technologies and monitoring strategies that can be applied in various combinations to achieve and maintain the required water purity.

2

Introduction

The goal of every clinical laboratory is to produce accurate results. Purified water constitutes the major component of many reagents, buffers, and diluents used in clinical laboratory testing. It can also become an indirect component of tests when it is used for washing and sanitizing instruments and laboratory ware, generating autoclave steam, etc. Inadequate control of contamination in purified water is an important potential cause of laboratory error. This guideline recommends measuring certain parameters of purified water used in clinical laboratory applications as a means of quality control for purification systems. The parameters are: resistivity, an indicator of ionic contamination; total organic carbon, an indicator of organic contamination; and viable plate counts, an indicator of microorganism contamination. Epifluorescence and endotoxin testing are included as optional approaches for measuring contamination from microbial sources. Particulate contamination is controlled by including appropriate filtration, or distillation, in the purification system. The guideline is not intended to assure the adequacy of purified water for a given laboratory application; rather, water of a specified purity must be validated as fit for use in a particular laboratory application. Any parameters used to specify a type of purified water, or to monitor the performance of a purification system, must be measured frequently enough to detect potential changes in the system, and the measurement results should be monitored for trends to anticipate maintenance before the water quality degrades to a point that will cause problems with laboratory testing. Other organizations have published guidelines and specifications for purified water intended for various applications. Water conforming to the guidelines and specifications of these organizations may or may not be equivalent to the types of purified water described in this CLSI guideline. Any type of purified water should be validated as fit for purpose before being used in clinical laboratory testing. ©

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Definitions

absorption – a process by which a substance is taken up chemically or physically in bulk by a material (absorbent) and held in pores or interstices in the interior; NOTE: See also adsorption, sorption. accuracy – closeness of agreement between a test result and the accepted reference value (ISO 3534-1)1; NOTE: Accuracy of a measurement is defined as the closeness of the agreement between the result of a measurement and a true value of the measurand (VIM93).2 activated carbon – porous carbon material used for removal of impurities; NOTE: See Section 6.5 for details. adsorption – adherence of molecules, atoms, and ionized species of gas or liquid to the surface of another substance (solid or liquid) as the result of a variety of weak attractions that involve ionic, Van der Waals, and surface-active (hydrophobic/hydrophilic) forces; NOTE: See also absorption, sorption. anion exchange resin – an ion-exchange resin with immobilized positively charged exchange sites, which can bind negatively charged ionized species. azeotrope – a blend of two or more components with equilibrium vapor phase and liquid phase compositions that are the same at a given temperature and pressure. bactericide – a chemical or physical agent that kills bacteria. biocide – a chemical or physical agent that kills microorganisms (as used in this document). biofilm – microorganisms, enclosed in a glycoprotein/polysaccharide matrix, that adhere to each other and/or to surfaces and may form macroscopic layers.3 CA membrane – a reverse osmosis membrane constructed of cellulose diacetate/triacetate. calibration – the set of operations that establish, under specified conditions, the relationship between values of quantities indicated by a measuring instrument or measuring system, or values represented by a material measure or a reference material, and the corresponding values realized by standards (VIM93).2 catalyst – a substance that increases the kinetics of a chemical reaction without being consumed in the reaction. cation exchange resin – an ion-exchange resin with immobilized negatively charged exchange sites, which can bind positively charged ionized species. colloid – small, solid particles that will not settle out of a solution. concentrate – the liquid containing dissolved and suspended matter that concentrates on one side of a membrane. condenser – the stage of a distillation system that removes sufficient heat from a vaporized liquid to cause the vapor to change to a liquid phase. conductivity – conductivity is the reciprocal of resistivity; NOTE: For water purification systems, conductivity is usually reported in microsiemens per centimeter (µS/cm). contactor membrane – a hydrophobic membrane used in removing dissolved gases from water. 2

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copolymer – a polymer formed from two or more different monomers. deadleg//dead volume – a region or volume of stagnation in an apparatus or distribution system. distillation – a purification process that utilizes changing the phase of a substance from liquid to vapor and back to liquid, usually at the boiling temperature of the substance, in order to separate it from other substances with higher or lower boiling points. electrodeionization (EDI) – technology combining ion-exchange resins and ion-selective membranes with direct current to remove ionic impurities from water and maintain the resin in regenerated condition. endotoxin – a thermostable lipopolysaccharide component from the cell wall of viable or nonviable gram-negative microorganisms. epifluorescence – method of fluorescence microscopy in which the excitation light is transmitted through the objective lens onto the specimen, and the fluorescence light is transmitted back through the objective lens to the eyepiece; NOTE: Fluorescence is the immediate emission of electromagnetic radiation, typically visible light, from molecules following absorption of light with a shorter wavelength. feed water – the water that is introduced into a purification process. filtration – a purification process in which the passage of fluid through a porous material results in the removal of impurities based on the physical interaction of the impurities with that porous material. fines – see particulates. gram-negative – refers to bacteria that do not retain the primary violet stain in the decolorization step in the procedure originally described by Gram. gram-positive – refers to bacteria that absorb and retain the primary violet stain in the decolorization step in the procedure originally described by Gram. ion exchange – a reversible chemical reaction between a solid containing immobilized ionic sites (ion exchanger) and a fluid (often water) by means of which ionized species may be exchanged from one substance to another. measurand – particular quantity subject to measurement (VIM93).2 microorganism – any organism that is too small to be viewed by the unaided eye, such as bacteria, viruses, molds, yeast, protozoa, and some fungi and algae. nonpurgeable organic carbon (NPOC) – the concentration of organic carbon remaining after sparging a sample to remove inorganic carbon. off-line – in water monitoring systems, referring to measurement devices that are not directly coupled to the water stream. on-line – in water monitoring systems, referring to measurement devices directly coupled to the water stream. particulates – discrete quantities of solid matter dispersed in water. permeate – substances passing through a semipermeable membrane. ©

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planktonic – a term used to describe aquatic microorganisms that float. plasticizer – a chemical component of plastics to make them softer and more flexible. polishing – in water purification systems, the final treatment stage(s) of the purification system. potable water – water that meets regulations as suitable for ingestion by humans. precision – closeness of agreement between independent test results obtained under stipulated conditions (ISO 3534-11); NOTE: Precision depends only on the distribution of random errors and does not relate to the true value or the specified value (ISO 3534-11). purgeable organic carbon (POC) – the concentration of carbon that escapes the sample in the gas phase during the process of sparging the sample to remove inorganic carbon prior to measuring the organic carbon. qualification – the act of establishing with documented evidence that the process, equipment, and/or materials are designed, installed, operated, and perform according to the predetermined specifications. reservoir – in water purification systems, a container holding quantities of purified water. resistivity – the electrical resistance between opposite faces of a one-centimeter cube of a given material at a specified temperature; NOTE 1: Resistivity is the reciprocal of conductivity; NOTE 2: For water analysis, resistivity is usually reported in megohm-centimeters (MΩ·cm). reverse osmosis (RO) – a process in which water is forced under pressure through a semipermeable membrane, leaving behind dissolved organic, dissolved ionic, and suspended impurities. risk – combination of the probability of occurrence of harm and the severity of that harm (ISO 151904); NOTE 1: risk assessment – scientific evaluation of known or potential adverse effects resulting from hazards. The process consists of the following steps: hazard, or potential hazard, identification; evaluation of the impact of the hazard; and assessment of the practicality of measures to mitigate the risk from the hazard. sanitization – chemical and/or physical processes used to kill microorganisms and reduce contamination from microorganisms. softening – a water treatment process whereby cations, notably divalent cations such as Ca++ and Mg++, are exchanged for sodium using cation-exchange resins in the sodium form. sorption – either or both of the processes of absorption and adsorption. sparging – injection of gas below the water surface to remove other dissolved gases and volatile organic compounds. stagnation – state of a liquid without current or circulation. sterilization – validated process used to render a product free from microorganisms (ISO 151904). TF membrane – a reverse osmosis membrane constructed of a thin film (TF) of polyamide materials. total carbon (TC) – total concentration of carbon (organic and inorganic) in a sample. 4

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total inorganic carbon (TIC) – total concentration of carbon as carbonates, bicarbonates, or dissolved carbon dioxide. total organic carbon (TOC) – total concentration of carbon in the form of organic compounds. validation – confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled; NOTE 1: The term “validated” is used to designate the corresponding status (ISO 90005); NOTE 2: The use conditions for validation can be real or simulated (ISO 90005); NOTE 3: A term used by the FDA for a study used to determine whether a test system meets user needs (12 CFR Parts 808, 812, and 820)6; NOTE 4: WHO defines validation as “the action of proving that a procedure, process, system, equipment, or method used works as expected and achieves the intended result” (WHO-BS/95.1793). verification – confirmation, through the provision of objective evidence, that specified requirements have been fulfilled; NOTE 1: The term “verified” is used to designate the corresponding status (ISO 90005); NOTE 2: Confirmation can comprise activities such as: performing alternative calculations; comparing a new design specification with similar proven design specifications; undertaking tests and demonstrations; and reviewing the document prior to issue (ISO 90005); NOTE 3: ISO 8402 defines verification as “confirmation, by examination and provision of objective evidence, that specified requirements have been fulfilled”); NOTE 4: The FDA defines verification as “a study used to determine whether a test system meets specifications” (21 CFR Parts 808, 812, and 820).6

4

Specifications

Specifications or recommendations are provided for six types of purified water intended for different needs in clinical laboratory testing. At some stage in the preparation of every type of purified water, the water must meet or exceed regulations for potable drinking water comparable to those of the European Union, Japan, or the United States. All of the parameters associated with a water specification must be measured while water purification systems are operating routinely. Insofar as it is reasonable to do so, samples must be obtained for measurement, or on-line measurements made, after the last purification component and as close to the purification system output as possible. Where any purification or storage components exist after an online measurement, the user should validate that the water remains fit for purpose. When systems include a recirculating loop to distribute water to remote points of use, samples must be obtained for measurement, or on-line measurements made, at or after the last port of the loop to ensure that contamination is not introduced by the loop or as the result of backflow at one or more ports on the loop. Using water that meets the specified limits for all of the parameters will reduce the probability of contaminants existing at levels that could interfere with clinical laboratory tests. However, the purpose of water specifications based on the parameters used by this guideline is to monitor for continued control of water purification systems, not to ensure that the water they produce is necessarily fit for specific applications. Purified water must be validated separately as fit for a particular laboratory application (see Section 5.1). To ensure the stability of a purification system, any parameters used to specify a type of purified water, or to monitor the performance of the system, should be measured frequently enough and the results monitored for trends to detect changes and initiate maintenance (see Section 5.2). Each laboratory must determine how often to measure the parameters of its purified water and purification system and how often to revalidate the water as fit for purpose, based on a balance of risk and practicality. Measurements made at intervals have the risk that an out-of-specification condition could have existed and adversely affected clinical testing during the interval between measurements. Risk will increase as the ©

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time between measurements, and between revalidations, increases with respect to the stability of the purification system. Setting an alert threshold for a measured parameter at a more stringent level than the validated water purity can reduce the risk from gradual drift; however, this strategy does not protect against abrupt changes. A risk assessment should be carried out to establish an appropriate monitoring program.

4.1

Organization of Water Purity Specifications

This subsection is provided only as an organizational outline to facilitate the use of this guideline; it is not intended as a summary nor is it intended to have any significance outside the context of the complete guideline. 4.1.1

General Concepts Applicable to All Types of Water

The product water meeting a set specification must be validated as fit for purpose for each laboratory procedure in which it is to be used (see Section 5.1). The system producing purified water must be validated to meet the user requirements specification (see Section 5.3). Regular monitoring and trending of appropriate measured parameters must be carried out and documented to verify that water purification technologies and systems are working effectively (see Sections 5.2, 6, and 7). Procedures must be established for system maintenance to keep the system in conformance with water purity specifications (see Sections 5.2 and 6). 4.1.2 4.1.2.1

Clinical Laboratory Reagent Water (CLRW) Ionic Impurities

Resistivity ≥10 MΩ·cm referenced to 25 °C (see Sections 4.2.1 and 7.1) NOTE: Pretreatment to remove CO2 may be needed prior to measuring resistivity for purified water that contains dissolved CO2 (see Sections 4.2.1 and 7.1.2.3). 4.1.2.2

Microbiological Impurities

Total heterotrophic plate count <10 CFU/mL (see Sections 4.2.2 and 7.2) NOTE: Cell counts by epifluorescence microscopy are an acceptable alternative to plate counts, provided the laboratory has established criteria for interpreting the results (see Sections 4.2.2 and 7.3). 4.1.2.3

Organic Impurities

TOC <500 ng/g (ppb) (see Sections 4.2.3 and 7.5) 4.1.2.4

Particulate Content

Purification systems must include a stage that blocks the passage of particles ≥ 0.22 µm at, or near, the output stage (see Sections 4.2.4 and 6.1). 4.1.3 • 6

Other Types of Purified Water for Use in the Clinical Laboratory

Special reagent water (see Section 4.3) ©

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Instrument feed water (see Section 4.4) Water supplied by a method manufacturer for use as a diluent or reagent (see Section 4.5) Commercially bottled, purified water (see Section 4.6) Autoclave and wash water applications (see Section 4.7)

4.2

Clinical Laboratory Reagent Water (CLRW)

Water that meets the specification for clinical laboratory reagent water (CLRW) should be pure enough to satisfy the requirements of most routine clinical laboratory testing. The specified parameters and limits are described in this section, and the methods for measuring the parameters are described in Section 7. Some laboratory testing applications, including those that are sensitive to dissolved CO2, require special reagent water (see Section 4.3). 4.2.1

Ionic Impurities

Ionic impurities are monitored by resistivity measurement. The resistivity specification for CLRW is a minimum 10 MΩ·cm, referenced to 25 °C, measured directly or after removal of carbon dioxide following the procedure specified in Section 7.1.2.3. The resistivity of water is a function of several factors, including the charge, concentration, and mobility of the dissolved ions. Resistivity is not an absolute measure of ionic impurity concentration, but is an effective means for monitoring and trending ionic impurity levels. As a point of reference, pure water has a theoretically achievable resistivity of 18.2 MΩ·cm, referenced to 25 °C; and a solution of NaCl in pure water with a concentration of 0.36 µmol/L will have a resistivity of 10 MΩ·cm, referenced to 25 °C. At the same molar concentration, solutions of most other inorganic, monovalent ions will have approximately the same resistivity. H+ and OH- ions will produce lower resistivity, at the same molar concentration, due to their higher mobilities. Inorganic polyvalent ions carry incrementally more current than monovalent ions, so fewer ions (i.e., lower concentrations) are required to produce a solution with resistivity of 10 MΩ·cm. Ions that move relatively slowly in water (i.e., low mobility ions) do not carry current as efficiently as Na+ or Cl-. Therefore, higher concentrations of low mobility ions are necessary to produce a solution with a resistivity of 10 MΩ·cm, referenced to 25 °C. If a substance is weakly ionized, only a fraction of molecules exist as ions at any given time, and the concentration of the substance required to produce a solution with resistivity of 10 MΩ·cm, referenced to 25 °C, will be higher. See Sections 6.4 and 7.1 for more details. When exposed to air, the resistivity of otherwise pure water will fall to about 1 MΩ·cm, referenced to 25 °C, because CO2 is somewhat soluble and its hydrated form, H2CO3, dissociates to CO3=, HCO3– , and H+ ions. As long as a purified water has a resistivity of at least 10 MΩ·cm, referenced to 25 °C, it meets the resistivity specification for CLRW, whether or not the water contains some CO2, because the total ion content will be low. However, if the resistivity of a purified water is <10 MΩ·cm, referenced to 25 °C, it will be necessary to remove some, or all, of any dissolved CO2 from a sample in order to verify that the water meets the resistivity specification for CLRW (see Section 7.1.2.3). It is impractical, if not impossible, to prevent CLRW and related solutions from being exposed to air as they are used in most laboratory applications. Fortunately, the great majority of laboratory applications are not adversely affected by the presence of dissolved CO2. Validation that a CLRW, as used in an application, is fit for purpose will verify that any dissolved CO2 present is not adversely affecting the results (see Section 5.1). If a procedure is adversely affected by dissolved CO2, a special reagent water with an appropriate resistivity specification may be required (see Section 4.3).

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Microbiological Impurities

The specification for microbial content of CLRW at the point where the water exits a purification system for use in the laboratory is a maximum of 10 CFU/mL, measured by the plate count method described in Section 7.2. Microbial content can also be measured by epifluorescence; however, there are no consensus specifications, so laboratories must establish appropriate specifications based on validation of the water as fit for purpose. See Section 7.3 for more information. 4.2.3

Organic Impurities

Organic impurities are measured as total organic carbon (TOC). The specification for TOC is a maximum of 500 ng/g (ppb). The TOC specification is a new requirement in this edition of the guideline. The 500 ng/g limit is a practical limit for organic contamination of CLRW that is consistent with the limit used by the pharmaceutical industry for purified water.7 A TOC specification is included because organic contamination of purified water can be an important source of interference in test procedures, and, as is true for all parameters, is dependent on both feed water composition and operating characteristics of water purification systems. TOC measurement provides a practical way to monitor organic contamination levels and to identify changes in levels that may require corrective action in the water purification system. Because the mole fraction of carbon is not the same for all molecules, a TOC value cannot be equated with the total mass of organic material present in an unknown water sample. See Section 7.5 for further information on TOC. 4.2.4

Particulate and Colloid Content

Particulate and colloid specifications are based on the process of filtration. A particle-rejecting stage (e.g., reverse osmosis, ultrafiltration, microfiltration, distillation) that will remove all particles greater than or equal to 0.22 µm must be included at or near the final output stage of the purification system. A 0.22-µm, or finer, screen (absolute) filter will remove most microorganisms and particulates, including those released from purification components (see Section 6.1.1). Reverse osmosis and ultrafiltration will also remove colloids (see Sections 6.1.2 and 6.2).

4.3

Special Reagent Water (SRW)

When applications require water of different purity than CLRW, clinical laboratories should specify a special reagent water. The parameters used to specify CLRW should be included in any SRW specification, but the limits may be different and additional parameters may be added if needed. Some examples of applications that may require SRW include: •

trace organic analysis, which may require a lower TOC or an ultraviolet (UV) spectrophotometric absorbance specification;

•

DNA and RNA testing, which may require specification for levels of nucleic acids, proteases, or nuclease activity;

•

trace metals analysis, which typically requires an acceptable blank response for each metal to be measured;

•

cell/tissue/organ culture and fluorescent antibody detection of microorganisms, which may require an endotoxin and/or sterility specification; and

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low CO2 water.

4.4

Instrument Feed Water

Instrument feed water is intended for the internal rinsing, dilution, and water bath functions of automated instruments. Use of CLRW for this application must be confirmed with the manufacturer of a specific instrument. Water meeting the manufacturer’s specifications must be used.

4.5

Water Supplied by a Method Manufacturer for Use as a Diluent or Reagent

Water that is provided as a diluent or reagent by the manufacturer of a particular analytic system is intended for use only as described in the product labeling for that system. Such water has been qualified by the manufacturer specifically for the uses stated in the product labeling, and is not an acceptable substitute for CLRW or SRW, unless it meets the appropriate specification. Such water is also not acceptable for other testing procedures, unless the laboratory validates it for those procedures.

4.6

Commercially Bottled, Purified Water

Commercially bottled, purified water must meet the required specifications for its intended use. The water should be packaged in a manner that protects it from environmental contamination or degradation during transportation and storage, and from the effects of the container itself. Containers must be cleaned to remove surface contamination and made of materials that do not leach significant inorganic or organic contamination. Containers must also be sufficiently impermeable to prevent significant external contamination from entering. Note that some types of plastic containers are sufficiently permeable to air to allow growth of autotrophic microorganisms, if they have a source of energy such as light. 4.6.1

Proper Labeling

Commercially bottled, purified water must include a lot number and expiration date as well as the values for resistivity, microbial count, TOC, and information on particulate control, plus other applicable parameters for the lot of water as it existed at the time of bottling. The user needs to be aware that the water may degrade during transportation and storage and must confirm the water is fit for purpose as used in the laboratory. 4.6.2

Validation in the Clinical Laboratory

Commercially bottled, purified water may have met the specifications for CLRW when it was bottled; however, at the point of use, the bottled water should be considered an SRW with specifications set by the laboratory, based on the intended use. The laboratory must validate that the bottled water is fit for its intended purpose in clinical laboratory testing. If a bottle is opened and reused over a period of time, the laboratory must validate the water remains fit for purpose throughout the entire period of use. Each new lot of bottled water must be validated for acceptable performance in the test procedures for which it will be used. Typical validation procedures are described in Section 5.1. In most cases, acceptable results for quality control samples will serve to validate the water purity. Bottled water should be purchased in a package size appropriate to the rate at which it is used to avoid prolonged storage of an opened container. Note that microbial and other airborne contaminants can be introduced during the period of use. The laboratory must establish an expiration date for an opened container, based on storage conditions and validation of fitness for purpose, to prevent accumulation of microbial or other contaminants. Note that types of purified water specified in the pharmaceutical industry (e.g., purified water, sterile water for injection, sterile water for irrigation) are not necessarily acceptable alternatives for CLRW. A ©

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pharmaceutical type of purified water must be validated as an SRW for the specific laboratory testing procedures in which it will be used.

4.7

Autoclave and Wash Water Applications

Autoclave and wash water is intended for use as feed water for autoclaves and for automatic laboratory dishwashers with heat drying cycles. This type of water is purified to low levels of inorganic, organic, and particulate impurities that otherwise could contaminate solutions and media in an autoclave or remain on washed laboratory ware. Various purification technologies can be used to produce this type of water. A consensus specification for this water is not available.

5

Validation and Trend Monitoring

5.1 Validation of Purified Water as Fit for Its Intended Purpose in Laboratory Procedures The laboratory must establish the chemical, microbial, and particulate quality requirements of purified water to be used for each of their applications and define one or more types of water that will meet those requirements. Once water purity specifications have been defined, the purified water must be validated as fit for its intended purpose in each laboratory procedure. Validation is confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled. Once validated, the acceptability of purified water should be monitored as described in Section 5.2 to ensure it continues to be fit for its intended purpose. In choosing a procedure for validation that a type of purified water is fit for its intended purpose, the technical details and potential sources of interference in a laboratory procedure must be considered. Approaches are listed below that, either individually or in combination, may provide objective evidence of validation. Other approaches may be adopted by a laboratory to satisfy the requirements of a test procedure. •

The candidate water can be used as a “blank” sample in the procedure to determine that the appropriate response is obtained for the test method. In many cases, the appropriate response will be no signal, a zero result, or absence of a negative effect on the method’s response.

•

The candidate water can be used to prepare reagents or media used in the test procedure. Recovery of expected results for quality control samples that have preassigned expected values (such as reference materials or calibrators provided by the method manufacturer) can be used to validate the suitability of the candidate water used in reagents or media. Recovery of expected results for patient samples and/or quality control samples assayed using reagents or media previously verified to give acceptable results (e.g., from another laboratory or from previous results in the same laboratory) can be used to validate the candidate water. The latter approach is also applicable to revalidation of a new lot of prepackaged bottled water, or revalidation of a water purification system following maintenance or sanitization.

•

The candidate water can be used to prepare quality control samples (when reconstitution with water is appropriate), and results for those quality control samples can be compared to results for the quality control samples prepared with an authenticated source of purified water. Authenticated sources of purified water may include: 1) a different lot that was previously validated; 2) a sample of validated water that was saved prior to a purification system sanitization, and was stored under conditions that would prevent contamination; or 3) a different supplier or an alternate purification system with specifications that provide water that is fit for purpose. If clinical laboratory results using either water source agree, it is likely the candidate water is acceptable.

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Water from an existing purification system can be validated by a review of existing quality control records for the laboratory testing procedures, confirmed by means of the laboratory’s quality assurance program that the laboratory results meet the clinical requirements of the healthcare system.

5.2

Trend Monitoring of Water Purity Specifications

After validation that a type of water (e.g., CLRW) is fit for purpose, it is critical to ensure that the water continues to meet its specifications. Verification of purity specifications is accomplished by measuring the appropriate parameters at intervals – consistent with risk and practicality – to prevent purity variations from impacting the accuracy of measurements, reliability of procedures, or quality of results. The data should be monitored using trend graphs analogous to Levey-Jennings7 or Shewhart8 control charts commonly used in clinical laboratories. The purpose of monitoring water quality is twofold: 1) to document the water is of the specified quality at a point in time; and 2) to detect deterioration of purification components before the deterioration impacts the acceptability of the water. Results from system monitoring must be evaluated to provide ongoing evidence that the preventative maintenance schedule is adequate to maintain the purified water specifications. The laboratory should also establish a procedure for responses to excursions or deviations from the established specifications that includes a review of past water testing results, an evaluation of the impact of the excursion or deviation, including the possible effect on released test results, and documentation of corrective action. The laboratory should also reevaluate the adequacy of the testing frequency when such an excursion from specifications occurs. It is important to recognize that deterioration in a measured parameter can indicate the need for system maintenance before the value for the parameter no longer meets the specification. For example, a purification system that uses mixed bed ion-exchange technology may produce water with a resistivity close to 18 MΩ·cm, referenced to 25 °C. For such a system, even though the resistivity is above the 10 MΩ·cm specification, a 1 to 2 MΩ·cm decrease in output resistivity may represent a significant change that may require prompt maintenance to replace deteriorating components. As an ion-exchange bed becomes exhausted, strongly ionized species may displace weakly ionized species that conduct little current but can reach unacceptable concentrations, even though the resistivity of the product water is greater than the minimum specification. Refer to Section 6.4 for more details on ion-exchange technology. 5.2.1

Maintenance of the Water Purification System

Procedures must be established for maintenance and/or replacement of water purification system components to ensure that the product water continues to meet specifications. Components can fail for a variety of reasons: UV lamps deteriorate with use; ion-exchange or sorption beds become exhausted as they take up contaminants; and filters can become blocked, perforated, or contaminated. Trend monitoring of parameters that measure product water specifications makes it possible to anticipate some maintenance. The measurement equipment itself requires calibration, and system controls require inspection and testing on a regular schedule. The frequency of maintenance activities should follow, as a minimum, the manufacturer’s recommendations. It is essential that the product water be tested to verify that it meets specifications following maintenance or component replacement. 5.2.2

Water Purification System Sanitization

Sanitization of the water purification and distribution system is critical to ensure microbial contamination is controlled within specifications. Sanitization frequency must be adequate to maintain the purity specifications and is established based on system usage, trend data for monitoring parameters, and the system manufacturer’s recommendation. Validation of the sanitization process should include verification that the system has been flushed sufficiently to remove or reduce traces of the sanitizing agent to levels that are consistent with the water being fit for purpose (see Section 5.1). ©

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Water Purification System Validation

Once a type of purified water has been validated as fit for purpose in the laboratory testing procedures, the purity specifications for that water should be incorporated into a validation procedure for the water purification system itself. A validation procedure for the water purification system is used to document the system’s ability to deliver adequate volumes of purified water of the stated specifications. 5.3.1 Validation of New and Major Upgrades to Water Purification Systems: A Master Validation Plan The master validation plan encompasses all major steps to be taken in order to successfully accomplish the project design, implementation, verification of fitness for use, and continuing use of the purified water system. The following formal documentation is recommended: •

user requirements specification (URS) for the purified water grade required by the laboratory to meet its laboratory procedure quality requirements;

•

documents relating to consultation with water system manufacturers/suppliers regarding design features, and system support by the manufacturer, as well as documentation (to be provided by the manufacturer) of the prospective system’s capability to meet or exceed the laboratory’s purified water requirements;

•

after the system has been installed, verification and documentation of installation qualification, operational qualification, and performance qualification over time;

•

written and, where appropriate, validated procedures and schedule for calibration, maintenance, sanitization, sampling, and testing;

•

schedule and criteria for requalification of the water system, for example, following replacement of critical components, following unexpected failures, when introducing new laboratory procedures, or as a periodic verification; and

•

training procedures and log of trained personnel on all pertinent procedures and practices.

Information gathered during the installation, operational, and performance qualifications can be used to determine initial operating parameters, monitoring procedures, and preventive maintenance schedules. 5.3.1.1

Installation Qualification

The installation qualification document establishes that the installed equipment meets all criteria and specifications detailed in the purchase order; that utilities (electricity, city water, or other feed water type) with the proper specifications are available; and that each component of the system (monitoring devices, alarms, gauges, motors, valves, switches) is correctly installed. An approved drawing of the system should be provided and validated for conformity to design. Evidence of calibration of the measuring and/or controlling instruments should be provided (if pertinent). 5.3.1.2

Operational Qualification

The operational qualification document establishes through testing and/or verification that each module or component of the system operates in full conformance with the agreed URS (mechanical, instrument, alarm, ON/OFF switches, applications software, etc.). Training of personnel involved in the testing and maintenance of the system should be planned at this stage. 12

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Performance Qualification

The performance qualification document demonstrates the capability of the system to deliver water of the specified purity under normal operation over time. The trend documentation and monitoring of performance parameters for a purification system in continuing use will provide ongoing performance qualification. 5.3.2

Retrospective Qualification of Existing Water Purification Systems

A retrospective qualification assumes that an existing water purification system has been in service for a period of time and that trend monitoring data is available. Retrospective qualification of an existing water purification system is based on review of existing parameter measurement data gathered since the beginning of the equipment’s installation. Review of existing trend monitoring data brings awareness of reliability issues, adequacy of maintenance and sanitization practices, and other useful facts about the system. The data review should include test results for the purified water collected on a regular basis over a significant time period (e.g., one to two years). The purpose of this data review is to assist with creation of a prospective validation plan. Generation of statistical trend graphs or use of other statistical monitoring tools are recommended for reviewing the retrospective data and for bringing attention to outliers. Follow-up of outlier data will identify conditions that may warrant modification in the parameter test frequency or changes in the system maintenance protocol.

6

Design Considerations

The purpose of this section is to provide laboratories with sufficient knowledge about water purification technologies to communicate effectively with water purification specialists and make informed decisions regarding the purchase and use of water purification systems. The section is not intended to be an in-depth review of water purification technologies; detailed information is available in texts and review articles.9,10 Complete systems are not discussed and recommendations for combining discrete technologies into systems have not been provided, because there are numerous effective configurations to produce water that meet the specifications for CLRW or SRW. However, there are a number of important generalizations that apply to any system. The contaminant levels of a municipal water supply can vary over the course of a year, and a contamination profile over a year or two can be helpful in the selection of a water purification system. The selection of the initial stages of a purification system, frequently referred to as “pretreatment stages,” will depend on the characteristics of the feed water. The primary purpose of initial treatment stages is to reduce damage to subsequent components to ensure reliable operation of a water purification system, and to decrease the cost of operation by preventing excessively frequent replacement of more expensive components. For example, a bench model purification system without pretreatment may be capable of producing CLRW when fed directly with water from the municipal supply; however, costly consumable components may exhaust so frequently that the system will be unnecessarily expensive to operate, and the purity of the product water may be inconsistent. Microorganisms and their by-products represent a particular challenge. In the absence of continuous biocidal conditions, microorganisms will grow as biofilms on all the wetted surfaces of water purification components, including storage tanks and the plumbing of a distribution system. A biofilm is a layer of microorganisms embedded in an organic matrix composed mostly of glycoproteins and heteropolysaccharides. Organisms in biofilms can multiply even when the concentration of nutrients in the main volume of water is very low, and the biofilm layer protects the organisms from periodic treatment with biocides that are primarily effective in killing planktonic (free-floating) microorganisms. The large surface areas of filters and beds of carbon or ion-exchange resins are likely to support the ©

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growth of microorganisms. Filters and beds also concentrate water contaminants that microorganisms can use as nutrients. Sloughing biofilm and by-products of microorganism growth and metabolism (e.g., endotoxins, alkaline phosphatase11) are always potential contaminants of water. The potential for microorganism contamination and its control are discussed for each type of water purification technology.

6.1 6.1.1

Filters Microporous Filters

There are two major types of microporous filtration media: depth filters, characterized by nominal particle size ratings, and screen filters, characterized by absolute particle size ratings. Depth filters are matted fiber or material compressed to form a matrix that retains particles by random adsorption or entrapment. Screen (also called absolute) filters have uniform molecular structures that, like sieves, retain all particles larger than the precisely controlled pore size on their surfaces. Depth filters (typically 1 to 50 µm) are commonly used as an economical way to remove the bulk of suspended solids and to protect downstream purification technologies from fouling and clogging. Screen filters (0.05 to 0.22 µm) are typically used as close as possible to the point of use to trap microorganisms and fine particulates. Very small microorganisms may pass through a filter. Trapped materials, including microorganisms, accumulate on filters and can lead to the release of contaminants over time. Suitable maintenance (regular sanitization and periodic replacement) is necessary to maintain desired levels of performance. Newly installed filters usually require rinsing before use to remove extractable contaminants to levels that meet testing parameter specifications. 6.1.2

Ultrafilters

“Ultrafilter” is a designation for a membrane filter that removes particles as small as protein macromolecules. Ultrafilters are characterized by molecular weight cutoffs (usually described in dalton units), and the efficiency with which they reduce the concentration of relevant contaminants to acceptable levels. Ultrafilters are usually installed near the outlet of a water purification system to reduce the concentration of microorganisms and large organic molecules, including nucleases and endotoxins. Ultrafilters must be regularly sanitized and/or replaced to maintain their effectiveness. Ultrafilters can be installed in traditional fashion, in which all the water flow is straight through the membrane, or in “cross-flow” (tangential flow) fashion, in which a portion of the input water flows across the membrane surface to reduce fouling by continuously rinsing away contaminants. 6.1.3

Vent Filters

Hydrophobic, microporous filters or ultrafilters are often fitted to water storage containers as vent filters in order to prevent particulates, including bacteria, from entering the stored water. By combining absorptive media with filter media, multicomponent vent filters can also minimize CO2 and organic contamination of stored water. Regular replacement is essential to maintain effectiveness; the manufacturer’s recommendations should be followed. Section 6.8 provides additional information on storage of purified water.

6.2

Reverse Osmosis (RO) Membranes

RO membranes are used to remove contaminants that are less than 1 nm (10 Å) nominal diameter. The RO process typically removes over 90% of ionic contamination, most organic contamination, and nearly 14

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all particulate contamination from water. RO removal of nonionic contaminants with molecular weights <100 daltons is dependent on many variables and can be low. Rejection rates increase toward complete removal as molecular weights increase above 100 daltons. In theory, new RO membranes will remove essentially all particulate material, including colloids and microorganisms, and molecules with molecular weights of >300 daltons; however, imperfections can occur that may permit some particulates to pass through. During the RO process, feed water is pumped past the input side of an RO membrane under pressure (typically 4 to 15 bar, 60 to 220 psi) in cross-flow fashion. Pretreatment of the feed water with microporous depth filters and activated carbon is usually required (see Section 6.5) to protect the membrane from large particulates, transition metals, and free chlorine. Typically, 15 to 25% of the feed water passes through the membrane as permeate, and the rest exits the membrane as concentrate that contains most of the salts, most of the organics, and essentially all of the particulates. The ratio of the volume of permeate to the volume of feed water is referred to as the “recovery,” which will vary with the water temperature. Operating an RO system with a low recovery will reduce membrane fouling, especially fouling that results from precipitation of low solubility salts. However, recoveries of 75% are possible, depending on the quality of the feed water and the use of filtration and softening pretreatment. The performance of the RO component of a water purification system is typically monitored by measuring the percent ionic rejection, which is defined as follows for either conductivity (Equation 1, more common) or resistivity (Equation 2) measurements. Both methods yield equivalent results, provided the conductivity (κinput and κpermeate) or resistivity (ρinput and ρpermeate) are expressed to a corresponding number of significant figures. Percent ionic rejection (conductivity) = RO =

Percent ionic rejection (resistivity) = RO =

κ input - κ permeate •100% κ input

ρpermeate - ρinput • 100% ρpermeate

(1)

(2)

Percent rejection is a useful indicator of RO membrane performance; however, it is important to bear in mind that it is a relative term and not a measurement of water purity. For the most part, well-maintained RO systems, operating with potable water as a source, will achieve percent rejections of >90%. RO membrane changes will affect the percent rejection; however, changes in the source water pressure, temperature, pH, and concentration of dissolved substances will also affect percent rejection. Percent rejection should be recorded when a new membrane is installed and trended thereafter. Abrupt changes suggest the need for immediate investigation and possible system maintenance. RO membranes deteriorate with use and can be rapidly damaged by misuse. Manufacturer guidelines should be followed regarding operation, maintenance, and replacement of RO membranes and pretreatment filters. RO membranes are commonly available as CA membranes, made of cellulose diacetate/triacetate materials, and TF (thin film) membranes, made of polyamide materials. TF membranes are stable over a greater pH range, reject monovalent ions somewhat more effectively, and provide approximately twice the throughput of CA membranes at equivalent operating pressures. However, oxidizing agents such as chlorine damage TF membranes, whereas CA membranes are relatively chlorine tolerant. The chlorine tolerance of CA membranes can be utilized in some systems. Chlorine can pass through RO membranes under appropriate conditions, so the growth of microorganisms on both sides of the membrane and in any components of a distribution system will be limited. The residual chlorine or chloramine in the RO water can be removed at the periphery of the distribution system during further steps of purification. ©

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Contactor Membranes

A contactor device uses a hydrophobic membrane filter to remove volatiles (e.g., CO2, O2) from water. The water stream passes on one side of the membrane, and a flush gas or vacuum removes volatiles from the other side of the membrane. The removal rate of a species is dependent on the permeability of the membrane, the contact area, contact time, and the difference in partial pressure across the membrane.

6.4

Ion-Exchange Resins

Beds of ion-exchange resins can efficiently remove ionized species from water by exchanging them for H+ and OH- ions. Ion-exchange resins are porous beads made of highly cross-linked, insoluble polymers with large numbers of strongly ionic exchange sites. Ions in solution migrate into the gel matrix (internal) and crevice (external) spaces within the beads where, as a function of their relative charge densities (charge per hydrated volume), they compete for the exchange sites. Strong cation resins are polysulfonic acid derivatives of polystyrene cross-linked with divinylbenzene. Strong anion resins are benzyltrimethyl quaternary ammonium hydroxide (Type 1) or benzyldimethylethyl quaternary ammonium hydroxide (Type 2) derivatives of polystyrene cross-linked with divinylbenzene. The degree of cross-linking in the resins can be controlled, with increased cross-linking producing a more rigid, less porous gel matrix. Resins can be specialized for certain applications by altering the nature of the charged groups, the degree of cross-linking, and the size of crevices; however, there are tradeoffs. Resins optimized for the removal of well-ionized inorganic salts are extremely efficient at this task, comparatively rugged, and regenerate well. On the other hand, macro reticular resins, which are loosely cross-linked and contain large crevices to improve the uptake of organic ionized species, are more fragile and difficult to regenerate. Ionexchange resins are available in a variety of types and grades, which are selected based on the source water and application requirements. Beds of ion-exchange resins are available as cartridges or tanks and are typically used for a period of time and then replaced with new, or regenerated, beds (batch operation). Keeping the anion and cation resins separated in their own tanks makes regeneration simpler; however, mixing the resins results in more efficient ion removal. When a bed of exchange resin is regenerated off-premise, it may be blended with beds from other locations during bulk regeneration, and the possibility of unexpected contamination should be considered and discussed with the supplier. Use of virgin resins avoids this potential problem. The limitations of ion-exchange resins arise primarily from their large surface area and the batch mode in which they are commonly operated. Ion-exchange materials must be highly porous and have extremely large surface areas in order to function effectively; however, this makes them a potential breeding place for microorganisms. The release of fines and soluble components depends on the initial quality of a resin. Regeneration can damage the molecular structures of resins, resulting in greater release of fines and soluble components. For all these reasons, bed volumes should be kept as small as reasonably possible. Filters are typically installed after the beds to trap fines and other particulate matter. Note that lower grade ion-exchange resins can contribute soluble organic contaminants that may not be removed by subsequent stages of purification. Beds should be designed and operated to minimize the separation of resin components and entrapment of air, which will substantially reduce the efficiency of performance. Ion-exchange materials shed more contamination when they are first installed than they do under steady-state conditions, so it is important to rinse them after installation. The amount of rinsing required varies with the type of resin used, but a minimum of several bed volumes is typically required. The manufacturer’s instructions for the rinse procedure should be followed when beds are exchanged.

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C3-A4 Release of Organics and Silica from Resin Pack on Exhaustion

90

18.5

80

18

60

Resistivity 17

50

Silica 16.5

40

TOC

30 16

Concentration (ppb)

70 17.5

20 15.5

10

15

0

Volume

Figure 1. Release of Organics and Silica From an Ion-Exchange Resin on Exhaustion. (From Whitehead P. High purity water—Are users getting the quality they expect? Ultrapure Water. 1996;13(6):37-40. Reprinted with permission.)

Because most ion-exchange beds used in batch mode operate in quasi-chromatographic fashion, they do not simply become less efficient as they exhaust; they release pulses of contaminants that have accumulated from the water stream since the time the beds were first placed in service. Strongly bound contaminants will displace weakly bound contaminants, so the first pulses of contaminants to be displaced from exhausting ion-exchange beds may be weakly ionized substances that have little effect on the resistivity of the product water. Thus, resistivity monitoring is unlikely to detect the initial release of these weakly ionized species, including charged organics, silicates, borates, etc. This situation is illustrated in Figure 1, which shows the relationship between resistivity, silica, and TOC measurements as an ionexchange bed begins to exhaust. It is impractical to monitor continually for the potential release of all types of weakly ionized substances; instead, purification systems should be designed, operated, and maintained to prevent their release in concentrations that can affect applications. A validated schedule for bed replacement, or an appropriate alert level, is essential to prevent release of impurities. Examples of approaches that have been reported to control the release of weakly ionized substances include: •

Use of ion-exchange beds in a passive, redundant fashion in which two identical beds are connected in series. When the first bed exhausts, as indicated by monitoring resistivity after the first bed, any weakly bound ionic substances that have been released from the first bed will have been bound by the second bed and not released into the product water. At this point, the first bed can be replaced with the second bed and a new bed installed in the second position, or both beds can be replaced at the same time.

•

Use of bed components that include specialized resins that are optimized to bind certain weakly ionized species.12

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Electrodeionization (EDI)

Electrodeionization is a technology combining ion-exchange resins and ion-selective membranes with direct current to remove ionized species from water and regenerate the resins.13 Ions that become bound to the ion-exchange resins migrate to a separate chamber under the influence of an externally applied electric field, which also produces the H+ and OH- necessary to maintain the resins in their regenerated state. Ions in the separate chamber are flushed to waste. The ion-exchange beds in EDI systems are regenerated continuously, so they do not exhaust in the manner of ion-exchange beds that are operated in batch mode. EDI beds are typically smaller and remain in service for longer periods than batch mode resins. EDI is typically combined with reverse osmosis or other pretreatment to ensure it is not overloaded with high levels of salts, and to minimize growth of microorganisms. The small volume of resins results in less bleed of organic molecules. EDI components are typically designed to produce product water with resistivity above 5 MΩ·cm referenced to 25 °C.14

6.5

Activated Carbon

Activated carbon is used to remove oxidizing biocides and organic compounds from water. Most activated carbon is produced from charcoal that is created by destructive distillation of coconut, pecan shells, wood, or coal at 450 °C. The charcoal is then ground and “activated” in a process involving roasting at 800 to 1000 °C in the presence of water vapor and CO2. Acid washing removes much of the residual alkaline oxide ash (e.g., MgO, CaO, Na2O, and K2O), iron, and other soluble material. Activated carbon can also be made from polymer beads (referred to as synthetic activated carbon) which contain fewer noncarbon contaminants than natural sources of carbon and are less likely to produce fines (see below). Activated carbon used in water treatment usually has a wide range of pore sizes and a surface area on the order of 1000 m2/g. Activated carbon takes up water contaminants through adsorption (to the surface) and absorption (into the spaces) by virtue of moderate attraction (ionic forces), weak attraction (polar and Van der Waals forces), and by surface-active attraction. The combination of adsorption and absorption is referred to as sorption. A major use of carbon is the removal of oxidizing biocides from water, so they do not damage membrane filters and ion-exchange resins. One gram of activated carbon will react chemically with 2 to 4 g of chlorine, oxidizing the carbon and producing chlorides. This reaction is very rapid, and small carbon filters can effectively remove chlorine from water. Organic fouling can interfere with the removal of chlorine by activated carbon. Such fouling is dependent on the local feed water supply, and this should be considered when sizing units. An increasing number of municipalities have begun using chloramines as the primary disinfectant, because they are safer to use and less likely to halogenate naturally occurring organic impurities to produce toxic substances in water supplies. Chloramines also cause much less damage to membranes. The breakdown of chloramine by carbon is a catalytic reaction producing ammonia, nitrogen, and chlorides, and the reaction is slow compared to the breakdown of chlorine. It is necessary to ensure adequate carbon volume for the feed water characteristics and application. The supplier should be consulted for sizing recommendations. The second major application of activated carbon is in the removal of organic compounds from purified water. Activated carbon does not remove all dissolved organic contaminants found in water, but its use can produce a significant reduction in TOC. Contact times necessary to remove organics vary with the structure of the molecules. Some organic molecules have large contact time requirements and will not be effectively removed by carbon bed sorption. The trace capacity number, the uptake of acetoxime, is a predictor of how well a particular type, or lot, of activated carbon can remove organics from water.15-17 Beds should be designed and operated to minimize the entrapment of air that will substantially reduce their efficiency and performance. Activated carbons have extremely large surface areas in order to function effectively, and they are a source of endogenous and accumulated inorganic and organic substances, which makes them a breeding 18

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place for microorganisms.18-20 Activated carbon is available with added biocides that are intended to retard the growth of microorganisms on the carbon. Activated carbon beds need to be changed frequently enough, based on monitoring microbial contamination, to limit bacterial build-up to levels that are acceptable for the purity of the final product water. Activated carbon beds are prone to releasing fines and soluble components into the water stream. Fine particles will be present in new beds of activated carbon, but they also develop as the result of mechanical and chemical effects during use. Molded and encapsulated activated carbon cartridges produce fewer fines than granular beds. Pulses of weakly sorbed organic contaminants will be displaced from beds of activated carbon by more strongly sorbed contaminants as the beds exhaust. However, activated carbon beds may also release pulses of previously bound contaminants as the result of abrupt changes in the input water (e.g., temperature; flow rate; concentration of ions, especially divalent ions; surface active contaminants).

6.6

Distillation

Distillation separates water from contaminants by changing the state of water from a liquid phase to a gas phase and then back to a liquid phase. Each of these transitions provides an opportunity to move pure water away from contaminants. Theoretically, distillation can remove most water contaminants, with the exception of those that have vapor pressures close to water (including azeotropes), and should not contribute organic or microbial contamination. Not all laboratory stills are designed to prevent CO2 in the air from dissolving in the distilled water they produce. Consequently, it may be necessary to remove dissolved CO2 from test samples of the purified water in order to confirm that it meets a resistivity specification (see Sections 4.2.1 and 7.1.2.3). 6.6.1

Central Stills

Single-stage, central stills are generally energy inefficient. Their boilers are commonly heated by boiler plant steam; and if a heat exchanger has developed pinholes, boiler steam containing azeotropic additives, such as morpholine, will mix with the boiler water. If the water feeding the boiler is not pretreated and boiler overflow is used to reduce scaling, overflow will increase the concentration of volatile contaminants in the distillate. Condensers of central stills must be well maintained or they may contribute metals, and possibly other contaminants, to the distilled water. Modern, centralized stills use multiple effects (ME) or vapor compression (VC) designs to achieve energy efficiency and improved purity. Such stills can reduce the total dissolved solids by more than 100 times, though they may not be especially effective at reducing the concentration of volatile water contaminants. The water feeding their boilers is usually pretreated to further improve purity and to minimize maintenance. 6.6.2

Laboratory (Small-Scale) Stills

Laboratory stills are frequently used to improve the purity of already purified water (e.g., RO, deionized, or centrally distilled). The great majority are continuous; as boiler water is distilled away, it is replaced with fresh feed water. Chemically resistant glasses (i.e., borosilicates) are the construction materials of choice, because they are stable and can be fashioned into the intricate, seamless shapes required. In order for a continuous still to take maximum advantage of the distillation process, it must boil the feed water under conditions that approach ideal and condense the product water in equilibrium with the most uncontaminated steam possible.

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Boiler and Boiler-Condenser Transition Stages

From a maintenance point of view, laboratory still boilers should be designed to withstand the aggressive dissolving power of pretreated water. Laboratory stills should not be fed with water that precipitates during boiling, because precipitates coat and damage heaters and creep along still surfaces. Contaminants that have vapor pressures lower than water must be excluded from the steam before it enters the condenser stage of a still, or they will not be removed. Therefore, the still boiler and boiler-condenser transition stage must be designed to minimize contamination of the steam entering the condenser. Contaminants with negligible vapor pressures compared to water (e.g., most inorganic salts; particulates, including microorganisms; colloids) should be eliminated by distillation. However, boiler water can enter the condenser stage by splashing, surface entrainment, and steam entrainment. •

Splashing – As water boils, splashing is inevitable; however, a still boiler can be designed to minimize splashing, and splashes that do occur can be prevented from reaching the condenser by providing enough height above the surface of the boiling water and by trapping rogue splashes in a transition stage between the boiler and condenser.

•

Surface Entrainment – The draft of steam in a still can propel a film of contaminated water along wetted surfaces from the boiler into the condenser. Such surface entrainment can be minimized by designing a still that avoids excessive steam velocities, places a vertical hydrophobic barrier in a transition stage between the boiler and condenser to interrupt the film of water, and condenses sufficient steam on vertical wetted surfaces to reverse the net flow of the water film.

•

Steam Entrainment – Bursting bubbles of boiler water will form mist that can be carried into a still’s condenser by the draft of steam flowing from the boiler to the condenser. This steam entrainment can be controlled by designing stills with boilers that have sufficient surface area, so that boiling is not unnecessarily vigorous, and by placing an effective mist filter/trap in a transition stage between the boiler and condenser. Mist particles can be very fine, and simple baffles are not likely to be adequate.

The concentration of contaminants in the boiler steam, which have significant, but lower, vapor pressures relative to water, will be a function of their concentration in the boiler water. Means must be provided for regulating their concentration in the boiler water, or the purity of the product water will be inconsistent. This can be accomplished by periodically draining the still boiler or by means of overflow. Overflow is less efficient than draining and may increase the boiler steam concentration of contaminants that have vapor pressures higher than water. 6.6.4

Condenser Stage

Contaminants that have vapor pressures higher than water are removed in the condenser stage of a still. Compound (multistage) condensers that equilibrate steam and boiling-hot water in multiple, specialized compartments are necessary in order to remove these contaminants efficiently. Such condensers are designed to equilibrate the product water with a compartment of steam containing low concentrations of contaminants, while concentrating the contaminants in a separate compartment of steam for efficient venting. Simple (single-stage) condensers cannot be effective, even if they operate close to the temperature of boiling water, because the distilled water product is in contact with the steam that is being vented. Increasing the concentration of contaminants in the vent steam of a simple condenser will necessarily increase their concentration in the product water. Condensers that operate at temperatures below the boiling point of water will also be ineffective, because they cannot concentrate contaminants for effective venting. Coil and jacket heat exchanges will have cold regions (potentially near freezing during winter at more polar latitudes) where the cooling water enters, unless they are fed from temperature-stabilized recirculating tanks. 20

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The purity of the product water will also depend on how effectively contamination in the ambient air (e.g., dust, volatiles) is prevented from entering the condenser. When a continuous still is not running, its condenser is not under a positive pressure of steam, and particulates in the ambient air can enter through the vents. Means must be provided for sanitizing a still’s condenser each time the still begins a cycle and for diverting any distilled water produced during the sanitizing period away from the storage/distribution system. Contaminants with vapor pressures higher than water will evaporate from boiling water relatively more quickly than water. Therefore, their concentration in the steam that enters a still’s condenser will be a function of the rate at which feed water enters the still’s boiler. Overflowing water through the boiler will increase the concentrations of these contaminants in the steam that enters the condenser above what their concentrations were in the boiler feed water. Means must be provided for regulating the rate at which water flows into the boiler of a continuous still, or the purity of the product water will be inconsistent. When water overflows from the boiler of a still designed with a simple coil or water jacket condenser, the concentration of contaminants with vapor pressures higher than water is likely to be greater in the product water than the feed water.

6.7

Ultraviolet Light

Ultraviolet light is used as a bactericide and to breakdown and oxidize organic contaminants. Various UV sources may be used, but the most common are low-pressure mercury lamps that give effective emissions predominantly at wavelengths of 185 and 254 nm. Medium-pressure mercury lamps are used for larger water treatment systems and produce higher intensities of broader band emissions. The intensity of UV radiation actually penetrating a purified water stream can be reduced by films, including biofilms, developing on the UV source window, or by the source lamp and protective sleeves becoming less transparent to UV light. A UV meter, placed on the opposite side of the flow chamber from the UV source window, may be used to provide quality control information for large systems but may be impractical for smaller point-of-use systems. When it is not practical to install a UV monitor, the lamps must be replaced on a schedule that will assure adequate performance. 6.7.1

Bacterial Contamination Reduction

UV light damages microbial DNA and enzymes (such as RNA polymerase) and is an effective biocide at sufficient dosages. A sufficiently high UV dose is needed to prevent the production of live, but nonreplicating, microorganisms that will not be identified by a plate count (see Section 7.2). Epifluorescence microscopy can be used to determine the actual number of living microorganisms in water that have been irradiated with UV light, and endotoxin testing will provide information regarding the concentration of cell wall material from living and fragmented microorganisms. 6.7.2

Organic Contamination Reduction

Passing water over a source of combined 185/254 nm UV light can effectively reduce residual organic contamination in water. The UV light breaks large organic molecules into smaller ionized components, which can then be removed by a downstream ion-exchange resin bed. This process must be engineered carefully to avoid reintroducing organic and microbial contaminants, which may bleed from the ionexchange resin. High purity ion-exchange resins that do not bleed significant levels of organic contaminants are necessary and must be monitored carefully for replacement at appropriate intervals.

6.8

Storage and Distribution

High-purity water is likely to become contaminated when it is stored or distributed through piping. Potential contaminants fall into the categories of soluble inorganic and organic compounds including dissolved gases, particles, and microorganisms. Contamination can result from contact with the materials ©

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from which storage and distribution systems are constructed, it can enter from the environment, and it can be produced by microorganisms within the system. The impact of potential contaminants should be assessed as part of the validation of a purified water as fit for purpose. Bacterial contamination is the most persistent problem in water storage and distribution systems. Bacteria have a great capacity for adjusting to nutrient-poor environments, such as pure water systems.21 Once bacteria establish, they provide the nutrients for other microorganisms to grow within the biofilm. Biofilms can exist in containers or distribution systems, and their products can have an insidious effect on a wide range of applications. Good design and proper maintenance programs are needed to minimize the impact of bacterial contamination. 6.8.1

Materials

The materials from which containers and distribution systems are constructed must have low permeability, must not contribute significant contamination to the water, and must withstand the agents and conditions used for sanitization without being damaged. Smooth surfaces and a minimum of crevices make controlling bacterial growth somewhat easier. Materials used to join plastic and metal pipes can be an unexpected source of chemical or microbiological contamination. Materials that are apparently inert in pure form can contaminate water with soluble substances, such as: a) polymerization catalysts, monomers, or short chains of polymers; b) plasticizers; c) substances used to facilitate release from production molds; and d) recycling contaminants. As a general rule, virgin, pure materials that have a history and data to support use with high-purity water are recommended. 6.8.1.1

Glass

Chemically resistant glass (i.e., borosilicates) may be useful for small-scale storage systems. It is nonporous, impermeable, and easily sanitized. Borosilicate glass will contribute trace amounts of ions to purified water. Soft glasses are not recommended because they contribute significant amounts of ions to purified water. The fragility of glass precludes its use for large containers or distribution systems. 6.8.1.2

Metals

Stainless steel distribution systems (types 304 and 316) have been widely used. Other metals are rarely used because of either expense or contamination issues. Welded, polished joints are preferable. After assembly, stainless systems must be polished and passivated with nitric acid, a process that dissolves exposed iron from the surface and produces an impervious chromium and nickel oxide surface layer. The surface finish should be inspected to determine when repassivation needs to be performed. Inadequate passivation can result in visible rust, called rouge. Biofilms have been credited with pitting and rusting stainless systems. Stainless steel is widely used when high pressure and high temperature steam sterilization is used for sanitization. 6.8.1.3

Plastics

Plastics have been widely used to construct high-purity water storage containers and distribution systems. Polyvinylidene fluoride (PVDF), acrylonitrile-butadiene-styrene (ABS), and clean grades of polyvinylchloride (PVC) are used for rigid piping systems. PVDF is suitable for high-temperature heat sterilization applications. Polyethylene (PE), polypropylene (PP), polyethylene terepthalate copolymers (PET), polyacetal, polyamide (PA), and high-density polyethylene (HDPE) are commonly used when flexible tubing is required. Less commonly used plastics include: polyether-ether-ketone (PEEK), perfluoroalkoxy vinyl ether (PFA), polytetrafluoroethylene (PTFE), and fluorinated ethylenepropylene (FEP). New polymers or combinations of polymers are constantly being developed. Composite multilayer pipes and tubes provide a variety of desirable features. 22

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In all cases, care should be taken to ensure that materials used are of suitable purity and do not contain additives or fillers that can contaminate the water. Many of these materials can be welded. Ultrasonic welding of plastic tubing can produce very clean connections. If solvent welding is used, allowance has to be made for an extended rinsing period. Compression or push-fit connections have been used successfully. The permeability of different plastics for liquids and gases spans a wide range. Contamination of purified laboratory water passing through plastic plumbing or stored in plastic containers by the gases in clean air does not represent a significant problem for the great majority of clinical laboratory applications. However, CO2 will have a measurable effect on the resistivity of purified water (see Section 7.1.2.2). Where CO2 is a potential interferent in assays, or there is a specific requirement to maintain product water at high resistivity values, suitable low-permeability plastics or multilayer composites should be selected and an appropriate system design adopted. There is also the possibility that gases, other than those normally found in air, as well as liquids can contaminate purified water when the external surface of plastic plumbing or containers is exposed to such substances. This possibility should be considered when plastic containers of purified water are stored in a closed space with, or in close proximity to, other chemicals. Suitable low-permeability plastics or multilayer composites should be selected and appropriate precautions taken to avoid contamination.22 6.8.2

Storage

Storage containers should be constructed of low gas permeability materials that contribute minimum contamination to the water and are not damaged by the sanitizing agents that will be used. The container should be opaque, or protected from light, to minimize the growth of autotrophic organisms. Storage containers that are small enough to be used to transport water should not be continually topped off. They should be sized to ensure that they are refilled at least every few days, drained between fillings to prevent stagnation, and sanitized on a regular maintenance schedule. It is good practice to equip the vents with hydrophobic filters (typically ≤0.22 µm) to prevent particulates and microorganisms from entering. Storage tanks that are part of permanent systems should be kept as small as practical. The water in a reservoir may be recirculated continuously or intermittently through water purification processes to maintain purity. If recirculation is used, attention should be paid to potential temperature increases that could contribute to microbial growth. It is good practice to minimize contact with ambient air by equipping storage tank vents with hydrophobic filters, and if applicable, with adsorbents/absorbents to remove organic vapors and carbon dioxide. Inert gas blanketing can help to maintain a high resistivity and, when make-up water is not already saturated with oxygen and nitrogen, reduce the growth of biofilm. High-purity argon, essentially free of CO2, has an advantage over nitrogen because many organisms can fix nitrogen. UV irradiation may also be used to control microorganism growth. Tanks should be designed so they can be easily drained to facilitate cleaning. Tanks should be drained regularly to avoid static conditions. When an overflow is used, it should be protected to avoid back contamination from the drain. 6.8.3

Distribution Systems

The design and choice of materials for distribution systems depends on the purity of the water being distributed, the size of the system, and the sanitization method to be used. The components used must withstand the sanitizing procedure that will be used, and should be constructed of materials that contribute a minimum of contamination to the water. Valves at dispense points in distribution systems should be designed to prevent backflow and introduction of contamination.

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Distribution systems should be designed so that water recirculates through as much of the piping as possible, with minimal dead-legs to prevent parts of the system from becoming stagnant. Ideally, deadlegs should be eliminated; however, when they exist, they should be flushed as often as necessary to mitigate stagnation effects. Recirculation should include enough water purification processes to maintain a consistent level of purity in the loop. The recirculation flow rate in larger distribution systems is usually continuous, while in smaller laboratory-based systems, intermittent flow may be used to control temperature build-up that could contribute to microbial growth. Historically, a reasonable linear flow rate has been considered advantageous from the standpoint of limiting biofilm growth; however, biofilm will develop at any flow rate and sanitization is the only way to combat it.23-27 The length and complexity of a distribution system is an important consideration in deciding on approaches to control biofilm and other sources of contamination. There are various approaches to controlling biofilm; including regular sanitization; distributing moderately pure water containing an easily removed biocide with final purification and quality monitoring at the periphery with point-of-use systems; and maintaining the water in the distribution system at 65 °C or above.28 6.8.4

Sanitization of Storage and Distribution Systems

Sanitizing storage and distribution systems must be performed often enough to prevent a significant buildup of biofilm. A variety of sanitization procedures are available and vary with system design.9,10 Validation of the sanitization process should include verification that the system, including all distribution legs to points of use, has been flushed sufficiently to reduce the concentration of any sanitizing agents to acceptable levels.

7

Testing

7.1

Resistivity

The measurement of electrolytic resistivity provides a useful, nonspecific assessment of the ionic content of purified water. 7.1.1

Summary of the Theory and Practice of Resistivity Measurement

Electrolytic conductivity, κ, and its reciprocal, resistivity, ρ, are related to the ionic content of water. At low concentrations, for a strongly ionized salt, the value of κ is approximately proportional to (and the value of ρ is approximately inversely proportional to) the concentration of the salt or its corresponding ion pair (e.g., NaCl and Na+ + Cl-, respectively) in solution, although the proportionality constant varies from ion to ion. The following relationship exists between κ and the ionic mobilities, u+ and u-, of the component ions, for a salt of 1:1 stoichiometry (e.g., NaCl, MgSO4):

κ=

1

ρ

= c z F (u+ + u− )

(3)

where F is the Faraday constant (96485.3383 C·mol-1), c is the molar concentration of the salt, and z is the charge on the cation or anion. For the commonly used units for κ (S·cm-1) and u (cm2·s-1·V-1), the conforming units of c are mol·cm-3. For mixtures of salts and for salts with non-1:1 stoichiometry, an analogous situation applies. In such cases, the form of Equation 3 becomes more complex, but the basic principles remain the same.29 To the extent that u+ and u- are independent of c, κ is linearly proportional to c. The proportionality is exact at infinite dilution, but the negative deviation increases with increasing c. 24

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The values of u+ and u- also depend strongly on the viscosity of the solution, hence on the temperature of the water. For many ions, the relative temperature coefficient of u is around +2%/°C. In addition to this dependence of u+ and u- on the temperature, t, the value of the equilibrium constant for the dissociation of water, Kw, is also temperature dependent. As a result, the net dependence of the conductivity of pure water, κΗ2Ο, on t is greater than that for solutions (see Section 7.1.2.4). Substances that carry electric current efficiently in water represent only a portion of the spectrum of water contaminants, and high-resistivity water can be significantly contaminated with a range of substances that interfere with laboratory applications. The measurement of ρ cannot indicate the presence or concentration of nonionized chemical species, nor of ionized chemical species at the ng/g level. Resistivity monitors consist of a conductivity cell (sensor) with cable and a meter or display unit with associated electronics, frequently provided with temperature compensation. The meter measures the resistance, R, between the sensing electrodes of the conductivity cell. R, ρ, and κ are related by Equation 4, where Kcell is the cell constant for the given cell [sensor]:

ρ=

1

κ

=

R K cell

(4)

Kcell has the dimensions of length-1. Its value is governed by the geometry of the cell (sensor). Owing to production tolerances, the value of Kcell typically varies up to 3% from unit to unit within a production run, with an average deviation of ±0.5%.30 The value of Kcell for the specific sensor is normally determined by the manufacturer and is entered in the electronics at installation of the sensor. Recalibration is required if any change occurs to the sensor (e.g., stress/bending at removal or installation) and periodically (typically annually) for verification. If a new sensor is installed, the new Kcell must be taken into account if a valid resistivity is to be displayed. Some conductivity systems may combine this Kcell correction with a revised calibration factor for the meter. In most high-purity water systems, the monitor is configured to provide readout in terms of resistivity. The following discussion is structured in terms of ρ to minimize the need for interconversion. 7.1.2 7.1.2.1

Equipment and Materials Resistivity Meters

Commercially available resistivity meters range from battery-operated, threshold-indicating meters to microprocessor-based digital display meters. Meters with analog display are also acceptable. Manufacturers of reagent-grade water systems frequently have built-in resistivity meters with temperature compensation that are designed to provide accurate measurements in the range of water quality produced. The manufacturer’s instructions should be followed in using all meters. 7.1.2.2

Effects of CO2 Contamination

CO2 dissolves in water, producing CO3=, HCO3– and H+ ions. If water with a resistivity of 18.2 MΩ·cm at 25 °C is exposed to air, its resistivity will fall rapidly at first and more slowly as the concentration of CO2 approaches equilibrium. The resistivity of otherwise pure water in equilibrium with air is about 1 MΩ·cm at 25 °C. For this reason, accurate resistivity measurements of high-purity water (CLRW or SRW) are only possible when resistivity cells are sealed to prevent contact with air. Air-tight resistivity cells are typically connected in line with water purification systems in order to prevent CO2 contamination. If significant CO2 contamination of purified water is unavoidable for some reason (e.g., bottling, storage, or ©

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method of purification), meaningful resistivity measurements can be obtained by removing some, or all, of the CO2 as described in Section 7.1.2.3. All plastic tubing has a finite permeability to CO2.31 Where plastic tubing is used in an inline system, the residence time of the purified water between the final purification stage and the resistivity sensor should be minimized and the flow rate maintained high enough to avoid significant contamination. Permeability values of CO2 for various plastics32 can be used to estimate, semiquantitatively, the level of CO2 that occurs for a given flow rate, system layout (i.e., distance from purification output to resistivity sensor), and wall thickness. 7.1.2.3

Verifying a Resistivity Specification When Purified Water Contains Dissolved CO2

Verifying that the resistivity of a purified water meets, or exceeds, a specification that permits possible CO2 contamination (such as CLRW) may require removing some, if not all, CO2 without adding ionic contamination. CO2 can be removed from purified water at room temperature by sparging with purified gas or by means of a contactor membrane. Heating a sample of purified water to just below the boiling point to drive off the CO2 is not practical because, at this temperature, the resistivity of pure water is nearly the same as that for water with significant ionic contamination. Sparging involves bubbling purified gas through the water in a closed resistivity measurement chamber to remove dissolved CO2 (see Figure 2). Sparging requires the use of very pure gas that will not introduce CO2 or other ionic contaminants to the water. Purified argon (99.9999%) contains <0.5 ppm CO2 and is sufficiently dense to blanket the surface of the sample water during sparging. Many varieties of sparging filters are available. Models commonly used for liquid chromatography have PTFE (polytetrafluoroethylene) bodies and 10 µm nominal porosity polyether-ether-ketone (PEEK) filter elements. Properly rinsed, they do not contribute ions and produce a dense stream of fine bubbles at low gas pressures. Positioning a resistivity cell inside the sparging chamber eliminates the need for connections with the potential for leaking CO2. Care must be taken to ensure that bubbles of sparge gas do not pass between the cell electrodes. If the resistivity cell is positioned external to the sparging chamber, the cell should have as small a volume as possible and be coupled to the sparging chamber as a closed loop. This arrangement will make it possible to efficiently circulate the water in the resistivity cell with the water in the sparging chamber by raising or lowering one with respect to the other. Sparging should be continued beyond the point that the resistivity reaches a maximum to confirm effective operation. A steady decline of the resistivity, after the maximum value has been attained, will indicate that either the gas or some hardware component of the measuring system is adding ion contamination to the sample. If air is leaking into the system, decreasing the flow rate of sparging gas will decrease resistivity, and increasing the flow rate of sparging gas will increase resistivity. Appendix A contains additional information on resistivity measurement in a sparging chamber.

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Sparging Chamber

Gas

Resistivity Cell

Figure 2. Configurations for Measuring Resistivity in a Sparging Chamber 7.1.2.4

Temperature Compensation

Resistivity meters in modern high-purity water systems normally incorporate a temperature sensor in the sensing unit. The temperature sensor (thermistor) measures the temperature of the water whose resistivity is simultaneously being measured. The meter corrects the measured value of ρ to a calculated value that would be obtained at 25 °C using a compensation algorithm. For the conductivity of aqueous ionic solutions at 25 °C, neglecting the contribution of the solvent water, ∆ρ/ρ is on the order of -2%/°C, owing to the temperature dependence of the mobility of the constituent ionized species. However, ∆ρ/ρ is approximately -5.3%/°C at 25 °C for pure water that contains no ionized impurities. This value derives from the temperature dependence of the dissociation constant of pure water and the inherent temperature coefficients of the mobilities of H+ and OH- ions that result from the dissociation of pure water. As the concentration of ionic impurities approaches zero, |∆ρ/ρ| at 25 °C increases from 2%/°C to 5.3%/°C, owing to the increasing contribution of the more temperaturedependent ρ of the solvent water. (NOTE: ∆ρ/ρ is expressed as an absolute value to avoid comparing two negative numbers.) The effect for practical measurements is that the temperature coefficient is also a function of ρ. An example showing this functional relationship is given in Appendix B. It is standard practice to correct resistivity measurements to 25 °C. The uncertainty component associated with the temperature correction increases as the actual sensor temperature deviates from 25 °C. Table 1 shows the impact of temperature on ρ for pure water and for a 0.36 µmol/L solution of NaCl, which has a resistivity of 10 MΩ·cm at 25 °C. An experimental value for ∆ρ/ρ can be measured and used for temperature correction (see Appendix B which describes examples of temperature correction or compensation). For the most accurate measurement in compensated mode, the cell should be held relatively close to 25 °C and an effective ρ - t compensation algorithm must be used.

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Table 1. Resistivity Temperature Table (Pure Water) Resistivity of 0.36 Temperature (°C) Resistivity of pure µmol/L NaCl in water water (MΩ·cm33) (MΩ·cm34) 0 86.19 28.21 5 60.48 22.66 10 43.43 18.30 15 31.87 14.87 20 23.85 12.15 25 18.18 10.00 30 14.09 8.28 35 11.09 6.90 40 8.85 5.79 45 7.15 4.89 50 5.85 4.15 7.1.3

Calibration

Calibrating resistivity instruments for measurements of high purity water is challenging. Stable standard solutions with resistivities of >1 MΩ·cm (conductivity <1 µS·cm-1) are not feasible, because of the potential for ionic contamination, especially by CO2 from the air (see Section 7.1.2.2). Traceable, stable standards are available at lower resistivities (higher conductivities). However, if such (high-conductivity) standards are used to calibrate most commercial cells designed for high-purity water, polarization at the electrodes becomes significant. This polarization, which is a function of the electrode materials used,35 results in an error in the corresponding calibration. An approach that avoids these problems involves producing water that is free of contaminating ions (including those related to CO2) in a closed-loop purification system and using this water as a calibration standard at 18.2 MΩ·cm at 25 °C. Manufacturers of resistivity measuring systems commonly use this approach; however, the instruments and equipment required to demonstrate that the water is, in fact, free of contaminating ions are not readily available in most laboratories. Readers desiring a detailed description should consult the original literature30,36 and/or the manufacturer of the given instrument. All the components of a resistivity instrument (i.e., the meter [electronics], cable, and cell) should be calibrated as a set in accordance with the manufacturer’s specific instructions to achieve the greatest accuracy. Resistivity meters use alternating current of various frequencies and waveforms to minimize polarization of cell electrodes and reject the reactive component of the cell current. Small changes in the impedance characteristics of individual cells can result in significant errors in calibration. For the same reason, installing a standard resistor in place of a cell only verifies the ability of the meter to correctly measure a pure resistance; it is not a meaningful calibration of the complete instrument.37 As noted in Section 7.1.2.4, measured ρ values are generally corrected from the actual measured value at the ambient temperature to the temperature-corrected value at 25 °C. Thus, calibration, and subsequent verification, of the temperature-measuring device at the temperatures of interest are critical to obtaining an accurate value for the temperature-corrected ρ value. The temperature-measuring device is most often a thermistor placed adjacent to, or within, the cell. Its accuracy over the applicable range is verified by placing it into a calibrated temperature bath or by comparison with a calibrated thermometer. The algorithm used to correct ρ to the temperature-corrected value at 25 °C is also critical. The exact algorithm used in the calculation varies among manufacturers. The general approach is illustrated in Appendix B using a simplified approach (Marsh-Stokes equation). A commonly used method for field verification of a resistivity instrument involves using a separate, calibrated resistivity instrument as a reference. The inline cell of the reference instrument is placed in 28

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series with, and close to, the cell of the instrument to be verified. The test and reference cells should be close enough to avoid significant contamination of the water stream or change in its temperature as it flows between the two cells, and simultaneously far enough apart to avoid mutual electronic interference between the calibration and test instruments. The validity of this approach depends on these factors and on how accurately the reference instrument has been calibrated.

7.2

Microbial Content by Colony Count

Clinical microbiology methods for growing pathogenic microorganisms emphasize handling samples to maximize the recovery of pathogens from clinical specimens. In clinical microbiology, this means providing a rich growth medium and optimal growing conditions that mimic physiologic conditions (temperature and nutrients). These same considerations are applied to recovering organisms that may be present in a purified water system. However, the growth conditions in purified water are different from those in clinical specimens. Purified water is nutrient poor, and is typically distributed and stored at ambient temperatures. Consequently, microorganisms growing in purified water are smaller, and are adapted to growing more slowly than organisms from clinical specimens. The procedures detailed here use conditions to maximize the recovery of microorganisms likely to be found in water systems, balanced with the need for timely results. 7.2.1

Total Heterotrophic Plate Count

The determination of total heterotrophic plate count (THPC) is an approximation of the viable number of microorganisms present in the system, and is expressed in colony forming units (CFU) per milliliter. Heterotrophic bacteria are organisms that require organic substrates to obtain carbon for growth and development. Water is tested for THPC by either the membrane filtration or spread-plate techniques. 7.2.1.1

Sample Collection

•

Water samples should be collected, using aseptic technique, into sterile specimen containers at each point-of-use site. Samples must be collected in a manner that prevents contamination by contact with skin or the environment.

•

Water samples intended to evaluate water as used in laboratory procedures should be collected from points of use in a manner consistent with the way it would be drawn normally.

•

If water is sampled from a port intended only for the purpose of microbial testing of the water within the distribution or storage system, then that port should be disinfected (usually with isopropyl alcohol) prior to sampling, and the port flushed thoroughly to remove the disinfectant before the water sample is drawn.

•

If testing cannot be accomplished within one hour, samples may be refrigerated at 2 to 8 °C for up to 24 hours. Samples should be allowed to reach ambient temperature before testing.

•

All samples should be gently and thoroughly mixed prior to processing.

7.2.1.2

Media and Incubation Conditions

The choice of media, filter size, incubation time, and incubation temperature should be validated to establish the adequacy of the methodology chosen. The recommended incubation temperature is 20 to 28 °C and the recommended incubation time is at least five days. If it is not practical to have an incubator controlled to this temperature, use a dust-free room temperature cabinet.

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The following low-nutrient agars are commercially available and suitable for water testing: • •

plate count agar; and R2A agar.

7.2.1.3

Membrane Filtration Technique

The membrane filtration technique is recommended for water samples that typically have less than 10 CFU/mL. The choice of water volume to test should be based on consideration of typical results obtained from the samples such that test results can be counted (i.e., filter is not overgrown). The sample is filtered through a sterile filter housing using a sterile 0.22 µm filter. Bacteria that are present in the sample are trapped on the filter. The filter is then placed face up on the surface of the nutrient media and incubated. The nutrients from the agar diffuse through the filter allowing the growth of colonies. (1) Pour up to 100 mL of a mixed water sample into the sterile filter unit with the vacuum off. (2) Turn on the vacuum to pull the sample through the filter. (3) Turn off the vacuum and rinse the filter housing by adding approximately 30 mL sterile 0.1% peptone to the unit. (4) Turn the vacuum on briefly to pull the peptone rinse through the filter. (5) Using sterile forceps, carefully lift the membrane out of the filter unit, holding onto the edge of the membrane. (6) Place the membrane (face up) onto the surface of an agar plate by gently rolling the filter membrane onto the surface of the media to avoid the formation of air pockets between the filter and the surface of the growth medium. (7) Incubate the plates for a minimum of five days at 20 to 28 °C. Plates should be inverted to prevent condensation on the agar surface. (8) After incubation, count the number of colonies and divide by the total volume tested to obtain the actual results in CFU/mL. (9) Membrane filtration results are valid up to 100 CFU/filter plate. Greater than 100 CFU represents an overcrowded plate, which can inhibit the formation of additional colonies. Results should be questioned if there is considerable clumping of colonies, growth only around the perimeter of the filter, and if duplicate plates are used (i.e., sample split between two plates) and there is significant differences between the two plates. 7.2.1.4

Spread-Plate Technique (Direct)

The spread-plate technique is suitable for samples that typically contain high numbers of microorganisms, such as feed water. The sample is applied directly to the surface of the media and then spread across the surface of the media with a “hockey stick.” (1) Using a sterile pipette, transfer 1 mL of sample onto the surface of one or two agar plates, depending on expected results, but maximum 1 mL per plate. The entire 1 mL of water must be spread on the agar surface. Two plates may be necessary to avoid water pooling on the surface.

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(2) Spread the sample over the entire agar surface using a sterile “hockey stick” to ensure proper sample distribution. (3) Incubate the plates at 20 to 28 °C for a minimum of five days. Plates should be inverted to prevent condensation on the agar surface. (4) Spread-plate technique results are valid up to 250 CFU/plate. Greater than 250 CFU represents an overcrowded plate, which can inhibit the formation of additional colonies. Results should be questioned if there is considerable clumping of colonies, growth only around the perimeter of the filter, and if duplicate plates are used (i.e., sample split between two plates) and there is significant differences between the two plates.

7.3

Microbial Content by Epifluorescence Microscopy

Epifluorescence microscopy is an effective and timely means of determining the concentrations of microorganisms in purified water.38-40 This technique can be used to detect and distinguish between living and dead microorganisms in less than an hour, which makes it useful when rapid corrective action is indicated. Epifluorescence counts are likely to differ from those obtained from plate counting, because microorganisms growing in laboratory water purification systems do not necessarily grow quickly, well, or at all on plate media.41 Cells that may not be capable of division when plated may be alive and capable of metabolic activity. UV treatment of water may damage replication mechanisms without causing death of a microorganism. Laboratories that adopt epifluorescence monitoring will need to establish thresholds of acceptable counts based on their laboratory requirements. It may not be possible to establish a consistent relationship between plate counts and epifluorescence counts, because plate counts are not necessarily a fixed fraction of the total concentration of planktonic microorganisms in a water system. 7.3.1

Method Overview: Total Direct Count Technique for Enumerating Bulk Water Bacteria

A water sample is filtered though a membrane filter, which is then appropriately stained and examined by epifluorescence microscopy. Depending on the concentration of microorganisms, the volume of water is adjusted (typically from 1 to 10 mL) to avoid significant overlap of cells. Staining with DAPI (4, 6Diamidino-2-phenylindole) does not distinguish viable and nonviable bacteria and yields a total count. Staining with a mixture of SYTO® 9a and PI (propidium iodide) dyes in DMSO (dimethylsulfoxide) will make it possible to distinguish between viable and nonviable bacteria with a high level of certainty.38-43 SYTO® 9 is a green-fluorescent nucleic acid stain that penetrates the walls of microorganisms, whether they are healthy or unhealthy; whereas, red-fluorescent PI nucleic acid stain only penetrates bacteria with damaged cell wall membranes. Cells with intact membranes will appear green and cells with damaged membranes will appear red. 7.3.2

Equipment and Consumables

The equipment needed to perform epifluorescence microscopy is relatively expensive, but is often available at university medical centers and microbiology laboratories. • • • • • •

epifluorescence microscope with UV light DAPI, FITC (fluorescein-isothiocyanate) and RITC (rhodamine-isothiocyanate), or equivalent filters digital or video imaging system (not required, but very helpful) membrane filtration apparatus for 25-mm diameter membrane filters consisting of a vacuum filter funnel manifold and vacuum filter holder/supports vacuum pump timer

a

SYTO® 9 is a registered trademark of invitrogen™.

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filter forceps for handling membrane filters

The consumable items required to enumerate microorganisms in laboratory water by epifluorescence microscopy are readily available. • •

black polycarbonate membrane filters (0.22 µm, 25 mm diameter) stains — DAPI solution – A solution of DAPI in CLRW (0.1 mg DAPI/mL CLRW) is filtered using a sterile, 0.22-µm pore size polyvinylidene fluoride (PVDF) syringe filter to eliminate large crystals or other particulates. Refrigerated, the solution can be stored in an amber bottle for a year or longer. — SYTO® 9/PI solution – Mix 1 mL of filter-sterilized CLRW with 15 µL of 3.34 mmol/L SYTO® 9 dye in DMSO, and 15 µL of 20 mmol/L PI dye in DMSO. The 1:1 ratio of the dyes gives good results in most applications; however, the ratio can be modified to optimize results for specific circumstances. The DMSO solutions of the dyes should be kept frozen, and the working solution should be used within a day and kept cool and in the dark. SAFETY NOTE: Nucleic acid stains should be handled with particular caution, and the user should bear in mind that DMSO is known to facilitate the entry of organic molecules into tissues. The stains should be disposed of by pouring them through charcoal, and the charcoal must then be incinerated to destroy the stains.

• • • • •

glass microscope slides glass coverslips nonfluorescing immersion oil 0.22-µm pore size PVDF syringe filters to eliminate large crystals or other particulates from stain solutions and CLRW used for rinse water 95% v/v ethanol

7.3.3

Procedure

The following procedure is not intended to cover every detail; laboratories are advised to consult with vendors of equipment and supplies regarding usage. 7.3.3.1

Sample Preparation

(1) Wet the stage of the filtration apparatus with filter-sterilized, CLRW type water and suction the water through with the vacuum until all of the water is gone. (2) Place a polycarbonate membrane filter on the stage with the shiny side facing up. (3) Place a filter funnel, which has been rinsed first with 95% v/v ethanol and then with filter-sterilized, CLRW type water, on the stage and clamp the filter into place. Ethanol helps disinfect the filter funnel and eliminate any residual particles that may interfere with microscopy; however, it must be rinsed away or it could have adverse effects on the bacteria and/or stains. (4) Obtain a freshly collected sample. If samples cannot be tested promptly, they can be refrigerated up to 24 hours. Vortex mix samples for at least 30 seconds to be certain the cells are well distributed.

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(5) For optimum volume to be determined, cells should not be clumped or layered on one another, so individual cells can be easily identified for enumeration; different volumes (e.g., 100 mL, 10 mL, and 1 mL) of the undiluted water sample might be filtered to avoid repeating the preparation. (6) Slowly add the volume of water sample to the filter, being certain to keep the entire surface of the filter covered, and turn on the vacuum to suction the sample through the filter. (7) Turn off the vacuum and release any residual suction on the filtration apparatus by gently lifting up the filter funnel. (8) The filter is ready to be stained for total direct counts or viability counts. 7.3.3.2

Staining

Total counts are performed using DAPI stain, and viable/nonviable counts are performed using the SYTO® 9/PI mixed stain. NOTE: The SYTO® 9/PI mixed stain will not produce good results with bacteria that have thick walls or membranes, such as Mycobacterium fortuitum. Comparing live/dead counts with total DAPI counts will establish whether the live/dead counts are missing a significant number of microorganisms. (1) Slowly (drop-wise) add staining solution to the filter until the filter is fully covered (approximately 0.25 mL/filter). (2) Cover the filter funnel with aluminum foil and incubate at ambient temperature for 15 to 20 minutes. The stains degrade when exposed to UV light, so covering the filtration funnels with aluminum foil during staining will help prevent photobleaching. (3) Use a vacuum pump to suction the staining solution through the filter. 7.3.3.3

Enumerating

Total counts are relatively unambiguous; however, viable/nonviable counts yield much more information, some of which is open to interpretation. Cells that cannot, or do not, replicate, but which are functioning in other respects, will be stained as viable. Cells with damaged walls, but which may be capable of recovery and replication, will be stained as nonviable. (1) Place a small drop of immersion oil on a slide and place the filter on the glass microscope slide, shiny side up. Place another small drop of immersion oil on top of the filter and cover the filter with a coverslip. Blot away any excess oil. (2) Use a 100 oil-immersion lens on an epifluorescence microscope with UV light and an appropriate filter (DAPI, FITC, RITC, or equivalent) to count the total number of cells in 20 random fields.44 Ideally, images of the fields can be stored using digital or video technologies. Record the appropriate information from the microscope (e.g., magnification, size of grid, zoom). Calculate the number of cells per volume based on the dilution factor, volume filtered, filter funnel surface area, etc. Close the shutter of the microscope whenever possible, because exposing the filter to UV light will photobleach the stains to the point that cells are no longer distinguishable from the background within approximately two minutes.

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Quality Control

A quality control check for the epifluorescence process can be completed by filtering known concentrations of fluorescent microscopic beads onto a membrane filter and processing the filter as though it were a routine sample. Known solutions of live and killed microorganisms can be mixed quantitatively and processed as samples.

7.4

Endotoxins

The majority of microorganisms present in water purification systems exist in biofilms and are not free floating in the effluent water. For this reason and because microbial cells can be killed, prevented from replicating, and filtered relatively easily, high-purity water may contain a greater mass of microbiological byproducts (e.g., toxins, metabolites, cell fragments) than of viable cells that can replicate and be detected by plate count or epifluorescence. Thus, endotoxin measurements are a useful adjunct to viable cell techniques to determine the levels of water contamination by cell wall debris from, primarily gramnegative, microorganisms. Endotoxins should be specifically tested when they may be an interfering substance in a measurement procedure. The Limulus amebocyte lysate, LAL reagent, is an enzyme activation cascade that can be initiated by both endotoxins (lipopolysaccharide), which react with cascade Factor C, and specific types of polysaccharide [(1,3)-beta-D-glucans], which react with cascade Factor G. Lipopolysaccharides are present in the cell walls of gram-negative bacteria. Beta-glucans are present in the cell walls of Eumycota (mushrooms, sac fungi, yeast, molds, etc.), certain bacteria, and green plants, including algae. Water samples that test positive with a glucan-sensitive endotoxin assay should be evaluated with a glucan-insensitive or glucanspecific assay. It is always important to rule out cross-reactions and interferences by performing standard additions of known concentrations of endotoxin and/or a (1,3)-beta-D-glucan, depending on which entity is of interest. The bacterial endotoxins test45 recognizes three major techniques to measure the reaction of an LAL test: 1) an increase in the turbidity of the reaction mixture with time (kinetic turbidimetric method); 2) an increase in intensity of color with time (kinetic or endpoint chromogenic method); and 3) presence or absence of gelation after a given incubation period (gel-clot method). Each method requires reagent formulated for the intended method. In purified water, the sizes and numbers of microorganisms tend to be small; therefore, the concentration of lipopolysaccharide, a function of the surface area of the cell, is relatively low. The most sensitive LAL method (kinetic turbidimetric) may detect the presence of fewer than 25 small cells (or their cell wall equivalents) per milliliter. Gel-clot testing is substantially less sensitive than kinetic turbidimetric testing but can be performed without specialized equipment. 7.4.1

Sample Collection

The terms, “sterile” and “nonpyrogenic” or “pyrogen-free,” do not define container characteristics that ensure suitability for endotoxin sample collection or storage. Containers must be certified or tested to demonstrate that they are free of detectable endotoxin and interfering substances, consistent with the sensitivity of the endotoxin assay being used. Containers must also be certified or tested to demonstrate that they will not adsorb a significant amount of the endotoxin in a sample at the temperature(s) and over the period of time that the sample will be stored prior to analysis. In order to reasonably minimize surface effects, it is recommended that sample containers be filled and that samples be at least 25 mL, though far less sample is actually required for testing. Samples should be collected from points of use in the same manner as water is collected for normal laboratory use.

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Endotoxins are stable and can be transported to an off-site laboratory; however, assays should be performed promptly to avoid microbial growth in the sample. Refrigeration slows the growth of most microorganisms, and therefore, reduces the chance that the concentration of endotoxin will increase during storage. Freezing and thawing is believed to promote adsorption of endotoxins to container surfaces. 7.4.2

Equipment and Procedure

Endotoxin tests are either performed as titrations to a gelation endpoint (gel-clot) or by measurement of optical density (absorbance). The tests are enzymatic assays, so instrumentation must provide a constant temperature to all reaction vessels during the period of incubation. The gel-clot method requires a water bath or dry-block incubator to maintain 37 °C for the required incubation period. The gels are physically unstable, so the incubation equipment must minimize disturbance to the reaction tubes (e.g., circulating water baths may cause too much vibration). Typically, gel-clot tests are sensitive to about 0.03 endotoxin units (EU)/mL; however, some reagents may be sensitive to as little as 0.015 EU/mL. When single-test vials (STVs) are used, water samples are added to small tubes containing premeasured, gel-clot test components, mixed, and incubated for a specified period of time. When multitest vials (MTVs) are used, water samples are added to reaction tubes and then reconstituted LAL components are added, the tube contents are mixed, and the tube is incubated for a specified period of time. A positive test is defined by presence of a clot that has formed in the bottom of a tube and that is sufficiently stable, so the tube can be inverted without causing the clot to dislodge. Any tube in which a solid gel has formed contains at least the concentration of endotoxin indicated by the reagent sensitivity. The concentration of endotoxin in a sample is measured by testing serial dilutions; as endotoxin is diluted to less than the sensitivity of the reagent, an endpoint, the last positive test in a series of dilutions, is reached. The dilution factor at the endpoint multiplied by the sensitivity of the reagent is the concentration of endotoxin in the undiluted sample. Gel-clot tests are standardized by testing a series of standard reference endotoxin concentrations that bracket the labeled sensitivity of the reagent. The endpoint observed in the standard series must be within the 50 to 200% error of the test for the test to be valid. Standard incubation time for this type of test is one hour. Tests based on reading absorbance use reagents formulated so the turbidity that develops during the gelation reaction or the intensity of color that develops on hydrolysis of an artificial substrate can be measured with a spectrophotometer. There are two methods of reading photometric tests—endpoint and kinetic. For endpoint methods, the absorbance is measured after a given period of incubation. The incubation period defines the sensitivity of the test; the shorter the incubation time, the less sensitive the test. Endpoint photometric tests are typically sensitive to 0.005 EU/mL. For kinetic methods, the rate of increase in absorbance is measured. Incubation proceeds until the data are sufficient to calculate the rate (or a value related to rate called a “time of onset”: the time taken to reach a predetermined threshold optical density). Both reading techniques use curves obtained by testing known concentrations of a standard reference endotoxin to compute endotoxin levels for unknown samples. Kinetic tests may be as sensitive as 0.001 EU/mL when read intervals are short and individual reactions precisely timed. Incubation times for endpoint and kinetic methods may range from 15 to 20 minutes for sensitivities around 0.25 EU/mL and 90 minutes for sensitivities as great as 0.001 EU/mL. The accepted error of photometric methods is typically 50 to 200%, the same as for gel-clot.45 Even though photometric methods allow calculation of a numeric value for the unknown concentration of endotoxin, the error is still broad because of inherent characteristics of LAL reactions. Therefore, it is very important to follow the instructions of the LAL manufacturer to obtain the best results from their reagents and instrumentation. Endpoint tests are usually done in 96-well, plastic plates that are incubated separately from the plate reader. Kinetic tests are usually done in 96-well, plastic plates, using an incubating plate reader, or in glass reaction tubes, using instruments that have been specialized for kinetic testing. In all cases, ©

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incubation temperatures must be uniform. Kinetic tests also require short read intervals (some instruments read all reaction vessels every ten seconds). Instruments used to make turbidity measurements must not disrupt the distribution of fine suspensions in the reaction vessel. 7.4.3

Standardization

Companies that provide LAL test materials usually provide standardized endotoxin for calibration purposes. The activity of these secondary standards is determined by reference to the United States Pharmacopoeia Reference Standard, which is currently the same preparation as the Second International Standard of the World Health Organization preparation (94/580) and that of the FDA (EC-6). The recommendations for performing potency determinations have been published.46 One would expect to standardize a kinetic LAL assay with a range of dilutions; however, it is also important to standardize each lot of gel-clot reagents. There are two reasons for doing so: 1) to confirm that a lot has the expected sensitivity; and 2) to become familiar with the appearance of the reaction mixture and clot over a bracketing range of dilutions.

7.5 Determination of Oxidizable Organic Substances, Expressed as Total Organic Carbon (TOC) Organic substances are monitored by oxidizing them and detecting the resulting oxidation products. By convention, the measurement is expressed as total organic carbon (TOC). When a water sample contains an unknown mix of organic molecules, as is usually the case, TOC cannot be related to the concentration of organic molecules in the unknown water because the amount of carbon is different in different molecules. For example, 100 ng/g (ppb) of carbon is present in a solution of 131 ng/g (ppb) phenol or 990 ng/g (ppb) chloroform, because phenol contains 76% by weight of carbon and chloroform contains 10% by weight of carbon. However, in practice, changes in the type or concentration of organic contaminants are likely to change the measured values for TOC. It is important to initiate investigation of the water purification system in response to changes in TOC, even if the values are below the specification or alert levels. A number of different approaches can be used to determine TOC, and each has merits and limitations that are discussed in the following sections. 7.5.1 7.5.1.1

Sampling Off-line

Measurements made on water samples that are collected into containers and transported to an instrument are considered to be off-line. Off-line measurement is not recommended when the TOC specification for purified water is below 50 ng/g (ppb), because the potential for contamination of the sample by organic and inorganic substances in ambient laboratory air, transfer systems, and containers will likely introduce significant error. Sample containers must be impermeable and free of organic substances. Narrow-mouth borosilicate glass containers are recommended. They should be carefully washed, rinsed, heated in an oven to 450 °C (below the strain point of the glass) with a clean air atmosphere for approximately one hour, and allowed to cool in the oven. This oven procedure is recommended, because it avoids a final rinse stage, which may introduce organic contaminants. Screw caps should be lined with metal foil or PTFE (polytetrafluoroethylene), and carefully cleaned. Ground glass stoppers can also be used. Alternatively, commercially available, precleaned, certified, low-TOC containers can be used. Containers should be validated as suitable for TOC determination by measuring blanks. 36

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Containers should be closed immediately after filling, and their closures should be protected from contamination that could enter the sample when they are opened (e.g., by placing the containers in plastic bags). Certified, precleaned containers are available with a secondary cover for their closures. If samples will not be analyzed within 24 hours, they should be protected from light and stored in a refrigerator but not frozen. 7.5.1.2

On-line

For on-line measurements, the instrument is connected directly to the purified water stream. On-line instruments permit continuous or semicontinuous determination of organic contamination. On-line measurement is recommended for TOC specification levels of <50 ng/g (ppb) and highly recommended when TOC specification levels are <20 ng/g (ppb).47 On-line measurements use a variety of sampling designs appropriate to the instrumentation, accuracy, and frequency of monitoring desired. The instrument can be connected to the main water stream by means of a connection designed to prevent contamination and to divert a sample portion of the stream for continuous or intermittent analysis. Depending on instrument design and other factors, the oxidized water sample can be directed to waste or recycled through an upstream stage of the water purification system to remove contaminants introduced by oxidation. Alternatively, a portion, or all, of the main water stream may pass through the measuring cell and continue on with the main stream, in which case there should be a subsequent repurification step to remove impurities introduced by oxidation. There must be verification that such a repurification step does not introduce significant organic contamination. 7.5.2

Oxidation of Organic Molecules

Purified water can contain a wide variety of organic compounds. Some organic molecules are oxidized more easily than others. Oxidation does not take place instantaneously, and the kinetics of oxidation depend on the nature of the organic material present and the oxidation conditions.48 The following techniques have been successfully used in various instruments for oxidation of organic species in purified water samples: •

185 nm and 254 nm UV light;

•

185/254 nm or 300 to 400 nm UV light combined with a catalyst;

•

persulfate and 185/254 nm UV light at room temperature or 90 °C;

•

persulfate at 100 °C;

•

ozone; and

•

high temperature (680 to 1050 °C) catalytic oxidation.

As a general rule, complete oxidation of organic molecules contributes to the accuracy of a TOC result. Some instruments oxidize for a fixed period of time that has been qualified to ensure that all organic molecules likely to be in the water sample are fully oxidized. Other instruments use dynamic techniques to determine when oxidation is complete by, for example, waiting until no more CO2 is produced or the resistivity of the sample reaches a constant level. Some instruments are designed to partially oxidize the organic contamination in a sample and are discussed further in Section 7.5.4. Laboratories must validate that the oxidation process used is effective for the purpose intended. The validation may be performed by the user or by the instrument manufacturer, and requires measuring the recovery of standard solutions containing molecules likely to be encountered in purified water, including ©

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molecules that are difficult to oxidize. For example, sucrose is frequently used as an easily oxidizable molecule, and p-benzoquinone as one that is more difficult to oxidize.49 7.5.3

Instruments That Determine TOC

Instruments that determine TOC are designed to detect CO2 selectively; their detectors respond minimally, if at all, to the other products of organic oxidation. 7.5.3.1

Terminology

•

TC (total carbon) is the total concentration of carbon (organic and inorganic) in a sample.

•

TOC (total organic carbon) is the total concentration of carbon contained in organic molecules in a sample. Elemental carbon is included as organic carbon.

•

TIC (total inorganic carbon) is the total concentration of carbon in the form of carbonate (CO3=), bicarbonate (HCO3–), and dissolved CO2 in a sample.

•

POC (purgeable organic carbon) is the concentration of carbon that escapes the sample in the gas phase during the process of sparging the sample to remove inorganic carbon prior to measuring the organic carbon. POC depends on the instrument design and operating conditions as well as the nature of specific organic molecules present.

•

NPOC (nonpurgeable organic carbon) is the concentration of organic carbon remaining after sparging a sample to remove inorganic carbon. NPOC depends on the instrument design and operating conditions as well as the nature of specific organic molecules present that can be lost as POC.

7.5.3.2

Nondispersive Infrared (NDIR) Detection

NDIR detectors measure CO2 in a dry gas phase and are specific for CO2. TOC instruments that use NDIR detectors can be designed to make two measurements, TIC and TC. First, the water sample is acidified (pH ≤2) to convert TIC to CO2, which is sparged by carrier gas through the NDIR detector in a recirculating loop. Because NDIR detectors are specific for CO2 molecules, the concentration of TIC can be determined, even though other inorganic gases and POC may be present in the recirculating loop. After the TIC has been determined, the recirculating gas loop is kept closed, the sample is oxidized, and the TC is determined. There is no loss of POC, because both NPOC and the recirculated POC are oxidized. Therefore, subtracting TIC from TC will equal TOC. As the TIC concentration increases with respect to TC, the uncertainty of TOC calculations will increase, because two independent measurements, each with an associated error, are being subtracted. Traces of moisture and impurities in the gas phase, the purity of the acidifying and oxidizing reagents, and the relative volume of the gas phase can also affect the limits of detection and accuracy. Alternatively, inorganic carbon can be removed from a water sample by acidifying and sparging without determining the inorganic carbon released. However, this approach determines NPOC, rather than TOC. Sparging to remove inorganic carbon also removes POC, which can include short chain aliphatics, alcohols, ketones, esters, halomethanes, and aromatic compounds such as benzene, toluene, and cyclohexane. Measuring NPOC can fail to detect significant organic contamination from POC molecules. When water purification systems produce water with a resistivity approaching that of pure water, and measurements are made on-line to prevent CO2 absorption from air, TIC will be very low and, depending on the TOC limit required, may be negligible and the step of an analysis that is intended to calculate, or 38

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remove, TIC can be omitted. In this special case, there will be no loss of POC, and instruments with this design will determine TOC. 7.5.3.3

Resistivity Detection

Resistivity detectors cannot be specific for CO2 unless ions other than CO3=, HCO3–, and H+ are excluded. Interposing a membrane that is selective for small gas molecules such as CO2 (e.g., Teflon®b AF, or similar) between a sample chamber and a resistivity cell can achieve the necessary selectivity. TOC instruments that use resistivity detectors combined with such membranes can be operated in much the same way as NDIR detectors (see Section 7.5.3.2). Some of these instruments split samples into two essentially matched paths to achieve greater stability. In one path, the sample is acidified to determine TIC, and in the other path the sample is oxidized and acidified to determine TC. The potential for error associated with increasing TIC is similar to that for instruments that use NDIR detectors. A contactor membrane may be used to reduce the impact of inorganic carbon and improve the accuracy of TOC determinations. As in the case of TOC instruments that use NDIR detectors, water with a resistivity approaching that of pure water will have a very low TIC and, depending on the TOC limit required, the step of an analysis that is intended to calculate, or remove, TIC can be omitted. 7.5.3.4

Calibration and Quality Control for TOC Instruments

TOC instruments are calibrated with external standards traceable to a recognized standards organization, preferably a national metrology institute (e.g., NIST in USA, IRMM in Belgium, and LGC in UK). Potassium hydrogen phthalate (KHP), sucrose, methanol, and p-benzoquinone are widely used. KHP is traceable for replaceable hydrogen content but not necessarily for carbon content. P-benzoquinone is more difficult to oxidize than some other molecules and thus serves as a more meaningful standard to verify the completeness of oxidation. A TOC instrument should be calibrated at a sufficient number of points to establish a calibration relationship that will satisfy the requirements for accuracy. Quality control procedures may vary with the technology used to determine organic contamination. In general, external quality control samples should be interspersed with purified water samples to confirm that instruments are stable. CLSI document C24—Statistical Quality Control for Quantitative Measurement Procedures: Principles and Definitions should be consulted for suitable techniques. If the controls indicate that an instrument’s calibration has drifted significantly, the instrument should be recalibrated. 7.5.4

Instruments That Are Not Carbon Specific

The term TOCEquivalent is used in this document to refer to values determined by instruments that are not carbon specific. TOCEquivalent instruments measure the change in resistivity of the water sample before and after UV oxidation, without a CO2-selective interface. They are most accurate when used with purified water that has a high resistivity, so the resistivity change due to the oxidation products can be distinguished from the baseline resistivity. TOCEquivalent instruments can be calibrated to give values consistent with TOC values when known organic compounds containing only carbon, hydrogen, and oxygen atoms are analyzed. TOCEquivalent values can differ from TOC values, because TOCEquivalent instruments respond to resistivity changes from all ions, not only ions related to CO2. Depending on the mix and concentrations of organic contaminants present in a water sample, different designs of TOCEquivalent instruments may give different values, and TOCEquivalent instruments may give values that are higher or lower than those from carbonspecific instruments.50 Values higher than a carbon-specific method are likely to be reported when organic b

Teflon® is a registered trademark of DupontTM.

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contaminants contain atoms other than carbon, hydrogen, and oxygen that oxidize to form strong ions. A chlorinated organic molecule producing H+ and Cl- ions is an example.51 TOCEquivalent instruments will report lower values when water samples contain ionized species, for example, carboxylic acids, that are oxidized to more weakly ionized species. However, in the carboxylic acid example, the difference becomes less significant as the resistivity of the water approaches 18 MΩ·cm, referenced to 25 °C, because the total concentration of ionized substances will be very low. Partial oxidation, used in some TOCEquivalent instrument designs, can also lead to TOCEquivalent values that may be higher or lower than TOC values. The oxidation products from a particular water sample may produce changes in resistivity that are different from the corresponding changes in resistivity that were produced by the standards on which the calibration was based. When TOCEquivalent instruments are used to monitor and trend organic contamination, a laboratory should establish a TOCEquivalent alert level that is consistent with the fit-for-purpose validation of the water. The TOCEquivalent alert level should be set to accommodate the potential differences between TOCEquivalent and TOC determinations, and the TOCEquivalent values should be periodically re-evaluated by comparison with values obtained by carbon-specific TOC measurements. In the case of SRW with low organic contamination, a TOCEquivalent specification may be established based on fit-for-purpose validation for the application and the measurement system used. 7.5.4.1

Calibration and Quality Control for TOCEquivalent Instruments

Calibration and quality control of TOCEquivalent instruments that are designed to permit the introduction of calibration or control solutions are analogous to those described in Section 7.5.3.4, except the organic substance(s) used as standards must not contain atoms other than carbon, hydrogen, and oxygen. In the case of instruments that do not have provision for introduction of calibration or control solutions, the manufacturer’s instructions for calibration and quality control of the instrument should be reviewed.

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References 1

ISO. Statistics – Vocabulary and Symbols – Part 1: Probability and General Statistical Terms. ISO 3534-1. Geneva: International Organization for Standardization; 1993.

2

ISO. International Vocabulary of Basic and General Terms in Metrology. Geneva: International Organization for Standardization; 1993.

3

Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Ann Rev Microbiol. 1995;49:711-745.

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ISO. Medical laboratories—Requirements for safety. ISO 15190. Geneva: International Organization for Standardization; 2003.

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ISO. Quality Management Systems – Fundamentals and vocabulary. ISO 9000. Geneva: International Organization for Standardization; 2000.

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Federal Register: Part VII Dept. of Health and Human Services, FDA. 12 CFR Parts 808, 812, and 820. Rules and Regulations. October 7, 1996;61(195):52222-52601.

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Levey S, Jennings ER. The use of control charts in the clinical laboratory. Am J Clin Pathol. 1950;20:1059-1066.

8

Shewhart WA. Economic Control of Quality of Manufactured Product. New York: Van Nostrand; 1931.

9

Meltzer TH. High Purity Water for the Semiconductor, Pharmaceutical, and Power Industries. Littleton, CO: Tall Oaks Publishing, Inc.; 1993:800.

10

Meltzer TH. Pharmaceutical Water Systems. Littleton, CO: Tall Oaks Publishing, Inc.; 1996.

11

Bole J, Mabic S, Long J. Impact and removal of bacterial alkaline phosphatase in water used in clinical analyzer chemistry. Clin Chem. 2005;51:A203.

12

Darbouret D, Kano I. Ultrapure water blank for boron trace analysis. J Anal At Spectrom. 2000;15:1395-1399.

13

Thate S, Specogna N, Eigenberger G. Electrodeionization - A comparison of different EDI concepts used for the production of high-purity water. Ultrapure Water. 1999;16(8):42-56.

14

Salem E. Deionization - Areas to consider when selecting an EDI system. Ultrapure Water. 2000;(6):72-76.

15

Megonnell N, McClure A. Cabon and MTBE – What’s a dealer to do? Water Conditioning & Purification. 2000;72-75.

16

ASTM D3860-98(2003). Standard Practice for Determination of Adsorptive Capacity of Activated Carbon by Aqueous Phase Isotherm Technique. ASTM International; www.astm.org. 2003.

17

Nowicki HG, Schuliger PE. Cabon software programs: sorbent performance evaluation ASTM aqueous phase isotherm program. Water Conditioning & Purification. 2000;102-106.

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MacLean RG. A new look at carbon: biological purification of drinking water. Water Conditioning & Purification. 1999:56-59.

19

Camper AK, LeChevallier MW, Broadaway SC, McFetters GA. Growth and persistence of pathogens on granular activated carbon filters. Applied Environmental Microbiology. 1985;50:1378.

20

Rollinger Y, Dott W. Survival of selected bacterial species in sterilized activated carbon filters and biological activated carbon filters. Applied Environmental Microbiology. 1987;53:777.

21

Adley CC, Saieb F. Biofilm formation in high-purity water: Ralstonia pickettii a special case for analysis. Ultrapure Water. 2005:14-19.

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http://www.nalgenelabware.com/techdata/Resin/index2.asp?m=&p=Permeability+CO2+%28metric%29&txtMError.

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Costerton JW, Stewart PS. Battling biofilms. Sci Am. 2001;285(1):74-81.

24

Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284(1):1318-1322.

25

Purevdorj-Gage LB, Stoodley P. Biofilm structure, behavior, and hydrodynamics. In: Ghannoum M, O’Toole GA, eds. Microbial Biofilms. Washington, D.C.: ASM Press; 2004.

26

Rickard AH, McBain AJ, Stead AT, Gilbert P. Shear rate moderates community diversity in freshwater biofilms. AEM. 2004;70(12):74267435.

27

Simoes M, Pereira MO, Vieira MJ. Action of a cationic surfactant on the activity and removal of bacterial biofilms formed under different flow regimes. Water Research. 2005;39:478-486.

28

Traeger H. Microbial control: how to protect against biofilm build up in loops and tanks. Ultrapure Water. 2005;22;24-30.

29

MacInnes DA. The Principles of Electrochemistry. New York: Reinhold Publishing Corporation; 1939:Chap. 3.

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30

Bevilacqua AC. Ultrapure Water - The Standard for Resistivity Measurements of Ultrapure Water. Proceedings of the 1998 Pure Water and Chemical Conference, Santa Clara, CA, USA, pp 141-172.

31

Carr G. The effect of plastic tubing type on oxygen and resistivity measurements in high-purity water. Ultrapure Water. December 2000:17-21.

32

Physical Properties of NALGENE Labware. Available at: http://nalgenelab.nalgenunc.com/techdata/resin/index2.asp?m=&p=Permeability +CO2+%28metric%29 and http://nalgenelab.nalgenunc.com/techdata/Resin/index2.asp?m=&p=Permeability+CO2. Accessed June 7, 2005.

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Morash KR, Thornton RD, Saunders CH, Bevilacqua AC, Light TS. Measurement of the resistivity of high purity water at elevated temperatures. Ultrapure Water. 1994;11(9):18-26.

34

Bevilacqua AC. The Effect of Temperature, Temperature Error, and Impurities on Compensated Conductivity Measurements. 16th Annual Semiconductor Pure Water and Chemicals Conference, Santa Clara, CA, March 3-6, 1997 (original data for Table 1 were provided by the author).

35

Light TS. High purity water – conductivity cell standard for low ionic strength solutions. Ultrapure Water. 1991;8(3):59-63.

36

Thornton RD, Light TS. A new approach to accurate resistivity measurement of high purity water. Ultrapure Water. 1989:14-21.

37

ASTM D5391, Vol. 11.01, April 2003. Standard Test Method for Electrical Conductivity and Resistivity of a Flowing High Purity Water Sample. Appendix A2.

38

APHA, AWWA and WEF. Standard Methods for the Examination of Water and Wastewater. 19th ed. Washington, D.C.: APHA; 1995:9216.

39

Kepner RL, Pratt JR. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiological Reviews. 1994;58:603-615.

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McFeters G, Pyle B, Lisle J, Broadaway S. Rapid direct methods for the enumeration of specific, active bacteria in water and biofilms. J Appl Microbiol. 1999;85:193-200.

41

McAlister MB, Kulakov LA, Larkin MJ, Ogden KL. Microbials - analysis of bacterial contamination in different sections of a high-purity water system. Ultrapure Water. 2001;18(1):18-26.

42

Boulos L, Prevost M, Barbeau B, Coallier J, Desjardins R. LIVE/DEAD BacLight: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J Microbiol Meth. 1999;37:77-86.

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McFeters G, Yu FP, Pyle BH, Stewart PS. Physiological assessment of bacteria using fluorochromes. J Micro Meth. 1995;21:1-13.

44

AWWA, AWWA and WEF. Standard Methods for the Examination of Water and Wastewater. 19th ed. Washington, D.C.: APHA; 1995:9216.

45

USP28-NF23 S1. General Chapters <85> Bacterial Endotoxins Test - The United States Pharmacopoeial Convention Inc.

46

Food and Drug Administration publication. Guideline on validation of the Limulus Amebocyte Lysate test as an end product endotoxin test for human and animal parenteral drugs, biological products and medical devices. December 1987.

47

Donovan RP, Painton Swiler LA, DeGenova J, Boswell T. Evaluating on-line TOC analyzers for high purity water recycle systems. Ultrapure Water. 1998:30-36.

48

Peyton GR. The free-radical chemistry of persulfate-based total organic carbon analyzers. Marine Chemistry. 1993;41:91-103.

49

USP28-NF23 S1. General Chapters <643> Total Organic Carbon - The United States Pharmacopoeial Convention Inc.

50

Rydzewski J. Instruments - identification of critical contaminants by applying an understanding of different TOC measuring technologies. Ultrapure Water. 2002;19:20-27.

51

McCurdy L. Implementing TOC testing for USP 23 – a case study. Pharmaceutical Engineering. 1997;17:2-7.

42

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Additional References Byrne W. Encyclopedia of Water Treatment – Interactive CD 17 Volumes. Littleton, CO: Tall Oaks Publishing; 2004. Hanselka R, Reinzuch KJ, Bukey M. Materials of construction for water systems, part II: real life failure modes of plastics. Ultrapure Water. 1987;4:50-53. Hanselka R, Williams R, Bukey M. Materials of construction for water systems, part I: physical and chemical properties of plastics. Ultrapure Water. 1987;4:46-50. Standard Methods for the Examination of Water and Waste Water. 20th ed. Water Environment Federation, Alexandria, VA; 1998.

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Appendix A. Resistivity Measurement in a Sparged Water Sample Argon Input Output Cin = 0, flow fAr

CAr(t), fAr

headspace

Input water

Co , f

V, C(t)

C(t), f

Output water

Stirrer Figure A1. Model for Argon Purge of Water in a Bottle NOTES: 1. 2. 3. 4. 5. 6. 7.

V is the volume of water in the bottle (assumed constant). Co, C(t), CAr(t) are the respective concentrations of CO2 at the locations noted in the figure. Perfect mixing is assumed in V and in headspace. f and fAr are flow rates at given locations, each assumed constant. Distribution coefficient: KH = C(t)/CAr(t), i.e., CAr(t) = C(t)/KH, (Henry’s Law) is assumed to hold at all t. The purge gas (Ar) is assumed to be CO2-free. Rate of transfer of CO2 from gas phase to aqueous phase or vice versa is governed by physical absorption only [i.e., any effect of the chemical kinetics of the reaction CO2(aq) ↔ H2CO3(aq) is neglected].

For the case where the removal of CO2 from solution or the absorption of CO2 into solution depends solely on physical absorption, i.e., transfer from the gas phase to aqueous solution or vice versa, the timedependent concentration of CO2 in solution may be derived from the initial value problem (IVP) defined by the following first-order differential equation:

C ' (t ) +

fC f Ar + fK H C (t ) = 0 K HV V

(A1)

with the boundary conditions for C(t) (general case):

C (0) = C0

C (t → ∞) = C∞

(A2)

The general solution to this IVP is given by Equation 3:

⎛ f + fK H ⎞ ⎟t C (t ) = C∞ + (C0 − C∞ ) exp⎜⎜ − Ar K HV ⎟⎠ ⎝

44

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Appendix A. (Continued) For the case with boundary conditions:

C ( 0) = 0

C (t → ∞) =

fK H C0 f Ar + fK H

(A4)

i.e., starting with CO2-free water in the bottle and introducing CO2-containing water with CCO2 = C0, Equation (3) becomes:

C (t ) = C0

⎛ f Ar + fK H ⎞ ⎤ fK H ⎡ ⎟t ⎥ ⎢1 − exp⎜⎜ − f Ar + fK H ⎣ K HV ⎟⎠ ⎦ ⎝

(A5)

For the case with boundary conditions:

C ( 0) = C 0

C (t → ∞) =

fK H C0 f Ar + fK H

(A6)

i.e., for purging that starts with water in the bottle having CCO2 = C0, Equation (3) becomes:

⎡ fK H ⎛ ⎞ ⎛ f + fK H ⎞ ⎤ f Ar ⎟⎟ exp⎜⎜ − Ar ⎟t ⎥ + ⎜⎜ C (t ) = C0 ⎢ K HV ⎟⎠ ⎦ ⎝ ⎣ f Ar + fK H ⎝ f Ar + fK H ⎠

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(A7)

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Appendix B. Methods for Correction or Compensation of Resistivity Measurements Following is a discussion of three methods used in the correction or compensation of resistivity measurements: In automatic temperature compensation, the meter’s probe cell contains a temperature compensation circuit element that internally adjusts the meter to read corrected resistance directly. 1. In manual temperature compensation, automatic compensation is not provided. An accurate temperature measurement should be taken simultaneously with the resistivity measurement. When the temperature of the water measurement is outside the 25 ± 2 °C range, correct the resistivity reading to 25 °C according to the meter instructions. In the absence of meter instructions for manual correction, determine the temperature coefficient empirically. Measure the subject solution at two different temperatures (preferably at 5 °C above and at 5 °C below the expected measurement temperature), then perform the following calculation:

ρt is the resistivity reading at temperature t. ρ1 is the resistivity reading at temperature t1, 5 °C above temperature t. ρ2 is the resistivity reading at temperature t2, 5 °C below temperature t. ρavg is the arithmetic average of the resistivity readings at temperatures t1 and t2. ρ25 is the calculated, temperature-compensated resistivity at 25 °C, temperature t25.

∆t is the absolute value of the difference between t25 and t, rounded to the nearest whole number. f is the average fractional change in resistivity per degree Celsius at temperature t. ρ avg

f =

=

ρ2

+ρ1

(B1)

2

ρ2

− ρ1

(B2)

(t1 − t 2 ) ρ avg

If t is above 25 °C, then use the following equation:

ρ 25 = ρ t (1 + f )∆t

(B3)

If t is below 25 °C, then use the following equation:

ρ 25 = ρ t (1 − f )∆t

(B4)

2. In the temperature range of 20 °C to 30 °C, the temperature-compensated resistivity at 25 °C calculated in this manner should be within 3% of the resistivity value, had it actually been measured at 25 °C. For larger temperature deviations, the Marsh-Stokes equation (or a similar curve fit) should be used instead of the linear (first-order) relationship assumed here. 3. The following temperature compensation equation is known as the Marsh-Stokes equation and illustrates the general approach used in many instruments. This equation converts any measured electrolytic conductivity reading in aqueous solution to its equivalent value at 25 °C. The equation assumes that the inherent (mobility-dependent) temperature coefficient of each ion is 2%/°C. NOTE: The Marsh-Stokes equation is phrased in terms of κ, not ρ. 46

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Appendix B. (Continued) κ 25 =

κ t - [0.05498 + 2.9326 • 10 -3 (t - 25) + 6.0629 • 10 -5 (t - 25) 2 + 4.2690 • 10 -7 (t - 25) 3 ] 1 + 0.02(t - 25)

+ 0.05498

where:

κt is the electrolytic conductivity in µS/cm, at temperature, t. κ25 is the electrolytic conductivity in µS/cm, at 25 °C.

t

is the temperature of measurement in °C.

κ25 is the calculated, temperature-compensated resistivity at 25 °C.

The Marsh-Stokes equation is based on the following compensation algorithm: a. Take the total measured electrolytic conductivity, κt, at the measurement temperature t. b. Subtract the conductivity of pure water at t from κt. The result (numerator of the first term of the Marsh-Stokes Equation) is the conductivity attributable to impurity ionized species in the water. c. Correct the result from (b) to the conductivity attributable to impurity ionized species in the water at t = 25 °C. This operation is performed by the denominator of the first term. d. Add κ for H2O at t = 25 °C to yield the corrected total conductivity at t = 25 °C, κ 25. State-of-the-art instrumentation may substitute a proprietary model in place of the Marsh-Stokes equation with a proprietary equation to model the κ - t relationship in the given instrument. Typically, the general approach is similar to the algorithm of the Marsh-Stokes equation, but the models for calculating the temperature-κ relationships (for pure water and for the impurity ion components) are more refined. Users should consult with the manufacturer or vendor to clarify the exact equation used and its validity range.

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Clinical and Laboratory Standards Institute consensus procedures include an appeals process that is described in detail in Section 8 of the Administrative Procedures. For further information, contact CLSI or visit our website at www.clsi.org.

Summary of Consensus and Delegate Comments and Working Group Responses C3-P4: Preparation and Testing of Reagent Water in the Clinical Laboratory; Proposed Guideline— Fourth Edition General 1.

I have a major concern with the removal of a silica specification CLRW. I understand that CLRW is a newly defined type of laboratory water; however, it is effectively the evolution of Type 1 reagent water from previous documents. The chemistry of silica, and especially colloidal silica, is extremely complex. The exact interference(s) that silica can cause are often unpredictable. The exact speciation of silica in water is very dependent on the pH, ionic strength, and specific anions and cations present in solution. One form of silica, for example, dissociated ionic silica, might not interact with other analytes, whereas the same level of colloidal might interact. Minor shifts in pH often significantly change the form of silica in solution. The only sure way to minimize the potential interactions of silica with clinical assays is to minimize the level of silica in the water— period. This document appropriately points out, and provides data showing that high resistivity water can contain significant levels of organic contaminants and silica (data in Figure 1). In many ways, silica contamination is like TOC in water. I’m sure that certain organic contaminants would have little impact on clinical assays, whereas other types of organics could have a very significant impact. Exact speciation of organics in water is often difficult and the best way to minimize potential problems is to minimize organic contaminants and to measure the level so that consistent water quality can be maintained. Silica levels should be minimized and monitored for exactly the same reasons. Some potable feed waters will have negligible organic content, and TOC will never be an issue with CLRW prepared from these waters. Other feed water can have significant levels of complex organics that can, and will, cause problems. Only by specifying a maximum TOC level can consistency be ensured. Silica levels in feed waters also vary considerably from location to location. I was pleased to see that a TOC specification has been included in this document. I find it ironic, and frankly troubling, that in the same document the silica specification has been removed. A silica specification is needed for CLRW.

•

A silica specification and test requirement were eliminated from the clinical laboratory reagent water designation in this edition for several reasons: there is no literature supporting silica interference in clinical laboratory assays; silica assays are difficult to perform with adequate sensitivity at low concentrations; if silica concentrations are high enough to be a significant concern, it is likely some aspect of the water purification system has failed and other substances will be at high enough concentrations to be detected by the testing that is included in the specifications. If a specific clinical laboratory measurement procedure is sensitive to silica, it would be appropriate to include a silica specification for a special reagent water designation; just as would be recommended for any substance that is of particular concern for a specific measurement procedure. Colloidal silica in the feed water can be a significant consideration in the design of a water purification system, and the guideline recommends that the local feed water be evaluated as part of the user requirements specification process (see Section 5). Colloidal silica can be a source of fouling of liquid handling components in automated measurement systems, and an instrument feed water specified by the manufacturer of such equipment should address all necessary requirements.

2.

The example of degradation of the resistivity of water in polypropylene tubing in Appendix A contains a number of significant flaws. I will provide details and the correct results later in my review. However, this appendix must be either completely reworked or removed from the document. I'm not exactly certain of the intended point of the analysis. If the objective was to show that resistivity will degrade in tubing in a matter of

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minutes, the correct analysis will show that this is not the case. In fact, the same level of degradation of resistivity (to about 3.5 mΩ·cm) will actually take many hours, not minutes. •

The original Appendix A has been deleted due to technical complexity; see response to Comment 68 for details.

3.

I have reviewed this document primarily from the perspective of a clinical laboratorian, in order to evaluate its ease of application in today’s clinical laboratory. I would like to point out that most hospitals in the US are less than 100 beds. In rural areas, over 80% of institutions are less than 100 beds; in fact 46% are less than 50 beds. Rural hospitals represent approximately half of all hospitals in the US. In addition, approximately ¼ of urban hospitals are less than 100 beds. [Loux SL et al. Comparing patient safety in rural hospitals by bed count. Adv in Pt Safety. 1:391-402 (2005)]. Hospital bed capacities continue to decrease in many areas, and staffing is approaching crisis levels with fewer well-trained technologists in the workforce pool. Even now, there is still incredible ignorance in the clinical laboratory when it comes to reagent grade water requirements. Having inspected many laboratories for the CAP, I note that laboratory staff still often refer to any water purification system as “the distilled water” system and write procedures referring to it as such. The level of ignorance will continue to increase as more well-trained professionals retire or leave the profession, leaving laboratory management to those who are already overburdened. This document does nothing to ensure that laboratories will be able to install and maintain water systems since everything is left up to the user to “validate.” While I understand this is meant to be an international document, it is my understanding that many foreign countries have adopted previous versions of the Water Guideline. I have no knowledge whether other countries face a similar crisis; however, CLSI still needs to keep in mind the crisis in the U.S. I note that the subcommittee did not include any representatives from the US manufacturers, except for Dr. Gibbs. Where was Millipore (US) or US Filter? While companies may be international, better representation from the US would have been desirable. Since this document is under review by two professionals whose expertise is water purification and monitoring, I will omit comments on definitions and technology issues to those who are better informed than I.

•

The committee agrees with the commenter that users will benefit from better understanding of purified water, how it is produced, and how purification systems need to be monitored to ensure suitable water quality. The document was revised to provide a user with adequate information to participate in an informed discussion with vendors of water purification systems for the purpose of making an appropriate selection of equipment to meet the laboratory’s needs. The guideline contains more information than the previous edition to assist a user with understanding water purification technology, and to enable the user to understand the importance of operating the equipment correctly. This edition includes new recommendations on validating the water as fit for its intended purpose in laboratory testing, and an important emphasis on monitoring for trends or changes in the parameters used to verify the quality of the water to ensure that purification systems continue to produce water meeting the purity requirements. The guideline has been edited in response to several reviewers’ comments to provide additional practical advice suitable for the novice user. It is important for the user of a large or small water purification system to validate system performance, and to monitor system performance in a manner similar to other equipment in the laboratory. Manufacturers of self-contained systems, commonly used in smaller laboratories, should provide appropriate information and protocols to users. Larger system installations should also include suitable instructions for the user to validate the system’s performance. The working group had members from both Millipore and US Filter, although they were from non-USA offices. In addition, the C3-P4 document was sent to seven special reviewers that included several USA manufacturers of water purification and water testing equipment. Four special reviewers, named in the acknowledgements, provided comments.

4.

©

I was disappointed to see extreme and major changes in the proposed C3-P4 document. This document is no longer serviceable to the user. Most laboratorians do not have a workable knowledge of a water treatment system and rely on a company salesperson for information. The salesperson may actually have limited training in how systems work. One very important table that provided a list of major contaminants and the types of treatment for their removal has been omitted.

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The text is too generalized and needs to be more concise. Terms are used without appropriate references. It appears that the guideline leaves quality assurance to outside sources or validation. What is meant by the term validation? In cases of litigation regarding use of product water in tests or other procedures, this could be a serious problem. •

See response to Comment 3 regarding improving the usefulness of information to the user and validation. Validation is used as defined by ISO and has been further clarified by edits in Section 5. The table in the previous edition was eliminated because it was an oversimplification. The technical detail in Section 6 has been expanded from the previous edition to provide the laboratory user with adequate information to understand the different technologies used in water purification and to engage a water purification system vendor to ensure the system selected will meet the laboratory’s requirements.

Scope 5.

Five types of “purity grade water” are listed. It states that procedures are provided for measuring and trending parameters. What is the scientific definition of a trending parameter? What is meant by the term “trending parameter to control”?

•

“Trending” is used as a verb to indicate the measured parameter is monitored by reviewing the trend in results over time and interpreting the current value in relation to previous results for the parameter. The text has been edited to more clearly reflect this usage.

Foreword 6.

The addition of TOC as a monitored contaminant is a positive change; however, the elimination of silica testing is problematic, since this has to be controlled in areas where high silica content in the feed water is an issue. Simply stating that the system should be designed to take this into account is hardly adequate. Our laboratory, for one, will continue to monitor this as long as I am responsible for water quality in our three institutions. The paragraph in the forward even admits that silica may be a problem when ion-exchange beds are nearing depletion.

•

See response to Comment 1.

7.

I take serious issue with the last paragraph on sterility of product water. It needs to be removed. Tissue culture work cannot be done with nonsterile water. Water defined by this document has never been restricted to only the clinical laboratory. Research and industrial laboratories have benefited from the guideline document. If it is intended for clinical only, then that needs to be underscored in document.

•

The text has been clarified regarding sterility requirements for culture in the Foreword and in Section 4.3, Special Reagent Water. The guideline is intended primarily for clinical laboratory requirements as stated in its title; however, the information should be useful in other laboratory settings.

Key Words 8.

Key words and purified water subgrades (see Scope) need to be added to glossary. Usage is confusing in text.

•

The water types have been added to key words; they have not been added to the glossary because they are defined by the specifications in Section 4, and a short definition could lead to misunderstanding.

Introduction 9.

Paragraph 1—This paragraph is poorly constructed and needs to be deleted.

•

The text has been clarified.

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10. Paragraph 2—The last sentence needs further detail. •

The text has been clarified by combining the first two paragraphs and editing the text.

11. Paragraph 3; The last sentence suggests that routine monitoring is optional. Maintenance and operation is key to the performance of a system. Persons without background in water treatment would benefit from specific information in this guideline. •

The text has been edited to reflect that monitoring and maintenance are requirements.

Section 3, Definitions 12. The units between brackets for “resistivity” should be corrected. •

The raised dot has been used between multiplied units in accordance with the American National Standard for Metric Practice, ANSI/IEEE Std 268-1992 (Institute of Electrical and Electronics Engineers, New York, NY, October 1992).

13. Electrodeionization (EDI): The phrase “impurity ionized species” seems cumbersome and confusing. Would “ionized impurities” be better? •

The suggested change has been made.

14. Softening: The phrase “whereby cations are exchanged” should read “whereby multivalent cations are exchanged.” •

The definition of softening has been edited to include this point.

15. Total organic carbon (TOC): To the best of my knowledge, the scientifically correct term for this measurement is “total oxidizable carbon.” In many ways, this is similar to the nearly interchangeable use of “pyrogens” and “endotoxin.” In both cases, there is a difference between what is scientifically correct and what term is commonly used. Since this is meant to be a scientific document, I would favor the more precise definition. •

Both definitions have been used in the past; the definition total organic carbon is used in this document because, although the measurement technologies oxidize organic molecules, the result is expressed as carbon content.

Section 4, Specifications 16. Paragraph 5—How would a laboratory set up a risk assessment for use of this guideline? •

The important point for the user is to be aware that risk must be considered relative to the use of the water and the quality monitoring that is necessary to maintain the water at an acceptable purity. A definition of risk assessment has been added to the definitions. It would not be practical to provide a list of risk assessment procedures that would be applicable to all laboratories. This guideline is intended to provide laboratories with appropriate information to determine if their water purification is adequate to meet the laboratory use requirements.

Section 4.1, Organization of Water Purity Specifications 17. The sentence is unclear. •

The text has been revised to improve the clarity.

Section 4.1.2, Clinical Laboratory Reagent Water (CLRW) 18. A maximum allowable silica level should be added as a specification (see discussion of this in Comment 1). ©

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See the response to Comment 1.

Section 4.1.2.1, Ionic Impurities 19. A sample either has a resistivity of >10 mΩ·cm or it does not. Further “processing” of a sample water to confirm that a specification is met only introduces error in the measurement. How does a user confirm that significant ionic contaminants were not added to the water in the process of removing CO2? I understand the objective of trying to differentiate dissolved CO2 (actually HCO3– is dropping the resistivity) from other ionic contaminants. I just don't think that the method described in this document is feasible in a real laboratory setting. •

As explained in the text, dissolved CO2 (as HCO3– plus H+) will lower the resistivity of otherwise pure water but has no effect on most clinical laboratory procedures. Consequently, removing CO2 prior to resistivity measurement allows a laboratory to verify the specification when the water may contain dissolved CO2, and it has been verified that the dissolved CO2 does not affect the laboratory procedures using that water. If dissolved CO2 did affect a procedure, then a special reagent water with suitable specification may be more appropriate. The method has been tested in one working group member’s laboratory. It is not anticipated this will be a common procedure, but it is provided for the laboratory that may find it useful to verify a resistivity specification. The text includes precautions to avoid contamination of the water sample to be tested. If ionic contamination were introduced in the procedure, the result would fail the specification, and the user would be expected to review the procedure to ensure there was no contamination introduced in the water sampling or measuring process.

Section 4.1.2.4, Particulate Content 20. There is a typographical error – [0.22 should read < 0.22] •

The text is correct as written; particles ≥ 0.22 µm are blocked by the filtration stage.

Section 4.1.3, Other Grades of Purified Water for Use in the Clinical Laboratory 21. “Grades” of purified water are defined by specifications. Only special reagent water, instrument feed water, and probably water supplied by a method manufacturer actually have specifications. As later defined, prepackaged bottled water could range from sterile water for injection to Poland Springs water. This variability does not define a “grade” of water. Likewise, tap water (if it had low levels of inorganic, organic, and particulate impurities) could meet the autoclave and wash water application “grade” of water. Who decides what level of impurities is a low level? •

“Grades” has been replaced with “types” to avoid confusion. The text has been edited to clarify that water in prepackaged bottles is not expected to meet the specifications for clinical laboratory reagent water at the time of opening the container. Such water may have met those specifications at the time of bottling, but at the time of use would be considered a special reagent water. The laboratory would need to establish acceptance criteria and validate the water as fit for purpose similarly to validation of other reagents used by the laboratory.

Section 4.2.1, Ionic Impurities 22. I think it would be good to mention the theoretical maximum achievable resistivity in this section in case a reader of the document does not read the later sections covering resistivity. •

The information has been added as suggested.

Section 4.2.1, Ionic Impurities and Section 7.1.2.4, Temperature Compensation 23. Is it the solution of 0.34 µmol/L (4.2.1) or the solution of 0.34 ng/g (Table 1, Section 7.1.2.4) that gives a resistivity of 10 megaohm-centimeters? •

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A solution of 0.36 µmol/L gives a resistivity of 10 MΩ·cm at 25 °C; the text has been corrected. ©

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Section 4.2.3, Organic Impurities 24. As stated before in Comment 15, I believe TOC should be defined as total oxidizable carbon. •

See response to Comment 15.

Section 4.2.4, Particulate and Colloid Content 25. I think a brief explanation of the difference between a depth filter and surface, or screen, filter should be included here. Not all laboratorians will read later sections of the document where these differences are detailed. A very brief mention that there ARE different types of filters with the same micron rating is important to include in this section. •

The text has been clarified.

Section 4.6, Prepackaged Water 26. Comments about this section are already included above in Comment 21 on Section 4.1.3. •

See response to Comment 21.

27. I interpret CLRW as the replacement for type I water. Such water cannot be bottled or prepackaged. The water simply cannot meet the requirements for resistivity. In the previous water documents, a reference was included that demonstrated the observed degradation of bottled water in both glass and plastic [Gabler R et al. Degradation of high purity water on storage. J Liquid Chrom. 6:2565-2570 (1983)]. Certainly the TOC will change over time in plastic stored water; resistivity will continue to decrease; and the microbial content is likely to increase. Such statements as, “If a bottle is opened and reused over a period of time, the laboratory must validate the water remains fit for purpose throughout the entire period of use.” Do members of the subcommittee honestly believe that busy laboratorians in those small institutions are going to do all the required testing? •

See response to Comment 21.

Section 4.6.1, Proper Labeling 28. The phrase “for the lot of water as it exists in the container” should be replaced with “for the lot of water as it existed at the time of packaging.” There is no way to know the values of these parameters, short of measuring them, as they exist in a container. •

The suggested change has been made.

29. Define expiration date. Who sets this date? •

Section 4.6.2 states, “The laboratory must establish an expiration date for an opened container, based on storage conditions and validation of fitness for purpose, to prevent accumulation of microbial or other contaminants.” An expiration date is commonly understood to be the date after which the water is not to be used.

Section 4.6.2, Validation in the Clinical Laboratory 30. Generally, high-purity water degrades very quickly. Mention is not made as to how the bottled water is maintained/stored. •

©

It would be difficult to anticipate all likely laboratory scenarios. The text emphasizes the laboratory is responsible to verify that the water in an opened bottle remains fit for purpose.

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Section 5.1, Validation of Purified Water as Fit for Its Intended Purpose in Laboratory Procedures 31. Under the third bullet, the statement “authenticated sources of purified water may include: 1) a different lot that was previously validated.” Since water cannot be stored for long periods, how is this practical? Likewise “2) a sample of validated water that was saved prior to a purification system sanitization, and was stored under conditions that would prevent contamination.” Isn’t this the same as statement 1? The document seems to contain many such repetitions. •

Depending on context, the two statements could be interpreted as very similar. Each statement, however, can apply to different situations. For example, (1) might apply to purchased, packaged water, while (2) might apply to a central purification system that requires periodic sanitization and verification that the wash was adequate. Both are included to suggest different approaches that could apply in different situations. Storage of water is challenging; thus the caution regarding conditions that would prevent contamination. Section 6.8.2 provides more information on storage considerations so the user can make appropriate judgments regarding suitability of a storage condition for a specific application.

32. Last sentence: “Validation is confirmed, through the provision of objective evidence...” is misleading. This needs to be more specific. For example, a technician may consider objective evidence as turbidity, taste, or odor. •

Examples of objective evidence are provided in the next paragraphs of Section 5.1. The text has been edited to clarify this point.

33. Last paragraph—Statement is misleading. Delete or rewrite. Omit “confirmation that the patient results from the procedures meet the clinical requirements of the healthcare system.” •

The phrase has been rewritten for clarity.

Section 5.1, Validation of Purified Water as Fit for Its Intended Purpose in Laboratory Procedures and Section 5.2, Trend Monitoring of Water Purity Specifications 34. The more stringent requirements for “Validation and Trend Monitoring” (Sections 5.1 and 5.2) may certainly be a challenge for small and medium sized laboratories. •

The several approaches to meeting the requirements for validation are provided to allow laboratories to understand different approaches for obtaining the necessary objective evidence of water performance in laboratory procedures. They should not be burdensome to a small laboratory because, in most cases, the quality control procedures already in place will be sufficient, when extended to consider water as a potential source of error. The trend monitoring requirements are analogous to those already in place to monitor quality control results, instrument function checks, maintenance, etc. Including water quality monitoring in the normal review process will help to avoid problems that may be caused by changes in water purity.

Section 5.2, Trend Monitoring of Water Purity Specifications 35. The document recommends testing at “established regular intervals.” The user needs guidance regarding testing frequency. No doubt, there will be sites that select quarterly or semiannual testing for microbial content. •

The term “established regular intervals” has been removed. The text has been edited to clarify that a laboratory needs to make judgments regarding the risk of water quality deterioration adversely affecting results, and establish the frequency of monitoring the quality parameters to provide adequate data for trend monitoring appropriate to mitigate that risk.

36. Words such as “trend monitoring and excursions” need to be defined. •

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The text has been edited so that trend monitoring is explained in the context of quality control results monitoring, which should be familiar to laboratorians. “Deviations” has been added to the initial phrase that used “excursions” to assist in clarifying the meaning. ©

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Section 5.3.1, Validation of New and Major Upgrades to Water Purification Systems: A Master Validation Plan 37. I could not find a definition for URS in the document. This term may be very common in US laboratories, but I don't know what it is, and laboratorians in other countries might not, either. •

The words “user requirements specification” have been added in the text to define the initialism URS.

38. First bullet – Where is the definition of URS? •

The words “user requirements specification” have been added in the text to define the initialism URS.

39. Section 5.3.1.3 suggests simulation of adverse conditions. Seasonal changes are likely difficult to simulate and I seriously doubt that the vendors of purification systems will be in a position to complete such a task without incurring enormous cost to already fiscally challenged clinical laboratories (again remember the average bed capacity of hospitals in the US). In addition, in many cases, the laboratory has little or no input into system design when a new system is selected. This is especially true in smaller institutions. Architects and engineers sell administrations the least expensive solution and the laboratory often lives with the consequences. •

This section has been revised to remove simulation and clarify how to identify potential adverse conditions.

Section 5.3.2, Retrospective Qualification of Existing Water Purification Systems 40. The document recommends collecting data over two years to set up validation parameters. While this may be ideal, I suggest that it is impractical. •

A retrospective qualification assumes the purification system has been in service for a period of time and that trend monitoring data is available. The text has been revised to state “over a significant time period (e.g., one to two years)”; thus, one to two years is a suggestion for a significant time period.

Section 6, Design Considerations 41. Replace the word “expendable” with “consumable,” i.e., “consumable component” instead of “expendable component.” •

The suggested edit has been made.

42. Last paragraph—Add “alkaline phosphatase” to “microorganism growth and metabolism endotoxins),” i.e., “microorganism growth and metabolism (e.g., endotoxins, alkaline phosphatase).” •

(e.g.,

The suggested edit has been made.

43. The recommendation that “a contamination profile over a year or two can be helpful in the selection of a water purification system” is, at best, impractical. What vendor has the information for the local area? None. This is an unrealistic recommendation. •

See the response to Comment 40.

Section 6.1.1, Microporous Filters 44. I think “surface filter” is a more accurate description than “screen filter.” •

©

The alternate term “absolute” has been added to the text. “Surface” was considered uncommon usage and potentially confusing because a surface is difficult to define.

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Section 6.1.3, Vent Filters 45. High purity water is made on demand. Reference is made here to water storage containers. Elaborate on this point. •

A reference to Section 6.8, Storage and Distribution, has been added.

Section 6.2, Reverse Osmosis (RO) Membranes 46. It would be helpful if the formulas at the bottom of page 14 said “conductivity” and “resistivity” in front of the appropriate formula. •

The suggested edits have been made.

47. Paragraph 2: Water softening to minimize scale potential is VERY common pretreatment to RO systems. It should be added. •

Water softening has been included as a potential pretreatment at the end of the second paragraph.

48. Last paragraph: TF membranes provide twice the throughput of CA membranes “at equivalent operating pressures.” •

The suggested edit has been made.

49. Last paragraph: Chlorine passage through RO membranes is pH dependent. Above about pH 7.8, very little chlorine will pass through an RO membrane. •

The text has been modified to state that chlorine can pass through a RO membrane under certain conditions, not that it always passes through.

50. Paragraph 1: RO are generally reliable, not “may be imperfect.” RO is a final stage in the purification system, not an early stage. •

Edits have been made to emphasize the general reliability of RO membranes. The sentence regarding placement of RO in a purification system has been deleted.

51. Last paragraph: RO membranes are available in three types. •

The two types of RO membranes mentioned in the document are the most common types used. There may be variants or modifications of these types that have been given different names.

Section 6.4, Ion-Exchange Resins 52. The second paragraph states “use of virgin resins avoids this potential problem” (of unexpected contamination). Vendors will not and cannot guarantee virgin resins. There is also a statement that “the manufacturer’s instructions for the rinse procedure should be followed when beds are exchanged.” Does the committee believe that laboratories will perform such procedures when such a process is not part of a service agreement? •

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Virgin resins are commonly provided by manufacturers of ion-exchange systems. It is a reasonable expectation that laboratories will use equipment according to the manufacturer’s instructions; it is expected that laboratories will follow recommendations for rinsing resins when putting them into service.

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Section 6.5, Activated Carbon 53. The pore size range given for activated carbon is VERY wrong. In addition to very large pores (10 000 A), high surface activated carbon will have a huge number of pores in the 10 A to 20 A range. In fact, most of the 1000 m2/gram of surface area is contained in these small pores. I assume that the “insoluble biocides” mentioned in the next-to-last paragraph includes silver impregnated into activated carbon. Silver impregnated in carbon is NOT insoluble. Its solubility is low (ppb range), but not zero. •

Specific pore sizes of activated carbon have been removed from the text. The word “insoluble” has been removed to avoid confusion.

Section 6.6, Distillation 54. The introduction to Section 6 states, “The section is not intended to be an in-depth review of water purification technologies; detailed information is available in texts and review articles.” Consistent with this statement, Sections 6.6.3 and 6.6.4 should be deleted. •

The sections have been retained because they contain useful information for users of distillation.

55. It seems that an inordinate amount of space is dedicated to this section. This process for water purification is not common in clinical laboratories nowadays, and will likely become less so with energy costs climbing. Stills are high maintenance equipment that cannot produce water with a resistivity of 18 megohm-centimeters due to high CO2 content. I do not believe any laboratorian will entertain sparging as an option. This is impractical. (I suggest deletion of Section 7.1.2.3 which describes, in detail, the sparging procedure). •

Distillation is a suitable technology in appropriate laboratory settings. The usefulness of the sparging technique was addressed in the response to Comment 19.

56. Define how distillation is used to prepare high purity water and/or other types of laboratory grade water. •

The document includes explanation of distillation technology in Section 6.6.

Section 6.6.2, Laboratory (Small-Scale) Stills 57. I am unaware of any clinical laboratories using stills for production of high purity water. Can you provide estimate of usage? •

It is estimated that 10% of clinical laboratories are using distillation technology.

Section 7.1.1, Summary of the Theory and Practice of Resistivity Measurement 58. I think the discussion of cell constant in resistivity measurements at the end of the section is well beyond the intended scope of this document. These paragraphs should be removed. •

The cell constant is the basis for traceability to the SI. The information is provided to ensure the accuracy of the measurement.

Section 7.1.2.2, Effects of CO2 Contamination 59. In light of the inaccuracies contained in Appendix A, the second paragraph of this section should either be rewritten or removed. •

©

The original Appendix A has been deleted due to technical complexity; see response to Comment 71 for details.

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Section 7.1.2.3, Verifying the Resistivity Specification When Purified Water Contains Dissolved CO2 60. This entire section describes a technique that is almost never practiced and is completely impractical for implement in a real clinical laboratory. Its removal should be considered. •

See response to Comment 19.

Section 7.2, Microbial Content by Colony Count 61. Section 7.2.1.1 states, “water samples should be collected from points of use in the same manner as water is collected for normal laboratory use.” Why is there no guideline provided to the user on what is acceptable laboratory practice, e.g., opening the spigot and allowing the water to flow freely for at least one minute before collection? This document seems to leave every decision up to the user and does not give adequate working procedures or guidance. •

It is important that the water sampled for parameter testing is collected in the same manner as the water used for laboratory testing procedures. See response to Comment 66.

62. I take issue with the fact that the Filtration Kit Method has been removed. This is an extremely reliable approach for detecting heterotrophic bacteria and in my experience has been more reliable than filtration methods in some cases. The committee should keep in mind those small laboratories that have very limited microbiology capability and limited resources. •

Membrane filtration is included in the document. The previous edition referenced a pre-packaged kit that included the components needed. Specific reference to a kit has not been included in this edition, but a user can order the components in this manner if desired.

63. Overall, I do not think this document will be helpful to users seeking guidance on how to test and with what frequency. My concern is that, as has happened in the past, CAP will adopt the new guidelines and create even more confusion than already exists. •

This guideline is expected to provide a sound basis for decisions regarding preparation and use of purified water in clinical laboratory procedures. It is expected that each laboratory director will use this information to make appropriate judgments regarding fitness for purpose of water used in the laboratory, and to establish appropriate monitoring and trending procedures to ensure the appropriate water quality is maintained.

64. Delete first and second sentences, nonscientific. Microorganisms recovered from contaminated high purity water do not “become smaller and grow more slowly.” •

The text does not refer to changes in size of microorganisms after they are recovered from purified water; it refers to “microorganisms growing in purified water.” The text has been edited to make this clearer and to distinguish the growth rate in purified water from the growth rate in culture.

Section 7.2.1, Total Heterotrophic Plate Count 65. Is the pour plate technique still recommended for the determination of heterotrophic plate count? •

The limitations of the pour plate make it a less desirable option because subsurface organisms may not grow, or may grow very slowly due to oxygen requirements. They may also be more difficult to count and to subculture.

Section 7.2.1.1, Sample Collection 66. First bullet: The sampling point should be disinfected (usually with isopropyl alcohol) prior to sampling and water flushed from the port for one to two minutes before the sample is drawn. The objective of the sampling is to determine the microbiological content of the water in the water system, not to identify contaminated sample ports.

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The committee disagrees. It is important to identify two situations: 1) contaminated points of use where water is put into a secondary container or used for a laboratory procedure, and 2) microbial contamination in the water itself. The text has been edited to clarify that if water is sampled from a port intended only for the purpose of testing for microorganisms in the water produced by a purification system, then that port should be disinfected (usually with isopropyl alcohol) prior to sampling, and flushed thoroughly to remove the disinfectant before the water sample is drawn. In the first situation, it is important not to disinfect to enable the microbial testing to identify ports on the system that may be contaminated and need to be sanitized.

67. Samples must not be brought to ambient temperature before testing. This will increase counts. •

At the typical low level of microbial content in purified water, the count will not substantially increase in the 15 minutes it takes to warm to room temperature. Failure to warm the water sample increases the risk of shocking the organism and having viable organisms that do not grow in culture.

Section 7.2.1.2, Media and Incubation Conditions 68. Temp is recommended at 25 to 37 ºC. •

The most representative counts will be seen when the cultures are incubated at a similar temperature at which the water system is operating, usually ambient; higher temperatures can result in lower counts.

Section 7.2.1.4, Spread-Plate Technique (Direct) 69. Temp is recommended at 25 to 37 ºC. •

See response to Comment 68.

Section 7.4, Endotoxins 70. First sentence: Biofilms can occur in connective lines within a water treatment system; however, only at a minimum when proper sanitation is conducted. The majority of microorganisms are isolated from final product water. Endotoxin testing is important to include in the test parameters. Make reference that endotoxin indicates presence of gram-negative bacteria but not the presence of a gram-positive organism or fungi. •

Biofilms occur on all surfaces of a water purification system, including the connecting lines between the purification system components and the piping of a distribution system. Bacteria also grow in the purification components. The frequency of exchange of components and the frequency of system sanitization are established to control microbial contamination of the output water. See Section 6 for more details on microbial growth in components and Section 6.8.4 on system sanitization recommendations. The text has been edited to remind the reader that endotoxin is produced primarily by gram-negative microorganisms.

Appendix A, Permeability of Plastic Piping to Atmospheric CO2 71. This section contains significant errors and needs to either be rewritten or removed. Error 1: I have no idea where a value of 12400 cm3·mm·m-2·(24 h)-1·bar-1 for the permeability of CO2 in polypropylene was obtained. It's very hard to know, since no reference is cited. A value that I found from a reputable source is: 26+2 cm3·cm·m-2·day-1·atm-1 Converting this to the same units used in the appendix, one obtains 257 cm3·mm·m-2·(24 h)-1·bar-1 (Source: Department of Macromolecular Science, Center for Applied Polymer Research, Case Western Reserve University. Obtained from www.elsevier.com/locate/polymer) ©

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The permeability used in this analysis is incorrect by a factor of greater than 48. This will obviously lead to inaccurate results and conclusions. The permeability used may, possibly, be for porous, polypropylene membranes; however, it is not correct for dense polypropylene used for tubing and bottles. Error 2: The inside surface area of the tubing will be the limiting area for transfer of CO2, not the outside surface area. The inside surface area is: A = pi x D x L A = 3.14159 x 0.4 cm x 100 cm = 125.7 cm2 Thus, the surface area used in the calculation is 50% higher than the appropriate value (188.5 vs. 127.5) Errors 1 and 2 will both cause an overestimation of CO2 diffusion through polypropylene tubing. Combining these two errors shows that the diffusion rate is overestimated by a factor of 72.4 Error factor = (12400/257) x (188.5/127.5) = 72.4 Thus, the degradation of resistivity from 18.2 MΩ·cm to 3.5 mΩ·cm will take 72.4 times longer than calculated in the appendix. Degradation time = 10 minutes x 72.4 = 724 minutes = 12 hours and 4 minutes Even this corrected analysis overstates how quickly degradation will occur. The diffusion rate of CO2 out of the tubing into the water will slow as the concentration of CO2 in the water increases over time, thus reducing the driving force. •

The permeability 12 400 cm3·mm·m-2·(24 h)-1·bar-1 was obtained from http://www.nalgenelabware.com/techdata/Resin/index2.asp?m=&p=Permeability+CO2+%28metric%29 &txtMError (accessed 29 May 2005). The actual permeability of different plastics varies over a wide range, and the suggested value of 26 ± 2 cm3·cm·m-2·day-1·atm-1 is also representative. The commenter’s recalculation appears to be in error by a factor of 100. In converting from cm3·cm·m·day-1·atm-1 to cm3·mm·m-2·day-1·atm-1, the former is the rate of permeation through a centimeter of plastic, the latter is through 1 millimeter of plastic. Consequently, the latter should be higher by a factor of 10, but the commenter has divided by 10. Therefore, the suggested permeability value converts to 26 000 rather than 260 (257), which is approximately twice the original permeability value used.

2

The commenter’s second point is correct that the inner surface area is the limiting factor and this will give a value 127.5/188.5 smaller. Therefore, the overall effect of these changes is (26 000/12 400) x (127.5/188.5) = an increase of 1.42 x in permeability (not a decrease of 72.4 x as stated by the commenter). Additional simplifying assumptions regarding cumulative diffusion effects and non-linear diffusion over time would be necessary for a correct quantitative estimate of permeability of a gas through plastic. The original Appendix A has been deleted, because it was complex and required a number of assumptions about technical details to provide an example of plastic permeability to CO2. The important qualitative conclusions regarding the effect of plastic permeability on the resistivity of water have been included in the text.

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C3-A4 NOTES

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The Quality System Approach Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality system approach in the development of standards and guidelines, which facilitates project management; defines a document structure via a template; and provides a process to identify needed documents. The approach is based on the model presented in the most current edition of CLSI/NCCLS document HS1—A Quality Management System Model for Health Care. The quality system approach applies a core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any healthcare service’s path of workflow (i.e., operational aspects that define how a particular product or service is provided). The QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The quality system essentials (QSEs) are: Documents & Records Organization Personnel

Equipment Purchasing & Inventory Process Control

Information Management Occurrence Management Assessment

Process Improvement Service & Satisfaction Facilities & Safety

Facilities & Safety

Service & Satisfaction

Process Improvement

Assessment

X

Occurrence Management

Process Control

X

Information Management

Purchasing & Inventory

Equipment

Personnel

Organization

Documents & Records

C3-A4 addresses the quality system essentials (QSEs) indicated by an “X.” For a description of the other documents listed in the grid, please refer to the Related CLSI/NCCLS Publications section on the following page.

Adapted from CLSI/NCCLS document HS1—A Quality Management System Model for Health Care.

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Related CLSI/NCCLS Publications* C24-A2

Statistical Quality Control for Quantitative Measurements: Principles and Definitions; Approved Guideline—Second Edition (1999). This guideline provides definitions of analytical intervals, planning of quality control procedures, and guidance for quality control applications.

EP7-A2

Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition (2005). This document provides background information, guidance, and experimental procedures for investigating, identifying, and characterizing the effects of interfering substances on clinical chemistry test results.

GP2-A5

Laboratory Documents: Development and Control; Approved Guideline—Fifth Edition (2006). This document provides guidance on development, review, approval, management, and use of policy, process, and procedure documents in the medical laboratory community.

*

Proposed-level documents are being advanced through the Clinical and Laboratory Standards Institute consensus process; therefore, readers should refer to the most current editions.

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Active Membership (as of 1 April 2006) Sustaining Members Abbott Laboratories American Association for Clinical Chemistry AstraZeneca Pharmaceuticals Bayer Corporation BD Beckman Coulter, Inc. bioMérieux, Inc. CLMA College of American Pathologists GlaxoSmithKline Ortho-Clinical Diagnostics, Inc. Pfizer Inc Roche Diagnostics, Inc. Professional Members AABB American Academy of Family Physicians American Association for Clinical Chemistry American Association for Laboratory Accreditation American Association for Respiratory Care American Chemical Society American Medical Technologists American Society for Clinical Laboratory Science American Society for Microbiology American Society of Hematology American Type Culture Collection, Inc. ASCP Associazione Microbiologi Clinici Italiani (AMCLI) British Society for Antimicrobial Chemotherapy Canadian Society for Medical Laboratory Science - Société Canadienne de Science de Laboratoire Médical Canadian Standards Association CISMEL-SIMel Clinical Laboratory Management Association COLA College of American Pathologists College of Medical Laboratory Technologists of Ontario College of Physicians and Surgeons of Saskatchewan ESCMID Hong Kong Accreditation Service Innovation and Technology Commission International Federation of Clinical Chemistry Italian Society of Clinical Biochemistry and Clinical Molecular Biology Japanese Committee for Clinical Laboratory Standards Joint Commission on Accreditation of Healthcare Organizations Minneapolis Medical Research Foundation National Academy of Clinical Biochemistry National Society for Histotechnology, Inc. Ontario Medical Association Quality Management Program-Laboratory Service RCPA Quality Assurance Programs PTY Limited SDS Pathology Sociedad Espanola de Bioquimica Clinica y Patologia Molecular Sociedade Brasileira de Analises Clinicas Sociedade Brasileira de Patologia Clinica Taiwanese Committee for Clinical Laboratory Standards (TCCLS) Turkish Society of Microbiology World Health Organization Government Members Association of Public Health Laboratories BC Centre for Disease Control Caribbean Epidemiology Centre Centers for Disease Control and Prevention Centers for Medicare & Medicaid Services

Centers for Medicare & Medicaid Services/CLIA Program Chinese Committee for Clinical Laboratory Standards Department of Veterans Affairs Deutsches Institut für Normung (DIN) FDA Center for Biologics Evaluation and Research FDA Center for Devices and Radiological Health FDA Center for Veterinary Medicine Iowa State Hygienic Laboratory Massachusetts Department of Public Health Laboratories National Center of Infectious and Parasitic Diseases (Bulgaria) National Health Laboratory Service (South Africa) National Institute of Standards and Technology National Pathology Accreditation Advisory Council (Australia) New York State Department of Health Ontario Ministry of Health Pennsylvania Dept. of Health Saskatchewan Health-Provincial Laboratory Scientific Institute of Public Health; Belgium Ministry of Social Affairs, Public Health and the Environment Industry Members AB Biodisk Abbott Diabetes Care Abbott Laboratories Access Genetics Acrometrix Corporation AdvaMed Advancis Pharmaceutical Corporation Affymetrix, Inc. Agilent Technologies, Inc. Ammirati Regulatory Consulting Anna Longwell, PC Arpida Ltd A/S ROSCO AstraZeneca Pharmaceuticals Axis-Shield Diagnostics Axis-Shield POC AS Bayer Corporation – Tarrytown, NY Bayer Corporation - West Haven, CT Bayer HealthCare, LLC, Diagnostics Div. - Elkhart, IN BD BD Diabetes Care BD Diagnostic Systems BD VACUTAINER Systems Beckman Coulter, Inc. Beckman Coulter K.K. (Japan) Beth Goldstein Consultant (PA) Bio-Development S.r.l. Bio-Inova Life Sciences International Biomedia Laboratories SDN BHD bioMérieux (NC) bioMérieux, Inc. (IL) bioMérieux, Inc. (MO) Bio-Rad Laboratories, Inc. Bio-Rad Laboratories, Inc. – France Bio-Rad Laboratories, Inc. – Irvine, CA Bio-Rad Laboratories, Inc. – Plano, TX Black Coast Corporation – Health Care Systems Consulting Blaine Healthcare Associates, Inc. Cepheid Chen & Chen, LLC Chi Solutions, Inc. Chiron Corporation The Clinical Microbiology Institute Comprehensive Cytometric Consulting Control Lab Copan Diagnostics Inc. Cosmetic Ingredient Review Cubist Pharmaceuticals Cumbre Inc. Dade Behring Inc. - Cupertino, CA Dade Behring Inc. - Deerfield, IL Dade Behring Inc. - Glasgow, DE Dade Behring Inc. - Marburg, Germany Dade Behring Inc. - Sacramento, CA David G. Rhoads Associates, Inc. Diagnostic Products Corporation Digene Corporation

Eiken Chemical Company, Ltd. Elanco Animal Health Electa Lab s.r.l. Enterprise Analysis Corporation F. Hoffman-La Roche AG Focus Bio-Inova, Inc. Future Diagnostics B.V. Gavron Group, Inc. Gen-Probe Genaco Biomedical Products, Inc. Genomic Health, Inc. Gentris Corporation Genzyme Diagnostics GlaxoSmithKline Greiner Bio-One Inc. Immunicon Corporation Instrumentation Laboratory International Technidyne Corporation I-STAT Corporation Japan Assn. of Clinical Reagents Industries Johnson and Johnson Pharmaceutical Research and Development, L.L.C. K.C.J. Enterprises LabNow, Inc. LifeScan, Inc. (a Johnson & Johnson Company) Medical Device Consultants, Inc. Merck & Company, Inc. Micromyx, LLC MultiPhase Solutions, Inc. Nanogen, Point-of-Care Diagnostics Div. Nippon Becton Dickinson Co., Ltd. Nissui Pharmaceutical Co., Ltd. NovaBiotics (Aberdeen, UK) Novartis Institutes for Biomedical Research Olympus America, Inc. Optimer Pharmaceuticals, Inc. Ortho-Clinical Diagnostics, Inc. (Rochester, NY) Ortho-McNeil Pharmaceutical (Raritan, NJ) Oxoid Inc. Oxonica (UK) Paratek Pharmaceuticals Pathology Services Inc. PathWork Informatics Pfizer Animal Health Pfizer Inc Pfizer Italia Srl Phadia AB Powers Consulting Services PPD, Inc. Predicant Biosciences Procter & Gamble Pharmaceuticals, Inc. QSE Consulting Radiometer America, Inc. Radiometer Medical A/S Rapid Laboratories Microsystems Reliance Life Sciences Replidyne Roche Diagnostics GmbH Roche Diagnostics, Inc. Roche Diagnostics Shanghai Ltd. Roche Laboratories (Div. HoffmannLa Roche Inc.) Roche Molecular Systems Sanofi Pasteur Sarstedt, Inc. Schering Corporation Schleicher & Schuell, Inc. Seneca Medical Lab, Inc. SFBC Anapharm Sphere Medical Holding Streck Laboratories, Inc. Sysmex America, Inc. (Long Grove, IL) Sysmex Corporation (Japan) TheraDoc Theravance Inc. Third Wave Technologies, Inc. Thrombodyne, Inc. THYMED GmbH Transasia Engineers Trek Diagnostic Systems, Inc. TrimGen Corporation Watin-Biolife Diagnostics and Medicals Wyeth Research XDX, Inc. YD Consultant YD Diagnostics (Seoul, Korea) Trade Associations AdvaMed Japan Association of Clinical Reagents Industries (Tokyo, Japan)

Associate Active Members 35 MDSS/SGSAL (APO) 59th MDW/859 MDT/MTL (TX) 78th Medical Group (GA) Academisch Ziekenhuis -VUB (Belgium) ACL Laboratories (IL) ACL Laboratories (WI) Alexandria Hospital (IL) All Children’s Hospital (FL) Allina Health System (MN) Allina Labs Alton Memorial Hospital (MN) American University of Beirut Medical Center (NY) Anaheim Memorial Hospital (CA) Antwerp University Hospital (Belgium) Arnett Clinic, LLC (IN) Aspirus Wausau Hospital (WI) Associated Regional & University Pathologists (UT) Atlantic Health System (NJ) Avista Adventist Hospital Laboratory (CO) AZ Sint-Jan (Belgium) Azienda Ospedale Di Lecco (Italy) Barnes-Jewish Hospital (MO) Barnes-Jewish St. Peters (MO) Barnes-Jewish West County Hospital (MO) BayCare Health System (FL) Baystate Medical Center (MA) BC Biomedical Laboratories (Surrey, BC, Canada) Bedford Memorial Hospital (VA) Boone Hospital Center (MO) British Columbia Cancer Agency – Vancouver Cancer Center (BC, Canada) Broward General Medical Center (FL) Calgary Laboratory Services (Calgary, AB, Canada) California Pacific Medical Center Canterbury Health Laboratories (New Zealand) Capital Health System Fuld Campus (NJ) Capital Health System Mercer Campus (NJ) Carilion Consolidated Laboratory (VA) Carolinas Medical Center (NC) Central Baptist Hospital (KY) Central Ohio Primary Care Physicians Central Texas Veterans Health Care System Centura Laboratory (CO) Chang Gung Memorial Hospital (Taiwan) Children’s Healthcare of Atlanta (GA) Children’s Hospital Medical Center (Akron, OH) Children’s Hospital of Pittsburgh (PA) Childrens Hospital of Wisconsin Christian Hospital/Northeast/ Northwest (MO) Christus St. John Hospital (TX) City of Hope National Medical Center (CA) Clarian Health - Methodist Hospital (IN) Clendo Lab (PR) Clovis Community Hospital (CA) CLSI Laboratories (PA) Commonwealth of Kentucky Community Care 5 (OH) Community College of Rhode Island Covance Central Laboratory Services (IN) Creighton University Medical Center (NE) Danish Institute for Food and Veterinary Research (Denmark) Dekalb Memorial Hospital (IN) Detroit Health Department (MI) DFS/CLIA Certification (NC) Diagnofirm Med Labs Diagnósticos da América S/A (Sao Paulo) Dianon Systems (OK) Dr. Everett Chalmers Hospital (New Brunswick, Canada) East Kootenay Regional Hospital Laboratory (BC) Evangelical Community Hospital (PA) Faith Regional Health Services (NE)

FasTraQ Inc. (NV) Firelands Regional Medical Center (OH) Fisher-Titus Memorial Hospital (OH) Fleury S.A. (Brazil) Florida Hospital East Orlando Fresno Community Hospital and Medical Center Gamma Dynacare Medical Laboratories (Ontario, Canada) Gamma-Dynacare Laboratories (Brampton, Ontario) Geisinger Medical Center (Danville, PA) Geisinger Wyoming Valley Medical Center (Wilkes-Barre, PA) General Health System (LA) Hamad Medical Corporation (Qatar) Harris Methodist Fort Worth (TX) Hartford Hospital (CT) Health Network Lab (PA) Health Partners Laboratories (VA) High Desert Health System (CA) Hoag Memorial Hospital Presbyterian (CA) Holy Cross Hospital (MD) Hôpital Maisonneuve - Rosemont (Montreal, Canada) Hôpital Sainte - Justine (Quebec) Hospital Albert Einstein (Brazil) Hospital Consolidated Laboratories (MI) Hospital for Sick Children (Toronto, ON, Canada) Hôtel Dieu Grace Hospital (Windsor, ON, Canada) Hunter Area Pathology Service (DE) Hunterdon Medical Center (NJ) Indiana University Interior Health Authority Island Hospital (WA) Jackson Health System (FL) Jackson South Community Hospital (FL) Jacobi Medical Center (NY) John C. Lincoln Hospital (AZ) John H. Stroger, Jr. Hospital of Cook County (IL) Johns Hopkins at Bayview (MD) Johns Hopkins Howard County General Hospital (MD) Johns Hopkins Medical Institutions (MD) Kadlec Medical Center (WA) Kaiser Permanente (CA) Kaiser Permanente (MD) Kantonsspital Aarau AG (Aarau, AG) Karolinska University Hospital Kelowna General Hospital Laboratory (BC) King Abdulaziz Medical City – Jeddah (Jeddah, WR, Saudi Arabia) King Fahad National Guard Hospital (Saudi Arabia) King Faisal Specialist Hospital (Saudi Arabia) Kootenay Boundary Regional Hospital Laboratory (BC) Kosciusko Laboratory (IN) LabCorp (NC) Laboratoire de Santé Publique du Quebec (Canada) Laboratory Alliance of Central New York (NY) Laboratory Corporation of America (NJ)

Lewis-Gale Medical Center (VA) L’Hotel-Dieu de Quebec (Quebec, PQ) LifeCare Hospital Lab (PA) Littleton Adventist Hospital Laboratory (CO) Long Beach Memorial Medical Center (CA) Long Island Jewish Medical Center (NY) Magee Womens Hospital of UPMCHS (PA) Magruder Memorial Hospital (OH) Malmo University Hospital (Sweden) Manipal Acunova Pvt., Ltd. (India) Martin Luther King/Drew Medical Center (CA) Massachusetts General Hospital (Microbiology Laboratory) MDS Metro Laboratory Services (Burnaby, BC, Canada) Mease Countryside Hospital (FL) Mease Dunedin Hospital (FL) Medical Centre Ljubljana (Slovinia) Medical College of Virginia Hospital Medical University of South Carolina (SC) Memorial Hospital (OH) Memorial Medical Center (Napoleon Avenue, New Orleans, LA) Memorial Regional Hospital (FL) Methodist Hospital (TX) Missouri Baptist Medical Center (MO) Montreal General Hospital (Canada) Mount Sinai Hospital (NY) Mountainside Hospital (NJ) MRL Europe (Zaventem) National Healthcare Group (Singapore) National University Hospital (Singapore) NB Department of Health & Wellness (New Brunswick, Canada) NC State Lab of Public Health (NC) The Nebraska Medical Center New England Fertility Institute (CT) New York University Medical Center New Zealand Diagnostic Group NHG Diagnostics (Singapore) Nichols Institute Diagnostics (CA) NorDx (ME) North Bay Hospital North Coast Clinical Laboratory (OH) North Shore Hospital Laboratory (Auckland, New Zealand) North Shore - Long Island Jewish Health System Laboratories (NY) Northern Plains Laboratory (ND) Northwestern Memorial Hospital (IL) Ochsner Clinic Foundation (LA) Orange Coast Memorial Medical Center (CA) Orlando Regional Healthcare System (FL) Overlook Hospital (NJ) Parker Adventist Hospital Laboratory (CO) Parkland Health Center (MO) Pathology Associates Medical Laboratories (WA) Pathology Associates of Boone (NC)

Pediatrix Screening Inc. (PA) Penn State Hershey Medical Center (PA) Penticton Regional Hospital Laboratory (BC) The Permanente Medical Group (CA) Piedmont Hospital (GA) Pitt County Memorial Hospital (NC) Porter Adventist Hospital Laboratory (CO) PPD (KY) Presbyterian Hospital of Dallas (TX) Prince George Medical Lab (Prince George, BC) Provincial Health Services Authority (Vancouver, BC, Canada) Provincial Laboratory for Public Health (Edmonton, AB, Canada) Quest Diagnostics, Inc (San Juan Capistrano, CA) Quintiles Laboratories, Ltd. (GA) Regions Hospital Research Medical Center (MO) Rhode Island Department of Health Laboratories Riverview Hospital (BC, Canada) Riyadh Armed Forces Hospital (Riyadh) Royal Inland Hospital Laboratory (BC) Rural Health Ventures (NE) SAAD Specialist Hospital (Saudi Arabia) SAE – Laboratorio Medico (Brazil) St. Agnes Healthcare (MD) St. Anthony Hospital Central Laboratory (CO) St. Anthony Hospital North Laboratory (CO) St. Anthony’s Hospital (FL) St. Barnabas Medical Center (NJ) St. Christopher’s Hospital for Children (PA) St-Eustache Hospital (Quebec, Canada) St. John Hospital and Medical Center (MI) St. John Regional Hospital (St. John, NB, Canada) St. Joseph’s Hospital (FL) St. Joseph’s Hospital and Medical Center (AZ) St. Joseph’s Hospital-Marshfield Clinic (WI) St. Jude Children’s Research Hospital (TN) St. Louis Children’s Hospital (MO) St. Margaret Memorial Hospital (PA) St. Mary Corwin Regional Medical Center Laboratory (CO) St. Michael’s Hospital (Toronto, ON, Canada) San Antonio Community Hospital (TX) San Francisco General Hospital (CA) Santa Clara Valley Medical Center (CA) Shands at the University of Florida SJRMC Plymouth Laboratory (IN) Sonora Quest JV (AZ) South Bend Medical Foundation (IN) South Florida Baptist Hospital (FL) South Texas Laboratory (TX) South Western Area Pathology Service (Australia) Specialty Laboratories, Inc. (CA)

OFFICERS Robert L. Habig, PhD, President Abbott Laboratories Gerald A. Hoeltge, MD, President-Elect The Cleveland Clinic Foundation Wayne Brinster, Secretary BD W. Gregory Miller, PhD, Treasurer Virginia Commonwealth University Thomas L. Hearn, PhD, Immediate Past President Centers for Disease Control and Prevention Glen Fine, MS, MBA, Executive Vice President

Starke Memorial Hospital Laboratory (IN) State of Washington Department of Health Stormont-Vail Regional Medical Center (KS) Sunnybrook & Women’s College Health Sciences Centre (Toronto, Ontario) Sunnybrook Health Science Center (ON, Canada) Taiwan Society of Laboratory Medicine Tan Tock Seng Hospital (Tan Tock Seng) Temple Univ. Hospital - Parkinson Pav. (PA) Texas Department of State Health Services (TX) Timmins and District Hospital (Canada) The Children’s University Hospital (Ireland) Tri-Cities Laboratory (WA) Tripler Army Medical Center (HI) Tuen Mun Hospital (Hong Kong) Tuttle Army Health Clinic (GA) UCSD Medical Center (CA) UCSF Medical Center China Basin (CA) UNC Hospitals (NC) Union Clinical Laboratory (Taiwan) Universita Campus Bio-Medico (Italy) University Medical Center (CA) University of Chicago Hospitals (IL) University of Colorado Hospital University of Debrecen Medical Health and Science Center (Hungary) University of Illinois Medical Center (IL) University of Maryland Medical System University of MN Medical Center Fairview University of the Ryukyus (Japan) University of Virginia Medical Center University of Washington UPMC Horizon Hospital (PA) U.S. Army Health Clinic – Vicenza (APO) US LABS, Inc. (CA) USA MEDDAC-AK UZ-KUL Medical Center (Belgium) VA (Asheville) Medical Center (NC) Valley Health (VA) Vejle Hospital (VA) Vernon Jubilee Hospital Laboratory Virginia Beach General Hospital (VA) Warren Hospital (NJ) Washington Hospital Center (DC) Waterford Regional Hospital (Ireland) Wellstar Health Systems (GA) West China Second University Hospital, Sichuan University (P.R. China) William Beaumont Army Medical Center (TX) William Beaumont Hospital (MI) Winn Army Community Hospital (GA) Women’s Health Laboratory (TX) Woodlawn Hospital (IN) York Hospital (PA)

BOARD OF DIRECTORS Susan Blonshine, RRT, RPFT, FAARC TechEd

Gary L. Myers, PhD Centers for Disease Control and Prevention

Maria Carballo Health Canada

Valerie Ng, PhD, MD Alameda County Medical Center/ Highland General Hospital

Russel K. Enns, PhD Cepheid Mary Lou Gantzer, PhD Dade Behring Inc. Lillian J. Gill, DPA FDA Center for Devices and Radiological Health Jeannie Miller, RN, MPH Centers for Medicare & Medicaid Services

Klaus E. Stinshoff, Dr.rer.nat. Digene (Switzerland) Sàrl James A. Thomas ASTM International Kiyoaki Watanabe, MD Keio University School of Medicine

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