Clsi Ila06 A

I/LA6-A
Vol. 17 No. 17
Replaces I/LA6-T and I/LA7-P
October 1997 Vol. 12 No. 24 and Vol. 4 No. 10
Detection and Quantitation of Rubella IgG Antibody: Evaluation

and Performance Criteria for Multiple Component Test
Products, Specimen Handling, and Use of Test Products in the
Clinical Laboratory; Approved Guideline
This guideline identifies performance specifications and criteria for products used to detect rubella
antibody. It also provides procedures for collecting and handling specimens submitted for rubella
serological testing as well as the evaluation and reporting of results.
ABC
NCCLS...
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October 1997 I/LA6-A

and Performance Criteria for Multiple Component Test Products,
Specimen Handling, and Use of Test Products in the Clinical
Laboratory; Approved Guideline
Abstract
Detection and Quantitation of Rubella IgG Antibody: Evaluation and Performance Criteria for Multiple
Component Test Products, Specimen Handling, and Use of Test Products in the Clinical Laboratory;
Approved Guideline (NCCLS document I/LA6-A) is presented in two parts in order to provide a complete
review of rubella serology testing. Part I is intended for use primarily by manufacturers to enable them to
meet the general product criteria outlined in Section 3, the general performance criteria and specific
performance criteria (Sections 4 and 5, respectively), and to implement the validation testing procedures
presented in Section 6. Part I should also be useful to clinical laboratories that participate in the
validation testing of these products and be of general interest to laboratories in which rubella serological
testing is carried out.
Part II (formerly NCCLS document I/LA7-P) is intended to create an awareness of the preanalytical factors
related to rubella serology tests that may affect patient care. Sections 8 through 12 recommend
procedures for collecting and handling specimens submitted for serological testing and describe the
reporting and interpreting of test results.
[NCCLS. Detection and Quantitation of Rubella IgG Antibody: Evaluation and Performance Criteria for
Multiple Component Test Products, Specimen Handling, and Use of Test Products in the Clinical
Laboratory; Approved Guideline. NCCLS document I/LA6-A (ISBN 1-56238-335-3). NCCLS, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 1997.]
THE NCCLS consensus process, which is the mechanism for moving a document through two
or more levels of review by the healthcare community, is an ongoing process. Users should
expect revised editions of any given document. Because rapid changes in technology may
affect the procedures, methods, and protocols in a standard or guideline, users should replace
outdated editions with the current editions of NCCLS documents. Current editions are listed in
the NCCLS Catalog, which is distributed to member organizations, and to nonmembers on
request. If your organization is not a member and would like to become one, and to request a
copy of the NCCLS Catalog, contact the NCCLS Executive Offices. Telephone: 610.688.0100;
Fax: 610.688.0700; E-Mail: exoffice@nccls.org.
NCCLS VOL. 17 NO. 17 i

I/LA6-A
ISBN 1-56238-335-3
October 1997 ISSN 0273-3099

and Performance Criteria for Multiple Component Test Products,
Specimen Handling, and Use of Test Products in the Clinical
Laboratory; Approved Guideline
Volume 17 Number 17
Laurence P. Skendzel, M.D.

David J. Kiefer, Ph.D.
Robert M. Nakamura, M.D.
Cathy D. Nutter
Lawrence E. Schaefer
John A. Stewart, M.D.
ABC
This publication is protected by copyright. No part of it may be reproduced, stored in a retrieval system,
or transmitted in any form or by any means (electronic, mechanical, photocopying, recording, or
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NCCLS hereby grants permission to reproduce limited portions of this publication for use in laboratory
procedure manuals at a single site, for interlibrary loan, or for use in educational programs provided that
multiple copies of such reproduction shall include the following notice, be distributed without charge,
and, in no event, contain more than 20% of the document's text.
Reproduced with permission, from NCCLS publication I/LA6-A— Detection and Quantitation of
Rubella IgG Antibody: Evaluation and Performance Criteria for Multiple Component Test Products,
Specimen Handling, and Use of Test Products in the Clinical Laboratory; Approved Guideline.
Copies of the current edition may be obtained from NCCLS, 940 West Valley Road, Suite 1400,
Wayne, Pennsylvania 19087-1898 USA.
Permission to reproduce or otherwise use the text of this document to an extent that exceeds the
exemptions granted here or under the Copyright Law must be obtained from NCCLS by written request.
To request such permission, address inquiries to the Executive Director, NCCLS, 940 West Valley Road,
Suite 1400, Wayne, Pennsylvania 19087-1898 USA.
Copyright © 1997. The National Committee for Clinical Laboratory Standards.
Suggested Citation
NCCLS. Detection and Quantitation of Rubella IgG Antibody: Evaluation and Performance Criteria for
Multiple Component Test Products, Specimen Handling, and Use of Test Products in the Clinical
Laboratory; Approved Guideline. NCCLS document I/LA6-A (ISBN) 1-56238-335-3. NCCLS, 940 West
Valley Road, Suite 1400, Wayne, Pennsylvania 19087-1898 USA, 1997.
Proposed Guideline
August 1985
Tentative Guideline
December 1992
Approved Guideline
October 1997
ISBN 1-56238-335-3
ISSN 0273-3099
NCCLS VOL. 17 NO. 17 iii

Committee Membership
Area Committee on Immunology and Ligand Assay
Robert M. Nakamura, M.D. Scripps Clinic and Research Foundation

Chairholder La Jolla, California
Linda Ivor Del Mar, CA

Vice Chairholder
Subcommittee on Rubella Serology
Laurence P. Skendzel, M.D. Traverse City, Michigan

Chairholder
David J. Kiefer, Ph.D. Quest International Inc.

Miami, Florida
Robert M. Nakamura, M.D. Scripps Clinic and Research Foundation

La Jolla, California
Cathy D. Nutter Food and Drug Administration Center for

Devices/Radiological Health
Rockville, Maryland
Lawrence E. Schaefer Abbott Laboratories

Abbott Park, Illinois
John A. Stewart, M.D. Centers for Disease Control and Prevention

Atlanta, Georgia
ADVISORS
Carole A. Golden, Ph.D. Microbiological Research Corporation

Bountiful, Utah
Elaine Harris Instrumentation Laboratory

Orangeburg, New York
Elaine Kruger Instrumentation Laboratory

Orangeburg, New York
Kenneth D. McClatchey, M.D., D.D.S. Loyola University Medical Center

Board Liaison Maywood, Illinois
Beth Ann Wise, M.T.(ASCP), M.S.Ed. NCCLS

Staff Liaison Wayne, Pennsylvania
Patrice E. Polgar NCCLS

Editor Wayne, Pennsylvania
NCCLS VOL. 17 NO. 17 iv

ACTIVE MEMBERSHIP (as of 1 October 1997)
Sustaining Members Commission on Office FDA Division of Anti-Infective

Laboratory Accreditation Drug Products
American Association for Institut für Stand. und Dok. im Federacion Bioquimica de la
Clinical Chemistry Med. Lab. (INSTAND) Provincia (Argentina)
Bayer Corporation International Council for Health Care Financing
Beckman Instruments, Inc. Standardization in Administration
Becton Dickinson and Company Haematology INMETRO
Boehringer Mannheim International Federation of Instituto de Salud Publica de
Diagnostics, Inc. Clinical Chemistry Chile
College of American Pathologists International Society for Instituto Scientifico HS. Raffaele
Coulter Corporation Analytical Cytology (Italy)
Dade International Inc. Italian Society of Clinical Iowa State Hygienic Laboratory
Johnson & Johnson Clinical Biochemistry Manitoba Health
Diagnostics Japan Assn. Of Medical Massachusetts Department of
Technologists (Osaka) Public Health Laboratories
Professional Members Japanese Assn. Of Medical Michigan Department of Public
Technologists (Tokyo) Health
American Academy of Family Japanese Committee for Clinical National Health Laboratory
Physicians Laboratory Standards (Luxembourg)
American Association of Joint Commission on National Institute of Standards
Bioanalysts Accreditation of Healthcare and Technology
American Association of Blood Organizations Ohio Department of Health
Banks National Academy of Clinical Oklahoma State Department of
American Association for Biochemistry Health
Clinical Chemistry National Society for Ontario Ministry of Health
American Association for Histotechnology, Inc. Saskatchewan Health-
Respiratory Care Ontario Medical Association Government of Saskatchewan
American Chemical Society Laboratory Proficiency Testing South African Institute for
American Medical Technologists Program Medical Research
American Public Health Ordre professionnel des Swedish Institute for Infectious
Association technologistes médicaux du Disease Control
American Society for Clinical Québec
Laboratory Science Sociedade Brasileira de Analises Industry Members
American Society of Hematology Clinicas
American Society for Microbiology Sociedad Espanola de Quimica AB Biodisk
American Society of Clinica Abbott Laboratories
Parasitologists, Inc. VKCN (The Netherlands) ABC Consulting Group, Ltd.
American Type Culture AccuMed International, Inc.
Collection, Inc. Government Members aejes
Asociacion Espanola Primera de Ammirati Regulatory Consulting
Socorros Armed Forces Institute of Asséssor
ASQC Food, Drug and Cosmetic Pathology Atlantis Laboratory Systems
Division Association of State and Avecor Cardiovascular, Inc.
Assoc. Micro. Clinici Italiani- Territorial Public Health Bayer Corporation - Elkhart, IN
A.M.C.L.I. Laboratory Directors Bayer Corporation - Middletown,
Australasian Association of BC Centre for Disease Control VA
Clinical Biochemists Centers for Disease Control and Bayer Corporation - Tarrytown,
Canadian Society of Laboratory Prevention NY
Technologists China National Centre for the Bayer Corporation - West
Clinical Laboratory Management Clinical Laboratory Haven, CT
Association Commonwealth of Pennsylvania Bayer-Sankyo Co., Ltd.
College of American Pathologists Bureau of Laboratories Beckman Instruments, Inc.
College of Medical Laboratory Department of Veterans Affairs Becton Dickinson and Company
Technologists of Ontario Deutsches Institut für Normung Becton Dickinson Consumer
College of Physicians and (DIN) Products
Surgeons of Saskatchewan FDA Center for Devices and Becton Dickinson
Radiological Health Immunocytometry Systems
NCCLS VOL. 17 NO. 17 v

Becton Dickinson Italia Higman Healthcare Rhône-Poulenc Rorer

S.P.A.Becton Dickinson Hoechst Marion Roussel, Inc. Roche Diagnostic Systems (Div.
Microbiology Hybritech, Incorporated Hoffmann-La Roche Inc.)
Systems Hycor Biomedical Inc. Roche Laboratories (Div.
Becton Dickinson VACUTAINER i-STAT Corporation Hoffmann-La Roche Inc.)
Systems Integ, Inc. ROSCO Diagnostica
Behring Diagnostics Inc. International Biomedical The R.W. Johnson Pharmaceutical
Behring Diagnostics Inc. - San Consultants Research Institute
Jose, CA International Remote Imaging Sarstedt, Inc.
bioMérieux Vitek, Inc. Systems (IRIS) Schering Corporation
Biometrology Consultants International Technidyne Schleicher & Schuell, Inc.
Bio-Rad Laboratories, Inc. Corporation Second Opinion
Bio-Reg Associates, Inc. Johnson & Johnson Clinical SenDx Medical, Inc.
Biosite Diagnostics Diagnostics Sherwood-Davis & Geck
Biotest AG Johnson & Johnson Health Care Shionogi & Company, Ltd.
Boehringer Mannheim Systems, Inc. Showa Yakuhin Kako Company,
Diagnostics, Inc. Kimble/Kontes Ltd.
Boehringer Mannheim GmbH Labtest Sistemas Diagnosticos Sienna Biotech
Bristol-Myers Squibb Company Ltda. SmithKline Beecham Corporation
Canadian Reference Laboratory LifeScan, Inc. (a Johnson & SmithKline Beecham (NZ) Ltd.
Ltd. Johnson Company) SmithKline Beecham, S.A.
CASCO Standards Lilly Research Laboratories SmithKline Diagnostics, Inc.
Checkpoint Development Inc. Luminex Corporation (Sub. Beckman Instruments,
ChemTrak Mallinckrodt Sensor Systems Inc.)
Chiron Diagnostics Corporation MBG Industries, Inc. SRL, Inc.
Chiron Diagnostics Corporation - Medical Device Consultants, Inc. Streck Laboratories, Inc.
International Operations Medical Laboratory Automation Sumitomo Metal Bioscience Inc.
Chiron Diagnostics Corporation - Inc. Sysmex Corporation
Reagent Systems MediSense, Inc. TOA Medical Electronics
Clinical Lab Engineering Merck & Company, Inc. Unipath Co (Oxoid Division)
COBE Laboratories, Inc. Metra Biosystems Vetoquinol S.A.
Control Lab (Brazil) Neometrics Inc. Vysis, Inc.
Cosmetic Ingredient Review Nissui Pharmaceutical Co., Ltd. Wallac Oy
Coulter Corporation Norfolk Associates, Inc. Warner-Lambert Company
Cytometrics, Inc. North American Biologicals, Inc. Wyeth-Ayerst
CYTYC Corporation Olympus Corporation Xyletech Systems, Inc.
Dade International - Deerfield, IL Optical Sensors, Inc. Yeongdong Pharmaceutical Corp.
Dade International - Glasgow, DE Organon Teknika Corporation Zeneca
Dade International - Miami, FL Orion Diagnostica, Inc.
Dade International - Ortho Clinical Diagnostics, Inc. Trade Associations
Sacramento, CA Otsuka America Pharmaceutical,
DAKO A/S Inc. Association of Medical
Diagnostic Products Corporation Pfizer Canada, Inc. Diagnostic Manufacturers
Diametrics Medical, Inc. Pfizer Inc Health Industry Manufacturers
Difco Laboratories, Inc. Pharmacia & Upjohn (MI) Association
Eiken Chemical Company, Ltd. Pharmacia & Upjohn (Sweden) Japan Association Clinical
Enterprise Analysis Corporation Procter & Gamble Reagents Ind. (Tokyo, Japan)
Donna M. Falcone Consultants Pharmaceuticals, Inc. Medical Industry Association
Fujisawa Pharmaceutical Co. Ltd. The Product Development Group of Australia
Gen-Probe Radiometer America, Inc. National Association of Testing
Glaxo Inc. Radiometer Medical A/S Authorities - Australia
Greiner Meditech, Inc. Research Inc.
Health Systems Concepts, Inc. David G. Rhoads Associates, Inc.
Helena Laboratories
NCCLS VOL. 17 NO. 17 vi

Associate Active Members Gulhane Military Medical New York University Medical
Academy (Turkey) Center
Affinity Health System (WI) Harris Methodist Fort Worth North Carolina Laboratory of
Allegheny University of the (TX) Public Health
Health Sciences (PA) Hartford Hospital (CT) North Shore University Hospital
Alton Ochsner Medical Health Alliance Laboratory (NY)
Foundation (LA) Services (OH) Olin E. Teague Medical Center
American Oncologic Hospital Heritage Hospital (MI) (TX)
(PA) Hopital Saint Pierre (Belgium) Omni Laboratory (MI)
Anzac House (Australia) Incstar Corporation (MN) Our Lady of Lourdes Hospital
Associated Regional & Integris Baptist Medical Center (NJ)
University Pathologists (UT) of Oklahoma Our Lady of the Resurrection
Baptist Memorial Healthcare International Health Managment Medical Center (IL)
System (TX) Associates, Inc. (IL) PAPP Clinic P.A. (GA)
Beth Israel Medical Center (NY) Kaiser Permanente (CA) Pathology Associates
Brazosport Memorial Hospital Kangnam St. Mary’s Hospital Laboratories (CA)
(TX) (Korea) Permanente Medical Group (CA)
Bristol Regional Medical Center Kenora-Rainy River Regional PLIVA d.d. Research Institute
(TN) Laboratory Program (Dryden, (Croatia)
Brooke Army Medical Center Ontario, Canada) Polly Ryon Memorial Hospital
(TX) Klinisches Institute für (TX)
Brooks Air Force Base (TX) Medizinische (Austria) Providence Medical Center (WA)
Broward General Medical Center Laboratoire de Santé Publique Puckett Laboratories (MS)
(FL) du Quebec (Canada) Quest Diagnostics (MI)
Canterbury Health Laboratories Laboratory Corporation of Quest Diagnostics (PA)
(New Zealand) America (NC) Reid Hosptial & Health Care
Central Peninsula General Laboratory Corporation of Services (IN)
Hospital (AK) America (NJ) Sacred Heart Hospital (MD)
Childrens Hospital Los Angeles Lancaster General Hospital (PA) St. Boniface General Hospital
(CA) Libero Instituto Univ. Campus (Winnipeg, Canada)
Children's Hospital Medical Biomedico (Italy) St. Francis Medical Center (CA)
Center (Akron, OH) Louisiana State University St. John Regional Hospital (St.
Clendo Lab (Puerto Rico) Medical Center John, NB, Canada)
Clinical Diagnostic Services (NJ) Maimonides Medical Center St. Joseph’s Hospital -
Commonwealth of Kentucky (NY) Marshfield Clinic (WI)
CompuNet Clinical Laboratories Maine Medical Center St. Luke’s Hospital (PA)
(OH) Massachusetts General Hospital St. Luke’s Regional Medical
Consolidated Laboratory MDS-Sciex (Concord, ON, Center (IA)
Services (CA) Canada) St. Luke’s-Roosevelt Hospital
Consultants Laboratory (WI) Melbourne Pathology (Australia) Center (NY)
Detroit Health Department (MI) Memorial Medical Center (LA) St. Mary of the Plains Hospital
Dhahran Health Center (Saudi Methodist Hospital (TX) (TX)
Arabia) Methodist Hospital Indiana St. Paul Ramsey Medical Center
Duke University Medical Center Methodist Hospitals of Memphis (MN)
(NC) (TN) St. Vincent Medical Center (CA)
Dwight David Eisenhower Army Montreal Children’s Hospital San Francisco General Hospital
Medical Center (Ft. Gordon, (Canada) (CA)
GA) Mount Sinai Hospital (NY) Seoul Nat’l University Hospital
East Side Clinical Laboratory (RI) Mount Sinai Hospital (Toronto, (Korea)
Easton Hospital (PA) Ontario, Canada) Shanghai Center for the Clinical
Federal Medical Center (MN) National Genetics Institute (CA) Laboratory (China)
Frye Regional Medical Center Naval Hospital Cherry Point (NC) Shore Memorial Hospital (NJ)
(NC) New Britain General Hospital (CT) SmithKline Beecham Clinical
Gila Regional Medical Center New Hampshire Medical Laboratories (GA)
(NM) Laboratories SmithKline Beecham Clinical
Grady Memorial Hospital (GA) The New York Blood Center Laboratories (TX)
Great Smokies Diagnostic New York State Department of South Bend Medical Foundation
Laboratory (NC) Health (IN)
New York State Library
NCCLS VOL. 17 NO. 17 vii

So. California Permanente University of the Ryukyus VA (Long Beach) Medical Center
Medical Group (Japan) (CA)
Southeastern Regional Medical The University of Texas Medical VA (Miami) Medical Center (FL)
Center (NC) Branch Venice Hospital (FL)
SUNY @ Stony Brook (NY) University of Virginia Medical Veterans General Hospital
Tampa General Hospital (FL) Center (Republic of China)
UNC Hospitals (NC) U.S. Army Hospital, Heidelberg Virginia Baptist Hospital
University of Alberta Hospitals UZ-KUL Medical Center Warde Medical Laboratory (MI)
(Canada) (Belgium) William Beaumont Hospital (MI)
University of Florida VA (Albuquerque) Medical Winn Army Community Hospital
University Hospital (Gent) Center (NM) (GA)
(Belgium) VA (Ann Arbor) Medical Center Wisconsin State Laboratory of
University Hospital (London, (MI) Hygiene
Ontario, Canada) VA (Denver) Medical Center Yonsei University College of
University Hosptial (Linkoping, (CO) Medicine (Korea)
Sweden) VA (Jackson) Medical Center York Hospital (PA)
University Hospital (IN) (MS) Zale Lipshy University Hospital
University Hospital of (TX)
Cleveland (OH)
The University Hospitals (OK)
University of Medicine &
Dentistry, NJ University
Hospital
University of Michigan
University of Nebraska Medical
Center
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, FAIC
Family Medical Care mvi Sciences
Sharon S. Ehrmeyer, Ph.D.
William F. Koch, Ph.D., University of Wisconsin David E. Nevalainen, Ph.D.
President Elect Abbott Laboratories
National Institute of Standards Elizabeth D. Jacobson, Ph.D.
and Technology FDA Center for Devices and Donald M. Powers, Ph.D.
Radiological Health Johnson & Johnson Clinical
F. Alan Andersen, Ph.D., Diagnostics
Secretary Hartmut Jung, Ph.D.
Cosmetic Ingredient Review Boehringer Mannaheim GmbH Eric J. Sampson, Ph.D.
Centers for Disease Control
Donna M. Meyer, Ph.D., Tadashi Kawai, M.D., Ph.D. and Prevention
Treasurer International Clinical Pathology
Sisters of Charity Health Care Center Marianne C. Watters,
System M.T.(ASCP)
Kenneth D. McClatchey, M.D., Parkland Memorial Hospital
Charles F. Galanaugh, Past D.D.S.
President Loyola University Medical Ann M. Willey, Ph.D.
Becton Dickinson and Center New York State Department of
Company (Retired) Health
John V. Bergen, Ph.D.,

Executive Director
NCCLS VOL. 17 NO. 17 viii

Contents
Abstract ......................................................... i
Committee Membership . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Active Membership ........................................................ v
Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
Part I
1 Scope ........................................................ 1
2 Nature and Structure of Rubella Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
3 Status of Hemagglutination Inhibition Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
3.1 Use of HAI in Establishing the Calibration Standard . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.2 Relationship of HAI and Other Methods: Clinical Implications . . . . . . . . . . . . . . . . . . . 1
3.3 Reporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.4 Establishing a Reference Point to Define Immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.5 Positive Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3.6 Negative Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4 General Criteria for Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4.1 Quality Control Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

4.2 Calibrators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4.3 Description of the Specific Type and Class of Antibody . . . . . . . . . . . . . . . . . . . . . . . 3
4.4 Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
5 General Performance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6 Specific Performance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6.1 Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6.2 Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6.3 Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
6.4 Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
6.5 Suitability of Reagent Set for Diagnosis of Acute Infection or Reinfection . . . . . . . . . . . 5
7 Product Evaluation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.1 Calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.2 Statements of Signal Suppression or Interference . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
7.3 Validation Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Part II
8 Introduction ........................................................ 6
9 Scope ........................................................ 6
NCCLS VOL. 17 NO. 17 ix

10 Clinical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
10.1 Nature of Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

10.2 Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
10.3 Complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
11 Specimen Collecting and Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
11.1 Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
11.2 Data Provided to the Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
11.3 Storing Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
11.4 Filing System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
12 Clinical Use of the Test(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
12.1 Legally Marketed Commercial Test Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

12.2 Establishing Immune Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
12.3 Diagnosis of Subclinical or Clinical Rubella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
12.4 Diagnosis of Congenital Rubella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
12.5 Record Keeping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Summary of Comments and Subcommittee Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Related NCCLS Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
NCCLS VOL. 17 NO. 17 x

Foreword
During the past 25 years, a variety of tests have been described for the detection and/or quantitation of
rubella antibody. Several variations of the same basic test methodologies have been developed. In 1969,
the Centers for Disease Control (CDC) (now the Centers for Disease Control and Prevention), Atlanta,
Georgia, initiated a voluntary premarket evaluation and testing program for manufacturers of commercial
in vitro rubella diagnostic products. Since 1982, the United States Food and Drug Administration (FDA)
has classified rubella test kits as Class III devices. The first release of this document established
standards for all multiple component products intended for the detection of rubella antibody. It defined
the performance specifications required for these products. Products meeting these criteria are suitable
for use by a laboratory in reporting results for medical management decisions. Scientific advancements
and significant change in methods used for the detection of rubella antibody have led to revision of this
document.
Development of this guideline was undertaken as a result of two suggestions: one, from the American
Society for Microbiology, who suggested that NCCLS do a comprehensive study of rubella serological
testing; the other, from the FDA Center for Devices and Radiological Health and several manufacturers of
rubella testing products, who suggested that voluntary guidelines specifying a panel of reference
preparations would be helpful to laboratories involved in rubella testing.
Initially, the two parts of this guideline were separate NCCLS consensus documents, I/LA6-T (Evaluation
and Performance Criteria for Multiple Component Test Products Intended for the Detection and
Quantitation of Rubella IgG Antibody; Tentative Guideline) and I/LA7-P (Specimen Handling and Use of
Rubella Serology Tests in the Clinical Laboratory; Proposed Guideline). On the recommendation of the
NCCLS Area Committee on Immunology and Ligand Assay, the guidelines have been combined in this
approved level publication to provide to the clinical laboratory testing community a comprehensive
resource focused on rubella serology.
In the previous edition of I/LA6-T, the subcommittee recommended the use of 10 IU/mL as the breakpoint
for defining the probability of immune status and reaffirms this view in I/LA6-A. This edition of the
guideline also incorporates a major revision in two respects. The product criteria outlines in greater detail
the requirement to inform the user on the intended use of the test, and the sensitivity, specificity and
reproducibility of the test. The second feature of this edition is expansion of sections on use of the test in
diagnosis of subclinical and clinical rubella and congenital rubella. These sections should assist the user in
interpretation of test results in unusual situations. Additionally, all the comments received on both
previous editions have been considered in this revision, and responses to the comments are appended to
this guideline.
The NCCLS Subcommittee on Rubella Serology recognizes the ongoing change in laboratory practices and
welcomes constructive suggestions for improvement of this document.
Universal Precautions
Because it is often impossible to know which might be infectious, all patient blood specimens are to be
treated with universal precautions. Guidelines for specimen handling are available from the U. S. Centers
for Disease Control and Prevention [MMWR 1987;36(suppl 2S):2S–18S]. NCCLS document M29—
Protection of Laboratory Workers from Infectious Disease Transmitted by Blood, Body Fluids, and Tissue,
deals specifically with this issue.
NCCLS VOL. 17 NO. 17 xi

Key Words
Rubella hemagglutination inhibition (HAI) antibody test, rubella serodiagnostic products, diagnostic
product, product quality control, rubella product evaluation, rubella test kit, rubella reagents, performance
criteria, clinical rubella, congenital rubella, immunity, IgG, IgM.
NCCLS VOL. 17 NO. 17 xii

Detection and Quantitation of Rubella IgG Antibody:

Evaluation and Performance Criteria for Multiple Component Test Products,
Specimen Handling, and Use of Test Products
in the Clinical Laboratory; Approved Guideline
Part I
1 Scope The HAI test lacks sensitivity at dilutions less

than 1:8. Nonspecific inhibitors of hemagglu-
Part I of this document establishes performance tination cannot be reliably removed without
guidelines and criteria for evaluation of multiple giving rise to other nonspecific reactions.12
component test products used to detect rubella
antibody. 3.1 Use of HAI in Establishing the
Calibration Standard
2 Nature and Structure of Rubella
Virus HAI serves as the reference method to establish
a calibration standard for other rubella methods.
Since relatively few or no laboratories in the
Rubella virus consists of at least three envelope
United States use HAI for routine testing,13 an
glycoproteins: E1, E2a, E2b; a nucleocapsid-
alternative for calibration is the use of the World
associated protein, C; and two nonstructural
Health Organization International Standard
proteins.1-5 The term "rubella antibody" de-
(WHO) for antirubella serum, human. The 3rd
scribes immunoreactivity to some or all of the
International Standard includes two other assay
virus components. Individuals may respond
methods, the enzyme immunoassay and single
immunologically in a diverse manner to the
radial hemolysis, in addition to HAI in evaluation
rubella virus. The reagent sets used for detec-
of the reference preparation. (See Section 7.1.)
tion of antibody may react differently depending
on the reactivity of the predominant antibody to
the rubella peptide. 3.2 Relationship of HAI and Other
Methods: Clinical Implications
Serum samples from patients with various forms
of rubella virus infection were tested for Other methods including the commonly used
antibody to each of three viral structural enzyme immunoassay (EIA) and agglutination
proteins.4,6-8 In most sera, antibody to E1 test, are capable of detecting levels of antibodies
protein was the predominant species. Sera from less than 1:8.14-16 Tests based on the principle
patients with congenital rubella syndrome, of EIA provide a continuous assay of antibody
however, contained significantly more E2 and permit identification of very low levels of
antibody relative to E1 antibody than did sera rubella antibody.13 Studies conducted by
from other rubella patients. Grangeot-Keros and associates17 substantiate the
increased sensitivity of EIA when compared with
HAI tests in the detection of rubella-specific IgG.
3 Status of Hemagglutination
They used immuno-chromatographed
Inhibition Method recombinant proteins containing the three
structural proteins of rubella virus which are
A standard rubella hemagglutination inhibition immunologically and morphologically identical to
(HAI) antibody test was originally adopted by wild type virons instead of viral lysates for
NCCLS in 1979 and by the World Health antibody detection. Using methods other than
Organization (WHO)9 as the reference rubella HAI, immune individuals with specific but
antibody assay method. Previous studies have minimal detectable rubella antibodies have been
established a high correlation between rubella identified.15,16,18-21 Most, if not all, individuals
HAI and traditional rubella neutralization test with minimal detectable levels of HAI antibody
results.10-11 produce secondary-type antibody responses
NCCLS VOL. 17 NO. 17 1

upon revaccination and are likely to be people following natural exposure to the wild
immune.15,22-23 Revaccination of these people did virus. Newer, more sensitive techniques detect
not induce viremia and little or no rubella-specific rubella specific IgG antibody at levels below 1:8.
IgM was produced.15,16 As more people with Numerous reports indicate that people who have
vaccine-induced immunity join the adult been previously vaccinated with low or
population, there is a need for more sensitive apparently absent levels of HAI antibody produce
test methods to accurately assess serological secondary-type responses upon revaccination
status.18 The issue of the protective effect of and are clinically immune.14,27–34 Epidemiologic
very low antibody levels is further complicated studies support these findings.18,19,35,36 Sporadic
by reports of rare instances of viremia in people reports of viremia and/or reinfection among
with low antibody levels given rubella previously immunized persons with low antibody
vaccine.22,23 (Refer to Section 3.4 for levels have been reported but cases of
information on the issue of defining the reinfection have also occurred in persons with
reference point for rubella tests.) antibody levels at or above the 15 IU/mL
cutoff.37–40 Alterations of the complex immune
3.3 Reporting Results system may explain variations in response
among some individuals. Results of antibody
In an attempt to standardize the system of tests for rubella, like other laboratory tests, must
reporting results, the subcommittee recommends be evaluated in the context of the clinical
reporting results of quantitative tests in setting.
International Units (IU) rather than in dilutions.
The World Health Organization has released the Based on epidemiologic data, studies on
third international standard for antirubella sera. vaccinated individuals with low levels of
Use of this standard, or subsequent standards antibody and anecdotal reports, the
provided by the World Health Organization, will subcommittee restates the recommendation of
help resolve uncertainty about the interpretation 10 IU/mL as the reference point for IgG
of antibody detected by tests more sensitive antirubella antibodies.41,42 The effectiveness of
than HAI.24 Refer to Section 7.1 for additional rubella vaccination is well-documented and the
information on the WHO standard. 10 IU/mL antibody level is protective in the vast
%
majority of persons. Recognizing that sporadic
3.4 Establishing a Reference Point to Define and sometimes conflicting reports suggest a
Immunity relationship between antibody level and
protection against the rubella virus, the
A reference point for tests to detect antirubella subcommittee did not advocate lowering the
IgG serves to establish the recommended lower breakpoint below 10 IU/mL until additional data
level of specific IgG antibody protective against has been collected.
the disease and provide a threshold value for the
test. Physicians use the threshold value in Although scientific data are the basis for
making clinical decisions. A reference point may selecting the reference point to define immunity,
originate from research data, study of large results of antibody tests for rubella, like other
population groups, or by determination of the laboratory tests, must be evaluated in the
predictive value of a test and is not necessarily context of the clinical setting.
inclusive of every individual in a population.
3.5 Positive Sera
The original recommendations from the
Subcommittee on Rubella Serology (first The subcommittee recommends reporting the
published in the tentative edition of this level of rubella antibody in quantitative units as
guideline) established 1:8 or 15 IU/mL as the IU/mL. For qualitative or semiquantitative
indicator of immunity.12,25-26 The early screening tests, the designation “positive”
experiments which led to this definition of
rubella immunity probably will never be repeated
since the protective effect of an IgG antibody
%
level of 1:8 or greater derived from study of A gray zone exists when defining the reference point. For
further discussion of the gray zone, see Section 6.

identifies sera with IgG rubella antibody equal to mean and standard deviation for each lot number
or greater than ($) 10 IU/mL. of control material.
3.6 Negative Sera The College of American Pathologists (CAP)

provides a certified rubella reference material
The subcommittee recommends reporting results suitable as an alternate independent control.
in quantitative units at levels at and above the The reference material is intended for periodic
equivalence zone. Antibody levels less than the use to validate the calibration procedure. The
lower limit of the equivalence zone may be reference materials may be obtained from the
reported in IU/mL, as “less than,” or negative College of American Pathologists, Standards and
depending on the practice adopted in each Reference Material Program, 3325 Waukegan
laboratory. For qualitative and semi-quantitative Rd., Northfield, Illinois 60093-2750, Telephone
screening tests, the designation “negative” 800.223.4040, Ext. 468.
identifies sera with antibody levels of less than
(<) 10 IU/ml. 4.2 Calibrators
4 General Criteria for Product Calibrators are required to substantiate the

continued accuracy of the test method
In vitro diagnostic products are highly regulated throughout the laboratory’s reportable range for
in many countries. France, Germany, Japan, and patient test results. The calibration materials
the United States have strict regulations that should be appropriate for their method and, if
apply to Rubella immunoassays. possible, traceable to a reference method. Refer
tothe most current editions of NCCLS
(In vitro diagnostic products intended for commercial documents I/LA18—Specifications for
distribution in the United States are medical devices Immunological Testing for Infectious Diseases,
regulated by the Food and Drug Administration (FDA). and C24— Internal Quality Control Testing:
Manufacturers of these products are subject to the general
controls and other controls of the Food, Drug, and Cosmetic
Principles and Definitions, for the appropriate
Act (FD&C Act or “the Act”). General controls of the Act definitions of calibrations and controls.
are baseline requirements that apply to all medical device
manufacturers. Unless specifically exempted, medical 4.3 Description of the Specific Type and
devices must be properly labeled and packaged, be cleared
for marketing by the FDA, meet their labeling claims, and be Class of Antibody
manufactured under Good Manufacturing Practices (GMP)
which is a mandated quality assurance program. For further The rubella virus consists of major antigenic
information regarding FDA regulations, contact the Division
of Small Manufacturers Assistance (DSMA) in the office of
components associated with the viral envelope
Health and Industry Programs, Center for Devices and and the ribonucleoprotein core. The manufac-
Radiological Health. Telephone 800.638.2041 or FAX turer must describe adequately the antigen and
301.443.8818.) the specific type and class of antibody detected
by the reagent set, i.e., whole virus vs. selected
4.1 Quality Control Sera viral proteins. The impact of the nature of the
antigen on the diagnosis of acute or congenital
Products (quantitative and screening tests) infection should be clearly stated.
designed to test for rubella antibody should
include a minimum of three control sera (one The manufacturer should state whether or not
negative, one low-positive, and one high- the reagents are sensitive to early or late-
positive) to be assayed in each test run. The appearing IgG antibodies, and the time it takes
control sera for quantitative tests should be for a test to become positive following infection
expressed in international units and the low- or exposure.
positive should be in the range of 10 to 20
IU/mL. These controls must be distinct from 4.4 Labeling
calibrators used in the test procedure (see
Section 4.2). For quantitative tests, the Labeling should conform to in vitro diagnostic
manufacturer should provide statistical data, i.e., products labeling regulations. (In the U.S. refer
to 21 CFR 809.10 [Code of Federal

Regulations].) The labeling of each component calculation of sensitivity, equivocal results in the
and of the entire unit should meet all federal “gray zone” may be excluded but the
requirements. On the label or package insert, manufacturer should indicate the frequency of
indicate the intended use of the reagent set, this occurrence in their product labeling.
e.g., determination of immune status, diagnosis
of subclinical or clinical rubella, diagnosis of 6.2 Specificity
congenital rubella, and, if applicable, the
sensitivity to early or late-appearing IgG Specificity is the ability of the assay to discern a
antibodies (see Sections 4.3, 12.2.2, 12.3.1, rubella-negative serum with an antibody level
and 12.4.2). Indicate the limitations of testing less than 10 IU/mL from a rubella-positive serum
for the assay, e.g., not intended for use in (see Section 3.6). To be acceptable for in vitro
diagnosis of congenital rubella. diagnostic use, the assay should demonstrate
specificity of 95% based on assay of 100 or
5 General Performance Criteria more rubella-negative sera with antibody levels
less than 10 IU/mL.
All components of the product should function
as claimed by the manufacturer and produce the The manufacturer may define a “gray zone” for
expected results with clinical specimens. For the test but the limits must fall within the
example, a manufacturer may claim that requirements for reproducibility (see Section
hemolysis, bilirubin, and lipemia do not interfere 6.3). When a result in the series of tests for
with the reactivity of a reagent set. Clinical specificity falls in the “gray zone,” the
studies should include a sufficient number of manufacturer should handle the results according
samples to verify the claim. to the recommendations it makes to users of the
test, e.g., retest, redraw a specimen, etc. In the
6 Specific Performance Criteria calculation of specificity, equivocal results in the
“gray zone” may be excluded but the
Rubella sero-diagnostic products should manufacturer should indicate the frequency of
demonstrate linearity with the WHO Standard this occurrence in their product labeling.
over the range of the assay.
6.3 Reproducibility
6.1 Sensitivity
Reproducibility is the ability of the product to
Sensitivity is the ability of the assay to detect show uniformity of antibody level in replicate
antirubella antibody in serum at the 10 IU/mL testing of positive and negative sera based on
level (see Section 3.5). To be acceptable for in within-run testing. The manufacturer should
vitro diagnostic use, the assay must demonstrate define the reproducibility of the test using three
sensitivity of 95% based on assay of 50 or more “manufactured” lots of sera. At the 10 IU/mL
specimens with antibody levels exceeding 20 level, the 3rd standard deviation (SD) interval in
IU/mL. The assay must also demonstrate repetitive tests (40 or more assays) should not
sensitivity of 95% based on assay of 50 exceed ±3 IU/mL. For qualitative and semi-
specimens in the low range of 10-20 IU/mL. quantitative tests the manufacturer must define
Samples should be drawn from the clinical the “gray zone” in terms of IU/mL. For reagent
population of interest including pregnant women, sets intended to evaluate immune status only
children, and adults. (i.e., reagent sets reporting test results as
positive or negative) the product should
The manufacturer may define a “gray zone” for demonstrate reproducibility of $95% based on
the test but the limits must fall within the assays in duplicate of 40 or more rubella-positive
requirements for reproducibility (see Section sera with a titer in the range of 10 to 20 IU/mL
6.3). When a result in the series of tests for and 40 or more negative sera.
sensitivity falls in the “gray zone” the
manufacturer should handle the results according For qualitative and semiquantitative assays, the
to the recommendation it makes to users of the manufacturer should indicate the imprecision of
test, e.g., retest, redraw a specimen, etc. In the the method at three concentration levels—

negative, low positive, and positive. (See NCCLS Centers for Disease Control and Prevention
document EP10, Preliminary Evaluation of NCID-DVRD-VEHB
Clinical Chemistry Methods.) Bldg. 7, Rm. 206, G-18
1600 Clifton Rd.
6.4 Stability Atlanta, GA 30333
The product should be shown to perform reliably 7.2 Statements of Signal Suppression or
throughout the term of its shelf-life as stated by Interference
the manufacturer and indicated in the product
labeling. If a component is lyophilized, the Manufacturers should state the concentration
manufacturer must indicate the shelf-life of the range, in IU/mL, of samples tested in clinical
reconstituted component. trials. Manufacturers should also indicate any
signal suppression or interference (prozone or
6.5 Suitability of Reagent Set for Diagnosis “hook” effect) observed during clinical or
of Acute Infection or Reinfection analytical studies.
The manufacturer must indicate if the reagent 7.3 Validation Testing

set is suitable to determine a “significant
change” in IgG antibody levels for the diagnosis The reagents or kits, and any required special
of acute infection or reinfection (Refer to Section equipment are to be evaluated by at least two
12 for additional information). The term independent laboratories.
“significant change” should be defined by the
manufacturer and should include consideration of 7.3.1 Evaluation Serum Panel(s)
the assay imprecision. The manufacturer should
concurrently recommend assay of specimens as Panels of known reactivity tested with at least
good laboratory practice. two other commercially available assays as a
reference must include an array of high positive,
7 Product Evaluation low positive, and negative specimens suitable to
allow evaluation of test sensitivity, specificity,
7.1 Calibration and reproducibility. Sera should be drawn from
the clinical population of interest. (See Section
The requirements for calibration include: 6.1.) Clinical histories on rubella IgG immune
individuals may be inadequate. The CDC
(1) Manufacturers are required to use the provides a panel of coded sera for evaluation.
World Health Organization International The panel may be procured from the CDC at the
Standards for antirubella serum, human (see address listed in Section 7.1.
Section 3.1). The 3rd International Standard is
currently available and may be procured by 7.3.2 Reagents, Controls, and Equipment
contacting:
All product components necessary to perform
International Laboratory the test as defined by the manufacturer should
Statens Seruminstitut be included in the evaluation. These
Copenhagen, Denmark components will be provided by the
Telephone: 45-32-68-31-50 manufacturer for the evaluation.
Fax: 45-32-68-34-66
E-mail: gahansen@standard.ssi.dk 7.3.3 Evaluation Design
(2) Calibration verification using the CDC low- The product will be evaluated by the designated
titer rubella antibody standard with an assay laboratories using:
value of 20 IU/mL. The calibration procedure
should include testing with a ½ dilution of the (1) Evaluation serum panel(s);
standard. For procurement of the CDC reference
preparation contact:

(2) A panel of coded serum provided interpretation of testing have received scant
through the CDC; attention. Misinterpretation of test results may
lead to improper clinical decisions. "Black and
(3) The CDC low-titer rubella antibody white" interpretations are used even though
standard, including a ½ dilution of the standard. certain questionable "gray" areas exist.
To procure the coded serum panel, contact the
CDC at the address listed in Section 7.1. The 9 Scope
manufacturer will provide serum panels and
standards for use by the designated laboratories. Part II of this guideline describes recommended
procedures for collecting and handling specimens
7.3.4 Test Method and discusses the reporting and the laboratory
interpretation of test results. The laboratory
The test method used is that stated by the interpretation of test results should be
manufacturer in the product package insert. distinguished from the clinical interpretation.
The physician should evaluate the laboratory
7.3.5 Reports and Analyses of Validation interpretation of the data along with the clinical
Testing history and physical examination of the patient
to determine immune status (particularly in
The manufacturer should summarize results of critical cases of pregnancy) and in establishing
these evaluations to validate product perform- the diagnosis of current infection or congenital
ance claims. infection with rubella virus. Part II should help
various laboratory scientists perform and report
(All new in vitro diagnostic multiple component test products
the results of rubella serologic tests.
intended for the detection and quantitation of rubella
antibody and headed for commercial distribution in the
United States must undergo regulatory review by FDA before 10 Clinical Background
marketing. Contact FDA’s Division of Small Manufacturers
Assistance (DSMA) for technical assistance and regulatory
guidance for complying with FDA requirements for medical 10.1 Nature of Virus
devices.)
Refer to Section 2 for information on the nature
(It is recommended that manufacturers seek assistance to
independently test these products and make data available to of the virus and Section 4.3 for the
the FDA for review. This service may depend on availability manufacturers requirement to describe the
of personnel and time at the CDC. Manufacturers should specific type and class of antibody detected by
contact the CDC well in advance of the anticipated date for the reagent set.
requesting this service.)
10.2 Immunology21
Part II
10.2.1 Acute Infection
8 Introduction
The incubation period of acute rubella is 14 to
Rubella tests are usually ordered for one of three 21 days. Initially, antibodies in both the IgG and
purposes: IgM classes can be detected. Immunoglobulin M
antibodies generally do not persist beyond 5 to 6
(1) to establish immune status, natural or weeks after the onset of illness, whereas IgG
vaccine-induced immunity antibodies usually persist for the lifetime of the
patient.
(2) to diagnose acute (recent or current)
infection of clinical or subclinical rubella A number of methods are currently used for
detecting rubella antibodies. The HAI test
(3) to diagnose congenital rubella detects early IgM and later-appearing IgG
antibodies. The passive hemagglutination test is
Although the analytical aspects of the various sensitive to late-appearing IgG antibodies, which
tests for detection of antibodies to rubella are may not be detectable until weeks after onset of
widely discussed, preanalytical factors and the a rubella infection. Other test systems, such as

enzyme immunoassay and latex agglutination, NCCLS documents H4, Procedures for the
have immunoglobulin specificities controlled by Collection of Diagnostic Blood Specimens by
the antiglobulin conjugate used in the indirect Skin Puncture and H18, Procedures for the
assay. Refer to Section 4.3 for additional Handling and Processing of Blood Specimens,
information on the requirement of the provide detailed information on specimen
manufacturer to specify the characteristics of the collection and handling. Collect and handle the
antigen in the package insert. specimen, usually serum, aseptically. Since
microbial growth may interfere with the accuracy
10.2.2 Serological Pattern After Vaccination of testing, use of aseptically collected specimens
is particularly important when testing is delayed
Immunization with the attenuated rubella virus and/or specimens are stored for testing at a later
vaccine induces the formation of rubella-specific date.
IgM and IgG antibodies similar to those observed
with natural infections. The antibody titers are (1) Aseptically remove the specimen, usually
usually lower than those observed in natural serum, and place it in sterile, capped tubes
infections and the complement-fixing antibodies labeled with the specimen submission number,
are definitely found in lower titer. patient’s name, and other required data. Sterile
serum separators are a suitable means of
10.3 Complications transferring sterile serum specimens for testing
and/or storage.
Several investigators have established the
relationship between congenital anomalies and (2) Use aseptic technique and equipment
maternal rubella infections. Infection with rubella when removing a portion of the specimen,
virus during childhood or adulthood is usually a usually serum, for testing.
benign self-limited disease. However, the rubella
infection may be complicated by transient 11.2 Data Provided to the Laboratory
arthralgia or arthritis. Rare, infrequent
complications such as encephalitis and/or The majority of tests are performed to determine
thrombocytopenia may occur. the need for vaccination in people without a
history of exposure and/or without related
Infection of the fetus during the first trimester clinical findings. In these instances, patient
and, to a lesser degree, the second and third information follows the requirements for other
trimesters may result in congenital rubella with laboratory tests.
birth defects such as neurosensory deafness,
heart anomalies, cataracts, and retardation of For the proper assistance to the physician in test
growth. In neonates with congenital rubella selection and interpretation of test results when
infection, the virus may persist for 6 to 9 the clinical setting or findings suggest rubella
months after birth. exposure and/or infection, additional patient
information includes:
11 Specimen Collecting and Handling
! immunization history for rubella,
11.1 Specimen
! date of onset of symptoms,
The vast majority of laboratories rely on reagents ! major clinical findings, and
in prepackaged diagnostic kits in testing for
antirubella antibodies. The manufacturers of ! purpose for which specimen is submitted, e.g.,
diagnostic kits, by federal regulation, must immune status evaluation, congenital infection,
establish the specimen type to be used in serologic diagnosis of infection.
testing. While testing with serum prevails, users
are advised to follow the manufacturer’s
package insert. For some reagent kits, plasma
may be an acceptable specimen.

11.3 Storing Specimens In the selection of a reagent set, refer to

sections 2 and 4.3 for information on the nature
(1) Separate plasma or serum from blood cells and structure of rubella virus and the
as soon as practical. Use sterile technique if manufacturers requirements to describe the
samples are to be stored longer than 48 hours. specific type and class of antibody detected by
A preservative such as sodium azide should be the reagent set. Note that in testing for
added if samples are to be stored at refrigerator congenital rubella syndrome, the reagents must
temperature for greater than 48 hours. Serum or demonstrate the ability to detect E2 antibody.
plasma may be stored at 2-8o C for up to one
week if separation has been performed as listed Refer to Section 6.5 for the requirement of the
above with a preservative added (written manufacturer to indicate if the reagent set is
communication, A.M. Johnson, M.D., December suitable to determine a “significant change” in
1996). IgG rubella antibody level for the diagnosis of
acute infection or reinfection.
(2) For long term frozen storage, tightly sealed
freezer tubes with minimal dead (air) space 12.2 Establishing Immune Status
should be used. Immunoglobulins are stable at -
20o C for at least a year if these 12.2.1 Specimen
recommendations are followed. For longer term
storage, -70o C storage is recommended. To To evaluate the immune status after a past
avoid repeated freeze-thawing cycles of samples infection or from immunization, test a single
likely to be used repeatedly, multiple small specimen, usually serum.
aliquots in separate small tubes are
recommended (written communication, A.M. To assess the immune status of a pregnant
Johnson, M.D., December 1996). woman exposed to rubella virus, obtain the
specimen within ten days after exposure and
(3) We recommend holding specimens for at test for IgG and IgM antibodies. The antibody
least one year. This is particularly important for from previous infection is of IgG class.
later follow-up studies of pregnant women who
are inadvertently exposed to rubella virus. 12.2.2 Appropriate Tests
NCCLS document H18— Procedures for the Qualitative, semiquantitative or quantitative tests
Handling and Processing of Blood Specimens, are acceptable in the routine assessment of the
provides detailed information on specimen immune status.
storage.
12.2.3 Reporting Results
11.4 Filing System
Follow the package insert accompanying reagent
Establish a filing system of specimen storage for sets for the manufacturer’s recommendation
easy specimen retrieval at a later date. regarding the threshold or “cut-off” point for
defining immunity and the units for reporting
12 Clinical Use of the Test(s) results. In reporting the results, indicate the
generic and proprietary name of the assay.
12.1 Legally Marketed Commercial These results derived by different assays are not
necessarily interchangeable. Provide the
Test Kits
physician with the threshold value to define the
immune status or interpret the test result,
The WHO International Rubella Standard is the
particularly when qualitative tests are used. For
reference for all reagent sets.
quantitative tests, report results in IU/mL.
(In the United States all reagent sets must be cleared for
marketing using the FDA’s premarket notification process; (1) Report results in quantitative units or
Refer to Sections 4.0 and 7.3. The subcommittee as “positive” (antibody at or greater than 10
recommends use of reagent sets that have included an IU/mL) and “negative.”
evaluation by the CDC.)

(2) If the manufacturer defines a borderline ! a significant rise in (IgG) antibody

zone around the threshold value of the test, concentration.
marginal results should be annotated.
Laboratories are advised to establish a definitive In cases in which reinfection is considered
procedure for dealing with the issue of several weeks after possible exposure and the
“borderline” results, i.e., additional testing by rise in IgG or IgM antibody may be missed, the
another method, additional specimen for assay, diagnosis of reinfection must rely on evidence of
physician notification, etc. The manufacturer pre-existing rubella antibody by retesting stored
may provide recommendations for dealing with specimens or laboratory reports of two or more
this issue. Refer to Section 6.3 for requirements previous positive rubella antibody tests. A
of the manufacturer to define the reproducibility documented history of rubella vaccination
of the test and Sections 3.5 and 3.6 for followed by one positive test is acceptable
additional information on reporting results. evidence of pre-existing antibody because the
possibility of vaccine failure combined with
12.2.4 Interpreting Results43 laboratory error is remote.39,48 Refer to the
Section 12.3 for recommendations on
(1) In instances in which there is no history of appropriate tests for diagnosis.
exposure or possible infection, the presence of
antibody at or above the threshold level is 12.3 Diagnosis of Subclinical or Clinical
evidence of previous exposure/infection with Rubella43
rubella virus at an undetermined time, either as a
result of immunization or natural infection. 12.3.1 Appropriate Tests to Use
(2) The presence of IgG antibody in a Rubella-specific IgM antibody tests are preferred
specimen from a pregnant woman taken less for confirmation of subclinical or clinical rubella.
than ten days from exposure to rubella virus is
presumptive evidence of a previous infection at Testing for rubella IgG antibodies has changed
some time. In instances of exposure to rubella dramatically with the introduction of newer
virus, the potential of reinfection should be methods and previous recommendations on use
considered on an individual basis. Refer to item of acute and convalescent-phase specimens to
(3) below for additional guidelines on the detect a change in antibody titer may not be
interpretation of test results, especially in applicable. Follow the package insert
pregnant women. recommendations in selection of the test for
diagnosis of asymptomatic or clinical rubella.
(3) Diagnosis of reinfection: Rare anecdotal For some quantitative IgG rubella tests, the
reports occurring in locations outside of the standard curves do not show a linear relationship
United States cite instances of viremia without between the optical readings and antibody levels
clinical symptoms, in vaccinated persons and attaching clinical significance to changes in
exposed to the live rubella virus.44-46 Rare reports quantitative values in these instances is less
document symptomatic rubella reinfection in than desirable.
vaccinated individuals or those with naturally
acquired clinical rubella.37,47 In these unusual For the diagnosis of subclinical and clinical
circumstances, additional testing may show an rubella, test selection and interpretation must be
IgM response or an accelerated IgG response made on an individual basis. The selection
without formation of rubella-specific IgM.37-39 includes:
Each case must be considered individually in
regard to the interpretation of test results. ! Use of an IgM antibody test to detect recent
infection. Rheumatoid factor may potentially
The criteria for diagnosis of reinfection cross react with IgM tests and the factor must
include39,48: be removed unless the manufacturer provides for
its removal in the assay protocol. Since IgM
! a specific but unusually low level IgM tests may be subject to false positive results,
response, and/or confirmation by alternate methods is

recommended, i.e., significant increase in “negative for IgM antibody” (IgM antibody not
antirubella IgG, detection of viral protein, or detected).
detection of viral nucleic acid.
For quantitative IgG tests, report results in
! Use of a quantitative IgG test to detect a quantitative units as IU/mL. Indicate the generic
significant difference in antibody concentration and proprietary name of the test. (The
between the acute- and convalescent-phase laboratory should retain detailed information on
samples when testing the pair in the same run to reagent set identification.)
avoid differences in between-run sensitivity.
Follow the package insert recommendations A laboratory interpretation should accompany
when selecting a quantitative IgG test for the report. NCCLS document I/LA18—
diagnosis of subclinical and clinical acute rubella. Specifications for Immunological Testing for
Refer to Section 6.5. Infectious Diseases, provides detailed
information on reporting results.
12.3.2 Specimens
12.3.4 Interpreting Results
Selection of specimens relates to the test
selected for diagnosis of subclinical or clinical (1) The presence of antirubella-specific
rubella. IgM antibody is presumptive evidence of a
current or recent infection.
(1) Acute- and convalescent-phase serum
samples are required to demonstrate a significant (2) For IgG antirubella tests used to
change in antibody concentration during the support the results of IgM testing, follow the
course of the infection. Obtain the acute-phase manufacturer’s recommendation for interpre-
sample early in the infection, preferably less tation of a significant change in antibody
than seven days from onset of symptoms, and concentration between acute- and convalescent-
the convalescent-phase sample one to two phase specimens.
weeks after the first sample, but not earlier than
ten days after onset of symptoms. (3) If a traditional serial dilution test is
used, a four-fold or greater rise in antibody titer
(2) During this period, and depending on the between the acute- and convalescent phase
test system used, antibody of both the IgM and specimens is evidence of a current or recent
IgG classes will be detected. Some solid-phase infection with rubella virus.
assays are specific for either IgG or IgM
antibody. IgG antibody concentration rises 12.4 Diagnosis of Congenital
during the first two weeks after onset of the Rubella 43,49,50,51
rash and remains elevated indefinitely.

12.4.1 Specimen
(3) A single specimen may be sufficient to
determine IgM antibody only. Obtain this The criteria for the laboratory diagnosis of
specimen early (within two weeks) after the congenital rubella include:
onset of the rash. IgM antibody concentration
rises during the first two weeks after the onset ! Rubella virus isolation from nasopharyn-geal,
of the rash and usually declines to undetectable urine, cerebrospinal fluid specimens
levels in two to three months or less.
! Testing blood for the presence of rubella
12.3.3 Reporting Results specific IgM antibodies
The system for reporting results relates to the • Persistence of rubella-specific IgG antibody at
test used for assay and the manufacturer’s quantitative levels above and beyond that
recommendations. The report of a qualitative expected from passive transfer of maternal
IgM antibody test should be either “positive for antibody (See Section 12.4.4)
IgM antibody” (IgM antibody present) or

! Western blot analysis of all three structural infection occurs very late in pregnancy, only
proteins. about 11% of infected babies will have
circulating IgM antibody.28
12.4.2 Appropriate Tests to Use
Because IgG antibody passes the placental
IgM antibody tests using cord blood is the barrier, most IgG antibody in cord or neonatal
preferred serologic method for the diagnosis of blood will be of maternal origin. If the IgG
congenital rubella. Rheumatoid factor may antibody was from the mother, the concentration
potentially cross-react with IgM tests and must will have significantly declined in the baby's
be removed unless the manufacturer provides for sixth month sample and may be below
its removal in the assay protocol. Confirmation detectable levels. If it was produced as a result
by an alternate method is recommended. of congenital infection, it will have persisted.
Virus isolation techniques are the preferred 12.5 Record Keeping

alternate method.
Maintain a dated and numbered record sheet for
Indirect tests for rubella specific IgG antibody each run. Enter all pertinent information related
may be used in instances in which the diagnosis to the run on the record sheet, such as:
is considered at an interval after birth when IgM
testing or virus isolation techniques are not (1) test used
applicable. (2) lot number of reagents and concen-
trations used
For the diagnosis of congenital rubella, the IgG (3) results of antigen titration (if applicable)
test must detect the whole virus as compared to (4) results of positive and negative control
tests which detect antibodies to selected viral sera
proteins. Sera from patients with congenital (5) standards or calibrators.
rubella have increased ratios of antibody E2
relative to E1 antibody.4,6-8,51 Tests that detect
predominantly E1 antibody should not be used.
Refer to Sections 2 and 4.3 for additional
information on the nature of the rubella virus and
the requirement of the manufacturer to indicate
the nature of the antigens in their assays.
12.4.3 Reporting Results
Report results as described for clinical infection.
12.4.4 Interpreting Results43,49,50
Because maternal IgM antibody does not pass

the placental barrier, IgM antibody in cord or
neonatal blood has been produced by the fetus,
and its presence in the blood is presumptive
evidence of congenital infection. Additional
testing may be required for confirmation.
However, the absence of IgM antibody does not
rule out congenital infection. Contamination of
cord blood with maternal blood negates the use
of this specimen for testing.
If infection of the fetus occurs in the first

trimester of pregnancy, 90% of infected babies
will have detected circulating IgM antibody; if

References of the hemagglutination-inhibition test and the

neutralization test for the detection of rubella
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rubella virus envelope components. Arch
Virology. 1980; 65: 1-13. 12. Haukenes G, Blom H. False positive
rubella virus hemagglutination inhibition
2. Waxham MN, Wolinsky JS. Immuno- reactions: Occurrence and disclosure. Med
chemical identification of rubella virus hemag- Microbiol Immunol. 1975;1651:99-102.
glutinin. Virology. 1983;126:194 -203.
13. Skendzel LP, Edson DC. Evaluation of
3. Oker-Blom C, et al. Rubella virus enzyme immunosorbent rubella assays. Arch
contains one capsid protein and three envelope Pathol Lab Med. 1985;109:391-393.
glycoproteins, E1, E2a, E2b. J Virology. 1983;
46: 964-973. 14. Buimovici-Klien E, et al. Low level
rubella immunity detected by ELISA and specific
4. Katow S, Sugiura A. Antibody lymphocyte transformation. Arch Virology.
response to individual rubella virus proteins in 1980;66:321-327.
congenital and other rubella virus infections. J
Clin Microbiol. 1985;21:449-451. 15. Kleeman KT, Kiefer DJ, Halbert SP.
Rubella antibodies detected by several
5. Forng R-Y, Frey TL. Identification of commercial immunoassays in hemagglutination
the rubella virus nonstructural proteins. inhibition-negative sera. J Clin Microbiol.
Virology. 1995;206:843-853. 1983;18:1131-1137.
6. Fitzgerald MG, Pullen GR, Hosking 16. Storch GA, Myers N. Latex-
CS. Low affinity antibody to rubella antigen in agglutination test for rubella antibody: Validity of
patients after rubella infection in utero. positive results assessed by response to
Pediatrics. 1988;81:812-814. immunization and comparison with other tests.
J Infect Dis. 1984;149:459-464.
7. Hedman K, Sepp AL. Recent rubella
virus infection indicated by a low avidity of 17. Grangeot-Keros L, Pustowoit B, Hobman
specific IgG. J Clin Immunol. 1988;8:214-221. T. Evaluation of Cobas Core rubella IgG EIA
recomb, a new enzyme immunoassay based on
8. Terry GM, Ho-Terry L, Londesbrough recombinant rubella-like particles. J Clin
P. A bioengineered rubella E1 antigen. Arch Microbiol. 1995;33:2392-2394.
Virology. 1989;104:63-75.
18. Serdula MK, et al. Serological response
9. World Health Organization. Evaluation to rubella revaccination. JAMA. 1984;251:
of candidate international reference methods for 1974-1977.
the rubella hemagglutination-inhibition tests:
Report of a collaborative study. WHO. 1982; 19. Orenstein WA, et al. Prevalence of
LAB/82.1. rubella antibodies in Massachusetts school
children. Am J Epidemiol. 1986;124: 290-298.
10. Lennette EH, Schmidt NJ, Magoffin
RL. The hemagglutination-inhibition test for 20. Steece RS, et al. Problems in
rubella: A comparison of its sensitivity to that of determining immune status in borderline
neutralization, complement fixation, and specimens in an enzyme immunoassay for
fluorescent antibody tests for diagnosis of rubella immunoglobulin G antibody. J Clin
infection and determination of immunity status. Microbiol. 1984; 19:923-925.
J Immunol. 1967;99:785-793.
21. Hancock EJ, Pot K, Puterman ML, Tingle
11. Field AM, Vandervelde EM, AJ. Lack of association between titers of HAI
Thompson KM, Hutchinson DN. A comparison antibody and whole-virus ELISA values for

patients with congenital rubella syndrome. J 32. Storch GA, Myers N. Latex-
Infect Dis. 1986;154:1031-1033. agglutination test for rubella antibody: Validity of
positive results assessed by response to
22. Balfour HH, Groth KE, Edelman CK. immunization and comparison with other tests.
Rubella viraemia and antibody responses after J Infect Dis. 1984;149:459–464.
rubella vaccination and reimmunization. Lancet.
1981;1:1078-1080. 33. Grangeot-Keros L, Badur S, Pons JC,
Duthe S, et al. Use of in vivo challenge to
23. O'Shea S, Best JM, Banatvala JE. assess rubella immunity determined by
Viremia, virus excretion, and antibody responses heamagglutination inhibition and latex
after challenge in volunteers with low levels of agglutination. Res Virol. 1989;140:437–442.
antibody to rubella virus. J Infect Dis.
1983;148:639-647. 34. Buimovici-Klein E, O’Beirne AJ, Millian
SJ, Cooper LZ. Low level rubella immunity
24. Centers for Disease Control. Rubella detected by ELISA and specific lymphocyte
Prevention. MMWR. 1984;33:301–318. transformation. Arch Virol. 1980;66: 321–327.
25. Haukenes G, Blom H. False positive 35. Herrmann KL, Halstead SB, Wiebenga
rubella virus hemagglutination inhibition NH. Rubella antibody persistence after
reactions: Occurrences and disclosure. Med immunization. JAMA. 1982;247:193–196.
Microbiol Immunol. 1975;161:99–106.
36. Chu SY, Bernier RH, Stewart JA,
26. Bradstreet CMP, Kirkwood B, Pattison Herrmann KL, et al. Rubella antibody persistence
JR, et al. The derivation of minimum immune after immunization. Sixteen-year follow-up in
titer for rubella hemagglutination inhibition (HI) the Hawaiian Islands. JAMA. 1988;
antibody. A Public Health Laboratory Service 259:3133–3136.
collaborative survey. J Hyg.1978;81: 383–388.
37. Braun C, Kampa D, Fressle R, Wilke E,
27. Horstmann DM, Schluederberg A, et al. Congenital rubella syndrome despite
Emmons JE, et al. Persistence of vaccine- repeated vaccination of the mother: a
induced immune responses to rubella: coincidence of vaccine failure with failure to
Comparison with natural infection. Rev Infec vaccinate. Acta Paediatr. 1994;83:674–677.
Dis. 1985;7:580–585.
38. Hornstein L, Levy U, Fogel A. Clinical
28. Mortimer PP, Edwards JMB, Porter rubella with virus transmission to the fetus in a
AD, Tedder RS, et al. Are many women pregnant woman considered to be immune.
immunized against rubella unnecessarily? J Hyg. (Letter to the Editor). New Eng J Med.
1981; 87:131–138. 1988;319:1415–1416.
29. Butler AB, Scott RMcN, Schydlower 39. Best JM, Banatvala JE, Morgan-Capner
M, Lampe RM, et al. The immunoglobulin P, Miller E. Fetal infection after maternal
response to reimmunization with rubella reinfection with rubella: Criteria for defining
vaccine. J Pediatr. 1981;99:531–534. reinfection. Br Med J. 1989;299:773–775.
30. Vaananen P, Makela P, Vaheri A. 40. Partridge JW, Flewett TH, Whitehead
Effect of low level immunity on response to live JEM. Congenital rubella affecting an infant
rubella virus vaccine. Vaccine. 1986;4: 5–8. whose mother had rubella antibodies before
conception. Br Med J. 1981; 282:187–188.
31. Safford JW, Abbott GG, Deimler CM.
Evaluation of a rapid passive hemagglutination 41. NCCLS. Evaluation and Performance
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hemagglutination inhibition and a vaccine Intended for the Detection and Quantitation of
challenge study. J Med Virol. 1985;17: Rubella IgG Antibody; Tentative Guideline, 1985;
229–236.

NCCLS document I/LA6-T, Vol. 12, No. 4, 47. Wolf JE, Eisen JE, Fraimow HS.
Wayne, PA. Symptomatic rubella reinfection in an immune
contact of a rubella vaccine recipient. South
42. Skendzel, LP. Rubella Immunity: Med J. 1993; 86: 91-93.
Defining the Level of Protective Antibody. Am J
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Child. 1990;165:820-821.
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asymptomatic infection with rubella virus during 513.
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Rubella-specific serum and nasopharyngeal
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46. O’Shea S, Best JM, Banatvala JE.

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antibody to rubella virus. J Infec Dis.
1983;148:639-647.

Summary of Comments and Subcommittee Responses
I/LA6-T: Evaluation and Performance Criteria for Multiple Component Test Products Intended for the
Detection and Quantitation of Rubella Antibodies; Tentative Guideline
General
1. The guideline should include the correlation of latex to EIA and the reason there may be
occasional differences.
! This NCCLS guideline is not intended to identify differences between methods. The methods
may have unique immunoglobulin specificities controlled by the antiglobulin conjugate.
2. Would it not be more in keeping with modern usage to report results in kIU/L rather than
IU/mL?
! The subcommittee recognizes that kIU/L units has increased in popularity, especially in
European countries. However, the subcommittee believes, based on the experiences of its
members, that reporting IU/mL remains an appropriate alternative.
Section 3.4
3. Using a fluid sample handler system we occasionally find considerable variation around the 15
IU/mL cut-off for rubella. I am concerned that 10 IU is too low, requiring ideal conditions.
Can a 12 to 13 IU/mL cut-off be considered?
! Each laboratory must consider the variation in testing when establishing the reference point of
“positive” versus “negative.” The subcommittee has recommended to manufacturers the need
for statistical data describing the expected variation in testing, especially around the
breakpoint (see Section 6.3). The subcommittee recognized the existence of a borderline zone
around the breakpoint value of any test and marginal results should be properly annotated
(refer to Section 12.2.3).
4. Reference #19 uses only an EIA of 10 IU/mL but a PHA of 7.5 IU/mL and an HAI of 5.0
IU/mL. Could not an EIA also go down to 5 or 7.5 IU/mL? If the loss of specificity was the
reason 10 IU/mL were selected as the cut-off value it should have been stated more clearly in
the NCCLS document. 10 IU/mL is not the obvious answer I read from reference #17. Also
reference #25 is a letter to the editor that doesn't say very much new i.e., it repeats that
some patients, seronegative with an HAI titer of < 1:8, are indeed immune based on the
earlier booster studies.
! Sections 3.2 and 3.4 address the process of selecting 10 IU/mL as the reference point for
routine rubella testing. Factors to consider include the variation in testing, especially in the
range of the reference point and the risk of false positive results if 7.5 IU/mL or 5.0 IU/mL
were selected.
5. The meaning of the sentence, "The majority of seropositive persons are detected with 10
IU/mL as the indicator of immune status." is not clear by itself. No references are given for
this sentence which would also be true if 5, 7.5, 12.0, 12.5, or 15.0 were substituted for 10
IU/mL. Again if the reason for not wanting to go to 5 or 7.5 IU/mL is concerned about
specificity this should be stated more clearly in the document.
! Refer to the response for Comment 4.

6. Since the early booster or vaccination studies were done before 5.0 - 12.5 IU/mL quantitative
information was available, 10.0 IU/mL as an arbitrary cut of is again raised. Also there is no
data available to scientifically select a cut-off value below 15 IU/mL at the present time.
Particular challenge studies on volunteers with well defined quantitative seronegative Rubella
values (below 15 IU/mL) have not been performed.
! Sections 3.2 and 3.4 and the response to Comment 4 address the process of selecting 10
IU/mL as the reference point.
7. We are wondering where exactly the committee stands on the recommendation of 10 IU/mL
as the indicator of immune response. Our company takes the position that selecting 15 IU/mL
is a more safe breakpoint for protective immunity, because considering pregnant women with
antibody level of 10–15 IU/mL as being non-immune would result in appropriate behavior
(avoid contact with rubella infected individuals) and appropriate vaccination strategy (vaccinate
post pregnancy). As rubella vaccines are inexpensive more vaccinations by choosing a higher
cut off are preferable to the risk of overestimating anti-rubella titer (levels below 10 IU/mL
overestimated with test result of 10 IU/mL up to 15 IU/mL) or false positive anti-rubella IgG
result (no rubella antibodies with test result > IU/mL due to unspecific reactivity).
In addition, recommending a cut off in IU/mL is a critical issue because different

manufacturer’s reagents do not necessarily correlate quantitatively. The papers of Dimech et
al: Multicenter Evaluation of Five Commercial Rubella Virus Immunoglobulin G kits which
report in International Units per Milliliter. JCM 30, 633–641 (1992) and Pustowoit et al.:
Evaluation of recombinant rubella-like particles in a commercial immunoassay for the detection
of anti-rubella IgG, Clinical and Diagnostic Virology 5(1996):13–20, discusses the issues of
different calibration of rubella IgG reagents leading to miscorrelation between assays.
! In this document, the subcommittee restates the recommendation of 10 IU/mL as the

reference point for use by the clinical laboratory to define immunity against rubella.
Epidemiologic data, studies on vaccinated individuals with low levels of antibody, and
anecdotal reports provide adequate evidence that the 10 IU/mL antibody level is protective in
the vast majority of individuals. Keep in mind that immunity in a given patient is a clinical
decision and the results of anti-rubella tests, like other tests, must be evaluated in the context
of the clinical setting. Sporadic reports of viremia and/or reinfection among previously
immunized individuals with low antibody levels have been reported but reinfection has also
occurred in persons with titers at or above the 15 IU/mL breakpoint. Despite the occasional
occurrence of rubella reinfection in individuals with low titers, the theoretical risk is small
especially when compared with the significantly greater risk in individuals who have not been
vaccinated. Refer to reference 40 for more information.
I/LA6-A includes the recommendation that the reproducibility (3 standard deviation interval) of
anti-rubella tests should not exceed 3 IU/mL at the 10 IU/mL level. This recommendation is
significant because, for the first time, reproducibility limits are defined and accreditating
agencies can establish firm guidelines when evaluating performance of this test.
The Subcommittee on Rubella Serology has taken the first step leading to improved testing.
Other problems remain and should receive attention. Manufacturers cite difficulty in use of the
WHO standard and the calibration procedure lacks uniformity. Results derived by various
commercially prepared reagent sets are not interchangeable.
The subcommittee welcomes additional dialogue on these issues and appreciates

recommendations to improve the quality of this laboratory test.

8. The reference point does not establish the protective level; that can be determined only by
following inflection rates at various levels.
! In laboratory medicine it is customary to establish decision levels to guide physicians in

medical diagnosis. The data currently available indicate that 10 IU/mL is a suitable indicator.
A recent report using a new EIA test and recombiant rubella-like particles showed that
borderline values of between 10 and 15 IU/mL were truly positive by Western blot analysis.17
Section 4.1
9. The Guideline requires that “products... should include a minimum of three control sera (one
negative, one low-positive, and one high-positive) to be assayed in each test run.” There is
current commercial product which performs acceptably that calls for only two control sera
(one negative, one low-positive). In addition, that product doesn’t supply the control sera as
part of the product package. We believe the guidance in this respect is overly prescriptive.
! The items cited are recommendations reflecting the professional experience, judgement, and
agreement of the subcommittee and are not intended to be prescriptive. The subcommittee’s
intent is that they serve as a conservative series of recommendations that do not preclude the
use of alternative limits or ranges when appropriate, based on a product’s design or a
laboratory’s quality control procedures.
Section 4.3
10. What in reality is meant by “normal antigen control?” The impression in the guideline
suggests that it is substrate which, like non-cultured rubella medium extract should be used as
a “blank” for each sample to prove that the sample in general can be analyzed with the
method. Is this an acceptable interpretation? How do we get such a control? Please explain
the use of the control in detail.
! A normal antigen control is not required with the new generation of rubella tests.
11. How many parallel studies must be used when performing the reproducibility test?
! Section 6.3 recommends repetitive testing of 40 or more sera. Three sites should be chosen to
evaluate the product. Reproducibility should be performed on one lot of the product with at
least 2 runs of testing per day for 20 working days. The runs should be separated by at least
2 hours. Within run, between run, and total reproducibility should be calculated using NCCLS
guidelines.
12. How many manufacturing lots shall be tested for performance criteria at the minimum?
! At least three lots of product should be evaluated during a clinical evaluation to evaluate lot-
to-lot difference.
13. In how many different laboratories must the performance characteristics be verified? Is it
possible to use either a research and development or quality assurance laboratory of our own
as one of the selected laboratories?
! Three different sites should be chosen to evaluate a test. Ideally, they should be located in
different geographic locations to provide some heterogeneity in population. One of the sites
may a research laboratory of the manufacturer but the evaluation provided by this laboratory
should not exceed one-third of the total evaluation data.

Section 6.1
14. As indicators of sensitivity, the low positive sera are of real significance. High positive sera
likely only detect false negative reactivity resulting from negative prozone effects, gross
insensitivity, or transcription errors. Including so many of them (150) in the panel is a waste
of reagents while they give and exaggerated sense of sensitivity.
! Section 6.1 of I/LA6-A has been revised and greater emphasis placed on sensitivity in the
range of 10-20 IU/mL.
Section 6.2
15. This is an absolute minimum standard. Realistically, 5 false positives in 100 tests is hardly an
acceptable standard unless they come from sera with borderline levels of antibody.
Considering the possible consequences of infection and CRS in the absence of true rubella
antibody, no company can afford to accept the legal liability of calling a result from a truly
susceptible individual as positive.
! All tests for antibodies against rubella show variation at the cutoff level of 10 IU/mL. False
position or false negative results may occur. For this reason, the guideline advises
manufacturers to establish a borderline range to indicate an antibody level in the equivocal
zone. Repeat testing or immunization may be advisable. The clinical situation also dictates
the response of the physician. If the patient is not pregnant, repeat immunization may be
advisable. In a pregnant patient, additional testing or observation may be the prudent
approach.
Section 6.4
16. This section is unclear as to its meaning.
! This section is intended to underscore the importance of documentation from the manufacturer
on the stability of the reagents. In addition, laboratories using the reagents may monitor
stability by using multiple controls. This has been clarified in Section 6.4.
Section 7.0
17. This section should reference the NCCLS document RS13-P, Rubella Antibody; Proposed
Summary of Methods and Materials Credentialed by the NRSCL (National Reference System
for the Clinical Laboratory) Council.
! This reference has been added to the list of NCCLS Related Publications.

Summary of Comments and Subcommittee Responses
I/LA7-P: Specimen Handling and Use of Rubella Serology Tests in the Clinical Laboratory; Proposed
Guideline
General Comments
1. I'd be inclined to caution against using IgM tests as part of a torch screen or indiscriminately
on "high titer" specimens. Each of these approaches is used and both are likely to yield more
false positives than true positives. The IgM test should be reserved for patients with a strong
clinical history for rubella or with highly suggestive clinical findings.
! Sections 12.2.4 and 12.3.1 of I/LA6-A utilize rubella IgM testing for diagnosis of subclinical or
clinical rubella and rubella reinfection. The document does not deal with the issue of rubella
IgM testing as part of the TORCH screen.
2. More discussion on immune status and single-serum tests should be included.
! This document provides an overview of rubella testing and is not intended to identify
differences between various methods.
3. The scope of the project should be expanded to define the difference in principles between
reference methods (HAF) and screening methods (PHA, complement fixation).
! Refer to response to Comment 2.
4. It would be helpful if information on the how to remove rheumatoid factor was included.
! The Area Committee on Immunology and Ligand Assay has considered establishing guidelines
for rubella IgM reagents and testing procedures. The interest in IgM testing is very limited and
therefore no project has been authorized to date.
5. Methods and principles as stated are very vague. I would like the guideline to go into greater
detail about the isolation of IgM.
6. The committee could include some statement about there liability and sensitivity of the various
methods used, such as a comparison of IFA and ELISA and recommendations on the use of
commercial systems.
! Sections 12.2.4 and 12.3.1 of I/LA6-A utilize rubella IgM testing for diagnosis of subclinical or
clinical rubella and rubella reinfection. The document does not deal with the issue of rubella
IgM testing as part of the TORCH screen.
7. It is recommended that the "limited value" be defined in this document.
! The term “limited value” does not routinely appear in the medical literature and has not been
used by the subcommittee. Its definition is uncertain to the subcommittee.

Section 12.0
8. In Section 2.0 (now Section 9.0), perhaps the first paragraph should be deleted. It appears to
be redundant in that all of the information in the first paragraph is also contained in the second
paragraph. The second paragraph is far more complete and more to the point.
! Section 9.0 has been modified to eliminate the redundant information.
Section 4.1.1
9. The use of a sterile serum separator tube should be considered as an alternative to aseptic
technique mentioned in this section (now Section 11.1).
! Section 11.1 has been revised to include use of sterile serum separators.
Section 4.2
10. Submission Slip Items 4-7 (now Section 11.2) are extremely difficult to obtain, especially on
an outpatient basis. Patient histories are difficult to obtain. We are lucky if we get the name.
! Section 11.2 has been revised to reflect the type of information provided with the specimen.
Medical staff education is recommended in situations in which rubella exposure or infection is
under consideration.
11. Date and time should be included on the submission slip.
! The subcommittee agrees that date and time are standard information, especially with the use
of computerized systems.
Section 4.3.1
12. Eliminate "if kept sterile" and replace it with "up to one week." (This now appears in Section
11.3.)
! Storage of specimens for one year presents a problem in some laboratories. The
recommendation for one year storage is intended to allow for retesting of pregnant women
who may be exposed to the rubella virus. This issue is particularly important for the diagnosis
of reinfection. See Section 12.2.4 of I/LA6-A.
Section 4.3.2
13. Change "Store nonsterile specimens" to "To store specimens longer than one week, freeze."
(This now appears in Section 11.3.2)
! Section 11.3 of I/LA6-A has been revised.
Section 4.3.3
14. Regarding frost-free freezers (now in Section 11.3), I am not certain that what is stated is so;
see the introduction to CAP publication PPSH Hematology fascicle for a nice discussion.
! The recommendation regarding frost-free freezers has been removed from this revision of the
document.

15. I question the recommendation of holding the specimen for at least one year (now mentioned
in Section 11.3) for the following reasons:
1. The test results would be part of the medical and laboratory record.
2. If there is a conflict between the past and current results, you cannot retrospectively
manage the patient.
3. The cost of maintaining the specimen, the relevant demographic information and a
retrieval system would be enormous.
! Refer to Comment 12 and its response.
16. Storing specimens for one year presents a real hardship for hospital labs with little storage
capacity.
! Refer to Comment 12 and its response.
Section 5.1
17. This section (now Section 12.1) indicates that the HAI is the validating test against which
other tests should be measured. Consider the following:
1. MMWR 30(4): 38, 1981 "There are now more sensitive measures than the HI test to
determine rubella immunity."
2. CDC's summary analysis for Public Health Immunology 1984-1991 "Although the
HI test is still considered the acceptable "standard" in the field of rubella testing, an
increasing amount of evidence is being collected to challenge that traditional
position."
3. Similar challenges have appeared in the scientific literature such as those reported in:
a. JAMA 251: 1974, 1984

b. J. Infect. Dis. 149: 459, 1984
c. Numerous other scientific papers
! Despite its limitations, the HAI test is the primary standard used for evaluation of the WHO
preparation. HAI served as the initial standard in extensive clinical studies. Eventually, a more
sensitive method probably will replace HAI. In current practice, the EIA is used as the
reference assay to establish cut-off values to define the immune state. The method of assay is
not the issue of immediate concern. Before discarding the HAI, attention should focus on two
issues: (1) various methods, including the more sensitive assays, show discordant results, and
(2) standardization of the procedure for reconstitution and storage of the WHO standard.
When these issues have been resolved, transition to another standard would be appropriate.
18. In last paragraph (of the new Section 12.1), "run the new test for a time in parallel" is
ambiguous. It should read "Use control specimens with negative, low titer (ex. 1:8) and high
titer (positive). When switching to a new test or lot of reagents evaluate performance on
control specimens and 20 patient specimens. If concordant results are found, proceed with
the new test or lot of reagents. Keep statistics on patient findings to monitor the incidence of
negative and positive findings including the various titers.
! Section 12.1 has been revised to address this comment.

Section 5.2.1.1
19. From the first sentence (now in Section 12.2.1) remove the word "previous" from "previous
past infection.”
! Section 12.2.1 has been modified as recommended.
Section 5.2.3.1
20. Remove the words "reactive" and "nonreactive" [now in Section 12.2.3(1)]. The tests are
designed to detect antibody and test result should be reported exactly as that — antibody, in
this case, "rubella antibody detected." Qualitative test results should be reported as
immune/not immune.
! Section 12.2.3 has been revised accordingly.
Section 5.2.3.3
21. This section [now 12.2.3(3)], states, "For an immune status report, a titer of 8 is the minimal
recommended titer for a report of "reactive" (antibody present)." Using the titer of 1:8
appears to be in conflict with:
1. MMWR 30(4): 38, 1981 "Any detectable titer, even if very low, protects against
subsequent viremic infection ..."
2. Report published in J. Infect. Dis. 149: 459, 1984
3. Data contained in the CDC Premarket Evaluation Program Support No. 13. in which
the Rubascan is approved for testing for immune status. Rubascan uses a 1:1 titer
to indicate the presence of antibody (immunity). Furthermore, this document (I/LA7-
P Section 5.1) recommends using kits that passed the product evaluation.
Section 5.2.3.4
22. The recommendation [now in Section 12.2.3(4)] is made that marginal results should be
reported as such. Perhaps some comment should be made about the need for retesting these
marginal or "grey area" specimens.
! Section 12.2.3 of I/LA6-A has been revised to address this comment.
Section 5.2.4
23. This section on interpretation of results (now Section 12.2.4) should be expanded. The
critical specimens are not considered in this document such as a pregnant woman whose first
serum is tested more than 10 days after exposure. The type of testing, (i.e., IgG or IgM)
along with the time interval of testing and interpretation should be included in this document.
! Section 12.2.4 has been revised to address this comment.

Section 5.3.2
24. This section (now 12.3.2) should indicate when a CF, HAI, etc. is indicated or not clinically
indicated. This omission is a serious deficiency.
Section 5.3.2.1
25. As I am sure you are aware, the numerous tests which are used have a variety of ways in
which titers and ratios are determined. You have a difficult challenge in trying to write a
paragraph describing appropriate tests to use (now Section 12.3.2).
! The trend in manufactured reagent sets is to use IU/mL and the document has been written to
reflect the new direction in reporting results.
Section 5.3.2.2
26. It should be noted that the reagents for IgM antibody testing are currently quite expensive.
! In developing standards and guidelines, NCCLS committees are concerned with cost
effectiveness, but, NCCLS documents generally do not deal with financial considerations.
27. Comments are made (now in Section 12.3.2) about the occurrence of rheumatoid factor and
the fact that this RF factor can give a false positive result in specimens which are being tested
for IgM antibody. The statement is made that the rheumatoid factor must first be removed
before testing for rubella-specific IgM antibody. A reference which supports these comments
and which defines the treatments which are necessary for removal of the rheumatoid factor
should be added to this document.
! The Area Committee on Immunology and Ligand Assay has considered establishing guidelines
for rubella IgM reagents and testing procedures. The interest in IgM testing is very limited and
therefore no project has been authorized to date.
28. This section (now Section 12.3.2) should include information on which commercial tests are
available and their relative sensitivity and specificity.
Section 5.3.3.4
29. I would suggest removing "reactive for IgM antibody" and "nonreactive for IgM antibody" from
this section [now Section 12.3.3(4)].
! Section 12.3.3 includes alternate terms to describe the results of testing, i.e., “IgM antibody
present, IgM antibody not detected.”
30. Include information on IgM isolation tests available and their relative sensitivity and clinical
relevance.
! See response to Comment 27.

Section 5.5.1
31. Define what is meant by "Conditions of the test" (now in Section 12.3).
! Section 12.3 has been revised to address this comment.

Related NCCLS Publications†
DI1-A2 Glossary and Guidelines for Immunodiagnostic Procedures, Reagents, and Reference
Materials—Second Edition; Approved Guideline (1992). Provides common terminology and
basic methodology for users and manufacturers of immunodiagnostic systems.
DI2-A Immunoprecipitin Assays: Procedures for Evaluating the Performance of Materials; Approved
Guideline (1986). Procedures for evaluating the performance of materials used in
immunoprecipitin analysis.
DI3-A Agglutination Analysis: Antibody Characteristics, Methodology, Limitations, and Clinical

Validation; Approved Guideline (1993). Describes specificities of antibodies and their required
potency, labeling information, and characteristics and limitations of agglutination methods.
DI4-T Enzyme and Fluorescence Immunoassays; Tentative Guideline (1986). Methods of achieving
reliable, reproducible results from enzyme and fluorescence immunoassays of macromolecular
analytes. Factors that contribute to reliable and reproducible results are emphasized in
sections that describe how to choose enzyme fluorescence systems, how to produce, process,
purify, and characterize antibodies and antigens, as well as reagent separation techniques,
instrumentation, and equipment.
I/LA18-A Specifications for Immunological Testing for Infectious Diseases; Approved Guideline (1994).
Guideline that outlines specimen requirements, performance criteria, algorithms for the
potential use of sequential or duplicate testing, recommendations for intermethod comparisons
of immunological test kits for detecting infectious diseases, and specifications for development
of reference materials.
RS13-P Rubella Antibody; Proposed Summary of Methods and Materials Credentialed by the NRSCL
Council (1989).
†
Proposed- and tentataive-level documents are being advanced through the NCCLS consensus process; therefore, readers
should refer to the most recent editions.

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I/LA6-A

Detection and Quantitation of Rubella IgG Antibody: Evaluation

PUBLICATIONS NCCLS standards and guidelines represent a consensus opinion

Most NCCLS documents are subject to two levels of

Proposed An NCCLS consensus document undergoes the first

Detection and Quantitation of Rubella IgG Antibody: Evaluation

NCCLS VOL. 17 NO. 17 i

Detection and Quantitation of Rubella IgG Antibody: Evaluation

Laurence P. Skendzel, M.D.

Copyright © 1997. The National Committee for Clinical Laboratory Standards.

NCCLS VOL. 17 NO. 17 iii

Area Committee on Immunology and Ligand Assay

Robert M. Nakamura, M.D. Scripps Clinic and Research Foundation

Linda Ivor Del Mar, CA

Subcommittee on Rubella Serology

Laurence P. Skendzel, M.D. Traverse City, Michigan

David J. Kiefer, Ph.D. Quest International Inc.

Robert M. Nakamura, M.D. Scripps Clinic and Research Foundation

Cathy D. Nutter Food and Drug Administration Center for

Lawrence E. Schaefer Abbott Laboratories

John A. Stewart, M.D. Centers for Disease Control and Prevention

Carole A. Golden, Ph.D. Microbiological Research Corporation

Elaine Harris Instrumentation Laboratory

Elaine Kruger Instrumentation Laboratory

Kenneth D. McClatchey, M.D., D.D.S. Loyola University Medical Center

Beth Ann Wise, M.T.(ASCP), M.S.Ed. NCCLS

Patrice E. Polgar NCCLS

NCCLS VOL. 17 NO. 17 iv

ACTIVE MEMBERSHIP (as of 1 October 1997)

Sustaining Members Commission on Office FDA Division of Anti-Infective

NCCLS VOL. 17 NO. 17 v

Becton Dickinson Italia Higman Healthcare Rhône-Poulenc Rorer

NCCLS VOL. 17 NO. 17 vi

NCCLS VOL. 17 NO. 17 vii

OFFICERS BOARD OF DIRECTORS

John V. Bergen, Ph.D.,

NCCLS VOL. 17 NO. 17 viii

Active Membership ........................................................ v

2 Nature and Structure of Rubella Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3 Status of Hemagglutination Inhibition Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.1 Use of HAI in Establishing the Calibration Standard . . . . . . . . . . . . . . . . . . . . . . . . . . 1

4 General Criteria for Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

4.1 Quality Control Sera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

5 General Performance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

6 Specific Performance Criteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

NCCLS VOL. 17 NO. 17 ix

10.1 Nature of Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

11 Specimen Collecting and Handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

12 Clinical Use of the Test(s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

12.1 Legally Marketed Commercial Test Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Summary of Comments and Subcommittee Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Related NCCLS Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

NCCLS VOL. 17 NO. 17 x

NCCLS VOL. 17 NO. 17 xi

NCCLS VOL. 17 NO. 17 xii

Detection and Quantitation of Rubella IgG Antibody: