H42-AVol. 18 No. 21Replaces H42-T

December 1998 Vol. 12 No. 6

Clinical Applications of Flow Cytometry: Quality Assuranceand Immunophenotyping of Lymphocytes; Approved Guideline

This document provides guidance for the immunophenotypic analysis of non-neoplastic lymphocytesby immunofluorescence-based flow cytometry; sample and instrument quality control; andprecautions for acquisition of data from lymphocytes.

ABC

The attention of users is called to thepossibility that adherence to this guidelinemay require the use of an invention coveredby patent rights held by Ortho-ClinicalDiagnostics, Inc., Route 202, Raritan, NJ08869.

By publication of this guideline, no positionis taken with respect to the validity of thisclaim or any patent rights in connectiontherewith. The patentholder has, however,filed a statement of willingness to grant alicense under these rights on reasonable andnondiscriminating terms and conditions toapplicants desiring to obtain such a license.Details may be obtained from NCCLS.

No representative or warranty is made orimplied that this is the only license that maybe required to avoid infringement in the useof this guideline.

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THE NCCLS consensus process, which is the mechanism for moving a document through two ormore levels of review by the healthcare community, is an ongoing process. Users should expectrevised editions of any given document. Because rapid changes in technology may affect theprocedures, methods, and protocols in a standard or guideline, users should replace outdatededitions with the current editions of NCCLS documents. Current editions are listed in the NCCLSCatalog, which is distributed to member organizations, and to nonmembers on request. If yourorganization is not a member and would like to become one, and to request a copy of the NCCLSCatalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100; Fax: 610.688.0700;E-Mail: exoffice@nccls.org.

Clinical Applications of Flow Cytometry: Quality Assuranceand Immunophenotyping of Lymphocytes; Approved Guideline

Abstract

NCCLS document H42-A was developed to address issues of procedures and quality assurance forclinical applications of flow cytometry. It is designed to aid clinical laboratorians in the developmentof quality assurance procedures and to establish the foundation for different laboratories usingdifferent commercially available instruments to obtain comparable results. Specific topics coveredinclude: specimen collection, transport, and preparation; sample quality control and stainingprocedures; instrument calibration; sample analysis; and data analysis, storage, and reporting.

(NCCLS. H42-A—Clinical Applications of Flow Cytometry: Quality Assurance andImmunophenotyping of Lymphocytes; Approved Guideline. NCCLS document H42-A [ISBN 1-56238-364-7]. NCCLS, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 USA,1998.)

December 1998 NCCLS

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H42-A ISBN 1-56238-364-7

December 1998 ISSN 0273-3099

Clinical Applications of Flow Cytometry: Quality Assuranceand Immunophenotyping of Lymphocytes; Approved Guideline

Volume 18 Number 21

Michael Borowitz, M.D., Ph.D.Kenneth D. Bauer, Ph.D.Ricardo E. Duque, M.D.Allan F. Horton, Ph.D.Gerald Marti, M.D., Ph.D.Katharine A. Muirhead, Ph.D.Stephen Peiper, M.D.William Rickman, Ph.D.

ABC

December 1998 NCCLS

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This publication is protected by copyright. No part of it may be reproduced, stored in a retrievalsystem, or transmitted in any form or by any means (electronic, mechanical, photocopying,recording, or otherwise) without written permission from NCCLS, except as stated below.

NCCLS hereby grants permission to reproduce limited portions of this publication for use inlaboratory procedure manuals at a single site, for interlibrary loan, or for use in educational programsprovided that multiple copies of such reproduction shall include the following notice, be distributedwithout charge, and, in no event, contain more than 20% of the document's text.

Reproduced with permission, from NCCLS publication H42-A—Clinical Applicationsof Flow Cytometry: Quality Assurance and Immunophenotyping of Lymphocytes;Approved Guideline. Copies of the current edition may be obtained from NCCLS,940 West Valley Road, Suite 1400, Wayne, Pennsylvania 19087 USA.

Permission to reproduce or otherwise use the text of this document to an extent that exceeds theexemptions granted here or under the Copyright Law must be obtained from NCCLS by writtenrequest. To request such permission, address inquiries to the Executive Director, NCCLS, 940 WestValley Road, Suite 1400, Wayne, Pennsylvania 19087 USA.

Copyright ©1998. The National Committee for Clinical Laboratory Standards.

Suggested Citation

NCCLS. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Lymphocytes; Approved Guideline. NCCLS document H42-A (ISBN 1-56238-364-7). NCCLS, 940West Valley Road, Suite 1400, Wayne, PA 19087 USA, 1998.

Proposed GuidelineDecember 1989

Tentative GuidelineMay 1992

Approved GuidelineDecember 1998

ISBN 1-56238-364-7ISSN 0273-3099

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Committee Membership

Area Committee on Hematology

Eugene L. Gottfried, M.D. San Francisco General HospitalChairholder San Francisco, California

Subcommittee on Flow Cytometry

Michael Borowitz, M.D., Ph.D. The Johns Hopkins UniversityChairholder Baltimore, Maryland

Kenneth D. Bauer, Ph.D. Genentech, IncorporatedSouth San Francisco, California

Ricardo E. Duque, M.D. Lakeland Regional Medical CenterLakeland, Florida

Allan F. Horton, Ph.D. Coulter CorporationMiami, Florida

Rebecca L. Johnson, M.D. Berkshire Medical CenterPittsfield, Massachusetts

Gerald Marti, M.D., Ph.D. Food and Drug AdministrationBethesda, Maryland

Katharine Muirhead, Ph.D. SciGro, Inc.Malvern, Pennsylvania

Stephen Peiper, M.D. University of LouisvilleLouisville, Kentucky

William Rickman, Ph.D. PerImmune Inc. Rockville, Maryland

Advisors

Kenneth A. Ault, M.D. Maine Cytometry Research InstituteSouth Portland, Maine

Charles Bagwell, M.D., Ph.D. Verity Software House, IncorporatedTopshaw, Maine

Raul Braylan, M.D. University of FloridaGainesville, Florida

Scott C. Buessow, Ph.D. SmithKline Beecham Clinical LaboratoriesSeattle, Washington

Irene J. Check, Ph.D. Evanston HospitalEvanston, Illinois

December 1998 NCCLS

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Advisors (Continued)

G. David Cross, M.S. Centers for Disease Control and PreventionAtlanta, Georgia

Bruce H. Davis, M.D. William Beaumont HospitalRoyal Oake, Michigan

Chester J. Herman, M.D., Ph.D. Grady Memorial HospitalAtlanta, Georgia

Henry A. Homburger, M.D. Rochester, Minnesota

Berend Houwen, M.D., Ph.D. Loma Linda University School of MedicineLoma Linda, California

Jonathan Kagan, Ph.D. National Institutes of HealthRockville, Maryland

Lawrence S. Lamb, Jr., Ph.D., R.N. Richland Memorial HospitalColumbia, South Carolina

Alan L. Landay, Ph.D. Rush-Presbyterian-St. Luke's Medical CenterChicago, Illinois

Thomas McHugh, M.S. Medical Center at the University of CaliforniaSan Francisco, California

Dominic Pantalony, M.D. Toronto General HospitalToronto, Ontario, Canada

William N. Rezuke, M.D. Hartford HospitalHartford, Connecticut

Howard M. Shapiro, M.D. West Newton, Massachusetts

Carleton Stewart, Ph.D. Roswell Park Memorial InstituteBuffalo, New York

Thomas J. Tinghitella, Ph.D. Bridgeport HospitalBridgeport, Connecticut

Raymond R. Tubbs, D.O. The Cleveland Clinic FoundationCleveland, Ohio

Robert F. Vogt, Ph.D. Centers for Disease Control and PreventionAtlanta, Georgia

Susan Zolla-Pasner, Ph.D. Veterans Administration Medical CenterNew York, New York

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David E. Nevalainen, Ph.D. Abbott LaboratoriesBoard Liaison Abbott Park, Illinois

Marc R. Schlank, M.T.(ASCP), M.S. NCCLSStaff Liaison Wayne, Pennsylvania

Jennifer K. McGeary, M.T.(ASCP), M.S.H.A. NCCLSStaff Liaison Wayne, Pennsylvania

Patrice E. Polgar NCCLSEditor Wayne, Pennsylvania

December 1998 NCCLS

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ACTIVE MEMBERSHIP (as of 1 OCTOBER 1998)

Sustaining Members

American Association for Clinical ChemistryBayer CorporationBeckman Coulter, Inc.Becton Dickinson and CompanyCollege of American PathologistsDade Behring Inc.Ortho-Clinical Diagnostics, Inc.Pfizer IncRoche Diagnostics/Boehringer Mannheim Corp.

Professional Members

American Academy of Family PhysiciansAmerican Association of BioanalystsAmerican Association of Blood BanksAmerican Association for Clinical ChemistryAmerican Association for Respiratory CareAmerican Chemical SocietyAmerican Medical TechnologistsAmerican Public Health AssociationAmerican Society for Clinical Laboratory ScienceAmerican Society of HematologyAmerican Society for MicrobiologyAmerican Society of Parasitologists, Inc.American Type Culture Collection, Inc.Asociacion Espanola Primera de SocorrosAsociacion Mexicana de Bioquimica Clinica A.C.Assoc. Micro. Clinici Italiani- A.M.C.L.I.Australasian Association of Clinical BiochemistsBritish Society for Antimicrobial ChemotherapyCanadian Society for Medical Laboratory Science—Société Canadienne de Science de Laboratoire MédicalCanadian Society of Clinical Chemists

Clinical Laboratory Management Association of State and Association Territorial Public HealthCollege of American Laboratory Directors Pathologists BC Centre for Disease ControlCollege of Medical Laboratory Centers for Disease Control and Technologists of Ontario PreventionCollege of Physicians and Chinese Committee for Clinical Surgeons of Saskatchewan Laboratory StandardsCommission on Office Commonwealth of Pennsylvania Laboratory Accreditation Bureau of LaboratoriesInstitut für Stand. und Dok. im Department of Veterans Affairs Med. Lab. (INSTAND) Deutsches Institut für NormungInternational Council for (DIN) Standardization in FDA Center for Devices and Haematology Radiological HealthInternational Federation of FDA Division of Anti-Infective Clinical Chemistry Drug ProductsInternational Society for Federacion Bioquimica de la Analytical Cytology Provincia (Argentina)Italian Society of Clinical Health Care Financing Biochemistry AdministrationJapan Assn. Of Medical Instituto Scientifico HS. Technologists (Osaka) Raffaele (Italy)Japan Society of Clinical Iowa State Hygienic Laboratory Chemistry Manitoba HealthJapanese Assn. Of Medical Massachusetts Department of Technologists (Tokyo) Public Health LaboratoriesJapanese Committee for Clinical Michigan Department of Public Laboratory Standards HealthJoint Commission on National Association of Testing Accreditation of Healthcare Authorities - Australia Organizations National Health Laboratory National Academy of Clinical (Luxembourg) Biochemistry National Institute of StandardsNational Society for and Technology Histotechnology, Inc. National Institutes of HealthOntario Medical Association Ohio Department of Health Laboratory Proficiency Testing Oklahoma State Department of Program HealthOrdre professionnel des Ontario Ministry of Health technologistes médicaux du Saskatchewan Health- Québec Provincial Laboratory RCPA Quality Assurance South African Institute for Programs PTY Limited Medical ResearchSociedade Brasileira de Analises Swedish Institute for Infectious Clinicas Disease ControlSociedade Brasileira de Patologia ClinicaSociedad Espanola de Quimica ClinicaVKCN (The Netherlands)

Government Members

Armed Forces Institute of Amersham Pharmacia Biotech Pathology Ammirati Regulatory Consulting

Industry Members

AB BiodiskAbbott LaboratoriesABC Consulting Group, Ltd.AccuMed International, Inc.Accumetrics, Inc.

Asséssor

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Avecor Cardiovascular, Inc. Diagnostic Products Pharmacia & Upjohn Avocet Medical, Inc. Corporation Procter & GambleBayer Corporation - Elkhart, IN Diametrics Medical, Inc. Pharmaceuticals, Inc.Bayer Corporation - DiaSorin The Product DevelopmentMiddletown, Eiken Chemical Company, Ltd. Group VA Enterprise Analysis Corporation Radiometer America, Inc.Bayer Corporation - Tarrytown, Fort Dodge Animal Health Radiometer Medical A/S NY Fujisawa Pharmaceutical Co. David G. Rhoads Associates, Bayer Corporation - West Ltd. Inc. Haven, CT Gen-Probe Rhône-Poulenc RorerBayer-Sankyo Co., Ltd. Glaxo-Wellcome, Inc. Roche Diagnostics/Boehringer Beckman Coulter, Inc. Greiner Meditech, Inc. Mannheim Corp. Beckman Instruments (Japan) Health Systems Concepts, Inc. Roche Diagnostic Systems Ltd. Helena Laboratories (Div. Hoffmann-La Roche Becton Dickinson and Company Hoechst Marion Roussel, Inc. Inc.)Becton Dickinson Consumer Hybritech, Incorporated Roche Laboratories (Div. Products Hycor Biomedical Inc. Hoffmann-La Roche Inc.)Becton Dickinson I-STAT Corporation ROSCO Diagnostica Immunocytometry Systems Integ, Inc. The R.W. JohnsonBecton Dickinson Italia S.P.A. International Biomedical Pharmaceutical ResearchBecton Dickinson Microbiology Consultants Institute Systems International Technidyne Sarstedt, Inc.Becton Dickinson VACUTAINER Corporation Schering Corporation Systems Kendall Sherwood-Davis & Schleicher & Schuell, Inc.bioMérieux Vitek, Inc. Geck Second OpinionBiometrology Consultants Labtest Sistemas Diagnosticos SenDx Medical, Inc.Bio-Rad Laboratories, Inc. Ltda. Showa Yakuhin KakoBio-Reg Associates, Inc. LifeScan, Inc. (a Johnson & Company,Biosite Diagnostics Johnson Company) Ltd.Biotest AG LifeSign, LLC SmithKline BeechamBoehringer Mannheim GmbH Lilly Research Laboratories CorporationBristol-Myers Squibb Company Luminex Corporation SmithKline Beecham (NZ) Ltd.Canadian Reference Laboratory Mallinckrodt Sensor Systems SmithKline Beecham, S.A. Ltd. Medical Device Consultants, SmithKline Diagnostics, Inc.CASCO•NERL Diagnostics Inc. (Sub. Beckman Instruments,Checkpoint Development Inc. Medical Laboratory Automation Inc.)ChemTrak Inc. Streck Laboratories, Inc.Chiron Diagnostics Corporation MediSense, Inc. Sysmex CorporationChiron Diagnostics Corporation Merck & Company, Inc. TOA Medical Electronics- International Operations Neometrics Inc. Vetoquinol S.A.Chiron Diagnostics Corporation Nissui Pharmaceutical Co., Ltd. Vysis, Inc.- Reagent Systems Nippon Becton Dickinson Co., Wallac OyClinical Lab Engineering Ltd. Warner-Lambert CompanyCOBE Laboratories, Inc. Norfolk Associates, Inc. Wyeth-AyerstCombact Diagnostic Systems North American Biologicals, Inc. Xyletech Systems, Inc. Ltd. OBC Associates YD ConsultantControl Lab (Brazil) Olympus Corporation Yeongdong Pharmaceutical Cosmetic Ingredient Review Optical Sensors, Inc. Corp.Cytometrics, Inc. Organon Teknika Corporation ZenecaCYTYC Corporation Ortho-Clinical Diagnostics, Inc. Dade Behring Inc. - Deerfield, IL (England)Dade Behring Inc. - Glasgow, Ortho-Clinical Diagnostics, Inc. DE (Raritan, NJ)Dade Behring Inc. - Marburg, Ortho-Clinical Diagnostics, Inc. Germany (Rochester, NY) Dade Behring Inc. - Miami, FL Otsuka AmericaDade Behring Inc. - Pharmaceutical, Sacramento, CA Inc.Dade Behring Inc. - San Jose, Oxoid Inc. CA Oxoid LTD (U.K.)DAKO A/S Pfizer Inc

Trade Associations

Association of Medical Diagnostic ManufacturersHealth Industry Manufacturers AssociationJapan Association Clinical Reagents Ind. (Tokyo, Japan)Medical Industry Association of Australia

December 1998 NCCLS

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Associate Active Members

20th Medical Group (ShawAFB, SC)67th CSH Wuerzburg, GE (NY)121st General Hosptial (CA)Acadiana Medical Laboratories, LTD (LA)Advocate Laboratories (IL)The Aga Khan University Medical Center (Pakistan)Alabama Reference LaboratoryAllegheny General Hospital (PA)Allegheny University of the Health Sciences (PA)Allina Laboratories (MN)Alton Ochsner Medical Foundation (LA)Anzac House (Australia)Associated Regional & University Pathologists (UT)Baptist St. Anthony’s Health Network (TX)Baystate Medical Center (MA)Brazileiro De Promocao (Brazil)Brazosport Memorial Hospital (TX)Bristol Regional Medical Center (TN)Brookdale Hospital Medical Center (NY)Brooke Army Medical Center (TX)Brooks Air Force Base (TX)Broward General MedicalCenter (FL)Bullhead Community Hospital (AZ)Calgary Laboratory Services (Calgary, AB, Canada)Cambridge Hospital (MA)Capital Health System at Fuld (NJ)Central Kansas Medical CenterChildren’s Hospital (LA)Children's Hospital Medical Center (Akron, OH)Clendo Lab (Puerto Rico)Colorado Mental HealthInstitute at PuebloCommonwealth of KentuckyCommunity Medical Center(NJ)CompuNet Clinical Laboratories (OH)Consolidated Laboratory Services (CA)

Covance CLS (IN) Loma Linda University Medical Detroit Health Department (MI) Center (CA)Duke University Medical Center Louisiana State University (NC) Medical CenterDuzen Laboratories (Turkey) Lutheran Hospital (WI)E.A. Conway Medical Center Maccabi Medical Care and (LA) Health Fund (Israel)Elmhurst Memorial Hospital (IL) Main Line Clinical Laboratories, Emory University Hospital (GA) Inc. (PA)Fairview-University Medical Massachusetts General Center (MN) HospitalFederal Medical Center (MN) MDS Metro LaboratoryFlorida Hospital Alta Monte Services Florida Hospital East Orlando (Burnaby, BC, Canada)Foothills Hospital (Calgary, AB, MDS-Sciex (Concord, ON, Canada) Canada)Grady Memorial Hospital (GA) Med-Chem Laboratories Ltd. Guthrie Clinic Laboratories (PA) (Scarborough, ON, Canada)Hacettepe Medical Center Medical Center Hospital (TX) (Turkey) Medical College of Virginia Harris Methodist Fort Worth Hospital (TX) Memorial Medical Center (LA)Harris Methodist Northwest Memorial Medical Center (IL) (TX) Mercy Health System (PA)Hartford Hospital (CT) Mercy Hospital (NC)Health Alliance Laboratory (OH) Methodist Hospital (TX)Health Sciences Centre Methodist Hospital Indiana (Winnipeg, MB, Canada) Methodist Hospitals ofHoag Memorial Hospital Memphis Presbyterian (CA) (TN)Holmes Regional Medical Monte Tabor-Centro Italo- Center Brazileiro De Promocao (Brazil) (FL) Montefiore Medical Center (NY)Holzer Medical Center (OH) Montreal Children’s HospitalHopital de Chicoutimi (Canada) (Chicoutimi, PQ, Canada) Mount Sinai Hospital (NY)Hopital Saint Pierre (Belgium) Mount Sinai Hospital (Toronto,Hunter Area Pathology Service Ontario, Canada) (Australia) National Genetics Institute (CA)International Health Naval Hospital Cherry Point Management Associates, (NC) Inc. (IL) Nebraska Health System Intermountain Health Care New Britain General Hospital Laboratory Services (UT) (CT)John Randolph Hospital (VA) New England Medical Center Kaiser Permanente (CA) Hospital (MA)Kenora-Rainy River Regional New Hampshire Medical Laboratory Program (Dryden, Laboratories Ontario, Canada) The New York Blood Center Klinicni Center (Slovenia) New York State Department ofLa Rabida Children’s Hospital Health (IL) New York University Medical LabCorp (NC) CenterLaboratoire de Santé Publique NorDx (ME) du Quebec (Canada) North Carolina Laboratory ofLaboratory Corporation of Public Health America (NJ) North Coast ClinicalLancaster General Hospital (PA) Laboratory, Langley Air Force Base (VA) Inc. (OH)

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North Shore University Hospital Sarasota Memorial Hospital (FL) VA (Denver) Medical Center (NY) Seoul Nat’l University Hospital (CO)Northwestern Memorial (Korea) VA (Indianapolis) MedicalHospital Shanghai Center for the Clinical Center (IL) Laboratory (China) (IN)Ohio State University Hospitals Shands Healthcare (FL) VA (Tuskegee) Medical Center Olin E. Teague Medical Center SmithKline Beecham Clinical (AL) (TX) Laboratories (GA) ViroLogic, Inc. (CA)Our Lady of Lourdes Hospital South Bend Medical Foundation ViroMed Laboratories, Inc. (NJ) (IN) (MN)Our Lady of the Resurrection South Western Area Pathology Walter Reed Army Institute of Medical Center (IL) Service (Austraila) Research (MD)Pathology Associates Speciality Laboratories, Inc. Warde Medical Laboratory (MI) Laboratories (CA) (CA) Warren Hospital (NJ)Permanente Medical Group Stanford Health Services (CA) Watson Clinic (FL)(CA) Stormont-Vail Regional Medical William Beaumont Hospital (MI)PLIVA d.d. Research Institute Center (KS) Williamsburg Community (Croatia) Sun Health-Boswell Hospital Hospital (VA)Polly Ryon Memorial Hospital (AZ) Wilford Hall Medical Center (TX) Sunrise Hospital and Medical (TX)Providence Health System (OR) Center (NV) Wilson Memorial Hospital (NY)Providence Medical Center Sutter Health (CA) Winchester Hospital (MA)(WA) The Toledo Hospital (OH) Winn Army CommunityQueen Elizabeth Hospital Tri-City Medical Center (CA) Hospital (Prince Twin Lake Regional Medical (GA) Edward Island, Canada) Center York Hospital (PA)Queensland Health Pathology UCSF Medical Center (CA) Zale Lipshy University Hospital Services (Australia) UNC Hospitals (NC) (TX)Quest Diagnostics (PA) Unilab Clinical LaboratoriesQuest Diagnostics Incorporated (CA) (NJ) United Clinical Laboratories (IA)Quintiles Laboratories, Ltd. University of Alabama - (GA) Birmingham HospitalRegions Hospital University of Alberta HospitalsResearch Medical Center (MO) (Canada)Riyadh Armed Forces Hospital University of Chicago Hospitals (Saudi Arabia) (IL)Saint Mary’s Regional Medical University of Florida Center (NV) University Hospital (IN)St. Alexius Medical Center (ND) University Hospital (Gent)St. Anthony Hospital (CO) (Belgium)St. Boniface General Hospital University Hospital (London, (Winnipeg, Canada) Ontario, Canada)St. Francis Medical Center (CA) University Hospital ofSt. John Hospital and Medical Cleveland (OH) Center (MI) The University Hospitals (OK)St. John Regional Hospital (St. University of Medicine & John, NB, Canada) Dentistry, NJ UniversitySt. Joseph Hospital (NE) Hospital St. Joseph’s Hospital - University of Michigan Marshfield Clinic (WI) University of the Ryukyus St. Luke’s Regional Medical (Japan) Center (IA) University of Virginia MedicalSt. Luke’s-Roosevelt Hospital Center Center (NY) University of WashingtonSt. Mary Medical Center (CA) UPMC Bedford Memorial (PA)St. Mary of the Plains Hospital U.S. Army Hospital, Heidelberg (TX) UZ-KUL Medical Center St. Vincent’s Hospital (Belgium) (Australia) VA (Albuquerque) MedicalSan Francisco General Hospital Center (NM) (CA)

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OFFICERS BOARD OF DIRECTORS

A. Samuel Koenig, III, M.D., Carl A. Burtis, Ph.D. Robert F. Moran, Ph.D., President Oak Ridge National Laboratory FCCM, FAICFamily Medical Care mvi Sciences

William F. Koch, Ph.D., University of Wisconsin David E. Nevalainen, Ph.D. President Elect Abbott LaboratoriesNational Institute of Standards Elizabeth D. Jacobson, Ph.D. and Technology FDA Center for Devices and Donald M. Powers, Ph.D.

F. Alan Andersen, Ph.D., Secretary Hartmut Jung, Ph.D. Eric J. Sampson, Ph.D.Cosmetic Ingredient Review Boehringer Mannheim GmbH Centers for Disease Control

Donna M. Meyer, Ph.D., Tadashi Kawai, M.D., Ph.D. Treasurer International Clinical Pathology Marianne C. Watters,Sisters of Charity Health Care Center M.T.(ASCP) System Parkland Memorial Hospital

Charles F. Galanaugh, Past D.D.S. Ann M. Willey, Ph.D. President Loyola University Medical New York State Department ofBecton Dickinson and Center Health Company (Retired)

John V. Bergen, Ph.D., Executive Director

Sharon S. Ehrmeyer, Ph.D.

Radiological Health Ortho-Clinical Diagnostics, Inc.

Kenneth D. McClatchey, M.D.,

and Prevention

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Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Committee Membership . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v

Active Membership . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viii

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

4 Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4.1 Blood Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.2 Safety Attire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.3 Biological Safety Cabinets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.4 Specimen Containers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.5 Centrifugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.6 Pipetting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.7 Sharp Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.8 Blood Spills . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.9 Waste Disposal and Specimen Inactivation . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.10 Specimen Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44.11 Unfixed Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54.12 Equipment Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5 Specimen Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5.1 Patient Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.2 Venipuncture Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.3 Anticoagulant of Choice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.4 Labeling of Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

6 Specimen Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

6.1 Handling Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56.2 Effects of Storage and Holding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

7 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

7.1 Visual Specimen Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67.2 Selection of a Sample Preparation Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . 7

8 Immunofluorescence Staining of Lymphocyte Surface Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

8.1 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78.2 Optimization of Staining Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

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Contents (Continued)

8.3 Optimization of Reagent Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

9 Sample Quality Control (QC) Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

9.1 Preparation of Negative Control Specimen(s) . . . . . . . . . . . . . . . . . . . . . . . . . 109.2 Preparation of Positive Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

10 Instrument Quality Assurance (QA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

10.1 Instrument Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1110.2 Monitoring Instrument Performance and Reproducibility . . . . . . . . . . . . . . . . . 18

11 Sample Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

11.1 Instrument Configuration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1911.2 Order of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1911.3 Verification of Acceptable Specimen Viability . . . . . . . . . . . . . . . . . . . . . . . . 1911.4 Verification of Representative Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2011.5 Establishment and Monitoring of "Lymphocyte" Light Scatter Gates . . . . . . . . . 2011.6 Analysis of Negative Control Sample(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2711.7 Analysis of Samples Stained for Subset Enumeration . . . . . . . . . . . . . . . . . . . 27

12 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

12.1 Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2812.2 Potential Sources of Difficulty in Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 2912.3 Single Color (Single Antibody) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2912.4 Dual Color (Dual Antibody) Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

13 Data Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

13.1 Recording Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3113.2 Information to Be Stored . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3113.3 Types of Data Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3213.4 Duration of Data Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

14 Data Reporting and Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

14.1 Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3214.2 Supervisory Check . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3214.3 Review of Data Displays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3214.4 Reporting of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3214.5 Interpretation of Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3314.6 Notation of Out-of-Range Control Samples . . . . . . . . . . . . . . . . . . . . . . . . . . 3314.7 Inclusion of Reference Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

Appendix A. Potential Sources of Artifacts in Immunophenotyping by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

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Contents (Continued)

Appendix B. Alternative Methods for Cell Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Appendix C. Instrument Log Book—Optical Alignment, Fluorescence Amplification, and Compensation Logs . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Appendix D. Laboratory Worksheet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Appendix E. Determination of Reference Ranges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Appendix F. Three-Color Lymphocyte Immunophenotyping Methods . . . . . . . . . . . . . . . . . . 47

Summary of Comments and Subcommittee Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Related NCCLS Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

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Foreword

Advances in the availability and reproducibility of monoclonal antibody reagents specific for a widerange of cell types, coupled with lower costs for increasingly automated flow cytometers withgreater data analysis capabilities, have made flow cytometry the method of choice forimmunophenotyping in the clinical laboratory. NCCLS document H42-A represents the effort of theNCCLS Subcommittee on Flow Cytometry appointed to establish guidelines for immunophenotypingby flow cytometry. The primary purpose of this document is to establish performance guidelines forlymphocyte immunophenotyping by flow cytometry, as applied to analysis of cells from adults withnonhematopoietic disease.

H42-A is designed to aid clinical laboratorians in the development of quality assurance proceduresand to establish the foundation for different laboratories using different commercially availableinstruments to obtain comparable results. This document should help minimize interoperator andinterlaboratory variability in the various components of flow cytometry. Specific topics coveredinclude specimen collection, transport, and preparation; sample quality control and stainingprocedures; instrument calibration; sample analysis; and data analysis, storage, and reporting.

Comments on the tentative guideline (H42-T) received by the subcommittee have been reviewed andchanges incorporated, where appropriate. All comments and the subcommittee’s responses to thesecomments are included at the end of the document.

Standard Precautions

Because it is often impossible to know which might be infectious, all patient blood specimens are tobe treated with standard precautions. For specific precautions for preventing the laboratorytransmission of bloodborne infection from laboratory instruments and materials; andrecommendations for the management of bloodborne exposure, refer to NCCLS documentM29—Protection of Laboratory Workers from Instrument Biohazards and Infectious DiseaseTransmitted by Blood, Body Fluids, and Tissue.

Key Words

Autofluorescence, CD system, color compensation, dual parameter display, fluoresceinisothiocyanate (FITC), fluorescence, forward angle light scatter, gate, histogram, immunoglobulin,immunophenotyping, linear amplification, logarithmic amplification, low angle light scatter,lymphocyte, ninety degree light scatter, phycoerythrin (PE), positive procedure control, singleparameter display (histogram), subclass.

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Clinical Applications of Flow Cytometry: Quality Assurance andImmunophenotyping of Lymphocytes; Approved Guideline

1 Introduction

Flow cytometry is a rapidly developingtechnology that has moved from the researchlaboratory into the clinical laboratory. The goalof this document is to establish qualityassurance procedures that will help ensureprecision and accuracy of flow cytometricresults appropriate for their use in the clinicallaboratory. Since at present thepreponderance of systems used forlymphocyte subset typing in clinicallaboratories are fluorescence-based, thisdocument is limited to specific issuessurrounding the use of such systems. Majorpoints of discussion include the following:

! Patient groups to be included

! Potentially biohazardous procedures Autofluorescence, n - The intrinsicand appropriate precautions fluorescence of unstained cells generally

! Type and frequency of methodologic The level of autofluorescence is a function ofcontrols required the excitation and wavelength and varies with

! Guidelines for retention of laboratory of cellular activation. Cultured cell lines andrecords macrophages usually demonstrate higher

! Analysis methods for discrete vs.continuous intensity distributions Cluster differentiation (CD) system, n - The

! Guidelines for definition of laboratory similar patterns of reactivity with human cellsreference ranges. has been the focus of five international

2 Scope

The scope of this document is to establishperformance guidelines for typing oflymphocyte subsets from adults withnonhematopoietic disease usingimmunofluorescence-based flow cytometry.

The subcommittee recognizes that other typesof flow cytometers also exist or are indevelopment (e.g., systems using absorbancerather than fluorescence detection orimpedance rather than scatter). Some of theissues discussed in this document arecommon to the use of any flow cytometer forlymphocyte typing (e.g., sample collectionand transport, data reporting, and

interpretation). However, issues such assample preparation, instrument calibration,and quality control differ significantly fornonfluorescence-based systems and are notdiscussed here.

At present, there are no universally acceptedstandards for precision, accuracy, andinterlaboratory comparability in lymphocytetyping by flow cytometry. It is beyond thescope of this document to establish generalperformance criteria and reference ranges. Therefore, each laboratory is responsible forestablishing performance criteria and referenceranges appropriate for its particular instrumentand population base.

3 Definitions

caused by pyrimidines and flavin nucleotides.

the cell type being analyzed and/or the state

levels of autofluorescence.

identification of monoclonal antibodies with

workshops. Each group of antibodies wasassigned a CD number. Not all antibodies in aCD group react with identical portions(epitopes) of their target antigen. An antigenrecognized by a given cluster of antibodies(e.g., CD4) is referred to as a "CD antigen"(e.g., CD4 antigen). An antibody belonging toa given cluster is referred to as CD"x," withthe manufacturer's nomenclature given inparenthesis (e.g., CD4 [Leu3a]). Color compensation, n - Electronic ormathematical subtraction of a fraction of onesignal from a second, typically used incorrecting for overlapping fluorescence fromone fluorochrome in the wavelength regionwhere the second is to be measured so thatpopulations stained exclusively with each

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fluorochrome appear at right angles to each linear amp could have output varying fromother. one to five volts as the input signal varies

Dual parameter display, n - (dot plot, contourplot, cytogram, two-parameter histogram): A Logarithmic amplification, n - A logarithmicgraphic representation of data in which amplifier produces an output signal valuecorrelated intensities for two different proportional to the logarithm of the inputparameters measured on the same cell are signal value. A four-decade log amp might,plotted on an x,y grid. for example, have an output of 0 V for a 1-

Epitope, n - (antigenic determinant) That 100-mV input; 7.5 V for a 1-V input; and 10portion of an antigen against which the V for a 10-V input. Log amps are usefulspecific binding region of a monoclonal when analyzing samples containing cellsantibody reagent is directed. Epitopes may be whose measured parameters differ by orderslinear sequences of as few as six amino acids of magnitude.or conformationally determined sections of theantigen; each antigen typically contains Ninety degree light scatter, n - (right anglemultiple epitopes. [90 LS], wide-angle, side scatter (ss), or

Ffluorescein isothiocyanate, FITC, n - The light at right angles to the incident lightmost common fluorochrome for cell source. This measurement is related tophenotyping. Fluorescein conjugates absorb cytoplasmic granularity, membranemaximally at approximately 490 nm, close to irregularity, and/or nuclear shape of a cell orthe 488 nm emission of argon lasers, and particle.emit near 525 nm. Each conjugatedfluorescein molecule adds a net negative Phycoerythrin, PE, n - One of severalcharge to the antibody and therefore may phycobiliprotein-based fluorochromes, derivedchange its potential binding characteristics. from algae or bacteria, which can be

conjugated to antibodies for use inForward angle light scatter, FALS, n - (low immunotyping. PE has a molecular weight ofangle; FSC; FS) Measurement of light at a 240,000 kD. Although it absorbs lightlow radial angle relative to the incident light maximally at 545 nm, there is sufficientsource. Measured values are a function of the absorption at the 488 nm excitation by argoncross-sectional area and refractive index of a lasers to provide a usable signal in thecell or particle and the wavelength used for red/orange (575 nm) emission range. (NOTE: measurement. It is commonly used as an Denaturation of PE does not cause a spectralestimate of the relative size of a cell or shift but instead quenches the fluorescence.)particle. Aggregation of PE/immunoglobulin conjugates

causes nonspecific binding.Gate, n - A set of parameters used toelectronically select a particular cells to be Procedure control, n - Specimen that isevaluated. Typically, a region of interest is drawn, prepared, and stained with the samedefined based on one set of parameters (such protocol used for patient specimens. It isas FALS vs. 90 LS) and other parameters used for the following: (1) in instrumento

(such as fluorescence) are evaluated only for setup and monitoring for setting test-specificcells within a defined region. instrument settings and establishing color

Immunophenotyping, n - Identification of cell specimen for sample and data analysis.surface antigens (markers) characteristic ofsubsets of leukocytes utilizing fluorescence- Propidium iodide, PI, n - A dye which maylabeled antibody reagents that recognize serve to identify nonviable cells by virtue ofcell-associated molecules. its ability to penetrate cells with damaged

Linear amplification, n - A linear amplifier double-stranded nucleic acid. PI will absorb(amp) produces a signal output proportional to 488 nm argon laser light and emit in the redthe input signal amplitude. For example, a spectrum near 620 nm. This can be detected

from 0.01 to 0.05 volts.

mV input; 2.5 V for a 10-mV input; 5 V for a

o

orthogonal light scatter). Measurement of

compensation settings; and (2) as the control

membranes and subsequently bind to

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independently of fluorescein-labeled cells, but to obtain information by means of a specificonly with appropriate filter/PMT conditions laboratory test.when other fluorescent dyes are present. Propidium iodide cannot be used as an Sensitivity, n - The ability to distinguishindication of viability after fixation of cells signal(s) of interest from background "noise." because all cells become permeable and take Note that "noise" may be instrumental (opticalup PI. or electronic signals arising when no

Quality assessment, external, n - External (autofluorescence, nonspecific reagentquality assessment refers to a system of binding, etc.).retrospectively and objectively comparingresults from different laboratories by means of Single parameter display, n - (histogram) surveys organized by an external agency. The Frequency distribution of measured signalmain object is to establish between-laboratory intensities (i.e., channel numbers) observedand between-instrument comparability, if for cells within a population.possible in agreement with a referencestandard where one exists. External quality Specimen, patient, n - A volume of wholeassessment schemes may be regional, blood, bone marrow, body fluid, lymph nodenational, or international. They may also be or other tissue, appropriately collected,limited to the users of a particular instrument. transported, and processed to provide aIt is sometimes also referred to as “proficiency sample for performing one or more laboratorytesting” especially when the external agency tests.is a regulatory agency.

Quality assurance, n - The practice that immunoglobulin of the same isotype (class orencompasses all procedures and activities subclass) as the monoclonal antibody ofdirected toward ensuring that a specified interest but without specificity for any knownproduct is achieved and maintained. human antigens. Most monoclonal antibodies

Quality control, internal, n - Internal quality various subclasses of IgG (IgG1, IgG2a,control is the set of procedures undertaken in IgG2b, or IgG3). This type of control isa laboratory for the continual assessment of typically used to estimate FC-mediated,work carried out within the laboratory and nonspecific binding for antibody reagents of aevaluation of the results of tests to decide given subclass.whether the latter are reliable enough to bereleased to the requesting clinician. The Threshold, n - The level of signal above whichprocedures should include tests on control a measured value is believed to bematerial and statistical analysis of patients' significantly different than backgrounddata. The main object is to ensure day-to-day "noise." Threshold for instrumentconsistency of measurement or observation fluorescence sensitivity is the level of signalthat is, if possible, in agreement with an found for what is believed to be aagreed indicator of truth such as control nonfluorescent object. Threshold formaterial with assigned values. determination of positive antibody staining is

Resolution, n - The ability to discriminate to react specifically with a given antibodybetween cells or particles having different reagent.signal intensities. The ability to resolvediscrete populations is a function of biological Viability dye, n - A dye that may serve tofactors (heterogeneity of signal within each identify nonviable cells by virtue of its abilitypopulation, difference in signal intensities to penetrate cells with damaged membranesbetween populations) as well as instrumental and subsequently bind to double-strandedfactors (sensitivity, measurement CV). nucleic acid. Propidium iodide (PI), is the

Sample, patient, n - A patient sample is 488-nm argon laser light and emits in the redprepared from the patient specimen and used spectrum near 620 nm. This can be detected

fluorochrome or cell is present) or biological

Subclass control, n - A subclass control is an

used in flow cytometry are either IgM, or

the level of signal found for cells not believed

most commonly used viability dye. It absorbs

independently of fluorescein-labeled cells but

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only with appropriate filter/photomultipliertube conditions when other fluorescent dyesare present. Propidium iodide (PI) cannot beused as an indication of viability after fixationof cells by most methods because all cellsbecome permeable and take up PI.

4 Safety

There are many etiologic agents that may bepresent in blood and body fluids. Because thesample may contain the active agent ofinfection with resulting morbidity or mortality,all specimens should be handled as thoughthey are potentially infectious. Standard1,2

precautions" should be observed.

4.1 Blood Collection

Blood should be collected as outlined in theNCCLS document M29 taking care to 2

prevent needlestick injuries. Needles shouldbe discarded in a puncture-resistant containerand should not be resheathed, purposefullybent, cut, or removed from the syringe.

4.2 Safety Attire

Gloves and laboratory coats should be wornby all personnel processing blood specimens. If splashing or spattering of the specimen isanticipated, masks and protective eyewearshould be worn to prevent exposure of eyes,nose, and mouth. Gloves should be changedand hands washed after completion of thework and before leaving the laboratory. Formore detailed information, see Reference 2.

4.3 Biological Safety Cabinets

If possible, all work should be done in abiological safety cabinet (BSC, Class I or II). If this is not possible, all procedures that havethe potential for creating droplets or aerosols(vortexing, opening evacuated tubes, etc.)should be performed in a BSC (Class I or II).

4.4 Specimen Containers

All specimens should be aliquotted into tubesthat have caps or microtiter plates that havecovers (preferably sealed covers). The capsand covers should be in place except whenadding to or removing from the containers.

4.5 Centrifugation

If specimens are centrifuged, care should betaken to avoid aerosol formation (e.g., insealed vessels or inside safety carriers).

4.6 Pipetting

Pipetting by mouth is not allowed. Manualpipetting devices must be used.

4.7 Sharp Devices

Use of "sharps" (e.g., needles, glass Pasteurpipets, glass containers, etc.) should beavoided as much as possible.

4.8 Blood Spills

If a blood spill occurs, it should bedecontaminated at once with an appropriateagent such as 1/10 volume dilution of 0.71mol/L sodium hypochlorite (i.e., undilutedhousehold bleach) or appropriately dilutedmycobacteriocidal hospital disinfectants.3

4.9 Waste Disposal and SpecimenInactivation

Specimens and all disposable laboratorysupplies should be collected in puncture-proofcontainers designed specifically for suchmaterials and disposed of by appropriatemeans (e.g., incineration, autoclaving).

An alternative technique for specimeninactivation is to mix samples with bleach,retain for 24 hours and dispose of properly.

4.10 Specimen Storage

It is preferable to assay only samples in whichinfectious agents have been inactivated. Fixation is also utilized for stabilization of thesample, so that it may be analyzed at a laterdate. For flow cytometric analysis, after thelast centrifugation step in the labelingprocedure, samples should be resuspended ina solution of buffered (pH 7.0 to 7.4)paraformaldehyde or formaldehyde (commonly0.5 to 2.0%) and stored at 4 C untilo

analysis. Because specimen preparation and4

fixation techniques may vary depending onlysing agents and type/concentration offixatives used, the minimum and maximum

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time the specimens should be stored infixative will also vary. For validity of theanalysis, the concentration ofparaformaldehyde/formal-dehyde andmaximum storage time should be verified. Minimum storage time in paraformal-dehyde/formaldehyde required to inactivatepotentially infectious agents is not clearlyestablished.5,6,7

4.11 Unfixed Specimens

When unfixed specimens are to be run on theflow cytometer, the laboratory must be ableto demonstrate that appropriate biosafetyprocedures are in place to minimize exposureto infectious material. Because aerosolgeneration in flow cytometers with astream-in-air is a potential hazard, specialattention must be exercised to operate theinstrument in accordance with themanufacturer's biosafety recommendations.

4.12 Equipment Disinfection

The waste container on the flow cytometer should contain 1/10 of its volume of 0.71mol/L sodium hypochlorite (undilutedhousehold bleach).

Laboratory equipment should be properlydisinfected with a solution known toinactivate infectious agents or freshly made10% solution of household bleach. It isparticularly important that instruments beproperly disinfected prior to repair ormaintenance (see NCCLS document I17-P).8

5 Specimen Collection

5.1 Patient Information

A test requisition should accompany allspecimens. This should include the date, age,gender, and unique identifier of the patient,name of requesting physician or originatinglaboratory, and a presumptive diagnosis. Some medications may interfere with cellpreparation, staining, or analysis. This shouldbe taken into consideration when interpretingabnormal results.

5.2 Venipuncture Technique

Venipuncture procedures as outlined inNCCLS document H3—Procedures for theCollection of Diagnostic Blood Specimens byVenipuncture should be followed when9

obtaining the blood specimen(s). Bloodspecimens must be regarded as potentiallyinfectious. Blood collection procedures asoutlined in Reference 2 should be followed forthe safety of the phlebotomist.

5.3 Anticoagulant of Choice

For immunophenotyping by flow cytometry,ethylenediaminetetraacetic acid (EDTA), acidcitrate dextrose (ACD, solution A), or heparinmay be used, although specimen stabilityvaries somewhat among the threeanticoagulants. If a white count and10-14

differential count is to be made from the samespecimen used for flow cytometry, EDTA isthe anticoagulant of choice.15

5.4 Labeling of Specimen Specimen(s) should be labeled with a uniquepatient identifier and with date and time ofcollection and processing.

6 Specimen Transport

6.1 Handling Specimens

All specimens are to be handled in accordancewith the understanding that they may transmithepatitis virus, human immunodeficiencyvirus, or other infectious agents. Thetransportation of specimens containingpotential infectious agents should includemeasures that minimize the risk of exposureof individuals to the potential etiologic agents. Specimens should be placed in well-stopperedtest tubes and the tubes should be placedwith adequate absorbent material in a securely closed container for local transportation. Interstate shipments of blood samples aresubject to the packaging and labelingrestrictions outlined in the InterstateQuarantine Regulations, Etiologic Agents,Code of Federal Regulations. These16

guidelines have been outlined in detail inNCCLS document H5—Procedures for theHandling and Transport of DomesticDiagnostic Specimens and Etiologic Agents.17

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6.2 Effects of Storage and Holding

The yield of T lymphocytes from bloodsamples is affected by a number offactors. Ideally, specimens should be 12,18-24

processed immediately after collection. Whenthis is not possible, the laboratory shouldverify that their anticoagulant (see Section5.3), holding temperature, and preparationmethod maintain specimen integritycomparable to freshly processed material. Thisvalidation protocol should include normal andabnormal specimens. Specimens should be maintained at 18 to 22C during transportation and storage foro

maximum flexibility in sample preparationmethods.

7 Sample Preparation

The goal of sample preparation is to process a blood specimen into a representative samplesuitable for introduction into the flowcytometer for analysis. All pertinent andrepresentative cellular and antigenicparameters for lymphoid cells should bemaintained. Irrelevant cells (e.g., erythrocytes)that may slow or interfere with analysisshould be removed. Avoidance of differentiallosses among lymphocyte subpopulationsduring cell preparation is critical. In general,the more a cell suspension is manipulated orprocessed the greater the opportunity for cellloss (which is seldom equivalent among allsubpopulations of lymphocytes). A wholeblood method is recommended as theprocedure of choice because it involves aminimum of manipulation. The advantagesand limitations of alternate preparationmethods are discussed in Appendix B.

7.1 Visual Specimen Evaluation Observable specimen problems are of twotypes:

(1) Those indicating that the sample is alteredor damaged and calling for immediatespecimen rejection.

(2) Those suggesting that specimenmishandling may have occurred andcalling for further evaluation during samplepreparation. Mishandling problems should

be recorded because they may prove to beinformative when preparing and analyzingthe specimen and interpreting the results.

7.1.1 Hemolysis

Hemolysis indicates that the blood has beenexposed to conditions that can causeerythrocyte lysis, suggesting that leukocytesmay be damaged, also. Severely hemolyzedspecimens should be rejected.

7.1.2 Clotted Blood

Even a partial clot may cause selective loss oralteration of certain subpopulations. Clottedspecimens should be rejected.

7.1.3 Partial Draw

For some anticoagulants, a partially filled tubemay produce hypertonic conditions deleteriousto cells (i.e., ACD). Depending on themagnitude of the underfill, one may accept orreject the specimen. If the specimen isaccepted, the laboratory should havepreviously demonstrated that the observedunderfill does not affect final results, and theunderfill should be recorded for considerationduring preparation, analysis, andinterpretation.

7.1.4 Temperature Extremes

If the specimen tube has been mailed to thelaboratory, it may have been exposed totemperature extremes and should beexamined upon receipt to determine if it isunusually warm or cold to the touch. Even ifall other evaluation criteria are satisfactory,this observation should be noted for furtherconsideration during preparation, analysis, andinterpretation. 7.1.5 Improper Specimen Labeling

Any specimen that has not been labeled witha unique patient identifier should be rejected.

7.1.6 Specimen Integrity

One should visually inspect the specimen forany other changes (e.g., lipemia). Thisobservation should be noted for furtherconsideration during separation, analysis, andinterpretation.

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7.2 Selection of a Sample PreparationProcedure

7.2.1 Whole Blood Lysis vs. DensityGradient Methods

The whole blood lysis method is generallya

recommended because it does not employdensity gradients or lengthy centrifugationwhich may lead to differential losses ofspecific subpopulations. However, thisprocedure assumes equivalent recovery of alllymphocyte subsets.

7.2.2 Selection of Lysing Agent

Several lysing techniques are available. Theseinclude tris-buffered ammonium chloride andhypotonic buffer. Several proprietary19,25

lysing reagents are also available frominstrument or monoclonal antibodymanufacturers. When using commercialreagents, the manufacturer's recommendedprotocol must always be followed. 7.2.3 Validation of Whole Blood Procedures

7.2.3.1 Storage Conditions

If specimens are to be stored, holding at 18 to22 C is recommended. The laboratory musto

verify that stored specimens give resultsequivalent to fresh specimens using theirstorage conditions and lysing procedure.

7.2.3.2 Specimen Age

Maximum acceptable specimen age is afunction of the anticoagulant, lysing agent,and storage conditions. The laboratory10,11

should verify the maximum allowablespecimen age for the anticoagulant and lysingreagent combination used.

7.2.3.3 Abnormal Lymphocyte Counts

Specimens with a pronounced lymphopeniamay not have sufficient lymphocytes in the

whole blood aliquot for flow cytometricanalysis. Conversely, normal concentrationsof antibody reagents may be insufficient tosaturate all binding sites in specimens withlymphocytosis, leading to possible falsenegative results. Each laboratory must thushave a way of determining the appropriatelymphocyte count in the specimen, either bydirect measurement or by having access tothe lymphocyte count from anotherlaboratory. Samples should be adjusted sothat the lymphocyte concentration is in theappropriate range for staining.

8 Immunofluorescence Staining ofLymphocyte Surface Antigens

8.1 Reagents

8.1.1 Reagents for Lymphocyte Phenotyping The following procedures assume the use offluorochrome-conjugated antibody reagents. Directly-labeled subclass controls are used toestimate the level of combined autofluor-escence and nonspecific binding found in agiven specimen.

Reagent Reactivity

CD3 T cells

CD4 T cell helper/inducer subset, monocytes

CD8 T cell suppressor/ cytotoxic subset, NK subset

CD19 B cells

CD56 or CD16 NK cells/T subsets

Subclass control nonspecific + auto- fluorescence

This panel identifies the major lymphocytesubsets (T, B, and NK). Together, thesereagents should account for essentially alllymphocytes in a healthy individual. Wherereagents identify more than one major subset(e.g., some CD3 T cells can express so calledNK markers) accurate enumeration of thesesubsets requires a two-color dual parameteranalysis. The laboratory's choice of reagentsshould be tailored to the clinical problemunder investigation. It is recognized thatother reagents or combinations may be usefulfor diagnosing or monitoring specific disease

U.S. Patent 4,284,412 issued to Ortho-Clinicala

Diagnostics, Inc. Ortho offers licenses under this patent ona nonexclusive basis. Manufacturers considering marketingof systems for carrying out the whole blood lysis methodshould see the note on the inside front cover of thisguideline.

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states. In some cases, more reagents thanare clinically indicated or requested may haveto be analyzed to assure that all relevantlymphocytes are included and that a minimalnumber of irrelevant cell types are analyzed(see below). Single antibody determinationsare not recommended under anycircumstances.

Other reagents or combinations may be usefulfor diagnosing or monitoring disease states. Itis beyond the scope of this document torecommend or to define reagents which haveclinical utility.

8.1.2 Reagents for Establishing orMonitoring Lymphocyte Gates

Reagent Reactivity

CD45 Pan leukocytesCD14 or MonocytesCD13 Myeloid Cells

Intrinsic light scatter properties commonlyused to assist in distinguishing lymphocytesfrom other events can be affected by sample26

age, choice of lysing agent, fixation,instrument optics, disease states, etc. (SeeFigures 1 and 6.) It is important to estimateboth the number of contaminating cell types (which may besources of false negative or false positiveevents) and the proportion of the totallymphocytes which are included within theselected lymphocyte gate; the reagents listedabove can assist in this task. Lymphocytesmay usually be identified as CD45 andbright+

CD14 . Contaminating granulocytes are–

CD45 and monocytes are CD14 anddim + +

CD45 while erythrocytes are essentially+

CD45 negative. See Section 11.5 for furtherdiscussion of the advantages and limitationsof this method of monitoring quality oflymphocyte gates.

An alternate marker to CD14 is CD13(antimyeloid) which identifies contaminatingmonocytes and granulocytes. However, theamount of CD13 antigen on monocytes istypically low, so the choice of reagent maydepend on instrument sensitivity. In someclinical situations lymphocytes may expresslower levels of CD45 antigen and in othersmonocytes may lose significant amounts ofCD14 antigen (upon cellular activation).

8.2 Optimization of Staining Protocol

When commercially available reagents have adetailed staining protocol, the protocol shouldbe followed exactly. This is also true for redcell lysis reagents (see Figure 1).

A

B

C

Figure 1. Sample preparation can also affectresolution of populations based on their lightscattering characteristics. Erythrocytes were lysedby (A) hypotonic lysis, (B) NH Cl, and (C) a4

commercial lysing reagent.

The following variables have typically beenexamined by the manufacturer in arriving attheir recommended procedure.

8.2.1 Volume

Where sample plus reagent volume exceeds0.2 mL, mixing during incubation and/or an

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increased time of incubation may be required. All of these procedure alterations should beA staining buffer (pH 7.2) containing at least documented in the record, and should only be1% protein is recommended for employed when there is a CBC available and adensity-purified cell preparations. clear cut separation exists between negative

8.2.2 Temperature example, can be lost if the antibody

Room temperature (18 to 22 C) is staining will remain defined.o

recommended for staining samples forimmunophenotyping. Some antigens (e.g., CD8 and CD16) are shed

8.2.3 Time of Incubation with Reagent instructions for such antibodies should be

Time to reach saturation is affected by sample recommended amount will be sufficient forvolume and fluorochrome. PE-conjugated staining unseparated blood. The amountsreagents may require longer incubation times required for separated cells will be less, andcompared with FITC conjugates. Typical using the recommended amounts may resultstaining times recommended by the in nonspecific staining.manufacturer will vary from 10 to 30 minutes.

Any deviation from the standard laboratory ormanufacturer's protocol should bedocumented in a laboratory protocol book togive results comparable to those obtainedusing the recommended procedure.

8.2.4 Cell Count

The amount recommended for staining withmost commercial reagents will be sufficient tostain 100 µL of blood, assuming a normalrange of approximately 4 to 10 x 103

leukocytes per mm (4 to 10 x 10 per L) or3 9

0.5 to 2 x 10 separated mononuclear cells6

per mm . However, there is usually sufficient3

leeway to provide adequate staining of bloodwith higher counts. The upper limit of theWBC which can be accepted is determined bythe percentage of lymphocytes in the sample,the reactivity of the monoclonals with otherblood elements (e.g., polymorphonuclearleukocytes, or PMNs) as well as the amountof antibody employed.

If a white count and differential is available,the amount of blood can be adjusted toensure optimum staining of samples with veryhigh lymphocyte counts (e.g., some leukemiasand infants). Similarly, if a sample isleukopenic, the amount of blood or theamount of antibody can be adjusted. Thebuffy coat of leukopenic samples can also beprocessed in order to acquire sufficient cellsfor staining and analysis.

and positive cells: HLADR positive T cells, for

concentration is too low, although the B cell

from the cell surface into the plasma. The

examined to determine whether the

8.3 Optimization of ReagentConcentration

As in Section 8.2 above, most commercialreagents have recommended amounts forstaining a fixed volume of blood (or number ofseparated cells) in a fixed time. Theserecommended amounts are typically derivedby determining the amount of reagent requiredto give:

! The maximum separation between thepositive/negative populations

! The maximum fluorescence intensity ofthe positive population.

For each lot of a given reagent, the laboratoryshould verify that the manufacturer's antibody gives comparable positive/negativeresolution and comparable positive stainingintensity with previous lots using thelaboratory's method of sample preparation.

For any deviation from the manufacturer'srecommended staining conditions (e.g., time,volume, temperature, or cell number), thelaboratory must determine the minimalamount (volume or weight) of reagentrequired to give optimal positive/negativeresolution and optimal positive stainingintensity.

NOTE: Excess reagent may cause increasednonspecific staining of negatives,resulting in decrease of positive/neg-

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ative resolution, and may causequenching, resulting in decreasedstaining intensity and percent positive 9.1.1.2 Other Approachescells.

9 Sample Quality Control (QC)Procedures

Both positive and negative controls are a vitalpart of quality assurance. Negative controlsestablish the background expected as a resultof combined autofluorescence and nonspecificstaining in the sample. Positive controls areneeded to demonstrate adequate performanceof reagents and labeling procedures.

9.1 Preparation of Negative ControlSpecimen(s) 9.1.1 Negative Reagent Control(s)

Negative reagent controls establish thebackground fluorescence expected as a resultof nonspecific reagent binding andnonreagent-related cellular autofluorescence,and are the minimum negative control whichshould be run.

9.1.1.1 Subset Subclass Controls

This control attempts to estimate nonantigen-related binding and is added to the cells in thesame manner as the test reagent. Althoughnot ideal in all respects, the best availablenegative reagent controls for murinemonoclonal reagents are murine monoclonalantibodies or myeloma proteins not specificfor human cells, but of the same or similarsubclass as the test reagent. Subclass controlreagents should be appropriately matched tothe test antibody's concentration andfluorochrome: protein ratio.

It should be noted that different negativecontrol reagents of the same subclass maygive variable staining intensities. Also, somenonspecific staining may result from bindingto cells without interactions with known Fcreceptors. This should be considered whenselecting an appropriate subclass control,which should perform consistently over awide range of clinical specimens (i.e.,nonspecific binding is similar for mostsamples).

In cases where the panel of monoclonalantibodies includes antibodies of more thanone subclass used with one fluorochrome, itmay be acceptable to use a pooled subclasscontrol. If this is done, it is necessary toverify that nonspecific staining with thepooled subclass control is no greater than thatwith the individual subclass controls.

Because most nonspecific binding is due tothe interaction of the Fc portion of theantibody used to the Fc receptor on cells,another approach to control for thisnonspecific binding is to block it in thestaining procedure. This is done by addingirrelevant antibody (commonly serum frommouse, horse, or goat) to the blood beforeaddition of the monoclonal antibody reagents.

9.1.2 Autofluorescence Control

To determine the backgroundautofluorescence of the cells, the appropriatecontrol is a tube containing unstained cellsprepared by the same method as the patientsamples. This control is not required but maybe helpful in identifying interferences fromnonantibody factors (e.g., medications or dyecarry-over) which affect cellular autofluoresc-ence.

9.2 Preparation of Positive Controls

Positive controls are needed to verify theperformance of reagents, preparationmethods, and staining procedures. They arealso used in performing instrument qualitycontrol. Two different types of positivecontrols may be used. Positive reagentcontrols verify that when cells with a givenmarker are present, the reagent is capable ofproperly identifying them. Positive procedurecontrols verify that the sample preparationand labeling procedures are adequate. A blood specimen from a healthy donor oftenserves as both positive reagent control andpositive procedure control.

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9.2.1 Positive Reagent Controls essential element of daily quality assurance

Freshly isolated lymphocytes, frozen lympho- performance must be monitored under thecytes, appropriately preserved mononuclear same conditions as are used to run testcells, or whole blood preparations may be samples. Instrument quality control thereforeused as reagent controls. Positive reagent involves two procedures:controls are used to validate proper binding ofcurrent reagent lots and to test new reagent (1) Instrument setup—identification of appro-lots, and should be run when reagent lots are priate conditions for analysis of testchanged. The accepted variability for new lots samples and establishment of acceptanceof reagents compared to current lots should values for materials to be used in dailynot be any greater than the variability found instrument monitoring (described infor replicate samples of the current lot. Section 10.1) One advantage of using frozen lymphocytes (2) Instrument monitoring—use of referencefrom the leukapheresis of a single donor or samples or materials with stable propertiesappropriately preserved lymphocytes is that to monitor instrument performance and tothey provide a cell source that is consistent reproduce test-specific analysis conditionsfrom day to day for comparison of reagent (described in Section 10.2).performance. For that reason, it is importantthat the cells are properly frozen and properly Table 1 summarizes the various tasks involvedthawed each day that they are used. Cell in instrument quality assurance and the12

viability and recovery should be established in general properties of samples useful inthe laboratory. A disadvantage of this type of performing each task.preparation is that it cannot also double as apositive procedure control when the wholeblood procedure is the method used forpatient specimens. If commercially available controls are utilized,the manufacturer's directions should befollowed exactly.

9.2.2 Positive Procedure Control

When performing whole blood lysisprocedures, adequacy of sample preparationand labeling procedures are assessed using aspecimen from a healthy donor which isdrawn, prepared, and stained using the sameprotocol as used for patient specimens.Ideally, a procedure control is prepared andrun with every batch of patient samples.

If commercially available controls are utilized,the manufacturer's directions should befollowed exactly.

10 Instrument Quality Assurance(QA)

There are at present no standards which canbe used to check the accuracy of flowcytometric test results. Hence, verifyingreproducibility of instrument performance is an

for the flow cytometry laboratory. Instrument

10.1 Instrument Setup

Instrument setup first establishes thatinstrument performance is acceptable undersome initial (frequently manufacturer-defined)set of conditions, and then identifies optimalconditions for analysis of representative testsamples. Once optimal test-specificconditions are identified, acceptance rangesfor materials to be used in daily performancemonitoring and reference ranges for specificmarkers are established under those sameconditions.

The following instrument setup procedures(Table 1) should be carried out when the flowcytometer is first received, when majormaintenance or repair is performed, and whenany significant change in specimenpreparation or staining protocols is made.

Table 1. Quality Assurance Overview

QA Function Why When What (sample properties)

INITIAL INSTRUMENT SETUP OPTIMIZE ANALYSIS NEW TEST OR INSTRUMENT SEE BELOWCONDITIONS CONDITIONS

Align optical system (10.1.1.1). Maximize intensity, minimize New instrument, major Homogenous signals in all channelsvariability. maintenance or repair. used for specimen analysis; long

term stability (e.g., uniformfluorescent plastic beads).

Establish acceptance values for Determine laboratory’s expected Whenever significant alignment See alignment.particles to be used in alignment range of values indicating changes are made. monitoring (10.1.1.2). acceptable alignment.

Establish test-specific instrument Optimize optically aligned Introduction of test; after any “Normal” specimens preparedsettings, giving: a) optimal light instrument for analysis of test significant change in specimen using laboratory’s standard methodscatter resolution (10.1.2.1) specimens. preparation or staining protocol. and labeled with i) reagent givingb) optimal fluorescence “bright” staining and goodresolution (10.1.2.2) c) optimal resolution of positive from negativecolor compensation cells (e.g., CD3), and ii) reagent(10.1.2.2[3]). giving “dim” positive staining (e.g.,

CD2 or CD56).

Establish acceptance values for Determine laboratory’s expected Whenever significant changes See test-specific instrumentmaterials to be used in intensity range of values indicating are made in test-specific conditions.monitoring (10.1.2.2[2]). acceptable ability to resolve dim instrument settings.

positive cells from negatives.

Establish acceptance values for Determine laboratory’s expected Whenever filters are replaced; Lymphocytes stained with mutuallymaterials to be used in range of values indicating whenever instrument exclusive antibodies bearingcompensation monitoring acceptable ability, in two-color parameters which affect relevant flurochromes (bright(10.1.2.2[4]). analysis, to resolve dual fluorescence amplification are reagents preferred, e.g., FITC-CD4

positive cells from cells labeled altered (e.g., detector & PE-CD8) OR plastic particlesonly with one reagent or the voltage/gain settings). with intensities and spectralother. characteristics similar to bright

antibody positive cells.

Table 1. (Continued)

QA Function Why When What (sample properties)

DAILY PERFORMANCE ASSURE ACCEPTABLE WHENEVER SAMPLES ARE STABLE: ACCEPTANCE VALUESMONITORING PERFORMANCE RUN PREVIOUSLY ESTABLISHED

Monitor optical alignment Verify acceptable alignment. Whenever samples are run or Reference material with acceptance(10.2.1) instrument malfunction is values established under conditions

suspected. of optimal alignment (e.g., uniformfluorescent beads). See alignmentprocedure, initial instrument setup.

Monitor intensity performance Verify reproducible intensity Whenever samples are run or Freshly prepared specimen stainedand fluorescence resolution measurements and acceptable instrument malfunctin is with relevant fluorochromes OR(10.2.2) sensitivity. suspected. plastic compensation particles with

acceptance values establishedunder appropriate compensationlevels (see Color Compensation,Initial Instrument Setup).

Verify system performance Verify performance of sample Whenever test specimens are Specimen from a healthy donor(10.2.4) preparation and staining method run; whenever sample prep or prepared according to standard test

using sample for which staining problems are protocol using reagent for whichexpected results are known. suspected. laboratory reference limits are

established.

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10.1.1 Optical Alignment (channel numbers) for the alignment particles,

10.1.1.1 Optimization of Optical Alignment instrument setting(s) required to achieve a

Proper alignment of the optical components of instrument performance varies on a daily basisthe flow cytometer (laser, flow cell, focusing as directly, but can nonetheless be used tolenses, collecting lenses, photodetectors, etc.) establish acceptance ranges for performanceshould be established using the monitoring.manufacturer's recommended alignmentmaterials and procedures. Alignment Whichever method is used, alignment particlesparticles are typically uniform plastic particles should be run under conditions of optimalincorporating a fluorescent dye; other alignment a total of 20 or more times over amaterials may also be recommended by the minimum of at least five separate days,manufacturer. collecting data for all parameters which will be

used to analyze test specimens. The rangesThe goal in optimizing alignment is to of expected values for mean channel numbersimultaneously achieve maximum signal and CV (Method 1) or for PMT settingsintensity (maximum mean channel number) required to achieve specified mean channeland minimum signal variability (minimum values (Method 2), along with relevantcoefficient of variation, CV) for all parameters instrument settings, should be recorded in anwhich will be used to analyze test specimens. instrument log book (Appendix C) for subsequent use as acceptance ranges in dailyFor instruments with fixed optical systems, monitoring. Any new lot of alignmentwhere alignment is not carried out by the user particles should be run in parallel with the oldon a daily basis, alignment should be lot to establish acceptance ranges for the newmonitored using manufacturer-recommended lot.particles to determine whether instrumentperformance meets specifications. 10.1.2 Test-Specific Instrument Settings

10.1.1.2 Establishment of Acceptance Alignment particles often have significantlyValues For Alignment Particles To Be Used In different light scatter and fluorescenceDaily Monitoring intensities than lymphocytes stained with

In some cases, manufacturers provide may be necessary to "fine tune" instrumentexpected values for their particles when used settings for analysis of test specimens. Thiswith specific instruments, instrument settings, is done using blood from healthy individualsfilters, etc. In other cases, the laboratory prepared using the laboratory's standardmust determine optimum settings for their method and labeled with: own instrument-alignment particlecombination and establish their own expected ! A reagent which gives relatively highvalues. staining intensity and good resolution of

Two different methods for monitoringreproducibility of instrument alignment are in ! A reagent which gives dim butcommon use. Method 1 monitors the consistently greater staining intensity thanreproducibility of mean intensities and CVs for unstained cells (e.g., CD2/E rosettealignment particles under fixed instrument receptor or CD56 on lymphocytes, CD13conditions (i.e., specified laser power, filters, on monocytes). PMT high voltages and gains) as shown inFigure 2, Panel A. Differences in measured Selection of appropriate test-specific settingsparticle intensity thus give a direct indication involves two steps:of how much instrument performance variesfrom day to day. Method 2 monitors the (1) Optimization of light scatter resolution toreproducibility of instrument conditions allow clear visualization of the lymphocyte(typically PMT high voltage and/or gain) population; appropriate amplification required to achieve specified mean intensities

as shown in Figure 2, Panel B. Differences in

specified intensity do not indicate how much

fluorochrome-labeled antibodies. Therefore, it

stained and unstained cells (e.g., CD3).

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Figure 2. Methods for Instrument Performance Monitoring. Squares and triangles represent valuesobtained by two different operators; dashed lines represent acceptance ranges for parameters shown(mean ± 2 S.D.).

A. Mean intensity and coefficient of variation (CV) of fluorescent polystyrene particles weremonitored under constant instrument conditions (filters, laser power, detector high voltage/gainsettings, linear amplification).

B. Detector high voltage required to reproduce mean channel number ± 1 channel for FITC-conjugated calf thymocyte nuclei was monitored using fixed laser power, filter sets, andlogarithmic amplification. NOTE: Out-of-range value in "red" channel on day 32 was due topropidium iodide carry-over.

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settings must be identified for both forward (2) Establishment of acceptance values forand 90 degree light scatter. materials to be used in daily intensity monitoring(2) Optimization of fluorescence resolution to

allow clear discrimination of negative, It has been suggested that level of expressiondimly stained, and brightly stained of specific cell surface antigens varies as apopulations: function of maturation, differentiation, and/or

C For single color analysis, appropriate clinical utility of such variations has not yetfluorescence amplification settings must be been reached, use of a consistent intensityidentified. scale from day to day is recommended as one

way to speed the process of gathering theC For dual color analysis, appropriate necessary information.

fluorescence amplification andcompensation settings must be identified. Two different approaches to daily intensity

10.1.2.1 Optimization of Light Scatter use freshly prepared blood specimensResolution following establishment of instrument

The lymphocyte population should be well If the intensity value recovered from theresolved from other leukocyte populations, "normal" sample falls within the laboratory'sand the shapes of all leukocyte clusters established range for the measured antibody,should be visible. (See figures in Section by using appropriate statistical analysis,11.5.1 for examples of a variety of light unknowns can then be analyzed. The valuesscatter amplification and display methods.) obtained for the normal sample that day, as

10.1.2.2 Optimization of Fluorescence must be recorded. Modification to instrumentResolution settings based on the "normal" must be

(1) Establishment of appropriate fluorescence population norm for the laboratory. Theintensity settings problem of variability can be reduced if daily

verification is done using a reagent whichFluorescence amplification/gain settings gives relatively stable intensities fromshould be chosen to place the positive peak of individual to individual (e.g., CD3).the "bright" control at or slightly above themidpoint of the fluorescence intensity scale; A second method, which leads to morehowever, no positive cells should accumulate reproducible intensity settings is to use ain the highest channel of the histogram. It is reference material with stable staining and/orpossible that significant numbers of unstained fluorescence intensity characteristics similar topatient cells will be as bright as "dim" those of antibody-stained lymphocytes. controls since they are totally unpredictable Ideally, it should contain populations withwith respect to their cytoplasmic content, fluorescence intensities similar to unstainedwhich is the primary source of fluorescence in and dimly antibody stained lymphocytes (i.e.,unstained cells. Amplification should be when the instrument is able to resolve theseselected to place as many of the unstained populations, it should also be able to resolvecells as possible in a higher position than in dimly stained lymphocytes). Acceptancethe lowest channel and still not allow values for the intensity reference material areaccumulation of positive cells in the highest then established by running the material underchannel. When dual color analysis is to be test-specific conditions a total of 20 or morecarried out, appropriate fluorescence times over a period of at least five days. amplification/gain settings should be verified These values and all relevant instrumentwith samples that represent "dim" and settings are then recorded in the instrument"bright" staining with each fluorochrome of log book (Appendix C) for use in dailyinterest. monitoring. Any new lot of reference

disease state. While consensus on the27,28

monitoring are possible. One approach is to

operating parameters with a standard material.

well as corresponding instrument settings

justified based on the statistics of the

29

material should be run in parallel with the old

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lot to establish acceptance ranges for the new Ideally, the antibody combination used tolot. establish compensation settings should be chosen from antibodies in the test panel(3) Establishment of appropriate color giving the highest fluorescence intensities

compensation settings (e.g., CD4/ CD8), since this will make

The goal of color compensation is to assure Figures 3 and 4, panel A). For example, ifthat when both fluorescein-(FITC) and normal lymphocytes are used as thephycoerythrin-(PE) conjugated antibodies are reference material for establishingused to label a cell sample, cells co-labeled compensation settings, the following sampleswith both reagents can be accurately might be evaluated:distinguished from cells labeled only with one reagent or the other. This corresponds to Tube 1: FITC-CD4 positiveselecting instrument conditions under which Tube 2: PE-CD8 positivecells labeled only with FITC give no greater Tube 3: admixture of Tubes 1 and 2.signal than unstained cells on the PEhistogram and vice versa. Electronic If higher values are found for percent dualsubtraction is typically used to correct positive in two-color (tube 3) than in single(compensate) for the fact that a small color samples (tubes 1 and 2),proportion of FITC fluorescence is detected undercompensation is occurring andusing "PE" filters and, conversely, a small compensation settings must be increased toproportion of PE fluorescence is detected avoid overestimation of dual labeled cells (seeusing "FITC" filters. Figures 3 and 4, panel A). Note that it is

Degree of electronic subtraction required (electronically overcorrect; see Figures 3 anddepends on spectral characteristics of the 4, panel C). Overcompensation will not affectfilters used and on level of signal detection of singly labeled cells, but mayamplification. Therefore, compensation cause underestimation of dual-labeled cells,settings must be re-evaluated whenever filters especially when they are dimly positiveare replaced or when instrument parameters (compare Figures 3 and 4, panels B and C). which affect fluorescence amplification are Therefore, the lowest compensation settingsaltered (e.g., photomultiplier high voltage which give satisfactory agreement betweenand/or gain settings). This is true whether single color and dual color samples should becompensation levels are established using selected to avoid overcompensation. cells or plastic particles. Note that if the Satisfactory agreement means that theintensity scale is reset daily to a constant difference in % positive determined fromposition using a stable intensity reference single vs. dual color samples should be nomaterial, this corresponds to maintaining a greater than the difference between replicateconstant system amplification from day to determinations on single color samples.day. Therefore, if this method is used, Equivalently, cells labeled only with FITCcompensation settings need only be re- should give no greater signal than the evaluated when filters are replaced. negative control at the PE detector.

(a) Use of cells to establish compensation give no greater signal than the negativesettings control 0at the FITC detector.

Lymphocytes stained with mutually exclusiveantibodies bearing the relevant fluorochromesare the proper reference materials forestablishing appropriate compensationsettings or for verifying that compensationsettings chosen using plastic particles areappropriate.

undercompensation more visually evident (see

also possible to overcompensate

Conversely, cells labeled only with PE should

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Figure 3. Effects of under- or overcompensation on two-color analysis. Beads labeled with no fluorochrome(N), FITC only (F), PE only (P), or both fluorochromes (FP) were analyzed at several different compensationsettings. LFL1=all fluorescence collected using 525±20 nm bandpass filter. LFL2=all fluorescence collectedusing 575±15 nm bandpass filter.

A. Slight undercompensation : average LFL2 signal of FITC beads greater than average LFL2 signalof unlabeled beads, average LFL1 signal of PE beads slightly greater than average LFL1 signal ofunlabeled beads.

B. Correct compensation: average LFL2 signal of FITC beads same as average LFL2 signal ofunlabeled beads, average LFL1 signal of PE beads same as average LFL1 signal of unlabeledbeads.

C. Slight overcompensation: average LFL2 signal of FITC beads less than average LFL2 signal of

unlabeled beads, average

LFL1 signal of PE beads less than average LFL1 signal of unlabeled beads.

D. Identification of dual-labeled events: compensation settings as in B; all dual-labeled events fallin region 2. This figure demonstrates appropriate compensation for identifying a lowfluorescence-intensity dual staining population.

E. Effect of overcompensation on dual-labeled events: compensation settings as in C; dual-labeledevents beginning to fall into region 4 (PE negative) and would be difficult to distinguish fromFITC only of the same intensity.

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If the plastic particles do not have precisely

A. No settings may be somewhat different thanCompensation those which would be chosen using stained

B. Correct If the laboratory uses plastic compensationCompensation particles with manufacturer-defined values to

C. OverCompensation If the laboratory uses plastic compensation

A. Uncompensated data

B. Correct spectral compensation

C. Overcompensated data

Figure 4. Effect of compensation on two-colorimmunofluorescence display.

(b) Use of particles to establish compen-sation settings

Plastic particles labeled with FITC and PEwhich have stable spectral characteristics andintensities similar to those of antibody-labeledcells may also be useful in initial choice ofapproximate compensation levels. For someparticles, the manufacturer provides expectedvalues for particle intensity which have beenestablished under test-specific conditions on aparticular type of instrument. In other cases,the laboratory assigns its own values forparticle intensities under test-specificconditions.

the same spectral characteristics as stainedleukocytes (something which is difficult toaccomplish), particle-based compensation

leukocytes. Therefore, compensationsettings initially chosen using any plasticparticles should always be verified usingstained lymphocytes as described in Section10.1.2.2 (3)(a).

establish compensation settings,manufacturer- recommended proceduresshould be followed. However, thecompensation levels chosen should still beverified using lymphocyte preparations suchas those described in Section 10.1.2.2 (3)(a).

particles without manufacturer-defined values,the particles should have fluorescenceintensities at least as bright as the brightestantibody-labeled samples under testconditions to avoid possibleundercompensation. Approximatecompensation settings should be chosen byrunning the particles under test-specificinstrument settings and then verified usinglymphocyte preparations such as thosedescribed in Section 10.1.2.2 (3)(a).

(4) Establishment of acceptance valuesfor materials to be used in dailycompensation monitoring

Expected values for compensation particlesmay be provided by the manufacturer forsome instruments with fixed optical systems. For other particles and/or instruments whichare user adjustable, expected values areestablished by running the material under test-specific conditions and at established intensityand compensation settings a total of 20 ormore times over a period of at least five days. Mean fluorescence intensities for eachpopulation of interest (FITC only, PE only, andnegative for both), along with all relevantinstrument settings and compensationsettings are recorded in the instrument logbook (Appendix C) for use in daily monitoring.Any new lot of compensation referenceparticles should be run in parallel with the old

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lot to establish acceptance ranges for the new handling, preparation, or staining should belot. investigated.

Expected values for compensation reference 10.2.1 Verification of Optical Alignmentparticles must be reestablished whenevercompensation settings are re-evaluated (i.e., To verify optical alignment perform thewhenever filters are replaced or when instru- following steps:ment parameters which affect fluorescenceamplification are altered significantly [e.g., (1) Run alignment particles under instrumentphotomultiplier high voltage and/or gain settings determined at the time of initialsettings]). instrument setup.

10.2 Monitoring Instrument Performanceand Reproducibility

For daily performance monitoring, control orreference materials are reassayed under test-specific instrument settings and the valuesobtained compared to the acceptance valuespreviously established under conditions ofoptimal performance. Consistent qualityassurance requires that each of the relevantfunctions listed in Table 1 be performedregularly. Note, however, that a separatesample is not necessarily required for eachfunction. One QC material may serve severalfunctions, or a mixture of several materialsmay be formulated to minimize the timerequired to verify proper instrumentperformance. For example, an intensitycontrol containing both cellular and plasticparticles of varying intensities has beendescribed which can be used to assessinstrument reproducibility with respect to lightscatter, fluorescence sensitivity, and amplifierresponse characteristics. Recording values30

for certain instrument settings (e.g., lasercurrent required to achieve a given poweroutput, laser power required to achieve agiven forward scatter intensity for a standardparticle) may also assist in identifying trendsrequiring preventive maintenance (e.g.,cleaning optics, laser tube replacement) and inavoiding down-time due to catastrophicfailure. In addition to improving confidence in testdata, information provided by the variousreference materials can be of significant valuein troubleshooting. If ambiguous orinconsistent test results are obtained,reassaying the various reference materials canimmediately establish whether faultyinstrument performance is responsible. If thisis not the case, possible errors in sample

(2) Record the mean channel number and CV,or instrument parameters (PMT settings)required to reproduce a specified meanchannel number, for all parameters to beanalyzed for test specimens in the dailylog book and on Levy-Jennings plots.

If values are not within the range ofacceptance, alignment should be optimizedbefore proceeding.

10.2.2 Verification of Fluorescence Resolution

To verify reproducibility of intensity scale andinstrument sensitivity, use the followingprocedures:

! If freshly stained samples are used toverify instrument sensitivity, establish thatthe dimly stained positive control can bedistinguished from the unstained control.

! If intensity reference particles (e.g.,fluorochrome-labeled beads or nuclei) areused to verify instrument sensitivity, runthem under test-specific settingsestablished at the time of initial setup andestablish that "dim" particles can bedistinguished from "blank" ones.

! If a constant intensity scale is being used,run intensity reference particles undertest-specific instrument settings andestablish that their mean channel valuesfall within the ranges established at thetime of initial instrument setup.

! Record mean fluorescence channel andCV for all populations of interest (e.g.,unstained and dimly stained) in the dailylog book and on Levy-Jennings plots.

If mean fluorescence values are not withinthe range of acceptance, reasons for

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altered instrument sensitivity should be determine the source of the problem. Anydetermined before the analysis of test problems identified using this sample must bespecimens and they should be corrected rectified before the analysis of testas necessary. specimens.

10.2.3 Verification of Appropriate ColorCompensation

If dual-color analysis is being run, verifyappropriate color compensation settings usingthe following procedures:

! If freshly stained samples are used toverify compensation settings, establishthat single-color and dual-color samplesgive equivalent results [see Section11.1.2.2 (3)(a)]

! If compensation particles (e.g., FITC- andPE-labeled beads) are used to verifycompensation settings, run them undertest-specific settings and establish thattheir values fall within the expectedranges determined at time of initialinstrument setup.

! Record mean fluorescence intensity foreach population of interest (FITC only, PEonly, and negative for both) in the dailylog book and on Levy-Jennings plots.

If particle values are not within the range ofacceptance, compensation settings should bere-evaluated using antibody-stained lympho-cytes.

10.2.4 Verification of Overall SystemPerformance

To verify overall system performance use thefollowing procedures:

(1) Run the positive procedure control.

(2) Verify that the LS resolution of allleukocyte populations is acceptable.

(3) Verify that the percent antibody-positivelymphocytes falls within laboratory-established ranges for the markersselected.

If this positive control does not meetlaboratory criteria, remedial action should betaken. Instrument performance and/orstaining procedure should be checked to

11 Sample Analysis

11.1 Instrument Configuration

Proper instrument configuration includes:

! Selection of operating conditions whichminimize biohazard to the operator andmaximize decontamination of fluid waste

! Reestablishment of test specificconditions and validation of acceptableinstrument performance under thoseconditions (Section 10.0).

11.2 Order of Analysis

11.2.1 Procedural Control Specimen

(1) Controls for establishing and monitoringlight-scatter gates used to define lympho-cytes

(2) Negative subclass controls for definitionof intensity threshold above which cellsare considered to be antibody positive

(3) Positive controls for verification of stain-ing and preparation methods.

11.2.2 Patient Specimens

(1) Controls for establishing and monitoringlight scatter gates used to define lympho-cytes

(2) Negative subclass control for definition ofintensity threshold above which cells areconsidered to be antibody positive

(3) Patient samples stained with monoclonalantibodies.

11.3 Verification of AcceptableSpecimen Viability

Because of potential biohazards, it isrecommended that all samples be fixed usingagents known to inactivate viruses and other

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infectious agents prior to introduction into the may indicate size and/or density differencesflow cytometer (see Section 4 on safety). which could affect relative sedimentation

However, nonviable cells are a significant verified by analyzing a well-mixed sample andsource of false positive staining and each comparing it to results of analysis of the samelaboratory should have an established method sample which has been allowed to settle for afor assessing sample viability after preparation time comparable to the longest expectedand staining. Where fluorescent viability sample analysis time.31-34

stains such as propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) are used tomonitor sample viability on the basis of dyeexclusion, it is essential to use unfixedsamples (see Section 5.11), since these dyesmay redistribute over all cells after fixationand storage. If alternative viability probes areused for postfixation viability discrimination,the laboratory must verify, usingrepresentative patient specimens, that suchprobes give results comparable to thoseobtained using a standard viability dye (PI,trypan blue) in unfixed samples. Whatevermethod is used, the laboratory is responsiblefor determining the concentration of viabilitydye to use to give optimal resolution betweenlive and dead cells. A minimum viability of 85% is recommend-ed. A more restrictive light scatter gate is19,35

sometimes used to exclude nonviable cells,which typically have lower forward scatterintensity; however, this may also bias againstcertain lymphocyte subtypes. Whenever25,35,36

restrictive light scatter gating is used, anattempt should be made to estimate theproportion of lymphocytes included andexcluded by the gate (see Section 11.5).

If specimen viability is below the establishedlaboratory minimum, test results are suspectand this should be noted on the test report.When possible, the specimen should bereprepared if less than 24 hours old and/orredrawn.

11.4 Verification of RepresentativeSampling

To avoid possible artifacts resulting from Figures 5 and 6 show "typical" dualdifferential sedimentation rates, samples parameter light scatter displays obtained fromshould be well mixed immediately prior to lysed whole blood preparations. Even underintroduction into the instrument and, if ideal conditions there is some overlappossible, during data accumulation. between cell types (e.g., between large

Verification of representative sampling is small lymphocytes and red cells or plateletespecially important when specimen light aggregates). In addition, the distinctionscatter characteristics are abnormal, since this between leukocyte clusters is affected by

rates. Representative sampling may be

11.5 Establishment and Monitoring of"Lymphocyte" Light Scatter Gates

The first step in sample analysis is toestablish the gating parameters which will beused in distinguishing lymphocytes from othercell types present in the specimens, mostcommonly forward and right angle lightscatter. Light scatter gates serve to define"what is a lymphocyte?" for the purpose ofcollecting immunofluorescence data, anderrors or biases resulting from improperlyselected light scatter gates can significantlyalter the resulting immunophenotype. Therefore, once light scatter gates have beenselected, the laboratory should attempt toevaluate the quality of the chosen gates withrespect to the following two issues:

! What fraction of the events within thechosen light scatter gate arenonlymphocytes?

! What fraction of the lymphocytes presentwithin the sample are excluded from thechosen lymphocyte gate?

11.5.1 Typical Methods for EstablishingLymphocyte Scatter Gates

Two different approaches are commonly usedto establish lymphocyte gates. Method 1relies on the observation that normal lympho-cytes characteristically exhibit forward and 90degree light scatter intensities lower thanthose of other leukocytes but higher thanthose of red cells, platelets and "debris.

lymphocytes and small monocytes, between

37-40

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instrumental, methodological, and biological to clinical condition. There are nofactors including: controls which can be used to define the

! Forward scatter detector geometry lympho-cyte, and therefore ability to(scattering angle subtended, stream-in-air identify a well-resolved lymphocyte clustervs. enclosed flow cell) (Figure 6). may be significantly affected by such

! Type of lysing agent used (Figure 1).

! Heterogeneity of light scatter properties between including all cells believed to beamong lymphocyte subsets. lymphocytes and excluding all contaminating

cell types (Figure 7). Typical compromises! Altered frequency of lymphocyte subsets are to:

having light scatter overlapping with smallmonocytes (e.g., NK cells). ! Set more conservative light scatter gates,

! Cell activation due to clinical condition, phocytes, in order to limit the number ofsample age or mishandling. contaminating cells.36-40

Method 2 relies on the observation that ! Correct for the proportion of contaminat-normal lymphocytes characteristically exhibit ing cells when immunophenotypes arehigher staining intensities with CD45 reported (i.e., if 20% CD4 cells areantibodies than do other leukocytes, and that observed when 85% of light scattererythrocytes, platelets, and debris do not stain events are lymphocytes and if all positivewith such antibodies. CD45 antibodies events are believed to be lymphocytes, a35,36,38

give overlapping intensity distributions for corrected value of 20/85=23.5% CD4lymphocytes and monocytes, and two-color lymphocytes would be reported). staining with CD14 antibodies is typically Unfortunately, since nonlymphocytes mayused to completely resolve lymphocytes from constitute either false positives (e.g.,monocytes. In this method, CD45 CD4 monocytes) or false negatives (e.g.,36,40 bright

CD14 cells with appropriate light scatter are CD4 erythrocytes) depending on the+ –

defined as lymphocytes: a dual parameter antigen of interest, simply correcting thelight scatter distribution gated on all CD45 denominator for contaminating cells maybright

CD14 cells is collected, and a light scatter still not give an accurate result. Using+ –

gate is established which includes as many of checks for self-consistency (see Sectionthese cells as possible. However, a 11.7.2.2) can help ensure that suchlymphocyte light scatter gate set broadly corrections are appropriate.enough to include most CD45 CD14bright + –

cells with low-moderate light scatter may also 11.5.2 Choice of Method for Establishinginclude a significant proportion of Lymphocyte Gatescontaminating cell types. Resolution ofCD45 CD14 lymphocytes from CD45 There is no ideal method for definingbright + dim–

CD14 granulocyte is affected by biological lymphocyte light scatter gates at present, but+ –

and methodological factors including: Methods 1 and 2 provide complementary

! Instrument optical configuration (Figure 6) requires no additional reagents and can be

! Relative frequency of specific cell types most instruments, but does not identify(e.g., CD45 CD14 cluster may be leukocytes with light scattering characteristicsbright+ –

difficult to identify in lymphopenic speci- overlapping those of lymphocytes (e.g.,mens; basophils exhibit light scatter basophils, monocytes, degranulatedsimilar to lymphocytes but are CD45 ) granulocytes). Moreover, technical factorsdim +

may affect separation of populations and! Variations in relative intensity of lympho- make Method 1 difficult to apply (see Figure 8)

cyte labeling with CD45 (e.g., due to particular CD45 antibody reagent used or

intensity threshold corresponding to a true

intensity variations).

Usually, a compromise must be made

which then will not include all lym-

+

+

+

–

advantages and limitations. Method 1

rapidly applied using software available on

December 1998 NCCLS

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Method 2 offers an alternative definition of 11.5.3 Monitoring of Lymphocyte Lightlymphocytes (CD45 CD14 ) helpful in Scatter Gates bright + –

attempting to check the proportion oflymphocytes excluded or of contaminating cell Regardless of the method used to selecttypes included. However, Method 2 assumes lymphocyte light scatter gates, the laboratorythat all monocytes are CD14 (which may not must attempt to evaluate the light scatter+

be valid in all specimens) and that a clearly gates used for analysis of each specimen withdefined CD45 CD14 cluster can be regard to the following:bright+ –

identified (see Figure 6).

It is to be expected that additional gating s.methods for defining "lymphocytes" will bedeveloped in the future, each having its own ! Frequency of lymphocyte exclusion.advantages and limitations (See Appendix Ffor an overview of three-color 11.5.3.1 Frequency of Contaminating Cellsimmunophenotyping methods which combineimmunofluorescence and light scatter gating Contaminating cell types include but are notmethods). It is the responsibility of the limited to the following:laboratory to determine the method orcombination of methods most appropriate for ! Nonleukocytes (e.g., CD45 cells; usuallyselection of lymphocyte light scatter gates in potential false negatives; Figure 9).their situation.

! Type and frequency of contaminating cell-

–

! Monocytes (e.g., CD14 cells; potential+

false negatives or false positives; Figure 9).

! Monocytes + granulocytes (e.g., potentialfalse negatives or false positives).

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Figure 5. Light Scatter Properties of Leukocytes. Whole blood from healthy individuals was lysed, washed, fixedovernight in buffered formaldehyde, and analyzed 18 to 24 hours later. Data from Laboratory 1 (Panels A and B) werecollected using linear amplification. Data from laboratory 2 were collected using linear amplification for forward scatter and

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linear (Panel C) or logarithmic (Panel D) amplification for 90 degree scatter. Instruments in both laboratories utilizeddetection in an enclosed flow cell; however, forward scatter collection angles differed somewhat.

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Figure 6. Effect of Optical Configuration and Alignment on Definition of Lymphocytes Using LightScatter or CD45/CD14 Staining.

Whole blood from a healthy individual was stained with FITC-conjugated CD45 and PE-conjugatedCD14, lysed, washed, and fixed overnight in 1% buffered formaldehyde. The same sample wasthen analyzed on instruments representing a typical range of optical configurations:

(1) Panels A and D: enclosed flow cell and relatively narrow forward scatter collection angle (.0.5o

to 4 ), fluorescence detection optimized using medium size (.5 Fm diameter) dimly fluorescento

particles

(2) Panels B and E: jet-in-air and larger forward scatter collection angle (.2 to 10 ), fluorescence o o

detection optimized using small (.2 Fm diameter) brightly fluorescent particles

(3) Panels C and F: jet-in-air and decreased forward scatter collection angle (.0.5 to 10 ), o o

fluorescence detection optimized using larger (.10 Fm diameter) brightly fluorescent particles.

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M = monocytes, G = granulocytes, L = lymphocytes, E = erythrocytes, D = debris, P = platelets, Eo =eosinophils

Figure 7. Comparison of Light Scattering and Immunofluorescence Methods for Identification ofLymphocytes.

Whole blood was stained with FITC-conjugated CD45 and PE-conjugated CD14, lysed, washed,fixed overnight in buffered 2% formaldehyde, and analyzed on an instrument utilizing detection in anenclosed flow cell. An ungated light scatter distribution was collected (Panel A) and used to identifya region believed to be primarily lymphocytes (region 1 = low-moderate scatter intensity). Simultaneously, an ungated immunofluorescence distribution (panel B) was collected and used todefine a region believed to be primarily lymphocytes on the basis of CD45 staining intensity (region2 = CD45 CD14 ). bright –

Cross-checking light scatter vs. immunofluorescence definitions of "lymphocyte" shows that someCD45 CD14 cells are clearly outside the lymphocyte scatter cluster (e.g., Eo in panel C). bright –

Conversely (panel D), some cells defined as lymphocytes by light scatter appear to be contaminatingcell types on the basis of their CD45/CD14 immunofluorescence (here estimated as 0.3%monocytes, 3% nonleukocytes, 5% basophils+granulocytes). It is also apparent (panel C) thatsome

CD45 CD14 cells with low-moderate scatter are not included within region 1; these represent anbright –

estimate of the number of lymphocytes excluded from analysis when region 1 is used as the lightscatter gate.

Note that neither definition of "lymphocyte" gives complete and unambiguous resolution from allother cell types. In optimizing light scatter gating of lymphocytes, operator and/or softwarealgorithms are attempting to minimize the number of lymphocytes excluded (i.e., to make region 1as large as possible) while still maintaining acceptable levels of contaminating cell types.

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Figure 8. Resolution between lymphocytes, monocytes, and granulocytes can be affected by severalfactors.

A. A well resolved normal blood sample. B. A sample held 24 hours before processing.

Light Scatter Gate Quality Control: Light Scatter Gate Quality Control:

Recovery: 100% of all lymphocytes Recovery: 97% of all lymphocytes (based on CD45/CD14 (based on CD45/CD14

staining) staining)

Contamination: 1% monocytes Contamination: 1% monocytes 2% granulocytes 5% granulocytes *

1% debris 5% debris

Too many nonlymphocytes in gate.*

C. A sample with improper erythrocyte lysing D. A sample containing leukemic blasts.and subsequent fixation.

Light Scatter Gate Quality Control: Light Scatter Gate Quality Control:

Recovery: 97% of all lymphocytes Recovery: 99% lymphocytes†

(based on CD45/CD14 (based on CD45/CD14 staining) staining)

Contamination: 1% monocytes Contamination: 1% monocytes 2% granulocytes 0% granulocytes†

1% debris 0% debris

CD45 intensity shows two brightly stained †

populations.

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11.5.3.2 Frequency of LymphocyteExclusion

(1) Estimate proportion of total lymphocytesincluded in chosen light scatter gates(e.g., determine fraction of total CD45bright

CD14 cells of low-moderate light scatter+ –

included in lymphocyte light scatter gates(Figure 7)

(2) For some abnormal specimens, lightscatter characteristics or CD45/CD14characteristics may not match those ofnormal lymphocytes (e.g., diseasesinvolving significant numbers of activatedor proliferating lymphocytes). Ifoverlapping intensities or other technicaldifficulties make estimation ofCD45 CD14 cells impractical for abright + –

given specimen, set lymphocyte gates asbroadly as possible consistent withacceptable levels of contaminating cellstypes (see Figure 8).36

11.5.3.3 Corrective Actions

(1) Each laboratory should establish limits ofcontaminating cell types, based ondocumentation that their inclusion doesnot significantly affect the measurementof interest.

If levels of nonleukocytes, monocytes,granulocytes exceed establishedlaboratory limits, corrective action shouldinclude adjustment of light scatter gates,reacquisition of data, and reanalysis ofimmunofluorescence data.

(2) If satisfactory values cannot be obtainedfor procedural control specimen, correctiveaction should include validation ofinstrument performance using appropriatecontrol materials

(3) If instrument performance parameters areacceptable but values for contaminatingcell types included or lymphocytesexcluded are still unacceptable, specimenshould be recorded in "Out of Range" logbook; sample preparation proceduresand/or reagents should be examined asother possible sources of the problem.

11.6 Analysis of Negative ControlSample(s)

Each patient sample must have its ownnegative reagent control (see Section 9.1.1). In analyzing negative control sample(s), it isnecessary to:

! Acquire single color or correlatedtwo-color immunofluorescence data usingdefined lymphocyte gates.

! Verify that mean signal intensities for thenegative cells do not exceed establishedlaboratory value.

Greater than expected intensity of thenegative reagent control may indicateincreased nonspecific binding due to reagentproblems or poor specimen viability; it mayalso indicate artifactual uptake offluorochromes remaining in the instrument,particularly when analyzing fixed specimens,nonspecific binding of reagents to dead cellsor binding of reagents to Fc receptors. Toevaluate whether intensity is greater thanexpected, it is necessary to reproduce theimmunofluorescence intensity scale(s) on adaily basis using an appropriate calibrationmaterial.

! Verify by comparing to the laboratoryestablished value that proportion of falsepositives determined using the selectedintensity threshold does not exceedlaboratory established maximum.

If the channel number or boundary is higherthan the normal negative control, that shouldbe noted on the test record.

11.7 Analysis of Samples Stained forSubset Enumeration

11.7.1 Acquisition of Single Color Histogramor Correlated Two Color ImmunofluorescenceDisplay

Counting errors for the estimation of positivecells are expected to be proportional to thesquare root of the number of positivescounted (e.g., if the population of interestcomprises 5% of the total number of cells and10,000 cells are evaluated, the expectedcount is 500±22; if 1000 cells are evaluated,

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the expected count is 50±7). Ideally, asufficient number of positive cells should becounted to ensure that variability due to thistype of error is no greater than the variabilityamong replicate preparations of a singlespecimen. This ideal may not be achievablein severely lymphopenic specimens.

11.7.2 Analysis of Samples Stained forSubset Enumeration (procedural controlspecimen, patient samples)

(1) Acquire single color or correlatedtwo-color immunofluorescence data usingdefined lymphocyte gates.

(2) Verify that data analysis results (Section10.0) for the positive process controlspecimen are within established laboratoryranges for each reagent and are self-consistent (e.g., %T [CD3+] + %B +%NK [CD3 , CD16 /CD56 ]= 100%±5).! + +

(3) If values for the procedural controlspecimen are out of limits for a reagent,record in "out of range" log book. If onlyone procedural control specimen wasprepared and it is out of limits, results oftest specimens prepared at the same timeare suspect and the fact that theprocedural control was out of limitsshould be noted on the test report.

(4) Observe special considerations foranalysis of patient samples.

(a) Analyze data using method consistentwith results of negative reagent controlfrom patient specimen. It is notacceptable to use "normal" specimennegative control to establish thresholds orintensities for definition of positivestaining in patient samples.

(b) If values for reagents examined are notself-consistent, this should be noted intest record. If possible, confirmation isdone by repreparing and reanalyzing therelevant sample(s) for that specimen.

12 Data Analysis

12.1 Goals

The tasks in data analysis are:

(1) To establish, in a reproducible manner, aboundary between cells of a population ofinterest (usually lymphocytes) believed tobe specifically labeled by a given antibodyreagent (positives) and those believed notto be so labeled (negatives).

(2) To determine the number of positive and negative events for each reagent ofinterest.

(3) To calculate the percentage of positivelylabeled cells for each reagent of interest.

Most of the recommended reagents (Section8.1) give fluorescence distributions with wellseparated populations of low intensitynegatives and higher intensity positives forspecimens from healthy individuals and formany patient specimens. The notableexception is CD8 which includes both dim andbright cells in the histogram. The analysisprocedures described below are mostappropriately used with reagents which givewell resolved positive and negativepopulations. Use of the same analysisprocedures with reagents giving incompletepositive/negative resolution may result in lessaccurate estimates of the true percentpositive.

If only a single antibody/fluorochrome reagentis used in each test sample, only two analysisresults are possible (positive vs. negative) andonly a single boundary must be chosen. Iftwo antibodies are used simultaneously ineach test sample, each conjugated to adifferent color fluorochrome, boundaries mustbe selected to define four distinct regions: cells labeled with neither antibody, cellslabeled with antibody #1 but not antibody #2,cells labeled with antibody #2 but notantibody #1, and cells labeled with bothantibodies.

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12.2 Potential Sources of Difficulty inAnalysis

It is the responsibility of the personperforming the data analysis to understandand take into account the effects ofdepartures from the ideal in order to obtain avalid analysis result.

12.2.1 Heterogeneous Intensity Distributions

Staining intensities of positive cells may bemuch more heterogeneous for abnormalspecimens than for healthy individuals due to

per cell. Fluorescence intensities of negativecells may also show increased heterogeneityin abnormal specimens due to enhancednonspecific binding of some (or all) antibodyreagents. Either cause may lead to moreoverlap between positive and negativepopulations and more difficulty in selecting anappropriate analysis boundary.

12.2.2 Inadequate Color Compensation

If proper compensation for spectral overlapbetween the two fluorochromes used in dualcolor (dual antibody) measurements is notobtained [Section 10.1.2.2(3)], the result maybe inaccurate.

12.3 Single Color (Single Antibody)Analysis

The fluorescence distribution of those cellswithin the lymphocyte gates is analyzed. Thedistribution of cellular fluorescence is normallycollected as a single parameter histogram ofcell count (frequency) vs. log of fluorescenceintensity.

12.3.1 Analysis Boundaries

Select an analysis method. In the mostcommonly used analysis technique, all eventsabove the channel number selected as thenegative/positive boundary in the negativereagent control are considered false positives(see Figure 9). Therefore, the boundary mustbe selected on the basis of what the laboratory defines as an acceptable falsepositive rate. The percentage chosen typicallyis in the order of 2%. The false positive19,41

rate chosen should be the same for allreagents and all specimens.

Various other methods have also beendescribed for selecting the boundary betweennegatives and positives. The most importantfeature of any method used is that thepositive/negative boundary should be reachedvia a well defined and reproducible procedurethat is consistently followed.

12.3.2 Analysis of Test Sample

(1) Determine the percentage of events at orabove the boundary channel (see Figure10).

(2) The boundary settings based on thenegative control may be observed to beinappropriate for a specific sample. Forexample, monocytes in the lymphocytegate may express low density CD4antigen, and CD20 may react weakly witha subpopulation of T cells. Under thesecircumstances, it may be appropriate tomove the boundary marker. It should benoted, however, that two-color analysis isoften a superior method for proving that adistinct population of dimly stainingevents should be included to the left ofthe boundary. Each laboratory shouldhave a written set of objective criteria todefine the appropriate placement ofmarkers to delineate the population ofinterest.

(3) The percentage of events above theboundary channel should, in mostinstances, be corrected by dividing by the% lymphocytes in the gate. However, itshould be recognized that if thecontaminating events are positive for thereagent of interest that this will result inan inaccurate estimate. It is theresponsibility of the laboratory todetermine which reagents cannot beappropriately corrected.

If separation between negative and positivepopulations is poorly defined (i.e., there issignificant overlap) the method of analysisdescribed in this section will give a minimumestimate of the true percent positive.

increased variability in the number of antigens

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Figure 9. Cursor is placed on X-axis such that # In establishing analysis boundaries using2% of the events are to the right of the cursor.

Figure 10. Demonstration of cursor setting forantibody-stained sample based on subclass control.

12.3.3 Recording Results

Record positive/negative boundaries andupper limit of range used to sum all positiveevents, as well as percent positive. This canbe accomplished graphically (by illustratingboundaries on hard copy) or numerically.

If there is poor positive/negative separation,this should be noted on the test record.

12.4 Dual Color (Dual Antibody)Analysis

In the most commonly used analysis method,data is displayed as a dual parameter displayof log FITC immunofluorescence and log PEimmunofluorescence. This dual parameterdisplay is then divided into rectangularquadrants by the use of two perpendicularboundaries. The quadrants are selected tocontain those cells which bind neither

antibody, bind only FITC-conjugated antibody,bind only PE-conjugated antibody, or bindboth antibodies (Figure 11).

If color compensation has not been accuratelyset [Section 10.1.2.2 (3)], use of rectangularanalysis boundaries may result in either falsepositives (Figures 3 or 4) or false negatives,particularly when low intensity dual positivepopulations are present.

12.4.1 Analysis Boundaries

negative reagent control for test specimen, allevents beyond the two perpendicularboundaries selected are again defined as falsepositives and the position of the boundariesdetermined based on what the laboratoryconsiders an acceptable false positive rate. As in single color analysis, the percent falsepositives (typically #2%) chosen should bethe same for all specimens and all reagents.

However, there are now three different typesof false positives: false positive for antibody#1 but not #2, false positive for antibody #2but not #1, and false positives for both. Generally, selection of boundaries giving #2%false positives for each antibody alone alsogives an acceptable number of false positivesin the dual stained category. If it does not,this may indicate a significant degree ofaggregation in the reagent resulting in dualcolor doublets. This possibility should beevaluated microscopically or by displayingforward scatter vs. fluorochrome dualparameter displays.

12.4.2 Recording Channel Numbers

If the channel numbers selected as analysisboundaries for a patient specimen aresignificantly different than those recorded forthe negative reagent control for the specimenfrom a healthy individual prepared at the sametime (Section 9.2), this should be noted in thetest sample record.

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A. Quadrants were established based onsubclass controls.

B. Two color immunofluorescence of CD3-FITC vs. CD8-PE in an HIV infectedindividual. The CD3 CD8 non T cells– +

appear in Quadrant 1 (12%). The CD3CD8 T cytotoxic suppressor cells are+ +

located in Quadrant 2 (42%). TheCD3 CD8 T cells are found in Quadrant 4+ !

(25%) while the double negativeCD3 CD8 cells are found near the origin– –

in Quadrant 3.

two color immunofluorescence using subclasscontrol.

12.4.3 Analysis of Test Sample

In analyzing the test sample, it is necessary todetermine the percentage of cells within eachquadrant of the dual parameter display, usingthe two perpendicular boundaries selected asdescribed in Section 12.4.1.

The boundary settings based on the negativecontrol may be observed to be inappropriate

for a given sample. Under thesecircumstances, it may be appropriate to moveeither the horizontal or vertical boundarymarker or both [see Section 12.3.2 (2)].

A correction for nonlymphocytes included inthe light scattering gate can be made bydividing this result by the percentage oflymphocytes in the gate.

If there is poor separation between thenegatives and positives in either color, thismethod will give a minimum estimate of thepercent positive.

12.4.4 Recording Results

Positive/negative boundaries and upper limitof ranges used to sum all positives, as well aspercent positives in each category, should berecorded.

If there is poor positive/negative separation,this should be noted in the test record.

13 Data Storage

The possibility of patients contestingdiagnostic implications derived in part fromlymphocyte phenotypic analysis makes itincumbent upon the laboratory to be able todemonstrate and verify the process used inarriving at the reported test results.

13.1 Recording Results

It is necessary to record the boundaries usedto identify the lymphocytes and the percent oftotal lymphocytes included within the lightscattering gate. It is also necessary to recordthe proportion and identity of nonlymphocytesincluded in the light scattering gate sincethese can affect subsequent calculations.

If the light scattering gate includes anunusually low proportion of lymphocytes orincludes an unusually high proportion ofnonlymphocytes, this should be noted.

13.2 Information to Be Stored

Patient data files should include all parametersused to obtain test results. These typicallyinclude:

Figure 11. Establishing fluorescence markers for

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! Low angle light scatter.

! 90 degree light scatter.

! One or two fluorescence parameters (forsingle or dual color analysis, respectively).

Patient data files should also include anygating or analysis regions used to obtainreported test results.

QA/QC data files should include allparameters, analysis regions, and analyticalresults used to verify system (instrument andmethod) performance.

13.3 Types of Data Storage

13.3.1 Paper Hard Copy

If data are stored as paper hard copy, allhistograms and analysis regions used inarriving at the reported test results (e.g.,lymphocyte gates, analysis boundaries) mustbe recorded on the hard copy of the relevantdata plot. A unique specimen identifier shouldalso be recorded with each data plot.

13.3.2 Archival Files

A variety of data storage media may be used,depending on the particular systems availablein the laboratory, including floppy disks,removable hard disks, optical disks, andmagnetic tape.

Data may be stored as data displays or in listmode (cell-by-cell list of all parameter values).

List mode has the advantage that the data canbe reanalyzed off-line or at a later time. Ifthere are problems with the sample, anexperienced individual or expert can be calledupon to reanalyze the data. List mode has thedisadvantage of using a large amount ofstorage space.

If data storage software does not provide forstorage with the data file of relevant gatesand analysis regions used to arrive at testresults, these must be recorded as paper hardcopy (e.g., amorphous lymphocyte gates) orin the test record (e.g., orthogonallymphocyte gates, analysis boundaries).

13.4 Duration of Data Storage

Patient data should be kept for a minimum oftwo years, or as required by state and federallaw, whichever is longer. After that, it is theresponsibility of the laboratory director todetermine whether to save or discard thedata. QA/QC data should be kept for aminimum of two years.

Data may be stored for as long thereafter asthe laboratory director considers necessary,providing storage space is available.

14 Data Reporting andInterpretation

14.1 Worksheet

A standard laboratory worksheet should bedeveloped for reporting immunophenotypingdata (see Appendix D for example).

14.2 Supervisory Check

Recording of laboratory data and calculationsshould be checked by supervisory personneland the report form should have thesupervisor's initials.

14.3 Review of Data Displays

Data displays for light scatter patterns,antibody staining profiles, and all gates andboundaries used to arrive at the test result(Section 12) should be reviewed by alaboratory professional when interpreting thedata.

14.4 Reporting of Data

All data should be reported as a percentpositive for a specific cell surface marker(Section 12) using a cluster of differentiationnumber with the commercial antibodydesignated in parentheses.

Absolute numbers for each of the lymphocytepopulations (e.g., T, B, NK) or subpopulations(e.g., CD4, CD8) should be reported only if anaccurate specimen white cell total anddifferential count has been obtained.

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When possible this count should be performedon a sample drawn at or about the same timeas the specimen drawn for phenotyping andassayed within six hours. When an accuratewhite count is not available, absolutelymphocyte counts cannot be reported.

14.5 Interpretation of Data

Data interpretation should be made using anestablished laboratory reference range.

Sample data that constitutes the referencerange should be obtained in a manner similarto that for the patient material (see Appendix E

14.6 Notation of Out-of-Range ControlSamples

If the results of analysis of positive methodcontrol samples are outside the laboratoryreference range and no reason has beendetermined, results on patient specimensprepared at the same time are potentiallysuspect and this should be noted on the testreport.

14.7 Inclusion of Reference Range

Written report of the data should includeestablished reference range for the laboratoryand signature of the laboratory professional.

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12. Nicholson JKA, et al. Comparison of Tand B cell analysis on fresh and aged blood. J. Immuno. Meth. 1984;73:29-40.

13. Hensleigh PA, Waters VB, HerzenbergLA. Human T lymphocyte differentiationantigens: Effects of blood sample storage onLeu antibody binding. Cytometry. 1983;31:453- 455.

14. Patrick CW, et al. Collection andpreparation of hematopoietic cells for cellmarker analysis. Laboratory Medicine.1984;15:659-665.

15. NCCLS. Reference leukocyte differentialcount (proportional) and evaluation ofinstrumental methods; Approved Standard. NCCLS document H20-A. Wayne, Pennsyl-vania:1992.

16. CFR, part 72. Interstate shipment ofetiologic agents. 1987;59-63.

17. NCCLS. Procedures for the DomesticHandling and Transport of DiagnosticSpecimens and Etiologic Agents—ThirdEdition; Approved Standard. AmericanNational Standard. NCCLS document H5-A3.Wayne, Pennsylvania: NCCLS; 1994.

18. Thornwaite JT, et al. In: Andreeff M,ed. Characteristics of monoclonal antibodymeasurements in human peripheral blood. Clinical Cytometry. Ann. N.Y. Acad. Sci.1986: (468)144-159.

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References (Continued)

19. Muirhead KA, et al. Methodologicalconsiderations for implementation oflymphocyte subset analysis in a clinicalreference laboratory. In Andreeff M, ed. Clinical Cytometry Ann. N.Y. Acad. Sci.1986: (468)113-127.

20. Shield CF, et al. Stability of humanlymphocyte differentiation antigens whenstored at room temperature. J. Immunol.Meth. 1983;62:347-352.

21. Grunow JE, et al. Preferential decreasein thymus dependent lymphocytes duringstorage at 4 C in anticoagulant.o

Transfusion.1983; 16:435-436.

22. Dzik WH, Neckers L. Lymphocytepopulations altered during blood storage. N.Eng. J. Med. 1983;(309):793.

23. Weiblen BJ, Debell K, Valeri CR."Acquired immunodeficiency" of blood storedovernight. N. Eng. J. Med. 1983;309:793.

24. Weiblen BJ, et al. Monoclonal antibodytesting of lymphocytes after overnightstorage. J. Immuno. Meth. 1984;70:179-183.

25. Loken MR, Meiners H, Terstappen LWM.Comparison of sample preparation techniquesfor flow cytometric analysis of immuno-fluorescence. Cytometry Supplement. 1988;2:53.

26. Ritchie AWS, Gray RA, Michlem HS.Right angle light scatter: A necessaryparameter in flow cytofluorometric analysis ofhuman peripheral blood mononuclear cells. J.Immunol. Meth. 1983;64:109-117.

27. Loken MR, et al. Flow cytometricanalysis of human bone marrow. II. Normal Blymphocyte development. Blood. 1987;70:1316-1324.

28. Thomas ML, Lefrancois L. Differentialexpression of the leukocyte common antigenfamily. Immunology Today. 1988;9:320-326.

29. Caldwell CW, et al. Fluorescenceintensity as a quality control parameter inclinical flow cytometry. Am. J. Clin. Pathol.1987; 88:447-456.

30. Vogt RF, et al. Model systemevaluating fluorescein-labeled microbeads asinternal standards to calibrate fluorescenceintensity on flow cytometers. Cytometry. 1989;10:294-302.

31. Horan PK, Loken MR. In Van Dilla M etal, eds. A practical guide for the use of flowsystems. Flow Cytometry: Instrument andData Analysis. Orlando, Florida: AcademicPress;1985:272. 32. Sasaki D, Dumas S, Engleman E. Discrimination of viable and nonviable cellsusing propidium iodine in two-color immuno-fluorescence. Cytometry. 1987;8:413-420.

33. Riedy MC, Muirhead KA, Jensen CP. Use of a photolabeling technique to identifynonviable cells in fixed homologous or hetero-logous cell populations. Cytometry. 1991;12:133-139.

34. Terstappen, LWMM, et al. Discriminating between damaged and intactcells in fixed flow cytometric samples.Cytometry. 1988;9:477-484.

35. Stewart CC. Clinical applications offlow cytometry: Immunologic methods formeasuring cell membrane and cytoplasmicantigens. Cancer. 1992;69:1543-1552.

36. Loken MR, et al. Establishing optimallymphocyte gates for immunophenotyping byflow cytometry. Cytometry. 1990;11:453-459.

37. Salzman GC, et al. Cell classification bylaser light scattering: Identification andseparation of unstained leukocytes. ActaCytologica. 1975;19:374-386.

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References(Continued)

38. Hansen NP, Hoffman RA. Method andApparatus for Automated Identification andEnumeration of Specified Blood Cell Subclass.U.S. Patent No. 4,284,412. Ortho DiagnosticSystems, Inc., Raritan, N.J., August 18,1981.

39. Stewart CC, Stewart SJ, Habersett RC. Resolving leukocytes using axial light loss. Cytometry. 1989;10:426-432.

40. Jackson AL, Warner NL. In: Rose NR, Friedman H, Fahey JL, eds. Preparation andanalysis by flow cytometry of peripheral bloodleukocytes. Manual of Clinical LaboratoryImmunology. Washington, D.C.: AmericanMicrobiology Association. 1986:226-235.

41. Herzenberg LA. In: Wier DM, ed.Analysis and separation using thefluorescence activated cell sorter. Handbookof Experimental Immunology. CellularImmunology. Oxford, England: BlackwellScientific Publications; 2:22.1-22.21.

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Appendix A. Potential Sources of Artifacts in Immunophenotyping by FlowCytometry

Cause Effect Resulting Artifact

Medications/drugs:

1. Zidovudine (AZT) Increased granulocyte Decreased light scatter resolution;fragility increased granulocyte contamination

of mononuclear preparations

2. Some antibiotics Increased cellular False positives if appropriate(e.g., cephalosporins) autofluorescence negative control is not used

3. Some chemotherapeutic Increased cellular False positives if appropriateagents autofluorescence negative control is not used(e.g., daunorubicin)

4. Nicotine Increased lymphocyte Lowered absolute values formargination, decreased lymphocyte subsetslymphocyte counts

5. Corticosteroids Decreased CD4 levels Overestimation of disease-related alterations

6. Antihuman lymphocyte Lymphocytopenia, Decreased labeling with antibodyantibodies (e.g., OKT3) modulation or blocking reagentor soluble CD4 of cell surface receptors

Biological factors:

1. Reticulocytosis Incomplete red cell Decreased light scatter resolution;lysis, increased RBC contamination of lymphocytecontamination of gatesmononuclear preps

2. Strenuous exercise Increased lymphocyte Lowered absolute values for margination, decreased lymphocyte subsetslymphocyte counts

3. Diurnal variation Variable absolute Variable absolute subset valueslymphocyte count

4. Specimen age and Variable granulocyte Increased granulocyte contaminationholding conditions preservation and/or of lymphocyte gates; false positive

leukocyte viability nonspecific staining of dead cells.

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Appendix B. Alternative Methods for Cell Preparation

B1 Buffy Coat And Lysis

In cases where the lymphocyte count is very low, the sample is centrifuged and the buffy coatremoved for staining with the Whole Blood Procedure. This step serves to concentrate the1

leukocytes for subsequent analysis. It is the responsibility of the laboratory to determine that such amethod does not give rise to selective enrichment of particular lymphocyte subpopulations.

B1.1 Factors with Adverse Effect

B1.1.1 Erythrocyte Concentration

If an excessive number of red cells is collected with the buffy coat, the aliquot stained will containan unusually large number of red cells. The excessive erythrocytes may overload the lysing reagentand result in incomplete lysis and a poorly delineated population for light scatter gating.

B2 Discontinuous Density Gradient Techniques

The general concept involves creating a discontinuous gradient by layering blood over a separationmedium of high density (•1.077 g/mL) followed by centrifugation. Erythrocytes and granulocytes,being of higher density, sediment to the bottom of the tube while lymphocytes and monocytes,being of lower density, are retained at the plasma-separation medium interface. The most commonpreparations consist of the synthetic polymer ficoll, which also causes erythrocyte agglutination,mixed with an iodinated x-ray contrast medium (reviewed in reference 2). The ultimate quality ofsuch a preparation is a function of cell size and, to a greater extent, cell density. Monocytes mayconstitute up to 20% of the cell suspension. Because all of these cell populations are defined by2,3

a limited range of sizes and/or densities, there is some overlap of adjacent populations. It is thisoverlap which creates a potential for inadequate separation, reduced yield, or biased cell loss if asample is suboptimal due to either extrinsic or intrinsic factors.4

B2.1 Factors with Adverse Effects

B2.1.1 Age of Specimen

Storage of a blood specimen results in a progressive increase in the proportion and type of cellscontaminating the mononuclear isolate. Within a few hours, the percentage of monocytecontamination begins to increase and, at about 24 hours, granulocyte contamination becomesapparent. For these reasons, density gradient separations should generally not be used on5,6

specimens over 24 hours old.7,8

B2.1.2 Storage Conditions

Storage of anticoagulated blood under some conditions but not others results in significant5,6,9 10-12

loss of certain lymphocyte subpopulations during the subsequent separation and up to a 70%decrease in the proportion of T cells. Therefore, specimen storage at room temperature is5

preferred.

If blood specimens are to be stored prior to density gradient isolation of lymphocytes, use of ACD-Aas anticoagulant or admixture with glucose-containing media prior to sample storage aids inmaintenance of specimen integrity.10,12

Specimen storage at 4 C may reduce the yield of CD4 lymphocytes in density gradient mediao

preparations.9,13-16

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B2.2 Storage of Separated Cells

Once isolated, the mononuclear cell suspension may be stored in an enriched (tissue culture grade)medium at 4 C for staining the next day.o 9

B2.3 Examination of Stained Smear

If the quality of a specimen is in doubt, and a density gradient separation is done, the finalpreparation should be examined in a stained smear to assess contamination by nonlymphoid cellsand a decision made concerning the relative suitability for analysis and the gating appropriate forthat specimen.

B3 Erythrocyte Agglutination Techniques

Dextran, Plasmagel (gelatin), methyl cellulose, and ficoll are high molecular weight polymers whichcause sedimentation of erythrocytes as a result of rouleaux formation when mixed with wholeblood. The technique is simple but leaves a leukocyte suspension consisting predominantly ofgranulocytes and is not generally recommended as a method for lymphocyte preparation.

B4 Monocyte Depletion

These procedures physically remove monocytes from either whole blood or enriched lymphocytesuspension based on functional properties of the monocytes. Incubation with iron particles permitsthe phagocytosis of the particles by the monocytes and subsequent removal with a magnet.Similarly, incubation of the cell suspension in a glass vessel at 37 C allows the monocytes too

adhere to glass. The nonadherent cells (lymphocytes) can then be washed off.

These procedures have variable effects on the lymphocytes, commonly resulting in the loss of up11

to 45% of the cells. Because of this loss, and the fact that some lymphocytes have limited ability2

for adherence, these techniques are generally not recommended for lymphocyte preparation.

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References to Appendix B

1. Hoffman RA, et al. Simple and rapid measurement of human T lymphocytes and theirsubclasses in peripheral blood. Proc. Nat. Acad. Sci. U.S.A. 1980;(77):4914-4917.

2. Boyum A. Separation of lymphocytes, granulocytes and monocytes from human blood usingiodinated density gradient media. Meth. Enzymol. 1984;(108):88-102.

3. Thompson JM, et al. The optimal application of forward and ninety-degree light scatter in flowcytometry for the gating of mononuclear cells. Cytometry. 1985;(6):401-406.

4. Pretlow TG, Pretlow TP. Sedimentation of cells: An overview and discussion of artifacts. CellSeparation: Methods and Selected Applications. 1982;(1):41-59.

5. Dzik WH, Neckers L. Mononuclear cell-surface antigens during storage of banked blood.Transplantation. 1984;(38):67-71.

6. Patrick CW, et al. Collection and preparation of hematopoietic cells for cell marker analysis. Lab. Med. 1984;(15):659-665.

7. Nicholson JKA, et al. Comparison of T and B cell analyses on fresh and aged blood. J.Immunol. Meth. 1984;(73):29-40.

8. Hensleigh PA, Waters VB, Herzenberg LA. Human T lymphocyte differentiation antigens: Effects of blood sample storage on LEU antibody binding. Cytometry. 1983;(3):453-455.

9. Weiblen BJ, et al. Monoclonal antibody testing of lymphocytes after overnight storage. J.Immunol. Meth. 1984;(70):179-183.

10. Ford WL. The preparation and labeling of lymphocytes. In: Weir DM, ed. Handbook ofExperimental Immunology. Cellular Immunology. Oxford, England: Blackwell Scientific Publications.2:23.1-23.22.

11. Iwatani Y, et al. Effects of various isolation methods for human peripheral lymphocytes on Tcell subsets determined in a fluorescence activated cell sorter (FACS), and demonstration of a sexdifference of suppressor/cytoxic T cells. J. Immunol. Meth. 1982;(54):31-42.

12. Muirhead KA, et al. Methodological considerations for implementation of lymphocyte subsetanalysis in a clinical reference laboratory. In: Andreedd M, ed. Clinical Cytometry. Ann. N.Y. Acad.Sci.1986;(468):113-127.

13. Shield CF, et al. Stability of human lymphocyte differentiation antigens when stored at roomtemperature. J. Immunol. Meth. 1983;(62):347-352.

14. Grunow JE, et al. Preferential decrease in thymus dependent lymphocytes during storage at 4C in anticoagulant. Transfusion. 1983;(16):435-436. o

15. Dzik WH, Neckers L. Lymphocyte populations altered during blood storage. N. Eng. J. Med.1983;(309):793.

16. Weiblen BJ, Debell K, Valeri CR. "Acquired immunodeficiency" of blood stored overnight. N.Eng. J. Med. 1983;(309):793.

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Appendix C. Instrument Log Book—Optical Alignment, Fluorescence Amplification, andCompensation Logs

Optical Alignment Log

Laser: Wavelength (nm) Power (mw) Current (amps)

Low Angle 90 Light Green Redo

PARAMETER: 1. Light Scatter 2. Scatter 3. Fluorescence 4. FluorescenceFILTERS: _____________ _________ ____________ ____________ GAIN: _____________ _________ ____________ ____________HIGH VOLTAGE: _____________ _________ ____________ ____________

Alignment Particle Source Lot Number

DATE OPERATOR

PARAMETER 1 PARAMETER 2 PARAMETER 3 PARAMETER 4 PARAMETER 5

CV MEAN CV MEAN CV MEAN CV PEAK CURRENT (AMPS)

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Appendix C. (Continued)

Fluorescence Amplification Log

Laser: Wavelength (nm) Power (mw) Current (amps)

Low Angle 90 Light Green Redo

PARAMETER: 1. Light Scatter 2. Scatter 3. Fluorescence 4. FluorescenceFILTERS: _____________ _________ ____________ ____________ GAIN: _____________ _________ ____________ ____________HIGH VOLTAGE: _____________ _________ ____________ ____________

Alignment Particle Source Lot Number

GREEN RED

DATE OPERATOR (Unstained) (Dimly Stained) (Unstained) (Dimly Stained)PEAK 1 PEAK 2 PEAK 1 PEAK 2

CV Mean CV Mean CV Mean CV Mean

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Appendix C. (Continued)

Compensation Log

Laser: Wavelength (nm) Power (mw) Current (amps)

Low Angle 90 Light Green Redo

PARAMETER: 1. Light Scatter 2. Scatter 3. Fluorescence 4. FluorescenceFILTERS: _____________ _________ ____________ ____________ GAIN: _____________ _________ ____________ ____________HIGH VOLTAGE: _____________ _________ ____________ ____________

Alignment Particle Source Lot Number

GREEN RED

DAT OPERATOR UNSTAINED GREEN RED Setting UNSTAINED GREEN RED SettingE ONLY ONLY (% Subtraction) ONLY ONLY (% Subtraction)

Compensation Compensation

CV Mean CV Mean CV Mean CV Mean CV Mean CV Mean

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Appendix D. Laboratory Worksheet

Accession #

Date

Prepared by Run by

Patient Date of Birth

Log # Date Collected Date Received

Date Prepared

White Blood Cell Count Lymphocyte%

Medication Diagnosis

Physician

Tube Antibody Commercial Lot # % Positive Absolute Source Number

1 CD45/CD14

2 SubclassControl

3 CD3/CD4

4 CD3/CD8

5 CD3/CD19

6 CD16 or CD56

Laboratory Remarks:

Analyzed by: Director:initials/date initials/date

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Appendix E. Determination of Reference Ranges

E1 Definitions

Reference values, n - A set of values for a measured quantity obtained from a group of individuals indefined state of health.

Reference range, n - Classically, the range of values found in 95% of a reference population ofhealthy individuals without overt clinical disease.

E2 Procedures For Determining Reference Ranges

Statistical methods, both parametric and nonparametric, and graphical methods are discussed indetail in references 1-3. Only a brief summary of the steps involved is presented here.

E2.1 Parametric methods

(1) Collect data on randomly chosen set of representative individuals (ideally up to 200 adultswithout disease or medication known to affect lymphocyte immunophenotyping).

(2) Inspect frequency distribution of values obtained.

(3) If frequency distribution is known, use appropriate statistical techniques to estimate the95% confidence interval and use the endpoints of 95% intervals as the reference range.

(4) If no satisfactory transform can be identified, use nonparametric methods which do notdepend on the exact distribution of the data.

(5) Age, gender, and race may influence the distribution of values which constitute alaboratory's reference range. Each laboratory must collect reference samples from theappropriate populations which reflect their patient population.

E2.2 Nonparametric methods

(1) Collect data on randomly chosen set of representative individuals.

(2) Arrange data in ascending or descending order.

(3) Use appropriate nonparametric techniques to identify desired limiting percentiles (e.g., 2.5and 97.5) to desired confidence level.

Nonparametric methods are most appropriate when data does not show a Gaussian distribution andcannot be so transformed. However, they are very sensitive to outliers, and final ranges chosenmay be highly dependent on methods used for removing outliers.1-3

E3 Pitfalls in Determining Flow Cytometric Reference Ranges

Because significant laboratory-to-laboratory differences related to sample preparation method andinstrumentation used have been reported, ideally each laboratory should establish its own referencerange.

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Appendix E. (Continued)

However, large data sets (>500 to 1,000 individuals) are required to accurately establish areference range covering 95% of healthy individuals, using either parametric or nonparametricmethods. This simply is not feasible for most individual laboratories, and confounding variablesother than sample size have also been described.4-5

One possible solution is accumulation of large data sets by several laboratories with such capability,analysis of these large data sets to determine whether they all fit the same statistical distribution. Ifthis were found to be true, it might be reasonable to assume that other laboratories' distributionsshould fit the same model and to use more moderate sample sizes to estimate the necessaryparameters to establish their own confidence intervals based on that model.

Another possible solution is generation of large databases by pooling of data among laboratoriesexhibiting similar performance, either as verified by participation in external quality control surveyprograms or on the basis of use of similar reagents, preparation methods and instrumentation.

Use of relatively broad reference ranges arising from small samples sizes will result in:

! Identifying more "healthy" individuals than appropriate as having "abnormal" values (andmay therefore affect patient care decisions).

! Reevaluating more samples than necessary if out of range samples are flagged for reruns orcloser scrutiny (and may therefore affect economics of testing).

Therefore, it is preferable to provide some indication of the precision of the estimated referencerange as well as the reference range.

References to Appendix E

1. Winkel P, Statlad BE. Reference values. In: Henry JB, ed. Clinical Diagnosis and Managementby Laboratory Methods, Philadelphia: W.B. Saunders Co; 1979:29-52.

2. Martin HF, Gudzinowic BJ, Fanger H. Normal Values in Clinical Chemistry. New York: MarcelDekker; 1975:102-236.

3. Henry RJ, Cannon DC, Winkelman JW. Clinical Chemistry. Principles and Technics. New York:Harper and Row; 1974:343-371.

4. Edwards BS et al. A comprehensive quality assessment approach for flow cytometricimmunophenotyping of human lymphocytes. Cytometry. 1989;(10):443-441.

5. McCarthy RC, Fetterhoff TJ. Issues in quality assurance in clinical flow cytometry. Arch.Pathol. Lab. Med. 1989;(113):658-666.

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Appendix F. Three-Color Lymphocyte Immunophenotyping Methods

Single color and two color immunophenotyping methods typically use forward and right angle lightscatter to distinguish lymphocytes from other cells present in the sample. However, many factorsincluding disease state, sample age, sample preparation artifacts, and instrument configuration canaffect ability to resolve a discrete lymphocyte cluster based solely on light scatter. This has lead todevelopment of strategies in which cell lineage specific antibodies are used to assist in selectinglight scatter gates which maximize the number of lymphocytes included while minimizing theinclusion of debris or interfering cell types (Section 11.5). 1,2

Three color immunophenotyping allows this strategy to be extended by using immunologic markersto augment, or even replace light scatter as the defining parameter for specific cell types. Twodifferent gating methods have been described for three color lymphocyte immunophenotyping, bothof which involve addition of internal gating reagent(s). One approach involves addition of a panleukocyte antibody such as CD45 to each tube, while the second involves addition of lineage3

specific antibodies. Both strategies seek to accomplish the following: 4-6

• Maximize the likelihood of including all lymphocytes within selected gates.• Minimize the likelihood of including debris or nonlymphocytes within selected gates.• Minimize interoperator and interlaboratory variability in selection of lymphocyte gates.• Improve ability to select acceptable lymphocyte gates in specimens with suboptimal light

scatter resolution.

Use of three-color immunophenotyping introduces additional complexities for the laboratory, sincethe instrument quality assurance principles described in Section 10 must be extended to ensureacceptable instrument sensitivity and color compensation for a third fluorescence detector. However, it also offers the opportunity to maximize measurement reliability and reproducibility whileminimizing the number of samples required for enumeration of all lymphocyte subsets (although notnecessarily amount of reagent). This appendix will outline the principles involved, particularly withregard to the two different gating strategies which have been described, highlighting the advantagesand limitations of each.

CD45 GATING METHOD

Section 11.5 of this document describes some of the common problems associated with use of lightscatter gating alone for lymphocyte subset enumeration. Nicholson et al. described addition of athird color CD45 pan-leukocyte antibody to two color mixtures of CD3/CD4 or CD3/CD8 antibodiesfor use as an internal gating reagent. In this method, lymphocytes are defined as CD45 cells3

bright

with low ninety degree scatter and are typically well resolved from debris and nonlymphocytes(Figure F1). This in turn minimizes the necessity to correct immunophenotypic results for theproportion of contaminating cells present within the lymphocyte gate (Section 11.5.1). For optimalresults, it is important that the CD45 reagent selected gives good resolution between lymphocytes,which are CD45 with low ninety degree scatter, and basophils, which are CD45 with lowbright dim

ninety degree scatter.

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Appendix F. (Continued)

Figure F1. T Cell Subset Analysis: Three Color Immunophenotyping with CD45 Gating. Samples werestained with FL-CD3/PE-CD4/PerCP-CD45 (upper panels) or FL-CD3/PE-CD8/PerCP-CD45 (lower panels). A lymphocytegate was set on CD45bright cells with low right angle scatter as shown (upper left and lower left). Debris and red cells areexcluded by virtue of being CD45 negative. Monocytes are excluded primarily based on their elevated ninety degree scatter,while basophils are excluded since they are CD45 . Neutrophils are excluded since they are CD45 and exhibit elevateddim dim

right angle scatter. Gated CD3/CD4 or CD3/CD8 immunofluorescence distributions were collected (upper and lower right,respectively) and used to enumerate percent dual positive lymphocytes (CD3 CD4 or CD3 CD8 , respectively. In this+ + + +

example, no isotype control was used, and cursor positions were set based on the events present in the dual negativepopulation.

Analysis of B cells and NK cells is done similarly to that described in Figure 1 for T cells, through theselection of appropriate antibodies. CD19 is an excellent B cell marker and can be combined withCD3 and CD45 for enumeration of CD3 (total T) and CD19 (total B) subsets. Note that if an+ +

isotype control is not used, this combination may give too few events in the dual negativepopulation to accurately set cursor positions for immunophenotypic analysis, particularly in samplesfrom HIV positive adults. In this case cursor position may have to be set based on the boundariesof the mutually exclusive CD3 and CD19 single positive populations. When NK cell reagent(s) arecombined with CD3 and CD45 antibodies, only events positive for the NK marker (CD16, CD56 orboth; see next section) but negative for CD3 should be reported as NK cells.

CD45 Gating—Advantages

• Pan-leukocyte gating reagent is included in every tube, allowing assessment of gatereproducibility from sample to sample

• Debris and red cells (CD45 ) and neutrophils (CD45 , elevated ninety degree scatter) are– dim

readily excluded from the lymphocyte gate

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Appendix F. (Continued)

• Basophils are resolved from lymphocytes based on CD45 intensity, whereas they are poorlyresolved by light scatter gating

• Improved ability to eliminate interfering cell types reduces need to correct immunophenotypicresults for poor lymphocyte gate purity

• CD4 and CD8 subsets can be enumerated using a two-tube panel• Provides duplicate CD3 determinations for evaluation of tube-to-tube reproducibility• The requirement for separate isotype controls may be less critical• A CD14 reagent is not required to gate out monocytes

CD45 Gating—Limitations and Potential Pitfalls

• Differences in ninety degree scatter are the primary means of distinguishing betweenlymphocytes and monocytes

• Use of "generous" lymphocyte gates to maximize lymphocyte recovery increases the likelihoodof monocyte contamination

• B cells may become CD45 in some HIV seropositive individuals and be excluded from thedim

lymphocyte gate 7

• Any immature CD3 4 8 cells will be counted twice+ + +

• Some three-color combinations may have insufficient dual negative cells to allow accuratepositioning of cursors to distinguish positive and negative populations

• Enumeration of all major lymphocyte subsets requires at least four tubes; only then can the "lymphosum" (%T + %B + %NK = 100) be used as a quality control check.

Lineage-Specific Gating Methods

An alternative approach utilizes lineage specific reagents rather than a pan-leukocyte antibody todefine improved gates for lymphocyte subset enumeration. The T-gating strategy described in 1992by Mandy et al. adds PerCP-CD3 to a tube containing FL-CD8 and PE-CD4 reagents. CD34

immunofluorescence in combination with ninety degree light scatter is used to define a cluster ofcells (T-Gate) which excludes debris, red cells, monocytes, and CD8 CD3 NK cells, and this gate is+ –

used to collect a CD4/CD8 immunofluorescence distribution (Figure F2). This method can be usedto determine CD4/CD8 ratios and to enumerate absolute CD4 and CD8 lymphocyte counts butdetermines CD4 and CD8 frequencies as percent of all T cells rather than percent of all lymphocytes. It is therefore best suited to instruments or reagent systems capable of generating absolute countsdirectly from gated cell clusters.

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Appendix F. (Continued)

Figure F2. T Cell Subset Analysis: Three Color Immunophenotyping with T-Gating. In this example, cellswere stained with FL-CD4/PE-CD8/Cychrome-CD3. A T-lymphocyte gate was set on CD3 cells with low ninety degree+

scatter as shown (upper panel). Debris and red cells are excluded by virtue of being CD3 negative. Monocytes, which mayexhibit low nonspecific levels of CD3 staining, are excluded as being CD3 with moderate ninety degree scatter. Neutrophilsdim

are excluded since they are CD3 negative and exhibit elevated right angle scatter. Gated CD4/CD8 immunofluorescencedistribution is collected (lower left panel) and used to enumerate CD4 , CD8 , CD4 CD8 (immature), and CD4-CD8- T+ + + +

lymphocytes as i) a percentage of total T cells or ii) absolute counts with instruments or reagent systems having thiscapability. CD3 lymphocytes may be enumerated as a percentage of total events (upper panel), as a percentage of low+

ninety degree scatter events (primarily lymphocytes) after collection of a single parameter CD3 histogram (lower right panel),or as absolute counts with instruments or reagent systems having this capability.

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Appendix F. (Continued)

Figure F3. T, B, and NK Cell Analysis: Three Color Immunophenotyping with Lineage Gating

In this example, cells were stained with FL-CD16/PE-CD19/Cychrome-CD3. As in Figure F2, a T-lymphocyte gate is set onCD3 cells with low ninety degree scatter as shown (upper right panel), to provide a replicate determination of absolute+

CD3 lymphocytes for purposes of quality control (upper right and/or lower right panels). An NK-gate is set on CD16 cells+ +

with low ninety degree scatter (center left panel), and used to enumerate total NK cells per L. The NK gate is also usedgenerate a CD3 histogram (center right panel) so that any CD3 CD16 cells included in the NK gate can be subtracted from+ +

the total NK count. Similarly, a B-gate is set on CD19 cells with low ninety degree light scatter (bottom left panel) and used+

to enumerate total B cells per L.

As a cross check, the lineage gated T, B and NK values can be summed and checked against the total lymphocyte countdetermined using a traditional light scatter gate (upper left panel). Alternatively, traditional lymphocyte gates can be used toenumerate % T, % B, and % NK cells using single (lower right panel) or two color (not shown) distributions and their"lymphosum" compared with that generated using the lineage gating method. As with two-color analysis (Section 11.5.3),10

the causes of any significant discrepancies between the results obtained by lineage gating and light scatter gating should beinvestigated before analysis results are reported.

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Appendix F. (Continued)

The concept of lineage specific gating reagents can be extended to encompass three color analysisof not only T and B cells but of all major lymphocyte subsets. When combined with reagent 4,8,9

systems and/or flow cytometers capable of directly providing absolute cell counts for enumeratedcell types, this approach can provide unambiguous cell cluster identification and enumeration of allmajor lymphocyte subsets in as few as two tubes if an isotype control is not used. When other5,9

lineage gates are added to T-gates, the results can be expressed as percent of total lymphocytesrather than as percent of total T cells. Figure F3 illustrates the type of reagent combination which4,9

would be used for the second tube in such a full lineage gating panel.

Lineage Gating—Advantages

• Debris and cell types not recognized by lineage specific antibodies (red cells, basophils) are excluded from the gated analysis

• Use of "generous" lineage gates is less likely to result in inclusion of contaminating celltypes, minimizing the need to correct results for contaminating cell types• Use of T-gating allows determination of absolute CD4 and CD8 T lymphocyte counts in + +

a single tube, and also allows enumeration of immature (CD4 CD8 ) and (CD4-CD8-) + +

T lymphocytes• Complete enumeration of all major lymphocyte subsets is provided by a two-tube panel• Duplicate CD3 determinations can be used for evaluation of tube-to-tube reproducibility• Comparison of Lymphosums obtained using lineage gates and traditional light scatter10

gates provides a quality control check.

Lineage Gating—Limitations and Potential Pitfalls

• Best suited to instruments and/or reagents capable of directly providing absolute cell counts• Enumeration of CD4 and CD8 as a percentage of total lymphocytes cannot be done+ +

using T-gating alone but requires a full two-tube panel• The Lymphosum (T+B+NK) calculated solely on the basis of lineage gating does notprovide an independent quality control check on gating recovery and purity

Other Considerations for Three Color Immunophenotyping

The Flow Cytometry Advisory Committee (FAC) for the National Institutes of Allergy and InfectiousDiseases, Division of AIDS (NIAID/DAIDS) has recommended that cursor positions for data analysisbe set using the negative population, making an isotype control tube unnecessary. For several ofthe reagent combinations described above (T-gate, Lineage gate, and potentially CD3/B-cell/CD45),there may be too few events in the negative population to allow accurate cursor placement. In thiscase, cursor position would be set using the lower boundary of antibody positive clusters. In someautomated analysis systems, the absence of an isotype control tube requires the user to manuallydefine gates and cursor settings for each sample. In such cases, the laboratory must assess thecost effectiveness of manual analysis versus inclusion of the isotype control.

A lineage-specific marker for NK cells has not yet been identified. CD16 alone, CD56, alone or thecombination of CD16 plus CD56 have been used as markers for NK cells. Each of these approacheshas advantages and limitations. The combination of CD16 plus CD56 should identify all cells havingNK activity but is more expensive than using either reagent alone and, when employed in a lineagegating strategy, requires a significant correction for CD3 cells which also express these markers. +

CD16 or CD56 used as single NK markers in combination with CD3 may fail to detect NK cells thatexpress one marker but not the other. However, such cells typically account for less than 2% oftotal lymphocytes (11-13).

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Appendix F. (Continued)

Fluorescence compensation for three color immunofluorescence analysis is more complex than fortwo color analysis and requires more controls be run to verify appropriate instrument performance. Laboratories that use three color analysis should provide their operators with specific expert trainingon methods for three color compensation on all instrument(s) to be used for sample analysis by thismethod.

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References to Appendix F

1. Loken MR, Brosnan JM, Bach BA, Ault KA: Quality control in flow cytometry: 1. Establishingoptimal lymphocyte gates for immunophenotyping by flow cytometry. Cytometry 11:453, 1990.

2. Letwin, BW, Wallace PK, Muirhead KA, Hensler GL, Kashatus WH, Horan PK: An improved clonalexcess assay using flow cytometry and B-cell gating. Blood 75:1178-1185, 1990.

3. Nicholson JKA, Jones, BM, Hubbard M: CD4 T-Lymphocyte determinations on whole bloodspecimens using a single-tube three-color assay. Cytometry 14:685-689, 1993.

4. Mandy FF, Bergeron M, Rectenwald D, Izaguirre CA: A simultaneous three-color T cell subsetsanalysis with single laser flow cytometers using T cell gating protocol. Comparison withconventional two-color immunophenotyping method. J. Immunol. Methods 156: 151-162, 1992.

5. FACSCount . The most complete system for measuring absolute CD4, CD8, and CD3 counts.™

FACSCount white paper, Becton Dickinson promotional literature. 1994.

6. Mercolino TJ, Connelly MC, Meyer EJ, Knight MD, Parker JW, Stelzer GT, DeChirico G:Immunologic differentiation of absolute lymphocyte count with an integrated flow cytometricsystem: A new concept for absolute T cell subset determinations. Cytometry (Comm. Clin. Cytom.)22: 48-59, 1995.

7. Hubbard MR, Jones BM, Nicholson JKA, Spira TJ: Presence of DIM CD45+B cells in HIV+individuals. Abstract P28, Clin. Appl. Cytometry Meeting. Charleston, SC 1995.

8. Peters RE, Janossy G, Ivory K, Al-Ishmail S, Campana D, Mercolino T: B cell gating withCD37P8. Leukemia 8: 1864-1870, 1994.

9. Connelly MC, Knight M, Giorgi JV, Kagan J, Landay AL, Parker JW, Page E, Spino C, WilkeningC, Mercolino TJ: Standardization of absolute CD4+lymphocyte counts across laboratories: Anevaluation of the Ortho Cytoron Absolute flow cytometry system on normal donors. Cytometry(Comm. Clin. Cytom.) 22: 200-210, 1995.

10. Schenker EL, Hultin LE, Bauer KD, Ferevas J. Margolick JB, Giorgi JV: Evaluation of dual-colorflow cytometry immunophenotyping panel in a multicenter quality assurance program. Cytometry14: 307-317, 1993.

11. Lanier LL, Testis R, Bindl J, Phillips JH: The relationship of CD16 (Leu-11) and Leu-19 (NKH-1)expression on human peripheral blood NK cells and cytotoxic T lymphocytes. J Immunol. 136:4480-4486, 1986.

12. Hultin LE, Tan S, Matud J. Giorgi JV: The relationship between CD16 and CD56 antigenexpression and NK activity in HIV-infected homosexual men. Cytometry (Suppl.) 6: 74(322D),1993.

13. Hu PF, Hultin LE, Hultin P, Hausner MA, Hirji K, Jewett A, Bonavida B, Detels R, Giorgi JV:CD16+CD56+ NK cell numerical deficiency and the presence of CD16 CD56-NK cells with lowdim

lytic activity suggest NK cells are continuously inactivated in HIV-infected donors. J. AIDs (In Press).

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Summary of Comments and Subcommittee Responses

H42-T: Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping ofPeripheral Blood Lymphocytes; Tentative Guideline

1. I have a problem with the use of the term "peripheral blood" rather than simply "blood." Thisis an old expression which is redundant and incorrectly implies differences from "central"blood. "Peripheral" adds no relevant information, and should be deleted wherever it occurs.

! The text of the document has been revised to address the comment.

2. A concern of mine is the use of obsolete units of expression inconsistent with SI units. Specifically, the use of "per mm ", instead of "/L" (e.g. Section 8.2.4) when referring to the3

concentration of cells.

!! The text has been revised as recommended. NCCLS subcommittees that draft standards havethe option of using units that are in common use; however, the IUPAC/IFCC-recommendedunits are also included parenthetically, where appropriate.

3. We are concerned with the section of the guideline dealing with the source of the differentialwhite blood count used for the calculation of absolute lymphocyte subset values. The currentguideline states that the performance of a differential by flow cytometry is unacceptable,which necessitates the use of a manual differential count when the white cell count is low. We have collected data from 60 control individuals where T cell immunophenotyping wasperformed. A count of 400 cells, as suggested in the CDC guidelines (MMWR Report May1992), is labor intensive and laborious, and our experience would suggest that a three-partdifferential from the flow cytometer using both forward vs right angle scatter and dual stainingfor CD45 and CD14, analyzing at least 10,000 cells, is more accurate. We feel theenumeration of T lymphocytes for following immune deficient patients and transplantrecipients requires the most accurate, efficient and economical method available and wecontend that use of the differential from the flow cytometer is the best way to provide this.While we understand that the guidelines cannot support the use of an unapproved method, wesuggest that there should be emphasis on the need to initiate and encourage thestandardization and collection of data necessary to obtain approval for this technology.

! The subcommittee agrees that a three-part flow differential based on forward and side scatter,dual staining with CD45 and CD14, and 10,000 events is as accurate as a hematologydetermined differential. Although the method is unapproved at this time, we encouragelaboratories to undertake such a comparative standardization. It should also be noted thatbead-counting methods for absolute counts are now appearing and will undoubtedlycomplement a flow-based differential. However, there can be definite differences betweenthe two methods as was recently pointed out for the white blood count as its relates toapoptotic cells. See Manion K and Frey T. Apoptosis of cells in aged samples as detected bythe proCOUNT reagent. Cytometry (Comm Clin Cytometry). 1996;26:317-3221.

4. The Centers for Disease Control (CDC) is proposing CD4 T-lymphocyte levels be included ascriterion for classifying HIV-infected persons by the newly revised CDC HIV classificationsystem. In addition, these levels are used as decision points for initiating Pneumocystis cariniipneumonia (PCP) prophylaxis and antiviral therapy, and as a prognostic indicator in patientswith human immunodeficiency virus (HIV) disease. The measurement of absolute CDx+

lymphocyte levels in whole blood requires three steps: (1)The total leukocyte count, (2) Thepercentage of leukocytes that are lymphocytes (differential), (3) The percentage oflymphocytes that are CDx (immunophenotyping by flow cytometry). Flow cytometry data+

should be reported both as a percentage of the total lymphocytes, corrected for thelymphocyte purity of the gate and absolute lymphocyte (subset) values. This document

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addresses the issue of reporting absolute counts only if an accurate white cell count (WBC)and differential is available. We suggest this be included as a requirement for reporting of flowcytometry data.

! The subcommittee does not agree with the proposal. We recognize the importance ofabsolute counting, but we are also aware of practical implications that a mandate on reportingonly absolute counts would have on the practice of flow cytometry. Given the current six-hour time limit for performance of hematology analysis, in many settings in which referencelaboratories perform lymphocyte subset analysis after overnight shipment of samples, thestandard (and appropriate) practice is for a local laboratory to perform the hematology portionof the analysis, and the reference laboratory to perform the flow cytometry. Requiring thislocal laboratory to report only absolute counts by including hematology results from anotherlaboratory would not only create logistical problems, but also have serious potentialimplications for the reference laboratory in that it would be required to use results withouthaving any control over quality assurance, proficiency testing, etc. Indeed an Expert AdvisoryPanel to the Centers for Disease Control and Prevention (unpublished observations) hasrecognized this problem and has recommended that any national proficiency testing programthat is established for absolute CD4 count determination reflect a laboratory’s current practice. Emerging technologies to permit direct absolute determination, while only beginning topenetrate the marketplace, will likely render this problem moot after a short period of time.

5. We have noted discrepancies between the immunocytochemical results obtained in ourlaboratory for CALLA (CD10) and the flow cytometric immunophenotype from our referencelaboratory. Specifically, for the three most recent pediatric acute lymphocytic leukemias, theimmunofluorescent studies have yielded negative or very low percentages of CALLA positivecells while the flow cytometric results have been 25, 72, and 97%. Other B-cell antigens(CD19 and CD24) are also strongly positive on flow cytometry. While I would expect thatimmunofluorescent studies would not be as sensitive a flow cytometry, I am surprised bythese results. Is there way we can improve our correlation with the reference laboratory'sflow cytometric studies?

! This is addressed specifically in NCCLS document H43—Clinical Applications of FlowCytometry: Immunophenotyping of Leukemic Cells.

6. We suggest that the third line of the abstract be changed to read "...have made flowcytometry a method of choice...," thus making the statement more general.

! While recognizing that circumstances may change with increasing acceptance of emergingtechnologies, the subcommittee notes that in current practice flow cytometry remains themethod of choice for immunophenotyping of lymphocyte subsets; changing “the” to “a” is notnecessary, even though it would still of course be correct.

7. In the summary of comments, comment 2, the statement is made that EDTA is theanticoagulant for specimens to be analyzed within six hours. What is said in the manual isthat EDTA is the anti-coagulant of choice for those in which a CBC is to be determined fromthe same specimen.

! The subcommittee agrees. The document refers to the use of EDTA for purposes ofperforming a white count and differential and does not state that it is necessarily preferable forlymphocyte immunophenotyping.

8. I question comment 4 in the summary of comments which refers to Section 7.2 on lysing ofwhole blood. I would agree that whole blood lyses is the method of choice for freshspecimens, however, specimens of twenty-four hours are better ficolled to remove dead cells.

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!! The subcommittee does not agree. Because of the known selective loss of lymphocytesubsets when aged specimens are subjected to density gradient separation, discussed inAppendix B and references therein, the subcommittee strongly discourages the use of densitygradient techniques, especially on older samples. In general, the reproducibility of lymphocytesubsets after 24 hours of storage is well established. If an older sample is found to have aviability problem the subcommittee believes it would be far better for the laboratory to rejectthe sample and request a replacement than to process it using density gradient techniques.

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Proposed- and tentative-level documents are being advanced through the NCCLS consensus process; therefore, readers should*

refer to the most recent editions.

59

Related NCCLS Publications*

GP5-A Clinical Laboratory Waste Management; Approved Guideline (1993). Guidance on safehandling and disposal of chemical, infectious, radioactive, and physical waste generated inthe clinical laboratory.

H3-A3 Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture—ThirdEdition; Approved Standard (1991). Methods for the collection of blood specimens byvenipuncture and appropriate training program aimed at increasing analyte integrity andminimizing laboratory error. Includes a 25-step protocol for specimen collection,recommendations for "order of draw" and considerations for performing venipuncture onchildren.

H5-A3 Procedures for the Handling and Transport of Domestic Diagnostic Specimens andEtiological Agents—Third Edition; Approved Standard (1994). American NationalStandard. Proper packaging, handling, and transport requirements for medical specimensincluding federal regulations.

H18-A Procedures for the Handling and Processing of Blood Specimens; Approved Guideline(1990). Addresses the multiple factors associated with the handling and processing ofspecimens, factors that can introduce imprecision or systematic bias into test results.

H20-A Reference Leukocyte Differential Count (Proportional) and Evaluation of InstrumentalMethods; Approved Standard (1992). Discusses automated differential counters andestablishes a reference method based on the visual (or manual) differential count forleukocyte differential counting to which an automated or manual test method can becompared, and an experiment to carry out the comparison. Describes procedures forcollecting specimens; preparing blood films and requirements for acceptable wedge andspun films; Romanowsky staining; the formed elements; variant leukocyte forms; and aprotocol for examining blood films. Details procedures for determining inaccuracy, andwithin-run and between-run imprecision; procedures for determining clinical sensitivity;and statistical methods for determining inaccuracy and imprecision.

H26-A Performance Goals for the Internal Quality Control of Multichannel Hematology Analyzers;Approved Standard (1996). Recommended performance goals for analytical accuracy andprecision based on mathematical models for the following measurements: hemoglobinconcentration, erythrocyte count, leukocyte count, platelet count, and mean corpuscularvolume.

M29-A Protection of Laboratory Workers from Instrument Biohazards and Infectious DiseaseTransmitted by Blood, Body Fluids, and Tissue; Approved Guideline (1997). Guidance onthe risk of transmission of hepatitis B virus and human immunodeficiency viruses in thelaboratory; specific precautions for preventing transmission of bloodborne infection duringclinical and anatomical laboratory procedures.

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NOTES

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NOTES

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