(Formerly NCCLS) Providing NCCLS standards and guidelines, ISO/TC 212 standards, and ISO/TC 76 standards

Transcription

1 (Formerly NCCLS) Providing NCCLS standards and guidelines, ISO/TC 212 standards, and ISO/TC 76 standards

2 Clinical and Laboratory Standards Institute Providing NCCLS standards and guidelines, ISO/TC 212 standards, and ISO/TC 76 standards 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. Report A document that has not been subjected to consensus review and is released by the Board of Directors. CONSENSUS PROCESS The CLSI voluntary consensus process is a protocol establishing formal criteria for: the authorization of a project 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. 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 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. VOLUNTEER PARTICIPATION Healthcare professionals in all specialties are urged to volunteer for participation in CLSI projects. Please contact us at customerservice@clsi.org or for additional information on committee participation.

3 ISBN Volume 25 Number 23 ISSN Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline Dorothy M. Adcock, MD William F. Brien, MD, FRCP(C) Stephen L. Duff, MBA Marilyn Johnston, ART Stephen Kitchen, FIBMS, PhD Richard A. Marlar, PhD Valerie L. Ng, PhD, MD Ton van den Besselaar, PhD Barry J. Woodhams, PhD Abstract Clinical and Laboratory Standards Institute document Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline is one in a series of guidelines that addresses methodology in blood coagulation testing. It is intended to provide guidance for both manufacturers and clinical laboratory personnel responsible for reporting patient INR results. H54 describes the use of certified plasmas to enhance performance of the prothrombin time (PT)/International Normalized Ratio (INR) system test; reviews limitations of the PT/INR system that may occur when a manufacturer-determined ISI is used without local verification or calibration; and provides a rationale for performing local ISI verification with recommendations as to when PT calibration may be indicated. This guideline is published in two parts. Part I provides an expanded account of the subject and Part II is an abbreviated version that may be useful in the clinical laboratory. Methods of calculating local ISI are provided and the procedure for creating a calibration line for direct INR determination is included. In the expanded guideline, the method of certified plasma preparation and method of INR value assignments are also described in detail. This guideline includes a recommended INR range that certified plasmas should cover and recommended number of certified plasmas required for local ISI calibration. A protocol for performing calibration of PT systems is provided. The objective of this guideline is to improve precision and trueness (accuracy) of PT/INR systems and enhance both laboratory standardization and patient care. Clinical and Laboratory Standards Institute (CLSI). Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline. CLSI document (ISBN ). Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA, 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: ; Fax: ; customerservice@clsi.org; Website: (Formerly NCCLS) Providing NCCLS standards and guidelines, ISO/TC 212 standards, and ISO/TC 76 standards DIN/NAMed

4 Number 23 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 Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline (ISBN ). Copies of the current edition may be obtained from Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania , 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 , USA. Copyright Clinical and Laboratory Standards Institute. Suggested Citation (Clinical and Laboratory Standards Institute. Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline. CLSI document [ISBN ]. Clinical and Laboratory Standards Institute, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA, 2005.) Proposed Guideline October 2004 Approved Guideline August 2005 ISBN ISSN ii

5 Volume 25 Committee Membership Area Committee on Hematology Bruce H. Davis, MD Chairholder Maine Medical Center Research Institute Scarborough, Maine Samuel J. Machin, MB, ChB, FRCPath Vice-Chairholder The University College London Hospitals London, United Kingdom Dorothy M. Adcock, MD Esoterix Coagulation Aurora, Colorado Frank M. LaDuca, PhD International Technidyne Corporation Edison, New Jersey Ginette Y. Michaud, MD FDA Center for Devices and Radiological Health Rockville, Maryland Albert Rabinovitch, MD, PhD Abbott Laboratories, Hematology Business Unit Santa Clara, California Maryalice Stetler-Stevenson, MD, PhD National Institutes of Health Bethesda, Maryland Advisors Charles F. Arkin, MD Lahey Clinic Burlington, Massachusetts J. David Bessman, MD University of Texas Medical Branch Galveston, Texas Sheila Clover, CPT(ASCP)(NCA) Phlebotomy West Brentwood, California Dennis J. Ernst, MT(ASCP) Center for Phlebotomy Education Ramsey, Indiana John A. Koepke, MD Durham, North Carolina Francis Lacombe, MD, PhD Hôpital Haut-Lévêque Pessac, France Kandice Kottke Marchant, MD, PhD The Cleveland Clinic Foundation Cleveland, Ohio Richard A. Marlar, PhD Oklahoma City VA Medical Center Oklahoma City, Oklahoma Diane I. Szamosi, MA, MT(ASCP)SH Greiner Bio-One North America Preanalytics Monroe, North Carolina Subcommittee on Calibration Plasmas Dorothy M. Adcock, MD Chairholder Esoterix Coagulation Aurora, Colorado William F. Brien, MD, FRCP(C) Princess Margaret Hospital Toronto, Ontario, Canada Stephen L. Duff, MBA Precision BioLogic Dartmouth, Nova Scotia, Canada Marilyn Johnston, ART Hemostasis Reference Laboratory Hamilton, Ontario, Canada Stephen Kitchen, FIBMS, PhD Royal Hallamshire Hospital Sheffield, United Kingdom Richard A. Marlar, PhD Oklahoma City VA Medical Center Oklahoma City, Oklahoma Valerie L. Ng, PhD, MD San Francisco General Hospital San Francisco, California Ton van den Besselaar, PhD Leiden University Medical Center Leiden, The Netherlands Barry J. Woodhams, PhD Stago R&D Gennevilliers, France Advisors John D. Olson, MD, PhD University of TX Health Center San Antonio, Texas Norbert F. Zander, PhD Dade Behring Marburg, Germany Staff Clinical and Laboratory Standards Institute Wayne, Pennsylvania John J. Zlockie, MBA Vice President, Standards David E. Sterry, MT(ASCP) Staff Liaison Patrice E. Polgar Projects Coordinator Donna M. Wilhelm Editor Melissa A. Lewis Assistant Editor iii

6 Number 23 iv

7 Volume 25 Contents Abstract...i Committee Membership... iii Foreword... vii 1 Scope Standard Precautions Definitions...1 Part I. Detailed Version Laboratory Monitoring of AVK Therapy Recommended Method Limitations of the INR System Optimizing Current PT/INR Test Systems (Prior to Local Verification or Calibration) Recommendations for Verification of the PT/INR Test System Verification of INR Results Recommendations for Further Improvement of the PT/INR Test System (INR Calibration) PT/INR Calibration General Guidelines for the Use of Certified Plasmas Frequency of Calibration Tissue Factor Species Effects PT/INR System Calibration to Determine Local ISI Procedure for Local ISI (ISI LS Determination) Number of Certified Plasmas Required for Local ISI Calibration Local System Verification Following ISI Calibration Criteria for Acceptance of Locally Calculated ISI Interpretation of Patient Test Results PT/INR System Calibration to Determine Direct INR Recommended Number and Preparation of Certified Plasmas Procedure for Preparation of a Direct INR Calibration Line Interferences Guidelines for Determining Result Acceptance Interpretation of Patient Test Results Choice of Verification/Calibration Material Method of Preparation of Certified Reference Materials Validation of Certified Plasmas Documentation Required by Manufacturers...23 v

8 Number 23 Contents (Continued) Part II. Abbreviated Version for Laboratorians Introduction Limitations of the INR System ISI Value Assigned by the Manufacturer Thromboplastin/Instrument Interaction Components of INR Determination Optimizing the Local PT/INR Test System Prothrombin Time Mean Normal Prothrombin Time Thromboplastin Reagent With a Generic ISI Thromboplastin Reagent With an Instrument/Reagent-Specific ISI Use of Good Laboratory Practices Recommendations for Verification of the Local Test System Verification of INR Results Local System Calibration General Guidelines for the Use of Certified Plasmas Procedure for Calculating Local ISI Procedure for Establishing Direct INR (Locally) Interpretation of Patient Test Results...33 References...34 Appendix A. Determination of the Geometric Mean Normal Prothrombin Time (MNPT)...37 Appendix B. Calculations for Local ISI Determination and Direct INR Calibration Line...38 Appendix C. INR Verification, ISI Calibration, and Direct INR Calibration Line Preparation Using Certified Plasmas...42 Summary of Consensus/Delegate Comments and Subcommittee Responses...43 The Quality System Approach...50 Related CLSI/NCCLS Publications...51 vi

9 Volume 25 Foreword The prothrombin time (PT) is the most widely performed coagulation assay. It is commonly used to monitor antivitamin K therapy (AVK). The wide use of the PT has resulted in the introduction of numerous thromboplastin reagents and coagulation instruments. Thromboplastin reagents can vary widely in their response to AVK therapy depending on the source material from which they are derived. This can result in a wide range of PT results on identical patient plasmas. Patient samples have shorter clotting times when less responsive PT reagents are used and longer clotting times when a more responsive reagent is used. A wide assortment of instruments is available to measure PT assays using a number of different technologies for endpoint detection (e.g., optical-based or mechanical clot detection). PT test results may vary depending on the clot detection mechanism employed and brand of coagulation analyzer used. Variation in both thromboplastin reagent and instrumentation has contributed to a lack of standardization in PT test results. In 1983, the World Health Organization (WHO) introduced the International Normalized Ratio (INR) for PT reporting in an effort to offset variation in thromboplastin reagent responsiveness and enhance standardization. An International Sensitivity Index (ISI) is assigned to each commercial lot number of thromboplastin reagent. The ISI is a measure of a reagent s responsiveness to depressed functional levels of vitamin K-dependent coagulation factors compared to the primary WHO International Reference Preparation (IRP). The INR is a mathematical conversion of the PT calculated as follows: INR = (Plasma PT MNPT) ISI. Though the INR system has improved PT reporting, it is still associated with unexpectedly high degrees of inconsistency in values between laboratories and significant variation in locally reported INRs, compared to expected or true results. Such variation may negatively impact patient care as inaccuracies in INR determination can result in chronic over- or under-anticoagulation, resulting in increased patient morbidity and mortality. Causes for variation in INR include, but are not limited to: 1) incorrect determination of the mean normal prothrombin time (MNPT) or PT; 2) difference in sodium citrate concentration employed for ISI determination compared to that used locally; 3) local effect of the reagent/instrument combination on the manufacturer s assigned ISI; 4) incorrect ISI value applied locally; 5) incorrect choice of IRP for reagent calibration, causing an inaccurate ISI value; and 6) inaccuracy and imprecision in the calibration of the commercial reagent against the appropriate IRP, causing an inaccurate and imprecise ISI value. In order to optimize performance of the PT, it is recommended that a thromboplastin with an assigned ISI value specific for the laboratory s thromboplastin/instrument combination be used. This is in preference to a thromboplastin lacking an instrument-specific ISI (generic ISI [see the Definitions section]). When using a reagent with a thromboplastin/instrument-specific ISI, local ISI verification should be performed to ensure the ISI value is correct for the laboratory. If different, local calibration should be performed. When using a generic ISI, local verification is mandatory and local ISI calibration is strongly recommended. Local PT calibration has been demonstrated to enhance the trueness (accuracy) and interlaboratory precision of INR determination. Local application of a conventional WHO calibration methodology is not feasible, as it is a labor-intensive, costly procedure using the manual tilt-tube method, and there is insufficient IRP available for individual laboratories. Instead, certified plasma samples with assigned PT/INR values can be used in individual laboratories to validate and if needed, calibrate the local instrument/reagent system. Local calibration can be achieved using certified plasmas by either calculating an ISI locally using orthogonal regression or by generating a calibration line upon which INR values are interpolated, a direct INR. vii

10 Number 23 With the use of certified plasmas for INR verification and/or calibration of the local PT/INR systems, it is anticipated that clinical laboratories will not only report more accurate INRs, but also enhance laboratory to laboratory consistency, leading to improvements in monitoring antivitamin K therapy and, ultimately, patient outcomes. A Note on Terminology Clinical and Laboratory Standards Institute (CLSI), as a global leader in standardization, is firmly committed to achieving global harmonization wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the global metrological community have evolved differently in the United States, Europe, and elsewhere; that these differences are reflected in CLSI, ISO, and CEN documents; and that legally required use of terms, regional usage, and different consensus timelines are all obstacles to harmonization. Despite these obstacles, CLSI recognizes that harmonization of terms facilitates the global application of standards and is an area that needs immediate attention. Implementation of this policy must be an evolutionary and educational process that begins with new projects and revisions of existing documents. In keeping with CLSI s commitment to align terminology with that of ISO, the following describes the metrological terms and their uses in : The term accuracy refers to the closeness of the agreement between the result of a (single) measurement and a true value of a measurand and comprises both random and systematic effects. Trueness is used in this document when referring to the closeness of the agreement between the average value from a large series of measurements and to a true value of a measurand. Precision is defined as the closeness of agreement between independent test/measurement results obtained under stipulated conditions. As such, it cannot have a numerical value, but may be determined qualitatively as high, medium, or low. For its numerical expression, the term imprecision is used, which is the dispersion of results of measurements obtained under specified conditions. In addition, different components of precision are defined in, primarily repeatability (i.e., the closeness of the agreement between results of successive measurements of the same measurand carried out under the same conditions of measurement ); while reproducibility describes the closeness of agreement of results of measurements under changed conditions. The CLSI Harmonization Policy recognizes ISO terms as the preferred terms. When appropriate, alternative terms are included parenthetically to help avoid confusion. Users of should understand, however, that the fundamental meanings of the terms are identical in many cases, and to facilitate understanding, terms are defined in the Definitions section of this guideline. All terms and definitions will be reviewed again for consistency with international use, and revised appropriately during the next scheduled revision of this document. Key Words Calibration, certified plasmas, international normalized ratio (INR), international sensitivity index (ISI), prothrombin time (PT), thromboplastin, verification Acknowledgment This guideline is being developed through the cooperation of the CLSI Area Committee on Hematology and its Subcommittee on Calibration Plasmas, and Technical Committee C5, Haemostaseology, of the Department for Medical Standards (Normenausschuss Medizin) at the German Standards Institute (Deutsches Institut für Normung [DIN]). Representatives of both CLSI and DIN are participating in the development of each organization s respective standard. It is expected that this effort will advance the international harmonization of this important hematology guideline, thereby improving healthcare delivery worldwide. The DIN representative for this project is Heinz Beeser, MD, PhD, Institute for Quality Management and Standardization in Transfusion Medicine, Teningen, Germany. viii

11 Volume 25 Procedures for Validation of INR and Local Calibration of PT/INR Systems; Approved Guideline 1 Scope CLSI document H54 reviews limitations of the INR system and provides a rationale for performing local PT/INR verification with recommendations as to when calibration may be indicated. The method of certified plasma preparation and method of INR value assignments are described in detail. Also included are the recommended INR range that certified plasmas should cover and recommended number of certified plasmas required for local ISI calibration. A protocol for performing calibration of PT systems is provided. Methods of calculating local ISI are included, as well as the procedure for creating a calibration line in order to interpolate a direct INR. To facilitate improved precision and accuracy of PT assay results, enhance laboratory standardization, and thereby optimize patient results and care, the guideline has been divided into two parts. Though both may be educational for all users, Part I is primarily intended for manufacturers of the reagents and instruments used in the PT/INR system, and for manufacturers of material such as certified plasmas to standardize the PT assay; Part II is written for laboratory professionals responsible for the performance of prothrombin time (PT) assays. 2 Standard Precautions Because it is often impossible to know what isolates or specimens might be infectious, all patient and laboratory specimens are treated as infectious and handled according to standard precautions. Standard precautions are guidelines that combine the major features of universal precautions and body substance isolation practices. Standard precautions cover the transmission of all infectious agents and thus are more comprehensive than universal precautions which are intended to apply only to transmission of blood-borne pathogens. Standard and universal precaution guidelines are available from the U.S. Centers for Disease Control and Prevention (Garner JS, Hospital Infection Control Practices Advisory Committee. Guideline for isolation precautions in hospitals. Infect Control Hosp Epidemiol. 1996;17:53-80). For specific precautions for preventing the laboratory transmission of all infectious agents from laboratory instruments and materials and for recommendations for the management of exposure to all infectious disease, refer to the most current edition of CLSI document M29 Protection of Laboratory Workers From Occupationally Acquired Infections. 3 Definitions accuracy (of measurement) closeness of the agreement between the result of a measurement and a true value of the measurand (VIM93) 1 ; NOTE: See measurand and trueness, below. antivitamin K (AVK) plasma plasma from an individual on antivitamin K (AVK) therapy; NOTE: See vitamin K antagonist, below. calibration set of operations that establishes, 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) 1 ; NOTE 1: According to the U.S. Code of Federal Regulations, calibration is the process of testing and adjustment of an instrument, kit, or test system, to provide a known relationship between the measurement response and the value of the substance being measured by the test procedure (42 CFR 493) 2 ; NOTE 2: The term is sometimes used to describe different situations; NOTE 3: See calibration line and direct INR determination, below. Clinical and Laboratory Standards Institute. All rights reserved. 1

12 Number 23 calibration line the graphic relationship (typically linear) between the clotting time in seconds and the INR of certified plasmas. certified plasmas normal or abnormal plasma samples assigned a PT/INR value by a manufacturer, using a manual method based on a WHO accepted protocol determined against an appropriate thromboplastin IRP (or manufacturer or standard reference reagent) that has been calibrated against the appropriate WHO standard in a multicenter study; NOTE 1: (i.e., a minimum of three laboratories for a primary standard and two laboratories for a secondary standard); NOTE 2: See standard reference reagent, below. direct INR determination INR determination from a PT/INR calibration line determined using certified plasmas without employing an ISI and MNPT; NOTE: See Figure 1 in Section 8.4. error (of measurement)//measurement error result of a measurement minus a true value (or accepted reference value) of the measurand (VIM93). 1 extrapolate to estimate (the value of a variable) outside the tabulated or observed range; NOTE 1: In this document, INRs above 4.5 would be extrapolated, while those between 1 and 4.5 would be interpolated; NOTE 2: See interpolate, below. generic ISI an ISI determined for a thromboplastin that is not instrument-specific (i.e., determined for a group of instruments that uses the same general method for endpoint detection, such as manual, photooptical, or mechanical methods); NOTE: See International Sensitivity Index and thromboplastin/instrument-specific ISI, below. imprecision dispersion of independent results of measurements obtained under specified conditions; NOTE: It is expressed numerically as standard deviation (SD) or coefficient of variation (CV). International Normalized Ratio (INR) patient s prothrombin time (PT) test result expressed as a ratio to a normal population (MNPT) which has been standardized (or normalized) for the potency of the thromboplastin used in the assay (revised from ISO/DIS 17593) 3 ; NOTE: INR = (Plasma PT MNPT) ISI. International Reference Preparation (IRP) a thromboplastin with defined biological activity used to calibrate other reference preparations and secondary or manufacturer s standards; NOTE 1: There are three species of IRP: bovine, rabbit, and human, which can be produced from original biological sources or other recombinant sources; NOTE 2: IRPs can only be used in combination with the manual technique (tilt-tube method, or other methods that have been validated; NOTE 3: WHO and European Union CRM standards are examples of IRPs; NOTE 4: IRPs are also sometimes referred to as primary standards. International Sensitivity Index (ISI) a quantitative measure, in terms of the first International Reference Preparation of thromboplastin, human, combined, coded 67/40, of the responsiveness of a prothrombin-time system to the defect induced by oral anticoagulants (WHO 880) 4 ; NOTE: See thromboplastin /instrument-specific ISI, below, and generic ISI, above. interpolate to insert, estimate, or find an intermediate term (in a sequence); NOTE 1: In this document, INRs above 4.5 would be extrapolated, while those between 1 and 4.5 would be interpolated; NOTE 2: See extrapolate, above. local test system ISI calibration determination of local test system ISI using certified plasmas. mean normal prothrombin time (MNPT) the geometric mean of the prothrombin times of the healthy adult population (WHO 880) 4 ; NOTE 1: For practical purposes, the geometric mean of the prothrombin time calculated from at least 20 fresh samples from healthy individuals, including those of both sexes, is a 2 Clinical and Laboratory Standards Institute. All rights reserved.

13 Volume 25 reliable approximation of MNPT. It is not necessary to collect and test all the individual samples in one session (WHO 880) 4 ; NOTE 2: It is recommended that each laboratory should determine MNPT using its own prothrombin-time system (WHO 880) 4 ; NOTE 3: The arithmetic mean should not be used. measurand particular quantity subject to measurement (VIM93). 1 precision (of measurement) closeness of agreement between independent test results obtained under stipulated conditions (ISO ) 5 ; NOTE: Precision is not typically represented as a numerical value but is expressed quantitatively in terms of imprecision the standard deviation (SD) or the coefficient of variation (CV) of the results in a set of replicate measurements. prothrombin time (PT) time in seconds required for a fibrin clot to form in a plasma sample after tissue thromboplastin and an optimal amount of calcium chloride have been added to the sample; NOTE 1: WHO defines PT in the following way: PT (tissue-factor-induced coagulation time) the clotting time of a plasma (or whole blood) sample in the presence of a preparation of thromboplastin and the appropriate amount of calcium ions (WHO 880) 4 ; NOTE 2: ISO defines the term as time required to clot a blood specimen once exposed to a thromboplastin reagent material (ISO 17593). 3 repeatability (of results of measurements) closeness of the agreement between results of successive measurements of the same measurand carried out under the same conditions of measurement (VIM93) 1 ; NOTE: It is often termed in the laboratory, within-assay, within-run, intra-assay, intra-run precision. reproducibility (of results of measurements) closeness of the agreement between the results of measurements of the same measurand carried out under changed conditions of measurement (VIM93) 1 ; NOTE: Two types of reproducibility are often used in laboratory medicine: between-assay, between-run, interassay, inter-run precision, and interlaboratory precision. The operative conditions should always be specified. responsiveness the degree of prolongation of the clotting time induced by a particular reagent/instrument system to plasma-related factors (e.g., decreased vitamin K-dependent factor activities or concentration of sodium citrate); NOTE: The longer the clotting time to a given factor activity, the more responsive the reagent/instrument system. sample (patient) one or more parts taken from a system and intended to provide information on the system, often to serve as a basis for decision on the system or its production (ISO 15189) 6 ; NOTE: A sample is prepared from the patient specimen and used to obtain information by means of a specific laboratory test. standard reference reagent//manufacturer s standard for this protocol, a preparation of thromboplastin reagent made by a similar procedure to that used for each individual lot of thromboplastin reagents; NOTE 1: Generally, it is one batch or part of a batch of the normal manufacturer s reagent that is retained as an internal standard. An ISI value is assigned according to the WHO guidelines for standard reference reagents by at least two laboratories. The ISI value may be assigned by either the tilt-tube method or be an instrument-dependent value. This material is then used for subsequent ISI assignment of each lot of similar reagents; NOTE 2: The term does not imply that the standard is developed directly from the primary standard (VIM93), 1 and it should be noted that a manufacturer s standard may in fact be three or more calibration steps away from the primary standard. standard deviation (of a random variable, or of a probability distribution) the positive square root of the variance σ = V(X) (ISO ). 5 test plasma plasma (from a patient or unknown source) that is analyzed by a specific laboratory test. Clinical and Laboratory Standards Institute. All rights reserved. 3

14 Number 23 test system a combination of thromboplastin, coagulation instrument, and technique used for prothrombin time determination. thromboplastin a reagent containing tissue factor and coagulant phospholipids (WHO 880) 4 ; NOTE 1: Many commercial thromboplastins are crude extracts prepared from mammalian tissues, in which tissue factor is only a minor component on a weight basis, and which also contain phospholipids (WHO 880) 4 ; NOTE 2: A preparation of a thromboplastin consisting of a tissue extract alone, either with or without added calcium chloride, is termed plain ; when the preparation contains adsorbed bovine plasma as a source of additional factor V and fibrinogen, it is termed combined (WHO 880) 4 ; NOTE 3: Thromboplastins may also be grouped into types, according to the tissue source from which they are derived (e.g., human, bovine, rabbit brain or lung, or human placenta) (WHO 880) 4 ; NOTE 4: The tissuefactor component of recombinant human thromboplastin reagents is produced in Escherichia coli, yeast, or insect cells by recombinant DNA techniques and then lipidated in vitro (WHO 880). 4 thromboplastin/instrument-specific ISI a thromboplastin ISI that is generated by the reagent manufacturer specific for the particular thromboplastin/instrument combination; NOTE: See International Sensitivity Index and generic ISI, above. trueness (of measurement) closeness of agreement between the average value obtained from a large series of test results and an accepted reference value (ISO ) 5 ; NOTE 1: Trueness is usually expressed numerically by the statistical measure bias that is inversely related to trueness; NOTE 2: See accuracy, above. validation confirmation, through the provision of objective evidence, that requirements for a specific intended use or application have been fulfilled (ISO 9000) 7 ; NOTE: WHO defines validation as the action (or process) of proving that a procedure, process, system, equipment, or method used works as expected and achieves the intended result (WHO-BS/ ). 8 verification confirmation, through the provision of objective evidence, that specified requirements have been fulfilled (ISO 9000) 7 ; NOTE 1: A one-time process completed to determine or confirm test performance characteristics before the test system is used for patient testing; NOTE 2: The ISO definitions for validation and verification are similar. vitamin K antagonists (AVK) a class of compounds that when taken orally, act as vitamin K antagonists and depending on the dose administered, result in diminished levels of functional factors II, VII, IX, and X giving an in vivo anticoagulant effect; NOTE 1: The broad class of compounds are referred to as coumarins; NOTE 2: Previously termed coumarin-derived oral anticoagulant therapy (OAT). 4 Clinical and Laboratory Standards Institute. All rights reserved.

15 Volume 25 Part I. Detailed Version NOTE: Clinical laboratorians should proceed to Part II. 4 Laboratory Monitoring of AVK Therapy 4.1 Recommended Method AVK drugs are widely used in the treatment of venous and arterial thrombotic disorders and function by inhibiting the hepatic biosynthesis of functional vitamin K-dependent coagulation factors. The dose of AVK drugs must be managed for each patient to provide the appropriate and safe level of anticoagulation and in the majority of patients, changes in dosage are based on the accurate results and interpretation of the clotting-based test termed the prothrombin time (ProTime, QuickTime, or PT). The INR system is the recommended method of reporting clotting times in patients on AVK. The INR is a mathematical conversion of the PT result based on the responsiveness of the thromboplastin reagent used in the assay Prothrombin Time (PT) The PT is a clotting test that utilizes citrated plasma from the patient, a method of clot detection such as mechanical or optical, and an initiating agent of the clotting cascade termed thromboplastin. Thromboplastin is a mixture of tissue factor (the specific protein responsible for initiating the extrinsic system of coagulation), phospholipid (necessary for surface assembly of the coagulation complexes), and calcium ions (necessary for the correct orientation and binding/assembly of complexes). Thromboplastins can be prepared by a variety of methods, such as tissue extraction, tissue culture, and molecular biological (genetic) technology, and includes human-derived thromboplastin, animal-derived thromboplastin, or thromboplastin made by tissue culture and molecular biological (genetic) technologies. Thromboplastins from different sources and methods of manufacture, different concentrations, and different mixtures of components will lead to different results of the PT assay. A mathematical equation was developed to provide a common scale for expressing PT results. The new procedure for standardizing PT results is the International Normalized Ratio (INR) and the equation that was developed for this system uses a calibration parameter termed the International Sensitivity Index (ISI) to normalize the results for the variation in thromboplastin responsiveness. WHO has established a set of guidelines to be used for determining the ISI assignment provided to each thromboplastin. A host of preanalytical and analytical variables may affect the results of the PT assay, and therefore determination of the INR. These variables must be minimized to reduce potential errors in results. The majority of these variables are discussed in two other documents (see the most current editions of CLSI/NCCLS documents H21 Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and H47 One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test) Mean Normal Prothrombin Time Determining the correct mean normal prothrombin time (MNPT) is vital to reporting accurate INR results, because INR is determined based on the following equation: INR = (Plasma PT MNPT) ISI. Using an inappropriate MNPT can have a clinically significant and systematic effect on the INR and can either inappropriately increase or decrease the results. The MNPT is defined as the geometric mean of the prothrombin times of the healthy adult population and can be approximated by the geometric mean of the prothrombin time calculated from at least 20 fresh samples from healthy individuals, including those of Clinical and Laboratory Standards Institute. All rights reserved. 5

16 Number 23 both sexes. 4 Arithmetic mean should not be used. Many laboratories still utilize the arithmetic mean; however, since a healthy population is considered to be lognormally distributed, the geometric mean is a more accurate value and it is the value used in the establishment of the INR of the certified plasmas. 9 The method for calculating the geometric mean is included in Appendix A. Individual samples used to determine MNPT are best tested over several days to take account of day-to-day variation in the measurement system. It is recommended that each laboratory should determine MNPT using its own thromboplastin/instrument combination, and its own blood collection system. Although the procedure as described above (i.e., the determination of the MNPT for each new batch of thromboplastin by each laboratory) is a laborious procedure, it is the recommended method for MNPT determination. This can be simplified, when necessary, by using a certified, lyophilized, pooled normal plasma. It should be realized that the PT of a lyophilized normal plasma is not identical to the MNPT, and a correction is required. For a description of this method, see Peters et al, INR Equation The INR equation is based on the combination of the instrument and reagent utilized for the patient s PT determination. The INR equation is: ISI PT (Plasma) INR = MNPT The ISI is a mathematical index of the responsiveness of the PT reagent and instrument combination to the degree of functional deficiency of the vitamin K-dependent proteins (which is an estimate of the degree of anticoagulation). The manufacturer may provide an ISI value, determined using a specific instrument brand and model (called the thromboplastin/instrument-specific ISI), or an ISI may be provided dependent on the methodology of clot detection (e.g., optical versus mechanical [generic ISI]). Different instruments may have different ISI values using the same reagent; for this reason, it is recommended that a thromboplastin with an instrument-specific ISI be used whenever possible. In this instance, local verification is recommended with each new lot of reagent instituted or following major instrument maintenance. If a PT reagent with a generic ISI is used, local verification using certified plasmas is required. If local verification fails with either a thromboplastin/instrument-specific ISI or generic ISI, corrective action, such as local test system ISI calibration, is required before patient results can be reported. 4.2 Limitations of the INR System The INR system generally works very well and should be the method of choice for laboratory monitoring of patients on AVK therapy. The universal acceptance of this procedure has made a major contribution to standardization of oral anticoagulant therapy. Failure to take into account the responsiveness of thromboplastin reagents by not applying the INR/ISI system or by using an incorrect ISI may severely compromise the clinical management of AVK patients. 11,12 In the last few years, there has been improvement brought about by a greater understanding of the laboratory requirements for the use of the INR system and as well by increased labeling by the manufacture of thromboplastin/instrument-specific ISI values. Some unexpectedly high degrees of inconsistency in INR values may still occur between laboratories (imprecision), causing significant variation in reported values compared to expected or true results. Outlined below are the common potential sources of imprecision and inaccuracy in the INR method of reporting ISI Value Assigned by the Manufacturer For each lot of thromboplastin reagent, manufacturers must assign an ISI value. This value should be both instrument-specific (and in some instances, instrument-model specific) and method-of-clot-detection- 6 Clinical and Laboratory Standards Institute. All rights reserved.

17 Volume 25 specific. However, it can, in some cases, be generic for the method of clot detection employed (e.g., optical versus mechanical). Due to limitations on availability of the IRP, a direct ISI assignment against an IRP is not feasible for every lot of thromboplastin reagent produced worldwide. Manufacturers generally calibrate each reagent lot using a standard reference reagent. All manufacturer-defined ISI values will be at least three calibration steps from the IRP. This is a limitation on the use of the ISI/INR system, as error can be introduced with each level of ISI assignment. For each calibration step, a 3 to 5% systematic error may occur. The accumulated variation of the reagent ISI is not an arithmetical addition of the subsequent calibration steps. The ISI is the product of the slopes of the individual steps. For example, if the CVs of the individual steps are 3%, the CV of the ISI is the square root of 9+9+9=27 (i.e., 5.2%). If the CVs of the individual calibration steps are all 5%, the CV of the ISI is the square root of =75 (i.e., 8.7%). The variation in the ISI value of the reagent ISI is paramount as it should be realized that the variation of the INR value will be greater than the variation of the ISI value. It is much more difficult to estimate the error in the INR, because of the three different error sources or variables that may contribute to the error (i.e., ISI error, MNPT error, and interaction between measurement system and patient individual factors). 9 Besides this inherent variation, there are a number of additional factors that can alter the ISI value of the thromboplastin assigned by the manufacturer and these are discussed below. Manufacturers should assign reagents ISI values according to guidelines established by the World Health Organization. 4 Any deviation from this procedure needs careful verification Variation in Manufacturer-Assigned ISI From IRP The ISI of each lot of thromboplastin is only as accurate as the procedure used to assign the value to the secondary or manufacturer standard and each subsequent lot of thromboplastin. Multiple factors may influence the ISI value assigned and a general list of these factors follows Choice of IRP The IRP materials have a formulation based on either recombinant or natural mammalian (human, rabbit, or bovine) extracted tissue factor. The source of phospholipids can be either natural or semisynthetic. Calibrations are more precise if performed against a reference preparation of similar composition and of the same species. The formulation of reagents may vary from one another and from the IRP or standard reference reagent by concentration and source of thromboplastin and phospholipid used, calcium concentration, and presence of additives such as heparin neutralizers. Comparison studies with a limited number of samples could show small although significant differences based on these variations in formulation between IRP and manufactured reagent Methods of PT and ISI Determination All ISI values of the IRP are defined by the tilt-tube method. This procedure therefore should be used for all secondary ISI assignments unless another method has been fully validated. Any error in this procedure will affect the accuracy of the ISI value assigned (usually to a standard). The method defined in the WHO guidelines for calculation of ISI must be followed. The slope and thus ISI must be determined using orthogonal regression analysis and based on a logarithmic-logarithmic plot. The manufacturer may use an automated method or methods with the secondary or manufacturer s standard, if more appropriate. Coagulation instruments, however, can have marked effects on the ISI of thromboplastins. ISI values are thus instrument- and reagent-specific. 13 Variation in performance of coagulation instruments of the same type does occur. Clinical and Laboratory Standards Institute. All rights reserved. 7

18 Number Quality of Blood Samples Used For correct collection and handling of blood specimens, refer to the most current edition of CLSI/NCCLS document H3 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture and the WHO publication Guidelines for thromboplastins and plasma used to control oral anticoagulant therapy. 4 The quality of the blood samples, and hence plasma used for ISI assignment, can affect the result. Some of the factors below may have differential effects on ISI assignment with different thromboplastin formulations. Blood samples should be collected using 105 to 109 mmol/l trisodium citrate as anticoagulant. 14 The proportion of blood to the trisodium citrate volume should be 9:1 (see Section ). Other collection tubes containing different additives, such as CTAD, may be used only when properly validated. A 129 mmol/l (3.8%) trisodium citrate concentration should not be used for PT/INR determination. See the most current edition of CLSI/NCCLS document H1 Tubes and Additives for Venous Blood Specimen Collection. Both the normal and AVK samples should be collected, processed, and tested by the same procedure within five hours (stored at room temperature) of collection of fresh samples, or within two hours of thawing/reconstituting if using frozen or lyophilized plasma. The five-hour restraint pertains only to ISI calibration according to the WHO document 4 and not to patient PT/INR testing. All AVK blood samples should be collected from individuals on stable AVK therapy and should have an INR that falls in the range of 1.5 to 4.5. All values falling outside this range should be discarded from any calculation, as inclusion could introduce error. AVK samples collected should be evenly spread across the whole 1.5 to 4.5 INR range. If plasmapheresis or blood bag collection is used, some differences in clotting times may be seen compared with syringe/blood collected into a vacuum tube (i.e., some blood collection sets contain a 4% trisodium citrate concentration). Any use of these alternate systems needs to be validated with the specific instrument reagent system in use. If lyophilized samples are used, addition of any additives to the plasma and the lyophilization process may affect results. 15 If depletion method is used to prepare artificially depleted vitamin K-dependent plasma, changes in reaction time of the plasma could occur due to removal of nonvitamin K coagulation factors, change in ph, or change in dilution. There is also the potential for differential matrix effects if normal and artificially depleted vitamin K-dependent plasmas are mixed to prepare samples with different INR values Transport/Storage Conditions of IRP and Thromboplastin Reagents The ISI values of the IRPs and reagent assigned ISI values may vary with time. Shelf life, storage, procurement, and shipping conditions can affect the ISI value assigned to each lot of reagents and also the standard reference reagent or manufacturer standard used for calibration. A number of studies have evaluated and documented the stability of the reference thromboplastins over many years. 16 All biological materials decay slowly with storage temperature and time. The rate of decay will depend on the physical state of the reagent (liquid or solid) and if solid, the amount of water contained in the solid. Intense light (if reagent is left in bright sunlight or too close to a light source) can cause chemical changes 8 Clinical and Laboratory Standards Institute. All rights reserved.