Ebook Clinical virology manual (4/E): Part 1

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Fourth Edition

Clinical Virology Manual

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Clinical Virology
Manual
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Fourth Edition

Editors

Steven Specter

Department of Molecular Medicine
University of South Florida College of Medicine, Tampa

Richard L. Hodinka

Clinical Virology Laboratory
Children’s Hospital of Philadelphia
and
Department of Pediatrics

University of Pennsylvania School of Medicine, Philadelphia

Stephen A. Young
TriCore Reference Laboratories
and
Department of Pathology
University of New Mexico, Albuquerque

Danny L. Wiedbrauk
Warde Medical Laboratory, Ann Arbor, Michigan

Washington, D.C.

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Copyright © 2009 ASM Press
American Society for Microbiology
1752 N Street, N.W.
Washington, DC 20036-2904

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Library of Congress Cataloging-in-Publication Data

Clinical virology manual / edited by Steven Specter . . . [et al.]. — 4th ed.
p. ; cm.
Includes bibliographical references and indexes.
ISBN 978-1-55581-462-5
1. Diagnostic virology—Handbooks, manuals, etc. I. Specter, Steven.
[DNLM: 1. Virology—methods. 2. Laboratory Techniques and Procedures. 3. Virus

Diseases—diagnosis. QW 160 C641 2009]
QR387.C48 2009
616.9′101—dc22

2009003225

All Rights Reserved
Printed in the United States of America
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Address editorial correspondence to: ASM Press, 1752 N St., N.W., Washington, DC
20036-2904, U.S.A.

Send orders to: ASM Press, P.O. Box 605, Herndon, VA 20172, U.S.A.
Phone: 800-546-2416; 703-661-1593
Fax: 703-661-1501
Email:
Online: estore.asm.org

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DEDICATION
We dedicate this edition of the Clinical Virology Manual to the memory of our colleague,
mentor, and friend, Herman Friedman, who passed away during the summer of 2007.
Dr. Friedman’s vision was responsible for the initiation of the first edition, and his foresight
and insights stimulated the dissemination of information through this virology manual and
the Clinical Virology Symposium for a field that continues to expand.
and
To our wives, Randie, Kitty, and Linda
and
Our children, Ross, Rachel, Ryan, Tyler, Brett, Jesse, and Eileen,
whose patience and support sustain us through all our endeavors

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Contents

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8 Immunoenzymatic Techniques for
Detection of Viral Antigens in Cells
and Tissue / 103

Contributors / ix
Preface to the Fourth Edition / xiii
Preface to the First Edition / xv

CHRISTOPHER R. POLAGE AND CATHY A. PETTI

SECTION I

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LABORATORY PROCEDURES FOR
DETECTING VIRUSES / 1
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2 Specimen Requirements: Selection,
Collection, Transport, and Processing / 18
THOMAS E. GRYS AND THOMAS F. SMITH

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10 Hemadsorption and
Hemagglutination-Inhibition / 119

Quality Assurance in Clinical Virology / 3

CHRISTINE C. GINOCCHIO

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STEPHEN A. YOUNG

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Immunoglobulin M Determinations / 124

DEAN D. ERDMAN AND LIA M. HAYNES

12 Susceptibility Test Methods:
Viruses / 134

Primary Isolation of Viruses / 36

MARIE LOUISE LANDRY

Neutralization / 110

DAVID SCHNURR

MAX Q. ARENS AND ELLA M. SWIERKOSZ

4 The Cytopathology of Virus Infection / 52
ROGER D. SMITH AND ANTHONY KUBAT

13 Application of Western Blotting to
Diagnosis of Viral Infections / 150

5 Electron Microscopy and Immunoelectron
Microscopy / 64

MARK B. MEADS AND PETER G. MEDVECZKY

RAYMOND TELLIER, JOHN NISHIKAWA,
AND MARTIN PETRIC

14 Nucleic Acid Amplification and Detection
Methods / 156

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DANNY L. WIEDBRAUK

Immunofluorescence / 77

TED E. SCHUTZBANK, ROBYN MCGUIRE,
AND DAVID R. SCHOLL

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7 Enzyme Immunoassays and
Immunochromatography / 89

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DIANE S. LELAND

JAMES J. MCSHARRY

Quantitative Molecular Techniques / 169

FREDERICK S. NOLTE

Flow Cytometry / 185

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CONTENTS

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SECTION II

Human Herpesviruses 6, 7, and 8 / 494

PHILIP E. PELLETT AND SHEILA C. DOLLARD

VIRAL PATHOGENS / 201

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Poxviruses / 523

CHRISTINE C. ROBINSON

VICTORIA A. OLSON, RUSSELL L. REGNERY,
AND INGER K. DAMON

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Respiratory Viruses / 203
Enteroviruses and Parechoviruses / 249

MARK A. PALLANSCH AND M. STEVEN OBERSTE

Parvoviruses / 546

STANLEY J. NAIDES

19 Rotavirus, Caliciviruses, Astroviruses,
Enteric Adenoviruses, and Other Viruses

Causing Acute Gastroenteritis / 283

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Measles, Mumps, and Rubella / 562

WILLIAM J. BELLINI AND JOSEPH P. ICENOGLE

TIBOR FARKAS AND XI JIANG

32 The Human Retroviruses Human
Immunodeficiency Virus and Human
T-Lymphotropic Retrovirus / 578

20 Waterborne Hepatitis / 311
DAVID A. ANDERSON

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JÖRG SCHÜPBACH

21 Blood-Borne Hepatitis Viruses: Hepatitis
Viruses B, C, and D and Candidate Agents of
Cryptogenetic Hepatitis / 325
MAURO BENDINELLI, MAURO PISTELLO,
FABRIZIO MAGGI, AND MARIALINDA VATTERONI

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Rabies / 363

23 Arboviruses / 387

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JOHN T. ROEHRIG AND ROBERT S. LANCIOTTI

Human Papillomaviruses / 408

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RAPHAEL P. VISCIDI AND KEERTI V. SHAH

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Human Polyomaviruses / 417

RAPHAEL P. VISCIDI AND KEERTI V. SHAH

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Herpes Simplex Viruses / 424

LAURE AURELIAN

Rodent-Borne Viruses / 641

BRIAN HJELLE AND FERNANDO TORRES-PEREZ

CHARLES V. TRIMARCHI AND ROBERT J. RUDD

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Chlamydiae / 630

CHARLOTTE A. GAYDOS

APPENDICES: REFERENCE LABORATORIES
Appendix 1
Virology Services Offered by the Federal
Reference Laboratories at the Centers for
Disease Control and Prevention / 659
BRIAN W. J. MAHY

Appendix 2
State Public Health Laboratory Virology
Services / 663
ROSEMARY HUMES

27 Cytomegalovirus, Varicella-Zoster Virus,

and Epstein-Barr Virus / 454

Author Index / 673

SONALI K. SANGHAVI, DAVID T. ROWE,
AND CHARLES R. RINALDO, JR.

Subject Index / 674

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Contributors

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DAVID A. ANDERSON

CHARLOTTE A. GAYDOS

Macfarlane Burnet Institute for Medical Research and Public
Health, 85 Commercial Road, Melbourne 3004, Australia

Division of Infectious Diseases, Dept. of Medicine, Johns
Hopkins University, Baltimore, MD 21205

MAX Q. ARENS

CHRISTINE C. GINOCCHIO

Dept. of Pediatrics, Washington University School of
Medicine, One Children’s Place, St. Louis, MO 63110

Microbiology, Virology and Molecular Diagnostics, North
Shore-LIJ Health System Laboratories, 10 Nevada Drive,
Lake Success, NY 11042

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LAURE AURELIAN

Virology/Immunology Laboratories, Dept. of Pharmacology
and Experimental Therapeutics, The University of Maryland,
School of Medicine, Baltimore, MD 21201

THOMAS E. GRYS

Division of Clinical Microbiology, Dept. of Laboratory
Medicine and Pathology, Mayo Clinic and Foundation,
Rochester, MN 55905

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WILLIAM J. BELLINI

Measles, Mumps, Rubella and Herpesvirus Laboratory Branch,
Division of Viral Diseases, National Center for Immunization
and Respiratory Diseases, Centers for Disease Control and
Prevention, Mailstop C-22, Atlanta, GA 30333

LIA M. HAYNES

MAURO BENDINELLI

BRIAN HJELLE

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Retrovirus Center and Dept. of Experimental Pathology,
University of Pisa, 37, Via San Zeno, I-56127 Pisa, Italy

INGER K. DAMON

Gastroenteritis and Respiratory Viruses Laboratory Branch,
Division of Viral Diseases, Centers for Disease Control and
Prevention, Atlanta, GA 30333
Center for Infectious Diseases and Immunity, Dept. of
Pathology, University of New Mexico, Health Sciences
Center, MSC08 4640, Albuquerque, NM 87131

Poxvirus Team, Poxvirus Rabies Branch, Division of Viral
and Rickettsial Diseases, National Center for Zoonotic,
Vector-Borne, and Enteric Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Road, NE, Mailstop
G-06, Atlanta, GA 30333

ROSEMARY HUMES
Association of Public Health Laboratories, 8515 Georgia Ave.,
Silver Spring, MD 20910

JOSEPH P. ICENOGLE
Measles, Mumps, Rubella and Herpesvirus Laboratory
Branch, Division of Viral Diseases, National Center
for Immunization and Respiratory Diseases, Centers for
Disease Control and Prevention, Mailstop C-22,
Atlanta, GA 30333

SHEILA C. DOLLARD
Division of Viral Diseases, Centers for Disease Control
and Prevention, 1600 Clifton Rd., Mailstop G18,
Atlanta, GA 30333

DEAN D. ERDMAN
Gastroenteritis and Respiratory Viruses Laboratory Branch,
Division of Viral Diseases, Centers for Disease Control and
Prevention, Atlanta, GA 30333

XI JIANG

TIBOR FARKAS

ANTHONY KUBAT

Cincinnati Children’s Hospital Medical Center, University of
Cincinnati College of Medicine, Cincinnati, OH 45299

Dept. of Pathology, Spectrum Health Butterworth Hospital,
Grand Rapids, MI 49506

Cincinnati Children’s Hospital Medical Center, University of
Cincinnati College of Medicine, Cincinnati, OH 45299

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CONTRIBUTORS

ROBERT S. LANCIOTTI

MARK A. PALLANSCH

Arboviral Diseases Branch, Division of Vector-Borne
Infectious Diseases, National Center for Zoonotic,
Vector-Borne, and Enteric Diseases, Centers for Disease
Control and Prevention, Fort Collins, CO 80521

Polio and Picornavirus Laboratory Branch, Centers for Disease
Control and Prevention, Atlanta, GA 30333

PHILIP E. PELLETT
Dept. of Immunology and Microbiology, Wayne State

University School of Medicine, 6225 Scott Hall, 540 East
Canfield Ave., Detroit, MI 48201

MARIE LOUISE LANDRY
Dept. of Laboratory Medicine, Yale University School of
Medicine, New Haven, CT 06520

MARTIN PETRIC
DIANE S. LELAND

BC Centre for Disease Control, Vancouver, BC,
Canada V5Z 4R4

Dept. of Pathology and Laboratory Medicine, Indiana
University School of Medicine, Indianapolis, IN 46202

CATHY A. PETTI
FABRIZIO MAGGI
Virology Unit, Pisa University Hospital, 37, Via San Zeno,
I-56127 Pisa, Italy

Dept. of Pathology, University of Utah School of Medicine,
ARUP Laboratories, Inc., Salt Lake City, UT 84108-1221

MAURO PISTELLO
BRIAN W. J. MAHY

Retrovirus Center and Dept. of Experimental Pathology,
University of Pisa, 2, SS Abetone e Brennero, I-56127
Pisa, Italy

Division of Emerging Infections and Surveillance Services
(DEISS), Mailstop D61, Centers for Disease Control and
Prevention, 1600 Clifton Road, Atlanta, GA 30333

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CHRISTOPHER R. POLAGE
ROBYN MCGUIRE

Dept. of Pathology, University of Utah School of Medicine,
ARUP Laboratories, Inc., Salt Lake City, UT 84108-1221

American Red Cross, Southern California Region,
100 Red Cross Circle, Pomona, CA 91768

RUSSELL L. REGNERY
JAMES J. MCSHARRY
Center for Emerging Infections and Host Defense,
Ordway Research Institute, 150 New Scotland Avenue,
Albany, NY 12208

MARK B. MEADS

PETER G. MEDVECZKY

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Dept. of Molecular Medicine, College of Medicine,
University of South Florida, 12901 30th Street,
Tampa, FL 33612

STANLEY J. NAIDES

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H. Lee Moffitt Cancer Center & Research Institute,
University of South Florida, 12902 Magnolia Drive,
Tampa, FL 33612

Immunology Research and Development, Quest Diagnostics
Nichols Institute, 33608 Ortega Highway, San Juan
Capistrano, CA 92675

JOHN NISHIKAWA

Poxvirus Team, Poxvirus Rabies Branch, Division of Viral
and Rickettsial Diseases, National Center for Zoonotic,
Vector-Borne, and Enteric Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Road, NE,
Mailstop G-06, Atlanta, GA 30333

Dept. of Pediatric Laboratory Medicine, The Hospital for Sick
Children, Toronto, Ontario, Canada M5G 1X8

FREDERICK S. NOLTE
Dept. of Pathology and Laboratory Medicine, Medical
University of South Carolina, Charleston, SC 29425

M. STEVEN OBERSTE
Polio and Picornavirus Laboratory Branch, Centers for Disease
Control and Prevention, Atlanta, GA 30333

VICTORIA A. OLSON
Poxvirus Team, Poxvirus Rabies Branch, Division of Viral
and Rickettsial Diseases, National Center for Zoonotic,
Vector-Borne, and Enteric Diseases, Centers for Disease
Control and Prevention, 1600 Clifton Road, NE, Mailstop
G-06, Atlanta, GA 30333

CHARLES R. RINALDO, JR.

Clinical Virology Laboratory, University of Pittsburgh Medical
Center, Pittsburgh, PA 15213, and Dept. of Infectious Diseases
and Microbiology, University of Pittsburgh Graduate School
of Public Health, Pittsburgh, PA 15261

CHRISTINE C. ROBINSON
Dept. of Pathology and Laboratory Medicine, The Children’s
Hospital, 13123 E. 16th Ave., Aurora, CO 80045

JOHN T. ROEHRIG
Arboviral Diseases Branch, Division of Vector-Borne
Infectious Diseases, National Center for Zoonotic,

Vector-Borne, and Enteric Diseases, Centers for Disease
Control and Prevention, Fort Collins, CO 80521

DAVID T. ROWE
Dept. of Infectious Diseases and Microbiology, University
of Pittsburgh Graduate School of Public Health,
Pittsburgh, PA 15261

ROBERT J. RUDD
Laboratory for Zoonotic Disease and Clinical Virology, New York
State Dept. of Health, Wadsworth Center, Albany, NY 12201

SONALI K. SANGHAVI
Clinical Virology Laboratory, University of Pittsburgh Medical
Center, Pittsburgh, PA 15213

DAVID SCHNURR
Viral and Rickettsial Disease Laboratory, California
Department of Health Services, Richmond, CA 94804

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CONTRIBUTORS

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DAVID R. SCHOLL

RAYMOND TELLIER

Diagnostic HYBRIDS, 350 West State St., Athens,
Ohio 45701

Dept. of Pediatric Laboratory Medicine, The Hospital for Sick
Children, Toronto, Ontario, Canada M5G 1X8

JÖRG SCHÜPBACH

FERNANDO TORRES-PEREZ

Swiss National Center for Retroviruses, University of Zurich,
CH-8006 Zurich, Switzerland

Dept. of Pathology and Dept. of Biology, University of
New Mexico, Health Sciences Center, CRF 327,
Albuquerque, NM 87131

TED E. SCHUTZBANK
CHARLES V. TRIMARCHI

Covance Central Laboratory Services, 8211 SciCor Drive,
Indianapolis, IN 46214

Laboratory for Zoonotic Disease and Clinical Virology,
New York State Dept. of Health, Wadsworth Center,
Albany, NY 12201

KEERTI V. SHAH
Dept. of Molecular Microbiology and Immunology, Johns

Hopkins University Bloomberg School of Public Health,
Baltimore, MD 21205

MARIALINDA VATTERONI
Virology Unit, Pisa University Hospital, 37, Via San Zeno,
I-56127 Pisa, Italy

ROGER D. SMITH
Dept. of Pathology and Laboratory Medicine, University of
Cincinnati Medical Center, Cincinnati, OH 45267-0529

RAPHAEL P. VISCIDI

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Dept. of Pediatrics, Johns Hopkins University School of
Medicine, Baltimore, MD 21287

THOMAS F. SMITH

DANNY L. WIEDBRAUK

Division of Clinical Microbiology, Dept. of Laboratory
Medicine and Pathology, Mayo Clinic and Foundation,
Rochester, MN 55905

Warde Medical Laboratory, Ann Arbor, MI 48108

STEPHEN A. YOUNG
ELLA M. SWIERKOSZ
Dept. of Pathology, Saint Louis University School of
Medicine, St. Louis, MO 63104

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TriCore Reference Laboratories, Albuquerque, NM 87102,
and Dept. of Pathology, University of New Mexico,
Albuquerque, NM 87131

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Preface to the Fourth Edition

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West Nile virus, bocaviruses, newer influenza and adenoviruses, and others. The information on the federal laboratory
organization at the Centers for Disease Control and Prevention (CDC) and state public health laboratories in the
Reference Laboratories section has also been updated.
This edition brings one other major change, the inclusion
of a new editor. We are pleased that the American Society
for Microbiology is continuing as our publisher for this edition and hope that ASM members as well as nonmembers
will find this manual a useful adjunct to the Manual for
Clinical Microbiology and Manual for Molecular and Clinical
Laboratory Immunology. There are a number of chapters for
which the authors have changed as a result of change of professional focus or retirement. We thank all of those authors
for their efforts. We hope that this edition is a credit to those
who preceded this effort, especially Jerry Lancz, who helped
to start this series.

The aims of this fourth edition of the Clinical Virology Manual

remain the same as the those for the first edition; thus, the
original preface is included to describe those goals. Updated
from the third edition, this new edition includes 34 chapters
and 2 appendices. The listing of laboratories offering viral
services has been deleted from the original section on Reference Laboratories in the appendix because there are now
many more virology services and the list changes too frequently. The reader is referred to his/her state health laboratories for assistance as needed. Many of the chapters in this
edition have been updated and expanded, while chapters on
some of the less used virology techniques of the past have
been deleted, including chapters on the interference assay,
radioimmunoassay, complement fixation, immune adherence
hemagglutination, and automation. This edition includes
separate chapters describing papillomaviruses and polyomaviruses, which were previously dealt with jointly as papovaviruses. The chapter updates are intended to address the
modernization that has occurred in the past several years,
with a strong emphasis on molecular diagnostics. In the Viral
Pathogens section we have included information on several
newly described viruses including human metapneumovirus,

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STEVEN SPECTER
RICHARD L. HODINKA
STEPHEN A. YOUNG
DANNY L. WIEDBRAUK

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Preface to the First Edition

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clinical technician for the diagnosis of a viral infection.

Each chapter includes information relating basic, pathogenic,
immunologic, and protective measures concerning each
virus group, as well as information on its isolation, propagation, and diagnosis. This section also includes a chapter on
Chlamydia. There are two reasons for including this family:
the clinical laboratory often isolates and diagnoses Chlamydia, and the techniques used in its isolation and diagnosis
are used in other instances.
The third section is designed to be used for reference.
Here we supply information about Federal Reference Laboratories at the Centers for Disease Control and their role in
the diagnosis of viral infection. The diagnostic and regulatory activities of state health laboratories and services available at individual hospital laboratories are provided in
survey form. This listing is somewhat incomplete in that it
contains information provided in response to an initial
questionnaire and follow-up.
The aim and scope of this volume are service: to the physician, as a source of basic and clinical information regarding viruses and viral diseases, and to the laboratories, as a
reference source to aid in the diagnosis of virus infection by
providing detailed information on individual techniques
and the impetus to expand services offered.

Clinical virology is an area that is undergoing rapid expansion. As a service for patient care, the utility of the clinical
virology laboratory has increased significantly in the past
decade. Due to the availability of commercial test kits, sophisticated yet simple diagnostic reagents, and the standardization of laboratory assays, accurate, reliable and, in many
instances, rapid protocols are currently available for the
diagnosis of a variety of viral agents producing human infections. Thus, the demands (on both the physician and the
clinical laboratory virologist) for the diagnosis of viral infections will continue to increase. With this in mind, this volume is written as both an aid to the clinician and as a guide
for the clinical laboratory.
This manual has three sections. The first describes laboratory procedures to detect viruses. The individual chapters deal
with quality control in the laboratory and specimen handling,
areas that are critical for an effective diagnostic laboratory.
This is followed by individual chapters that provide information or a detailed protocol on how to set up and test samples
for viral diagnosis using this technique. Both classical and the
newer, more experimental techniques are described in detail.

The second section focuses on the viral agents. Viruses
are grouped into chapters based on a target organ-system
categorization. In this way, viruses producing infection in a
particular organ or tissue are discussed and compared in a
single chapter. This approach more accurately reflects the
problems and choices faced by the attending physician and

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STEVEN SPECTER
GERALD LANCZ

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LABORATORY
PROCEDURES FOR
DETECTING VIRUSES

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Quality Assurance in Clinical Virology
CHRISTINE C. GINOCCHIO

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Effective September 1992, with the implementation of the
federal Clinical Laboratory Improvement Amendments
(CLIA) of 1988 (CLIA-88), all laboratory testing performed
on humans (except research) in the United States is regulated by the Centers for Medicare and Medicaid Services

(CMS), formerly known as the HCFA, unless the state health
department regulations exceed and are approved by CMS
(Centers for Disease Control and Prevention [CDC], 1992;
HCFA, 1992). In total, CLIA covers approximately 198,000
laboratory entities. The Division of Laboratory Services,
within the Survey and Certification Group under the Center
for Medicaid and State Operations, has the responsibility
for implementing the CLIA program (HCFA, 1992). The
CLIA program is designed to ensure quality laboratory testing, and all clinical laboratories must be properly certified to
receive Medicare or Medicaid payments.
The provisions of CLIA-88 include licensure, inspections
conducted by CMS or approved organizations, such as the
College of American Pathologists (CAP) and JCAHO, and
sanctions for failure to meet mandated standards (Warford,
2000). The stated purpose of CLIA-88 regulation of laboratories is to improve laboratory quality and achieve accurate
and reliable laboratory results. The main quality standards
of the regulatory and accrediting organizations can be categorized as (i) personnel qualifications, responsibilities, and competency assessment; (ii) proficiency testing for all analytes
and staff; (iii) written and approved procedures; (iv) method
verification and validation; (v) test reagent and equipment
quality control (QC) and preventive maintenance; and (vi)
patient test management. The process must include ongoing
assessment, and the goal is to provide for improvement of all
laboratory services. Please refer to references listed that provide in-depth information regarding U.S. clinical laboratory
regulations and accreditation requirements as well as useful
QA resources.

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REGULATORY REQUIREMENTS

Today, medical practitioners rely on the ability of the clinical
laboratory to provide key scientific data used for the diagnosis, treatment, and monitoring of persons with viral diseases.
Therefore, the accuracy of test results is critical, and ongoing
quality assurance (QA) and quality improvement programs
are key factors in maintaining service excellence. All members and levels of the laboratory staff, from lab assistants to
directors, are responsible for QA and improvement and
must play an active role on a daily basis. QA programs must
be comprehensive so that all aspects of the laboratory testing are monitored, including the preanalytical, analytical,
and postanalytical phases. Another key component of QA
is maintaining staff proficiency and competency. Quality
improvement programs are necessary to provide a surveillance
mechanism that will identify specific phases of the testing
that are suboptimal and need improvement. In Nutting’s 1996
report of a prospective study of the type and frequency of
laboratory testing problems in primary care physicians’ offices
during a 6-month period, a rate of 1.1 problems per 1,000 visits was found (Nutting et al., 1996; Warford, 2000). Twentyseven percent of these test problems had an impact on patient
care, including serious effects such as unnecessary hospitalization, prolonged hospital stay, more-invasive diagnostic
procedures, and delays in treatment. However, only 25% of
the laboratory problems involved test analysis or inconsistent results; 75% of errors occurred in specimen collection

and transport (43%) or timely provider notification of results
(32%). This and other studies (Boone et al., 1982; Bartlett
et al., 1994) confirm the need for laboratory involvement
in improving the total testing process if laboratory services
are to be meaningful and beneficial in patient health care
(Warford, 2000).
Finally, the laboratory must establish and comply with
written policies and procedures that maintain an ongoing
process to monitor, assess, and when indicated, correct problems identified in all phases of testing. The assessment must
include documentation of problems, communication with
appropriate persons, a review of the effectiveness of corrective
actions taken to resolve problems, revision of policies and
procedures necessary to prevent recurrence of problems, and
discussion of assessment reviews with appropriate laboratory
and clinical staff (Health Care Financing Administration
[HCFA], 1992; National Committee for Clinical Laboratory
Standards [NCCLS] 1998a, 2004a; Joint Commission on
Accreditation of Healthcare Organizations [JCAHO], 1998).

STAFF REQUIREMENTS, TRAINING,
AND COMPETENCY
Quality viral diagnostic services are highly dependent on welltrained and competent laboratory personnel (Bartlett et al.,
1994; JCAHO, 1998; NCCLS, 1998a, 2004a, 2004c; Sharp,
2003; Sewell, 2005). The laboratory director is responsible for
providing written qualifications, duties, and responsibilities
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LABORATORY PROCEDURES

for all staff positions in accordance with local, state, and federal requirements and ensuring that staffing levels are adequate for the type and volume of testing performed. Excessive
workloads are not consistent with quality, particularly with
subjective tasks requiring judgment, such as microscopy
(Warford, 2000). Staff qualifications for education, experience, training, and licensure or certification vary greatly
among the regulatory and accrediting agencies, with CLIA-88
having the minimum requirements (HCFA, 1992; Sewell,
2005). Several states require specific licensure for both laboratory directors and technical staff.
CLIA-88 categorized virology testing as moderate- and
high-complexity testing, with only a few infectious mononucleosis serology kits and rapid antigen immunoassays (e.g.,
for influenza virus and respiratory syncytial virus [RSV]) listed
as “waived” tests, i.e., exempt from many CLIA-88 regulations. Virology laboratories can offer level-one testing, which
consists of immunoassays for antigen detection without
microscopy, or level-two high-complexity testing for virus
isolation and identification and all other viral diagnostics.
Because most virology methods are complex and subjective,
requiring independent analysis and decisions, adequate education and training in theory and methods are essential for
quality results. Several studies have correlated the level of
education, training, and certification or licensure with laboratory performance quality, as measured in proficiency surveys (Gerber et al., 1991; Woods and Bryan, 1994; CDC,
1996; Shahangian, 1998; Warford, 2000; NCCLS, 2004c;
CLSI, 2007a).
Training verification and ongoing competency of laboratory staff is mandated by CLIA-88 and is another of the
main CMS inspection deficiencies cited (Table 1). The laboratory should have a comprehensive training manual and
documentation that all personnel have read and understand
the preanalytical, analytical, and postanalytical test requirements, have been trained to perform the procedure, are proficient in the testing, and are able to report patient results
accurately (Bartlett et al., 1994; JCAHO, 1998; NCCLS,
1998a, 2004a, 2004c; Sharp, 2003; Sewell, 2005). Training

verification and competency assessment can be documented
(Table 2) by observation, use of training checklists created
from the major and critical steps of the procedure manual,
written tests, and requiring the staff to test and pass a blinded

TABLE 1 Top CMS inspection CLIA-88 deficiencies cited,
1996 to 1998
PT program for each specialty and subspecialty inadequate
QA plan; lack of comprehensive written plan for maintaining
quality of overall testing process, identifying problems, and
implementing corrective action
QC not documented with at least two levels of controls for each
day of testing
Preventive maintenance and function checks of instrumentation
inadequate
Competency assessment program of staff performance inadequate
Daily supervisory review of QC, PM, and patient test results not
performed
Procedure manual and job descriptions without lab director’s
written designation of responsibilities and duties of staff
Correlation of multiple tests methods for same analytes not
documented
a

Sources: Chapin and Baron, 1996; Belanger, 1998.

Technical supervisor must assess and verify staff performance of
procedures at least annually by use of the following:
•

•
•

•

•

•

•

•

Direct observation of routine test performance, instrument
maintenance and function checks, and microscopy and
interpretation
Monitoring worksheets, result recording, and reporting
Testing proficiency samples, previously analyzed
specimens, blind controls, and/or reference samples
Daily review of QC records and preventive maintenance
records
Monitoring of failed test runs, unacceptable QC results, and
run contamination
Correlation of preliminary results with final or repeat results
for patterns of inconsistencies
Additional procedures such as written or verbal tests,
continuing education, problem solving of test failures,
evaluation of critical incidents, error reports, or client and
staff complaints
Reevaluation required with each change in methods

a

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proficiency panel prior to reporting clinical results. Training
can be provided by other trained laboratory staff or by test
vendors at their facilities or onsite. The manual should
also address training and competency with the laboratory
information systems, reporting policies, and institutional
mandatory topics, such as Health Information Portability
and Accountability Act requirements, fire and safety, administrative policies, emergency management, corporate compliance, and infection control.
Once laboratory staff have been initially trained, it is
essential to ensure ongoing competency and education, not
only with current testing procedures but also with the rapidly
changing field of virology in general. There are several methods that can be used to provide continuing education, including in-house lectures, teleconferences, educational internet
programs or CDs, and attendance at local, state, or national
workshops or scientific conferences. Competency assessment
is a daily process and should be formally documented at least
yearly for each staff member. Methods to assess individual
competency (Table 2) must include (i) direct observation
of test performance, instrument maintenance and function
checks, microscopy, and interpretation; (ii) monitoring worksheets, result recording, and reporting; (iii) testing of proficiency samples, previously analyzed samples, and blind controls
or reference samples; and (iv) daily review of QC and preventive maintenance records (Warford, 2000). Competency
can also be evaluated by monitoring (i) failed test runs and
QC failures; (ii) numbers of corrected reports due to technical or clerical errors; (iii) correlation of preliminary results
with final or repeat results for a pattern of inconsistencies;

and (iv) client and staff complaints. Finally, technical staff
should be evaluated for the ability to recognize unusual
results and solve problems when test failures occur. Deficiencies must be documented, a corrective action plan followed,
and the outcome assessed. The identification of common
deficiencies would indicate that the laboratory’s processes
for recruiting, staffing, training, continuing education, and
retention of a qualified staff need to be critically reviewed
and corrected appropriately (Warford, 2000).

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TABLE 2 Staff training verification and competency
assessment documentationa

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1. Quality Assurance

PROCEDURE MANUAL
A clear and concisely written procedure manual for all tests
performed by the laboratory must be available at the bench
and followed by the laboratory personnel. The procedures

should be written according to the guidelines established by
the CLSI (CLSI, 2006b), formerly known as the NCCLS.
Manuals should also address specific safety issues, such as
the use of biohazard hoods, protective personnel equipment,
transport and shipping of biological specimens or infectious
agents, agents of bioterrorism, disposal of infectious waste,
and emergency management. Textbooks, package inserts, and
manufacturer’s operator manuals may be used as supplemental references but do not replace the laboratory’s written test
procedures.
The required components of the laboratory procedure manual must address the preanalytical, analytical, and postanalytical phases of testing and are listed in Table 3 (HCFA, 1992;
CLSI, 2006b). Instructions that assist medical personnel in
appropriate test selection and ordering, patient preparation,
sample collection, storage, and transport must be available
either electronically or as laboratory testing manuals for distribution to all clients, including outreach physicians’ offices,
long-term care facilities, clinics, or hospital nursing units.
In addition to manuals that contain specific test protocols,
the virology laboratory must have written protocols that
describe the QA and quality improvement programs. The
policies should include (i) requirements for assessment of the
preanalytical, analytical, and postanalytical phases of testing; (ii) ongoing verification requirements for proficiency
testing; (iii) safety; (iv) technical training; and (v) ongoing
competency assessment. Yearly, the laboratory should select
and monitor specific quality indicators and document outcomes and corrective actions.
All procedures should be maintained under a document
control system according to the International Organization

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Requirements for patient preparation
Specimen collection, labeling, storage, preservation, transportation, processing, and test referral
Criteria for specimen acceptability and rejection
Background and significance of the test
Explanation of the test methodology
Detailed step-by-step performance of the procedure, including test
calculations and interpretation of results
Preparation and storage of slides, solutions, calibrators, controls,
reagents, stains, and other materials used in testing
Calibration and calibration verification procedures
Established and verified reportable range for test results for the
test system
Control procedures and corrective action to take when calibration
or control results fail to meet the laboratory’s criteria for
acceptability
Limitations in the test methodology, including interfering
substances
Reference intervals (normal values)
Reporting formats
Critical or alert values
Pertinent literature references
Alternative method if test or system is inoperative
a

Sources: HCFA, 1992; CLSI, 2006b.

for Standardization (ISO) 3500 regulations. This includes
records of date of initial use, dates of procedural changes,
and date of discontinuance. All new procedures and changes
in procedures must be approved, signed, and dated by the

current laboratory director before use. Discontinued procedures must be maintained by the laboratory for 2 years. There
must also be documentation that all technical staff who perform the procedures have read the procedure and any supplemental modifications. All procedures must be reviewed
yearly by the laboratory director.

PROFICIENCY TESTING
CLIA-88 has adopted external, graded proficiency testing
(PT) programs as the main indicator of the quality of laboratory testing performance (HCFA, 1992). In addition, PT
can serve proactively as a quality management tool (Boone
et al., 1982; Hoeltge and Duckworth, 1987; CDC, 1992;
ISO and International Electrotechnical Commission [IEC],
1997; JCAHO, 1998; Shahangian, 1998; CLSI, 2005c, 2007a).
All laboratories must participate in PT programs for each
analyte or test for which patient testing is performed. CLIAapproved 2007 PT programs and analytes offered are listed
in Table 4. CLIA-approved PT programs must include samples for viral antigen detection (rapid antigen tests for influenza viruses A and B, RSV, and rotavirus; antigen detection
by immunofluorescence for influenza viruses, parainfluenza
viruses, RSV, adenovirus, herpes simplex virus, varicella-zoster
virus, and cytomegalovirus) and virus isolation and identification. The PT program must include the more commonly
identified viruses, and the specific organisms found in the PT
samples may vary from year to year. The PT program must
provide a minimum of five samples per testing event, with a
minimum of three testing events at approximately equal
intervals per year. Guidelines for PT by laboratory comparisons also have been established by the ISO and IEC (ISO
and IEC, 1997). Information in the guidelines addresses the
development and operation of PT schemes and the selection
and use of PT schemes by laboratory accreditation bodies.
For any analyte that is not evaluated or scored by a CMSapproved PT program, the Clinical Laboratory Improvement Advisory Committee recommends that the laboratory

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TABLE 3 Mandatory components of procedure manualsa

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TABLE 4 CLIA-approved PT programs as of August 2007
Name and location
American Academy of Family
Physicians, Leawood, KS
American Association of
Bioanalysts, Brownsville, TX
American Proficiency Testing
Programs, Traverse City, MI
CAP, Northfield, IL

Medical Laboratory Evaluation,
Washington, DC
Wisconsin State Laboratory of
Hygiene, Madison, WI
New York State Department of
Health, Albany, NY

Analytes offered
Direct viral antigen detection
Direct viral antigen detection

Direct viral antigen detection
Direct viral antigen detection,
viral isolation and
identification, molecular
detection
Direct viral antigen detection
Direct viral antigen detection
Direct viral antigen detection,
virus isolation and
identification

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6

LABORATORY PROCEDURES

develops an in-house PT program to ensure that the laboratory tests five sample challenges, preferably three times per
year, to verify the accuracy of the test or procedure it performs
(HCFA, 1992; NCCLS, 2002; CLSI, 2005c). The testing
of in-house PT samples must comply with the same testing
guidelines as those required for CLIA-approved PT programs.
Sources for in-house PT materials can include blinded commercial panels of known reactivity, samples split with a reference laboratory, or previously tested samples of known
reactivity. Several studies have demonstrated that the best
measurement of laboratory routine performance can be accomplished with samples split and relabeled prior to receipt in
the laboratory (Boone et al., 1982; Farrington et al., 1995;
Gray et al., 1995a, 1995b; Yen-Lieberman et al., 1996;
Shahagian, 1998). The CDC also offers test panels for both
standard and rapid human immunodeficiency virus antibody testing, twice yearly, as part of its Model Performance

Evaluation Program. Although they are not graded challenges, results are provided with comparisons to those
obtained by other participating institutions.
Written policies defining the process for PT must be
clearly defined and understood by all personnel. PT must
be incorporated into the routine daily laboratory testing
and performed in the same manner and with the same staff
as routine patient samples. PT challenges should be rotated
among shifts and personnel who perform the testing. The
laboratory must identify viruses to the same extent it performs these procedures on patient specimens. Supervisory
personnel should ensure that their staff do not perform
“extra” testing (testing not normally performed for routine
clinical samples or duplicate testing) to confirm that their
initial results were correct. During the testing period, interlaboratory communication, comparison of results, and referral of the sample to a reference laboratory for identification
or confirmation is strictly prohibited. All submitted result
forms and final grading reports should be reviewed and
signed by the testing personnel, laboratory supervisor, and
laboratory director.
The accuracy of a laboratory’s response for a CLIAapproved PT challenge is determined by comparison of the
laboratory’s response for each sample with the response that
reflects agreement of either 80% of 10 or more referee laboratories or 80% or more of all participating laboratories
(HCFA, 1992; CAP, 2007). Unsatisfactory scores can also
result from a failure to participate in a testing event or failure to return PT results to the PT program within the time
frame specified by the program. However, consideration may
be given to laboratories if the PT program was notified as to
the circumstances of the failure. Failure to attain an overall
testing event score of at least 80% is unsatisfactory performance, and laboratories that fail consecutive challenges or
two of the three annual testing events are subject to severe
sanctions. For in-house-developed PT challenges, similar
guidelines should be developed for grading of results. For an
unsatisfactory testing event, the laboratory must review its

policies for PT, provide appropriate training, and employ
the technical assistance necessary to correct problems associated with the PT failure. Any type of PT assessment is
useless without investigation and efforts to improve system
problems. The investigative process and documentation of
any remedial action must include and be completed by all
persons involved in the PT failure, including the laboratory director. Documentation must be maintained by the
laboratory for 2 years from the date of participation in the
PT event.

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Inadequate PT performance is the most common postCLIA-88 inspection citation (Table 1) (Chapin and Baron,
1996; Belanger, 1998; Warford, 2000). PT failures provide an
opportunity for evaluation of factors contributing to test
performance problems (Table 5) (Warford, 2000), and use of
total quality management methods with staff input from all
sections and levels is recommended and outlined by CLSI
and others (Engebretson and Cembrowski, 1992). Investigations by CDC and CAP showed that approximately 20%
of repeated PT failures have no cause identified by the laboratory and that on-site technical consultation was required
for performance improvement (Boone et al., 1982; Hoeltge

and Duckworth, 1987).
Unfortunately, known PT samples are an imperfect measure of a laboratory’s performance accuracy and reliability
because they (i) are recognized challenges which have penalties for failure and are prone to special attention; (ii) test
only the analytical phase of testing, not specimen collection, transport, or usual result reporting; (iii) consist of
laboratory-adapted virus(es) or pooled, processed body fluids spiked with an analyte which may have a matrix effect,
which renders them inaccurate with certain methods; and
(iv) cannot test analyte concentrations near the assay cutoff
due to nonconsensus results with borderline levels (Warford, 2000). However, PT samples do still detect staff human
errors, serve as a form of competency testing, and identify
technical problems and some poorly performing methods.
Residual PT samples are excellent resource materials for
new test validations and technical training. Proficiency
sample testing and the analysis of results provided by programs such as CAP PT surveys also provide an educational
resource for the laboratory (Warford, 2000).
TABLE 5 Troubleshooting unacceptable patient or
PT resultsa
Procedure or method
•
•

•

Equipment, reagents, standards, QC materials
Limitations of methodology—sensitivity, specificity, precision,
linear range
Written procedure erroneous

Technical factors
•
•

•

Incubation time, temperature, humidity, carbon dioxide
Pipetting, dilutions, calculations
Misinterpretation, not following written protocol

Staff or staffing
•
•
•

Training, experience, continuing education
Use of overtime, per diem, rotating staff
Workload-to-staff ratio

Clerical error(s)
•

Mislabeling, transcription, units, computer entry

Sample or sampling
•
•
•

Transport time and/or temperature
Interfering substances, contamination
Organism or analyte not present or not viable on receipt

Obtain input on preventive measures from lab staff and others

a

Source: Warford, 2000.

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1. Quality Assurance

QA
Preanalytical Phase
QA for the preanalytical phase of testing begins with laboratory compliance, with the regulation that an appropriate
electronic or paper test requisition form is submitted from
an authorized person and, if a verbal request is made, that the
appropriate authorization is received within 30 days (HCFA,
1992). The components of the requisition need to be compliant with the general CLIA laboratory requirements; however, for virology specimens, it is especially essential to have
an accurate and specific sample type listed, indication of suspected virus(es), and any clinical information (e.g., immune
suppression) that may influence the selection of the tissue
culture medium. The date and time of specimen collection
are critical for assessing the quality of the specimen and the
ability to recover viruses, as a delay in delivery to the laboratory can significantly reduce the recovery of many labile
viruses, such as RSV. Entry of the test requisition information into a record system or a laboratory information system
can be a source of error, so the laboratory must ensure the
information is transcribed or entered accurately. This can be
monitored by performing a second-pass inspection of each
requisition. Alternatively, although most random errors will
not be detected, consistent errors may be detected by routinely selecting random requisitions and verifying that the
information (e.g., patient identification, medical record number, date of birth, tests ordered, specimen source) entered
into the patient’s sample record and the tests ordered were
accurate.

The laboratory must provide to clients written policies
and procedures for patient preparation, specimen collection,
specimen labeling (including patient name or unique patient
identifier), specimen storage and preservation, conditions
for specimen transportation, specimen processing, specimen
acceptability, and rejection. Clients must adhere to all of these
policies. This is to ensure that the specimen is collected properly and is appropriate for the targeted virus(es) and that the
integrity of the specimen is maintained. Specialized instructions are also essential, considering that at any time the
laboratory could receive specimens that may contain highly
infectious agents, such as avian influenza virus or the severe
acute respiratory syndrome (SARS) coronavirus, viruses that
should not be cultured in routine clinical laboratories. If
referral is required, laboratories should follow any local,
state, or federal regulations and can only refer testing
to a CLIA-certified laboratory or a laboratory meeting equivalent requirements, as determined by CMS (HCFA, 1992;
NCCLS, 1998b).
Specimens that are inappropriately collected, stored, or
preserved are one of the major sources of poor quality results
in the virology laboratory (Lennette, 1995; CLSI, 2005a).
Labile viruses die quickly, thus reducing virus titers and recovery. Nucleic acids can quickly degrade, resulting in falsenegative results with molecular-based assays. The laboratory
should reject these samples and request a new specimen.
Examples of common specimen rejection criteria are listed
in Table 6. If it is not possible to collect a new sample, the
laboratory must assess the integrity of the sample and determine if testing could still be performed, and a notation
should be included along with the test results indicating the
potential impact on the test results. This type of information
is important for monitoring and assessing the laboratory’s
procedures for sample collection, transport, and test performance. For example, if a significant discordance is noted with

poor virus recovery in culture and better detection by testing

that does not require live virus, such as direct immunofluorescence (direct fluorescent-antibody assay [DFA]) or rapid
membrane enzyme immunoassay (MEIA), an evaluation of
site compliance with appropriate specimen collection, storage, and transport protocols would be indicated. These data
may also guide the selection of the most appropriate test
methods for particular viruses and lead to an improvement
in specimen collection tools and methods.

Analytical Phase
Verification and Validation
Verification and/or validation studies of new tests, kits, or
reagents are essential prior to clinical use and the reporting
of patient results (Elder et al., 1997; Association for Molecular Pathology [AMP], 1999; NCCLS, 2003, 2004b; CLSI,
2005b, 2006c, 2006e, 2007b). Validation studies, performed
by the test manufacturer (U.S. Food and Drug Administration [FDA]-cleared assays) or by the laboratory (kits labeled
research use only [RUO] or investigational use only [IUO] or
as analyte-specific-reagent [ASR]-developed assays) determine the performance characteristics (i.e., precision, accuracy,
sensitivity, specificity, reportable range, etc.) of the test. Verification is defined as the ongoing process that confirms the
specified performance characteristics of the assay, as previously determined by the manufacturer or laboratory during
the validation studies. For FDA-cleared instruments, kits,
and test systems (both nonmolecular and molecular based),
the laboratory must verify the manufacturer’s performance
claims for accuracy, precision, and reportable range, as stated
in the package insert. The laboratory must also verify that
the manufacturer’s reference intervals (normal values) are
appropriate for the laboratory’s patient population. Verification usually consists of parallel testing the new product with
a standard method of known performance characteristics.
For qualitative assays, a minimum of 20 known positive
clinical samples and 50 negative clinical samples have been
recommended for this evaluation by McCurdy and colleagues (Elder et al., 1997). Negative samples should include
those containing other nontarget viruses commonly isolated

from the same source (e.g., other respiratory viruses) or genetically similar viruses that may cause a false-positive result
due to cross reactivity (e.g., enteroviruses and rhinoviruses).
For quantitative assays, in addition to clinical samples of
known reactivity, the laboratory must also verify the lower and
upper limits of detection, linearity across the dynamic range
of the assay, reproducibility, and precision of the assay. Verification must include all FDA-approved sample types (or
matrices) that will be used for testing with the assay (HCFA,
1992; NCCLS, 2003). The use of alternative sample types,
not FDA cleared, requires more extensive validation studies, as described below for non-FDA-cleared tests. Clinical
samples are essential for accurate verification studies. However, a sufficient quantity of positive or negative clinical
material may not always be available to the laboratory. Alternative sources can include split samples sent to a reference
laboratory, interlaboratory exchange of samples, proficiency
materials, and control material spiked into appropriate matrices at clinically relevant concentrations. When possible,
verification studies should be performed blinded to comparator results across multiple days, multiple runs, or batches
and using several technical personnel. Verification testing
must be performed in the same manner as patient samples,
following the manufacturer’s kit instructions, and include

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TABLE 6 Examples of specimen rejection criteriaa
Action
Problem
Delay in transit

Specimen

Test

Clotted blood
Whole blood (unspun)
Serum or plasma (RT)
Serum or plasma
(cold)
PPT tube (unspun)
PPT tube (spun)
PPT tube (spun)
Stool

Serology

Culture/PCR
PCR
PCR

PCR

Hemolysis

Serum

Serology

Lipemia/icterus
Mislabeled or unidentified

Serum
Any (except surgery)

Serology
All

Dry swab, wood, calcium
alginate, or charcoal swabs
Container gross external
contamination
Duplicate (<24 h)

Swab

Culture

Any (except surgery)

Any

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Sputum or stool for
respiratory viruses

Culture/DFA

QNS

Any

Any

Inadequate cellular material

Lesions, swabs

DFA

>24 h
6–12 h (whole blood)
25–72 h (RT)
>72 h (cold packs, refrigerated)
6–12 h (unspun, whole blood)
25–72 h (RT)
>72 h (cold packs, refrigerated)
>4 h C. difficile stool

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Reject and recollect

Any
Culture/DFA

Process and test with
disclaimer

>48 h (refrigerated) for
viral cultures

Any, cannot use
for PCR
Looks like whole blood

Any

Nonstandard source or

collection

a

>12 h (unspun, whole
blood)
>72 h (RT)

PCR
PCR
Clostridium
difficile toxin
Viral culture

Whole blood

Fixative (Formalin)
Non-VTM
(Bacti, Culturette)

>12 h (whole blood)
>72 h (RT)

PCR

Heparin (green top)

Any except surgery
(BAL fluid, biopsy
specimen, CSF)

Any
Swab

Reject (phone for
new sample)

Reject and recollect

Reject duplicate
blood, urine, stool

Reject and recollect
Cannot use Culturette
for DFA/EIA or
Chlamydia
Reject, recollect NP,
BAL fluid, or
NP-OP combination

Mild/moderate hemolysis
(note serum appearance in
computer)
Note appearance in computer
Tissue/CSF (have physician
identify and sign, add
disclaimer)
Note unsatisfactory swabs in
computer with disclaimer
Tissue/CSF (disinfect with
bleach)

Process if requested by physican

Can use Culturette for viral
culture (transfer to VTM as
soon as possible)
Add disclaimer

Call for physician’s test
priority list
Call for recollection

Abbreviations: RT, room temperature; PPT, plasma preparation tube; VTM, viral transport medium (SP buffer); NP, nasopharyngeal; OP, oropharyngeal;
BAL, bronchoalveolar lavage; CSF, cerebrospinal fluid; QNS, quantity not sufficient. Source: Warford, 2000.

the appropriate controls, as designated by the manufacturer
and regulatory agencies. Once studies are completed, the
laboratory can provide test results within the laboratory’s
stated performance specifications for each test system (HCFA,
1992).
The validation of new tests, kits, or reagents labeled for
RUO or IUO or as ASRs or in-house-developed assays
requires a significantly more extensive validation study, as
no manufacturer’s claims can be made regarding their use,
performance, or interpretation of the data (NCCLS, 2003,
2004b; CLSI, 2005b, 2006a, 2006c, 2007b). In addition,
for ASRs and in-house-developed assays, the laboratory
must also develop and optimize the actual testing protocol.

For non-FDA-approved methods, establishing the performance characteristics of accuracy, precision, reproducibility,
analytical sensitivity (limit of detection and/or limit of quantitation), specificity, interfering substances, and reportable and

reference ranges is CLIA mandated. The validation process
must include all phases of the testing and, for optimal evaluation of the results, be performed across multiple days, multiple
runs, or batches and using several technical personnel.
For molecular-based assays, validation studies must determine the most appropriate nucleic acid extraction procedures
for all matrices to be tested and assess the ability of the procedure to remove amplification inhibitors and the potential
for sample-to-sample cross-contamination (CLSI, 2005b).

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