MM22AE

February 2014
MM22-A
Microarrays for Diagnosis and Monitoring of
Infectious Diseases; Approved Guideline
This document provides guidance for the laboratory development

and use of qualitative nucleic acid microarray methods for the
diagnosis and monitoring of infectious diseases. It also presents
recommendations for validation and verification, quality control, and
interpretation of results.
A guideline for global application developed through the Clinical and Laboratory Standards Institute consensus process.
Licensed to: Kuzya Kuzmich

This document is protected by copyright. CLSI order #Ord-703180, Downloaded on 1/15/2022.
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ISBN 1-56238-951-3 (Print)
ISBN 1-56238-952-1 (Electronic)
ISSN 1558-6502 (Print) MM22-A
ISSN 2162-2914 (Electronic) Vol. 34 No. 3
Microarrays for Diagnosis and Monitoring of Infectious Diseases;
Approved Guideline
Volume 34 Number 3
Yi-Wei Tang, MD, PhD, D(ABMM) Francisco Martinez-Murillo, PhD
Sherry A. Dunbar, PhD Steven A. Miller, MD, PhD
Joseph D.C. Yao, MD, D(ABMM) Li Li, MS
Sridar V. Chittur, RPh, PhD Greg Peterson, MS, PhD
Sabrina Conoci, PhD Teresa J. Raich, PhD, MBA
Judy A. Daly, PhD Mark Poritz, PhD
Subhash Dhawan, PhD Wanda C. Reygaert, PhD
Shuguang Huang, PhD Hossein Salimnia, PhD, D(ABMM)
Abstract
Clinical and Laboratory Standards Institute document MM22-A—Microarrays for Diagnosis and Monitoring of Infectious
Diseases; Approved Guideline discusses the wide variety of nucleic acid microarray technologies that a growing number of
clinical laboratories have adopted for diagnostic testing. The different types of microarrays and their uses in various types of
laboratories have grown tremendously. MM22 specifically addresses infectious disease detection, identification, and genotyping,
as well as drug resistance determinants. This guideline describes types of microarray platforms and general considerations in
microarray development. It also provides recommendations for validation and verification of microarray performance and QC
and QA considerations, and discusses parameters for data analysis and interpretation.
Clinical and Laboratory Standards Institute (CLSI). Microarrays for Diagnosis and Monitoring of Infectious Diseases; Approved
Guideline. CLSI document MM22-A (ISBN 1-56238-951-3 [Print]; ISBN 1-56238-952-1 [Electronic]). Clinical and Laboratory
Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2014.
The Clinical and Laboratory Standards Institute consensus process, which is the mechanism for moving a document through
two or more levels of review by the health care community, is an ongoing process. Users should expect revised editions of any
given document. Because rapid changes in technology may affect the procedures, methods, and protocols in a standard or
guideline, users should replace outdated editions with the current editions of CLSI documents. Current editions are listed in
the CLSI catalog and posted on our website at www.clsi.org. If you or your organization is not a member and would like to
become one, and to request a copy of the catalog, contact us at: Telephone: 610.688.0100; Fax: 610.688.0700; E-Mail:
customerservice@clsi.org; Website: www.clsi.org.

Number 3 MM22-A
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Suggested Citation
CLSI. Microarrays for Diagnosis and Monitoring of Infectious Diseases; Approved Guideline. CLSI
document MM22-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.
Approved Guideline
February 2014
ISBN 1-56238-951-3 (Print)

ISBN 1-56238-952-1 (Electronic)
ISSN 1558-6502 (Print)
ISSN 2162-2914 (Electronic)
ii
Volume 34 MM22-A
Committee Membership
Consensus Committee on Molecular Methods
Frederick S. Nolte, PhD, Tina M. Hambuch, PhD Yi-Wei Tang, MD, PhD,
D(ABMM), F(AAM) Illumina, Inc. D(ABMM)
Chairholder San Diego, California, USA Memorial Sloan Kettering Cancer
Medical University Hospital Center
Authority Charles E. Hill, MD, PhD New York, New York, USA
Charleston, South Carolina, USA Emory University Hospital
Atlanta, Georgia, USA Zivana Tezak-Fragale, PhD
Barbara Zehnbauer, PhD, FDA Center for Devices and
FACMG Penny Keller, BS, MB(ASCP) Radiological Health
Vice-Chairholder Centers for Medicare & Medicaid Silver Spring, Maryland, USA
Centers for Disease Control and Services
Prevention Baltimore, Maryland, USA Emily S. Winn-Deen, PhD
Atlanta, Georgia, USA Rx Dx Advisors, Inc.
Thomas J. Lenk, PhD San Diego, California, USA
Helen Fernandes, PhD Celera Diagnostics
Weill Cornell Medical College Alameda, California, USA
New York, New York, USA
Document Development Committee on Microarrays for Diagnosis and Monitoring of Infectious

Diseases
Yi-Wei Tang, MD, PhD, Subhash Dhawan, PhD Staff

D(ABMM) FDA Center for Biologics
Co-Chairholder Evaluation and Research, Office of Clinical and Laboratory Standards
Memorial Sloan Kettering Cancer Blood Research and Review Institute
Center Rockville, Maryland, USA Wayne, Pennsylvania, USA
New York, New York, USA
Francisco Martinez-Murillo, PhD Luann Ochs, MS
Sherry A. Dunbar, PhD FDA Center for Devices and Senior Vice President – Operations
Co-Chairholder Radiological Health
Luminex Corporation Silver Spring, Maryland, USA Tracy A. Dooley, MLT(ASCP)
Austin, Texas, USA Staff Liaison
Steven A. Miller, MD, PhD
Joseph D.C. Yao, MD, D(ABMM) University of California San Marcy Hackenbrack, MCM,
Committee Secretary Francisco M(ASCP)
Mayo Clinic and Mayo Medical San Francisco, California, USA Project Manager
Laboratories
Rochester, Minnesota, USA Greg Peterson, MS, PhD Megan L. Tertel, MA
Kansas Department of Health and Editor
Judy A. Daly, PhD Environment
University of Utah – Primary Topeka, Kansas, USA Joanne P. Christopher, MA
Children’s Medical Center Assistant Editor
Salt Lake City, Utah, USA Mark Poritz, PhD
BioFire Diagnostics Inc.
Salt Lake City, Utah, USA
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Acknowledgment
CLSI and the Consensus Committee on Molecular Methods gratefully acknowledge the following
individuals for their help in preparing this document:
Sridar V. Chittur, RPh, PhD

University of Albany, SUNY
Rensselaer, New York, USA
Sabrina Conoci, PhD

STMicroelectronics
Catania, Italy
Shuguang Huang, PhD

Precision Therapeutics, Inc.
Pittsburgh, Pennsylvania, USA
Li Li, MS
FDA Center for Devices and Radiological Health
Silver Spring, Maryland, USA
Teresa J. Raich, PhD, MBA

Nanosphere, Inc.
Northbrook, Illinois, USA
Wanda C. Reygaert, PhD

Oakland University William Beaumont School of Medicine
Rochester, Michigan, USA
Hossein Salimnia, PhD, D(ABMM)

Detroit Medical Center University Laboratories
Detroit, Michigan, USA
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Contents
Abstract ....................................................................................................................................................i
Committee Membership........................................................................................................................ iii
Foreword .............................................................................................................................................. vii
1 Scope.......................................................................................................................................... 1
2 Introduction ................................................................................................................................ 1
3 Standard Precautions .................................................................................................................. 1
4 Terminology............................................................................................................................... 2
4.1 A Note on Terminology ................................................................................................ 2
4.2 Definitions .................................................................................................................... 2
4.3 Abbreviations and Acronyms ..................................................................................... 12
5 General Considerations in Developing Microarray Assays ..................................................... 13
5.1 Microarray Test Workflow ......................................................................................... 13
5.2 Assay Design Considerations ..................................................................................... 14
5.3 Probe Design and Update............................................................................................ 14
5.4 Preexamination Requirements .................................................................................... 15
5.5 Examination Requirements ......................................................................................... 15
5.6 Postexamination Requirements................................................................................... 18
5.7 Contamination Control ............................................................................................... 20
6 Platform Overview ................................................................................................................... 21
6.1 Printed Solid Surface Microarrays .............................................................................. 22
6.2 In Situ–Synthesized Oligonucleotide Microarrays ..................................................... 24
6.3 High-Density Silicon Microarrays .............................................................................. 29
6.4 Electronic Microarrays ............................................................................................... 31
6.5 Suspension Liquid Bead Microarrays ......................................................................... 32
6.6 Small Volume Polymerase Chain Reaction Arrays .................................................... 36
7 Clinical Applications ............................................................................................................... 39
7.1 Overview ..................................................................................................................... 39
7.2 Detection of Pathogens in Clinical Samples ............................................................... 39
7.3 Pathogen Genotyping .................................................................................................. 41
7.4 Detection of Antimicrobial Resistance Genes ............................................................ 41
7.5 Detection of Bioterrorism Pathogens .......................................................................... 42
7.6 Other Uses of Microarray Technology ....................................................................... 42
8 Assay Performance Verification and Validation ..................................................................... 42
8.1 Assay Verification and Validation .............................................................................. 42
8.2 Considerations Related to Microarrays ....................................................................... 52
9 Quality Control and Quality Assurance ................................................................................... 55
9.1 Laboratory Quality Assurance and Quality Indicator Considerations ........................ 55
10 Data Analysis and Interpretation ............................................................................................. 58
10.1 Challenges of Adapting Microarray Technologies to Detection of Pathogens ........... 58
10.2 Microarray Data Analysis Steps ................................................................................. 59
10.3 Threshold Value Determination .................................................................................. 64
10.4 Classifier Development (Supervised Learning) .......................................................... 66
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Contents (Continued)
References ............................................................................................................................................. 68
The Quality Management System Approach ........................................................................................ 80
Related CLSI Reference Materials ....................................................................................................... 82
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Foreword
At the time of the publication of CLSI document MM12,1 nucleic acid microarrays were not a major part
of the diagnostic test menu in clinical laboratories. Today, a growing number of clinical laboratories have
adopted a wide variety of microarray platforms for clinical testing and, as a result, the types and
associated purposes of such arrays have grown tremendously.
The scope of this subject is now too large to be covered in CLSI document MM12,1 and users of CLSI
documents would find separate guidance documents on human genetics and oncology and infectious
pathogens to be more useful than one large, comprehensive document. Toward that goal, the Consensus
Committee on Molecular Methods recommended revision of CLSI document MM121 to focus on
expression arrays and to be directed toward assay manufacturers.
Two new CLSI documents were proposed for development to provide guidance to clinical laboratories.
MM22 focuses on the use of nucleic acid microarrays in clinical microbiology and immunology
laboratories (pathogen profiling, etc.) and specifically addresses detection, identification, and genotyping
of infectious pathogens, as well as antimicrobial drug resistance determinants. The second document, in
development concurrently with MM22, focuses on the use of microarrays for human genetics and
oncology.
Key Words
Clinical diagnostics, infectious diseases, microarray analysis, nucleic acid testing, pathogen detection
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Microarrays for Diagnosis and Monitoring of Infectious Diseases; Approved

Guideline
1 Scope
Microarrays can be distinguished from each other based upon characteristics such as the nature of the
probe, the solid surface support used, and the specific method used for probe addressing and/or target
detection. MM22 focuses on the use of nucleic acid microarrays in microbiology and immunology
laboratories and specifies the requirements and recommendations for the use of microarrays for diagnosis
and monitoring of infectious diseases. It also covers infectious disease pathogen detection, identification,
and genotyping (strain characterization) as well as virulence and resistance genetic markers.
The intended users of this guideline are clinical laboratories performing qualitative, multiplexed nucleic
acid–based testing, which includes, but is not limited to, bacteriology, mycobacteriology, mycology,
parasitology, and virology. This guideline is not intended for use by research laboratories, but the research
community may use this guideline in order to translate their findings more easily. MM22 is not intended
for in vitro diagnostic (IVD) device manufacturers or to provide manufacturing guidelines, and does not
cover protein microarrays nor address microarray applications for unknown pathogen discovery, host or
microbial gene expression profiling, or host genomic polymorphism determination related to microbial
infections.
2 Introduction
The use of molecular detection techniques continues to increase in clinical microbiology and immunology
laboratories. The implementation of in vitro nucleic acid amplification techniques, led by PCR, in
diagnostic laboratories has transformed viral detection and specific microbial pathogen detection. The
continued advancement of molecular infectious disease diagnostics depends on the ability of multiplexing
technologies to easily and reliably detect multiple pathogens in a single clinical specimen. One approach
to multiplex detection, characterization, and monitoring of infectious diseases is microarray analysis.
Simply defined, a microarray is a collection of microscopic features (most commonly DNA) that can be
probed with target molecules to produce either quantitative (gene expression) or qualitative (detection and
identification) data. Largely due to advances in fabrication, robotics, and bioinformatics, microarray
technology has continued to improve in terms of efficiency, reproducibility, sensitivity, and specificity. In
addition, microarray platforms have expanded to include three-dimensional or suspension arrays. These
improvements have allowed for the transition of microarrays from strictly research settings to clinical
diagnostic applications. This has led to optimization of the diagnostic potential of microarrays and to the
development of commercially available qualitative detection platforms. A series of microarray-based
diagnostic devices are commercially available, with some having achieved regulatory agency clearance.
Some platforms can achieve multiplex analysis through parallel detection in multiple reaction chambers
as well. Thus, a new era in molecular diagnostics has arrived where the use of microarray technology in
the clinical microbiology laboratory is a reality.
3 Standard Precautions
Because it is often impossible to know what isolates or specimens might be infectious, all patient and
laboratory specimens are treated as infectious and handled according to “standard precautions.” Standard
precautions are guidelines that combine the major features of “universal precautions and body substance
isolation” practices. Standard precautions cover the transmission of all known infectious agents and thus
are more comprehensive than universal precautions, which are intended to apply only to transmission of
blood-borne pathogens. The Centers for Disease Control and Prevention address this topic in published
guidelines that address the daily operations of diagnostic medicine in human and animal medicine while
©
Clinical and Laboratory Standards Institute. All rights reserved. 1
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encouraging a culture of safety in the laboratory.2 For specific precautions for preventing the laboratory
transmission of all known infectious agents from laboratory instruments and materials and for
recommendations for the management of exposure to all known infectious diseases, refer to CLSI
document M29.3
4 Terminology
4.1 A Note on Terminology
CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization

wherever possible. Harmonization is a process of recognizing, understanding, and explaining differences
while taking steps to achieve worldwide uniformity. CLSI recognizes that medical conventions in the
global metrological community have evolved differently in the United States, Europe, and elsewhere; that
these differences are reflected in CLSI, International Organization for Standardization (ISO), and
European Committee for Standardization (CEN) documents; and that legally required use of terms,
regional usage, and different consensus timelines are all important considerations in the harmonization
process. In light of this, CLSI’s consensus process for development and revision of standards and
guidelines focuses on harmonization of terms to facilitate the global application of standards and
guidelines.
In keeping with CLSI’s commitment to align terminology with established ISO standards, the following
terms are used in MM22: measurand (quantity intended to be measured) is used in combination with the
term analyte (component represented in the name of a measurable quantity) when its use relates to a
biological fluid/matrix. Trueness is used in this document when referring to the closeness of agreement
between the average of an infinite number of replicate measured quantity values; the measurement of
trueness is usually expressed in terms of bias. Measurement procedure has replaced the term analytical
method for a detailed description of a measurement according to one or more measurement principles and
to a given measurement method that is based on a measurement model and includes any calculation used
to obtain a measurement result. The term diagnostic sensitivity is combined with the term clinical
sensitivity because in some jurisdictions, the term “clinical” often refers to clinical studies of drugs under
stringent conditions. For this document, validation is primarily a manufacturer’s responsibility to ensure
that design goals are met and performance claims are stated for a commercially developed assay or device
and a laboratory’s responsibility for a laboratory-developed test (LDT). Verification is an end-user
(clinical laboratory) responsibility to confirm that manufacturer’s claims are met on the specific device in
use by the laboratory and that medical needs are met. Validation is the establishment of analytical and
clinical performance characteristics whereas verification is the confirmation of previously established
performance characteristics.
In order to align the usage of terminology in this document with that of ISO and CLSI document
QMS01,4 preexamination, examination, and postexamination have replaced preanalytical, analytical, and
postanalytical, respectively, when referring to the testing phases within the laboratory path of workflow.
4.2 Definitions
accreditation – procedure by which an authoritative body gives formal recognition that an organization
or person is competent to carry out specific tasks (modified from ISO/IEC 17000).5
accuracy (measurement) – closeness of agreement between a measured quantity value and a true
quantity value of a measurand (JCGM 200:2012)6; NOTE: The concept “measurement accuracy” is not a
quantity and is not given a numerical quantity value. A measurement is said to be more accurate when it
offers a smaller measurement error (JCGM 200:2012).6
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2 Clinical and Laboratory Standards Institute. All rights reserved.
Volume 34 MM22-A
allele – one of the alternative forms of a gene that may occupy a given locus; NOTE 1: In genetics, any
of several forms of a gene that is responsible for hereditary variation; NOTE 2: One of the alternate
forms of a polymorphic DNA sequence that is not necessarily contained within a gene.
allele-specific primer extension – a solution-based, sequence-specific enzymatic reaction technology

that can be used to assay multiple alleles in a single tube; an enzymatic DNA polymerization reaction that
determines the genotype of a target.
amplicon – (amplification product) the relatively low-molecular-weight products created from a target
amplification reaction; NOTE: Amplicons are double-stranded DNA if created by a PCR and are
primarily single-stranded RNA if created in a nucleic acid sequence–based amplification or transcription-
mediated amplification reaction.
amplification – the enzymatic replication in vitro of a target nucleic acid; NOTE: PCR is a commonly
used method of amplification.
analyte – component represented in the name of a measurable quantity (ISO 17511)7; NOTE: See
measurand.
analytical sensitivity – quotient of the change in an indication and the corresponding change in the value
of a quantity being measured (ISO 15193)8; NOTE 1: For the purposes of microbial detection, analytical
sensitivity is equivalent to limit of detection; NOTE 2: In qualitative testing, the test method’s ability to
obtain positive results in concordance with positive results obtained by the reference method; NOTE 3:
The sensitivity depends on the imprecision of the assay.
analytical specificity – ability of a measurement procedure to determine solely the quantity it purports to
measure (ISO 15193)8; NOTE 1: The ability of a measurement procedure to measure solely the
measurand (ISO 17511)7; NOTE 2: The ability of a measurement procedure to distinguish the target
sequence(s)/allele/mutation(s) from other sequences/alleles in the specimen/genome.
annealing – the hybridization of two complementary strands of nucleic acid, as in the hybridization of a
probe or primer with the target DNA.
assay – a quantitative determination or measurement of the amount, activity, or potency of a constituent

or characteristic; NOTE 1: A qualitative assay reports only the presence or absence of the analyte,
without quantification; NOTE 2: A positive test result implies only that the assay signal exceeds the
analytical threshold (detection limit), or a cutoff point set to give an arbitrary combination of sensitivity
and specificity; NOTE 3: For the purpose of this document, assay is also known as the measurement
procedure.
bead array – created by either impregnating silica or polystyrene beads with different concentrations of
fluorescent dye, or by some type of bar-coding technology. The beads are addressable and used to identify
specific binding events that occur on their surfaces.
bias (of measurement) – estimate of a systematic measurement error (JCGM 200:2012) 6; NOTE: In
general, the deviation/difference is based on replicate measurement using an accepted (definitive,
reference, or designated comparison) method and the method being tested, and expressed in the units of
the measurement or as a percentage.
blank – (no template control) the apparent amount or concentration of a measurand measured by an
analytical process when a measurand is omitted from a tested sample or when the tested sample is known
to contain none of the measurand; a combination of the individual “blanks,” such as the extraction blank
and the reagent blank, as appropriate for the assay.
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calibration – operation that, under specified conditions, in a first step, establishes a relation between the
quantity values with measurement uncertainties provided by measurement standards and corresponding
indications with associated measurement uncertainties and, in a second step, uses this information to
establish a relation for obtaining a measurement result from an indication (JCGM 200:2012).6
CE marking – symbolizes the conformity of the product with the applicable European Community
requirements imposed on the manufacturer. The CE marking affixed to products is a declaration by the
person responsible that the product conforms to all applicable Community provisions, and the appropriate
conformity assessment procedures have been completed.9
clinical laboratory – laboratory for the biological, microbiological, immunological, chemical,

immunohematological, hematological, biophysical, cytological, pathological, genetic, or other
examination of materials derived from the human body for the purpose of providing information for the
diagnosis, management, prevention, and treatment of disease in, or assessment of the health of, human
beings, and which may provide a consultant advisory service covering all aspects of laboratory
investigation, including the interpretation of results and advice on further appropriate investigation (ISO
15189)10; NOTE: In some jurisdictions, the term “medical laboratory” is used.
clinical sensitivity – the ability of a test under study to give a positive result for subjects having the
disease/state in question or the proportion of subjects with a well-defined clinical disorder whose test
values are positive or exceed a defined decision limit (eg, a positive result and identification of the
patients who have a disease); NOTE: “Diagnostic sensitivity” is used in Europe and “clinical sensitivity”
is used in the United States.
clinical specificity – the proportion of subjects who do not have a specified clinical disorder whose test
results are negative or within the defined decision limit; NOTE: The term “clinical specificity” (United
States) is equivalent to “diagnostic specificity” (Europe).
clinical utility – value or benefit assigned to a particular outcome or state; diagnostic information that
contributes to the identification of a particular condition or disease; NOTE: Clinical utility can
encompass prognosis and monitoring response to therapy as well as diagnosis.
complementary – describing the property of two strands of nucleic acid that can hybridize by specific-
base pairing between the nucleotides.
control – (control material) a device, solution, or lyophilized preparation intended for use in the QC
process; NOTE 1: The expected reaction or concentration of measurands of interest are known within
limits ascertained during preparation and confirmed in use; NOTE 2: Control materials are generally not
used for calibration in the same process in which they are used as controls.
deletion – loss of one or more nucleotides from a nucleic acid sequence.
diagnostic accuracy – the ability of a test system to obtain the correct result.
diagnostic sensitivity – the proportion of patients with a well-defined clinical disorder (or condition of
interest) whose test values are positive or exceed a defined decision limit (ie, a positive result and
identification of the patients who have a disease); NOTE: The term “diagnostic sensitivity” (Europe) is
equivalent to “clinical sensitivity” (United States).
diagnostic specificity – the proportion of subjects who do not have a specified clinical disorder (or
condition of interest) whose test results are negative or within the defined decision limit; NOTE: The
term “diagnostic specificity” (Europe) is equivalent to “clinical specificity” (United States).
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diagnostic test – a measurement or examination of a diagnostic specimen for the purpose of diagnosis,
prevention, or treatment of any disease or the assessment of health or impairment of health of an
individual patient.
expression – the conversion of the genetic instructions present in a DNA sequence into a unit of
biological function in a living cell; NOTE: Expression typically involves the process of transcription of a
DNA sequence into an RNA sequence followed by translation of the RNA into protein; the RNA may be
spliced before translation to remove introns.
expression profiling – the measurement of the activity (ie, expression) of thousands of genes at once, to
create a global picture of cellular function, often using DNA microarrays.
external control – control material that mimics patient specimens and monitors the testing process from
specimen application to result interpretation.
false negative – a negative test result for a disease or condition when the disease or condition is present.
false positive – a positive test result for a disease or condition when the disease or condition is not
present.
feature – a single miniaturized hybridization reaction area on the solid surface of the microarray where
multiple copies of the single nucleic acid probe are immobilized.
fluorochrome – reagent that emits visible light when irradiated with excitation light of a shorter
wavelength.
fluorophore – fluorescent molecule that absorbs light energy and is promoted to an excited state that is
released as fluorescence, in the absence of a quencher, when the fluorophore falls back to the ground state
and releases the excess energy.
gel electrophoresis – separation of molecules, according to size and charge, in an electric field within a
matrix of agarose or polyacrylamide.
genotype – 1) the genetic makeup of an organism, or group of organisms, with reference to a single trait,
set of traits, or an entire complex of traits; 2) the specific allelic composition of a gene, or set of genes,
established at the DNA level.
guanine-cytosine (GC) content – the percentage of nitrogenous bases on a DNA molecule that are
guanine or cytosine.
hybridization – a base pairing of complementary strands of nucleic acid by hydrogen bond formation;
the binding of probe to specific nucleic acid sequences or amplification products; NOTE: Hybridization
can be performed with both nucleic acid target and probe in solution, or with either one bound to a solid
support such as a microtiter plate, glass, or membrane.
imprecision – dispersion of independent results of measurements obtained under specified conditions;

NOTE: It is expressed numerically as standard deviation or coefficient of variation.
in silico – an analysis performed on a computer or via computer simulation.
in vitro diagnostic (IVD) device – term used to describe those reagents, instruments, and systems
intended for use in the diagnosis of disease or other conditions, including a determination of the state of
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health, in order to cure, mitigate, treat, or prevent disease or its sequellae. Such products are intended for
use in the collection, preparation, and examination of specimens taken from the human body.
informed consent – the process by which a person voluntarily confirms the willingness to participate in a
particular medical procedure, after having been informed of all aspects of the procedure that are relevant
to the decision to participate.
inhibition – components within the clinical specimen or exogenous substances introduced during
specimen collection/processing that reduce amplification or detection.
interfering substances – endogenous (eg, blood components, acidic polysaccharides) or exogenous (eg,
talc, anticoagulant) substances in clinical specimens that can cause incorrect (false-positive or false-
negative) results in a test system.
internal control – a nontarget sequence present in the same sample tube, which is coamplified to identify
inhibition due to thermal cycler malfunction, suboptimal reagents or polymerase activity, or presence of
inhibitory substances in the sample matrix; a defined amount of internal control can be used to quantify
the target; NOTE: An internal control is placed in the same reaction tube as the specimen being analyzed.
Thus, an internal control will be subjected to exactly the same internal conditions and external parameters
as any measurand present in the tube.
laboratory-developed test (LDT) – assay that is researched, developed, prepared, and validated within
the laboratory institution that employs it for patient testing (eg, “in-house developed,” “user-developed”);
NOTE: This includes tests that are developed in the laboratory with measurands obtained from a single
source or multiple sources and validated for clinical use.
limit of blank (LoB) – the highest measurement result that is likely to be observed (with a stated
probability [alpha]) for a blank reaction; NOTE: LoB is not an actual concentration from which
measurements are obtained; the actual concentration at which a positive signal is ensured is called the
limit of detection. LoB is the highest apparent analyte concentration expected to be found when replicates
of a blank reaction (containing no analyte) are tested.11
limit of detection (LoD) – lowest amount of measurand in a sample that can be detected but not
quantified as an exact value; NOTE 1: In molecular methods and quantitative molecular methods, the
lowest concentration of measurand that can be consistently detected (typically, in greater than or equal to
95% of samples tested under routine clinical laboratory conditions) and in a defined type of sample;
NOTE 2: This concentration must yield an assay value that can be reproducibly distinguished from
values obtained with samples that do not contain the measurand.
locus – 1) the position of a gene on a chromosome; 2) the position on a chromosome of a DNA sequence
that is not necessarily contained within a gene; NOTE: Different forms (alleles) of the gene may occupy
the locus.
lysis – use of mechanical (eg, sonication) and/or enzymatic (eg, lysozyme) treatments, detergents, or
chaotropic agents to disrupt protective cellular structures, such as capsules, spores, cell walls, and
membranes in order to release endogenous nucleic acids; NOTE 1: Resulting lysates contain host and
microbial nucleic acids that must be stabilized to protect from cellular nucleases that are also released
during the lysis process; NOTE 2: Selected methods are chosen according to source organisms and
starting material, and may combine both cell disruption and inactivation of intracellular nucleases; NOTE
3: Ideal lysis procedures must be rigorous enough to disrupt the target cell type and gentle enough to
preserve target nucleic acid.
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matrix – (of a material system) components of a material system, except the measurand (modified from
ISO 15193,8 ISO 1519412).
measurand – quantity intended to be measured (JCGM 200:2012)6; NOTE: See analyte.
measurement procedure – detailed description of a measurement according to one or more measurement

principles and to a given measurement method, based on a measurement model and including any
calculation to obtain a measurement result (JCGM 200:2012)6; NOTE 1: A measurement procedure is
usually documented in sufficient detail to enable an operator to perform a measurement (JCGM
200:2012)6; NOTE 2: A measurement procedure can include a statement concerning a target
measurement uncertainty (JCGM 200:2012)6; NOTE 3: A measurement procedure is sometimes called a
standard operating procedure, abbreviated SOP (JCGM 200:2012)6; NOTE 4: See assay.
measuring interval – set of values of quantities of the same kind that can be measured by a given
measuring instrument or measuring system with specified instrumental measurement uncertainty, under
defined conditions (JCGM 200:2012)6; NOTE 1: This represents the interval of in vitro diagnostic (IVD)
examination results over which the performance characteristics of the IVD medical device were validated
by the manufacturer; NOTE 2: The measuring interval over which the performance characteristics of an
IVD medical device have been validated has been called the reportable range (ISO 18113-1)13; NOTE 3:
See reportable range.
medical laboratory – see clinical laboratory.
melting temperature (Tm) – temperature at which one half of complementary DNA molecules exist as a
double-stranded complex and the other half as single strands.
microarray (resequencing) – a microarray using specific nucleotide match/mismatch probes that allows
for the sequence of a genomic region to be determined.
microarray (tiling) – a microarray using overlapping or adjacent probes to interrogate a specific genomic
sequence region.
mismatch – a base pair difference between two nucleic acid sequences that renders them less than 100%
complementary.
multiplex – simultaneous detection of two or more nucleic acid targets in a single reaction.
mutation – an alteration in DNA sequence, whose molecular and clinical consequences are not
established, which could range from having no consequences to deleterious or beneficial consequences.
negative predictive value – the likelihood that an individual with a negative test result does not have the
disease, or other characteristic, that the test is designed to detect; NOTE: From predictive value theory,
the proportion of all negative tests that are true negatives.
nucleic acid extraction – the separation of nucleic acids (RNA, DNA) from biological materials to
perform amplification and analysis of nucleic acids; NOTE: Initially, cell membranes, walls, and other
protective structures must be lysed and cellular nucleases inactivated before separating desired nucleic
acids from the cellular debris. Extraction procedures may combine purification or isolation of nucleic
acids according to level of purity needed for downstream applications.
oligonucleotide – a short molecule, usually 6 to 100 nucleotides, of single-stranded nucleic acid, which is
usually synthesized chemically.
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oligonucleotide ligation assay (OLA) – a solution-based, sequence-specific enzymatic reaction

technology based on the joining of two adjacent oligonucleotide probes using a DNA ligase while they
are annealed to a complementary DNA target (eg, PCR product); an enzymatic DNA ligation reaction that
determines the genotype of a target.
pathogenic – capable of producing disease and can be used to describe both pathogenic bacteria and a
pathogenic mutation.
phenotype – the observed biochemical, physiological, and/or morphological characteristics of an

individual, as determined by the genotype and the environment in which it is expressed.
polymerase chain reaction (PCR) – a technology used to synthesize multiple copies of a defined region
of target DNA; NOTE: A common method of DNA amplification, using pairs of oligonucleotide primers
as start sites for repetitive rounds of DNA polymerase-catalyzed replication alternating with denaturation
in successive heating-cooling cycles.
polymorphism – a common variation in the sequence of DNA among individuals resulting in the
occurrence together in a population of two or more alternative genotypes, each at a frequency greater than
that which could be maintained by recurrent mutation alone.
positive predictive value – the likelihood that an individual with a positive test result has a particular
disease, or characteristic, that the test is designed to detect; NOTE: From predictive value theory, the
proportion of positive tests that are true positives.
precision (measurement) – closeness of agreement between indications or measured quantity values

obtained by replicate measurements on the same or similar objects under specified conditions (JCGM
200:2012)6; NOTE: Measurement precision is usually expressed numerically by measures of imprecision,
such as standard deviation, variance, or coefficient of variation under the specified conditions of
measurement (JCGM 200:2012).6
primer – an oligonucleotide usually of 18 to 25 bases which, when hybridized to a complementary strand

of target DNA and in the presence of DNA polymerase and nucleoside triphosphates, can act to initiate
DNA synthesis from its 3ʹ-OH end.
probe – defined segment of single-stranded nucleic acid used to identify specific DNA or RNA
molecules bearing the complementary sequence; NOTE 1: The probe often carries a label (radioisotopic,
chemical, or fluorescent), to allow detection of the probe and/or facilitate detection of the probe
recognizing its target; NOTE 2: In one usage, the probe is the molecule (eg, complementary DNA,
oligonucleotide) immobilized on the solid support to form the array.
proficiency testing (PT) – determination of laboratory testing performance by means of interlaboratory

comparisons; NOTE 1: Commonly, a program periodically sends multiple specimens or samples to
members of a group of laboratories for analysis and/or identification; the program then compares each
laboratory’s results with those of other laboratories in the group and/or with an assigned value, and
reports the results to the participating laboratory and others; NOTE 2: In some countries, PT can use
government-approved providers, which are different from interlaboratory comparisons. Also, and more
formally, known as “external quality assessment”; NOTE 3: In the United States, PT is also a
requirement for compliance with federal regulations for clinical laboratories.16
purification – separation of nucleic acids from other components of the sample and sample matrix;
NOTE: Methods applied are often combinations of extraction, precipitation, chromatography,
centrifugation, electrophoresis, or affinity separation.
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quality assurance (QA) – part of quality management focused on providing confidence that quality
requirements will be fulfilled (ISO 9000)14; NOTE 1: The practice that encompasses all procedures and
activities directed toward ensuring that a specified quality of product is achieved and maintained. In the
testing environment, this includes monitoring all the raw materials, supplies, instruments, procedures,
sample collection/transport/storage/processing, recordkeeping, calibrating and maintenance of equipment,
QC, proficiency testing, training of personnel, and all else involved in the production of the data reported;
NOTE 2: These activities include monitoring, evaluating, taking preventive and corrective actions, if
necessary, and monitoring the corrective actions for the preexamination, examination, and
postexamination phases; NOTE 3: QA is a comprehensive set of policies, procedures, and practices used
to monitor the laboratory’s entire testing process and ensure that the testing site’s results are reliable;
NOTE 4: QA is also described as a planned and systematic set of quality activities including risk
assessment.
quality control (QC) – part of quality management focused on fulfilling quality requirements (ISO
9000)14; NOTE 1: In health care testing, the set of procedures designed to monitor the test method and
the results to ensure appropriate test system performance, including testing control materials, charting the
results and analyzing them to identify sources of error, and evaluating and documenting any remedial
action taken as a result of this analysis; NOTE 2: The set of procedures undertaken in a laboratory for the
continuous assessment of work carried out in the laboratory and evaluation of tests to decide whether
these are reliable enough for release of results to the requesting health care provider. The procedures
should include tests on control material, results of which may be plotted on a quantitative control chart
showing upper and lower standard deviation (SD)–based ranges (eg, ± 1 SD, ± 2 SD, ± 3 SD), and may
also include statistical analysis of patient data (eg, moving averages). The main objective is to ensure day-
to-day consistency of measurements or observations, if possible, in agreement with an indicator of truth,
such as a control material with end-user assigned values.
real-time polymerase chain reaction (PCR)//homogeneous, kinetic polymerase chain

reaction (PCR) – method that combines PCR and fluorescent probe detection of amplified product in the
same reaction vessel; NOTE: The presence of amplified product is determined with each PCR cycle.
reference interval – interval between, and including, the lower reference limit to the upper reference
limit of the reference population (eg, 95% of persons presumed to be healthy [or normal]); NOTE: See
reference range.
reference material – material, sufficiently homogeneous and stable with reference to specified
properties, which has been established to be fit for its intended use in measurement or in examination of
nominal properties (JCGM 200:2012)6; NOTE: Material or substance, one or more of whose property
values are sufficiently homogeneous and well established to be used for the calibration of a measuring
system, the assessment of a measurement procedure, or for assigning values to materials (ISO 15195).15
reference method – a thoroughly investigated method, in which exact and clear descriptions of the
necessary conditions and procedures are given for the accurate determination of one or more property
values, and in which the documented trueness and precision of the method are commensurate with the
method’s use for assessing the trueness of other methods for measuring the same property values or for
assigning reference method values to reference materials.
reference range – the range of test values expected for a designated population of individuals (42 CFR
493)16; NOTE 1: For example, 95% of individuals that are presumed to be healthy (or normal); NOTE 2:
See reference interval.
repeatability (measurement) – measurement precision under a set of repeatability conditions of

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repeatability condition (of measurement) – condition of measurement, out of a set of conditions that
includes the same measurement procedure, same operators, same measuring system, same operating
conditions and same location, and replicate measurements on the same or similar objects over a short
period of time (JCGM 200:2012).6
reportable range – a set of values of measurands for which the error of a measuring instrument is
intended to lie within specified limits; NOTE 1: In this document, the term “reportable range” (United
States) is equivalent to “measuring range” (Europe); NOTE 2: The range of test-result values over which
the laboratory can establish or verify the accuracy of the instrument, kit, or test system measurement
response; NOTE 3: See measuring interval.
reproducibility (measurement) – measurement precision under reproducibility conditions of

reproducibility condition (of measurement) – condition of measurement, out of a set of conditions that
includes different locations, operators, measuring systems, and replicate measurements on the same or
similar objects (JCGM 200:2012).6
reverse transcription – the process of enzymatically synthesizing complementary DNA from an RNA
template.
sample – one or more parts taken from a primary sample (ISO 15189)10; NOTE 1: A sample is prepared
from the patient specimen and used to obtain information by means of a specific laboratory test; NOTE
2: A sample is any type of sample obtained from a patient, not limited to blood.
sensitivity (of a measuring system) – quotient of the change in an indication of a measuring system and
the corresponding change in a value of a quantity being measured (JCGM 200:2012)6; NOTE: Sensitivity
of a measuring system can depend on the value of the quantity being measured (JCGM 200:2012).6
sequence – the order of nucleotides (adenine [A], cytosine [C], guanine [G], thymine [T], or uracil [U]) in
a stretch of DNA or RNA.
sequencing – methods and procedures that determine the sequence of amino acids (in a protein) or
nucleotide residues (in DNA).
signal – a quantity that represents the measurand and which is functionally related to it; NOTE 1:
Generally, a signal is the chemical, radioactive, luminescent, or colorimetric output of an assay detection
system; NOTE 2: In microarray analyses, signal is the fluorescence intensity information captured by the
detection system.
signature – one or more nucleic acid sequences unique to an organism of interest that allows
discrimination from other organisms (either those closely related or those expected to be present in the
same sample type).
single-base chain extension (SBCE) – a solution-based enzymatic method for determining the identity of
a nucleotide base at a specific position along a nucleic acid; an oligonucleotide primer annealed to a
complementary sequence is enzymatically extended a single base by a nucleotide terminator
complementary to the nucleotide being identified.
Southern blot – a solid-phase membrane to which DNA, transferred from a gel after electrophoresis, is
bound so it can be hybridized with a labeled nucleic acid probe.
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specificity – (analytical) in quantitative testing, the ability of a measurement procedure to determine only
the component (measurand) it purports to measure or the extent to which the assay responds only to all
subsets of a specified measurand and not to other substances present in the sample;
NOTE: For qualitative or semiquantitative tests, the method’s ability to obtain negative results in
concordance with negative results obtained by the reference method.
specimen – (patient) discrete portion of a body fluid or tissue taken for examination, study, or analysis of
one or more quantities or characteristics, to determine the character of the whole (ISO 18113-1).13
spot – the physical location of a single hybridization event on the microarray solid surface with the final
fluorescent signal.
target – the area of the nucleic acid to be detected or amplified for detection or analysis; NOTE: As used
in microarrays, the target is the molecule in solution (new usage) not attached to the glass support
(original usage).
target-specific polymerase chain reaction (TS-PCR) – PCR method in which one or both primers of
the pair are designed to amplify a specific sequence to determine the presence or absence of a target
and/or differentiate a specific target within a mixture of target nucleic acids.
target-specific primer extension (TSPE) – a solution-based, sequence-specific enzymatic reaction

technology that can be used to assay multiple targets in a single tube; an enzymatic DNA polymerization
reaction that determines the presence of a specific target.
template – the portion of nucleic acid that acts to provide sequence information for the synthesis of a new
strand of DNA or RNA.
test system – a unit or device used to measure or assess the presence or absence of a particular substance,
or to quantify that substance, found in blood or body fluids; NOTE: A test system includes instructions
and all of the instrumentation, equipment, reagents, and/or supplies needed to perform an assay or
examination and generate test results.
trueness (measurement) – closeness of agreement between the average of an infinite number of replicate
measured quantity values and a reference quantity value (JCGM 200:2012)6; NOTE: Closeness of
agreement between the average value obtained from a large series of results of measurements and a true
value (modified from ISO 3534-1).17
unidirectional workflow – the manner in which testing personnel and patient specimens move through
the molecular amplification process in order to minimize cross-contamination and backflow of amplified
nucleic acid from previous reactions.
validation – confirmation, through the provision of objective evidence, that the requirements for a
specific intended use or application have been fulfilled (ISO 9000)6; NOTE 1: The use conditions for
validation can be real or simulated (ISO 9000)6; NOTE 2: A term used by the US Food and Drug
Administration for a study used to determine whether a test system meets user needs (21 CFR 820)18;
NOTE 3: The World Health Organization defines validation as “the action (or process) of proving that a
procedure, process, system, equipment, or method used works as expected and achieves the intended
result” (WHO-BS/95.1793)19; NOTE 4: Examples include validation of the process to use a new
diagnostic tool, such as an automated laboratory test system; information system; or evidence-based
medicine; NOTE 5: It is primarily the manufacturer’s responsibility to ensure that design goals are met
and performance claims are stated for a commercially developed assay or device, and a laboratory’s
responsibility for a laboratory-developed test.
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verification – confirmation, through the provision of objective evidence, that specified requirements have
been fulfilled (ISO 9000)6; NOTE 1: The ongoing process that confirms specified requirements
(predetermined by validation) are fulfilled; NOTE 2: In the context of this document, this is an end-user
(clinical laboratory) responsibility to confirm that manufacturer’s claims are met on the specific device
being used, and also that medical needs are met.
4.3 Abbreviations and Acronyms
A adenine
bp base pair(s)
C cytosine
CCD charge-coupled device
cDNA complementary DNA
CSF cerebrospinal fluid
CV coefficient of variation
DMD digital micromirror device
DMT 4,4ʹ dimethoxytrityl (an acid-labile protecting group used in oligonucleotide synthesis by
the phosphoradimite method)
DNA deoxyribonucleic acid
dsDNA double-stranded DNA
EDTA ethylenediaminetetraacetic acid
FDA US Food and Drug Administration
G guanine
GC guanine-cytosine
HA hemagglutinin
HBV hepatitis B virus
HCV hepatitis C virus
HIV human immunodeficiency virus
HPV human papilloma virus
ISO International Organization for Standardization
IQ installation qualification
IVD in vitro diagnostic
LDT laboratory-developed test
LoB limit of blank
LoC “lab-on-chip”
LoD limit of detection
MA data variability vs mean
OH hydroxyl
OLA oligonucleotide ligation assay
OQ operational qualification
PCR polymerase chain reaction
PGA photogenerated acid
PLPG photolabile protecting group
PQ performance qualification
PT proficiency testing
QA quality assurance
QC quality control
RNA ribonucleic acid
SBCE single-base chain extension
SD standard deviation
ssDNA single-stranded DNA
T thymine
Tm melting temperature(s)
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TS-PCR target-specific polymerase chain reaction

TSPE target-specific primer extension
UV ultraviolet
5 General Considerations in Developing Microarray Assays

Microarray testing has several potential applications in the clinical laboratory, particularly in pathogen
detection and genotyping assays.20 Although microarray test developers and manufacturers maintain the
responsibility for test platform and microarray design, clinical laboratories implementing these tests
should have a firm understanding of the principles of microarray testing in order to adopt these methods
successfully. This section describes the process of microarray test development and provides an overview
of the best practices with respect to assay design.
5.1 Microarray Test Workflow
Most microarray tests will consist of a combination of steps that may include:
 Nucleic acid extraction/purification

 Amplification
 Labeling
 Hybridization and washing
 Detection
 Data analysis and interpretation
For additional information on each step of the process, refer to the CLSI documents listed in Table 1.
Table 1. CLSI Documents Pertaining to Microarray Analysis Workflow

Microarray Test Step CLSI Document
Nucleic Acid Extraction/Purification MM0621
MM0922
MM121
MM1323
MM1724
MM1925
Amplification MM0326
MM0621
MM0922
MM121
MM1724
MM1925
Labeling MM0326
MM121
Hybridization and Washing MM0326
MM121
Detection MM0326
MM0621
MM121
Data Analysis and Interpretation MM0621
MM121
MM1724
MM1925
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Each of these steps requires careful design and control considerations. This section deals specifically with
those design aspects that are specific to microarray testing, particularly the target selection criteria
affecting hybridization, data analysis, and interpretation. Additional information on microarray
measurement procedures can be found in CLSI document MM12.1
5.2 Assay Design Considerations
5.2.1 Target Selection
The selection of microarray targets should be based on the clinical significance of the infectious agents or
sequence variants for a defined patient population. Candidate nucleotide sequences need to be submitted
to rigorous bioinformatics analysis to ensure adequate selectivity based on in silico hybridization
conditions.27 This can be accomplished by searching nucleotide sequence databases for sequence
homology to selected targets or by performing whole genome analysis for specific sequence selection de
novo.28 For organism detection, the selected target should be shown to be conserved within clinically
relevant strains while maintaining sequence differences from other organisms including those of
genetically related species. Multiple probes can be designed to accommodate sequence variation in order
to differentiate species with similar genetic elements. Using this multitarget approach, microarray testing
platforms can be used to distinguish pathogenic species from closely related nonpathogenic organisms.
Often, highly conserved targets such as ribosomal RNA, RNA polymerase, or other housekeeping genes
are useful organism or strain markers, but each selected sequence must be shown to function
appropriately when compared to available databases. Finally, a general database search (eg, GenBank
available from the National Center for Biotechnology Information at
http://www.ncbi.nlm.nih.gov/genbank) should be done to determine possible unexpected sequence
homology to other organisms and to the human genome.
5.2.2 Primer Assessment
In most microarray designs, probe sequences would not be expected to interact and do not need to be
analyzed for homology to each other. However, primer sequences for nucleic acid amplification may be
used in solution in a multiplex reaction and such potential primer-primer interactions need to be analyzed.
Several bioinformatics tools are available to aid in the design of multiplex primer sets in order to avoid
nonspecific primer amplification.29-31 While bioinformatic approaches can provide valuable information
about potential primer interactions, dissimilar systems and assays will be affected differently by primer
interactions. Therefore, empirical testing of the PCR reaction or multiplex should be the final measure for
determining if primer interactions are having a negative effect on the PCR reaction.
5.3 Probe Design and Update
5.3.1 Physical Probe Arrangement
The specifications of each microarray platform will typically determine the physical arrangement of
nucleotide probes, including the binding surface, attachment chemistry (ie, probe attachment method
chemistry), linker region (ie, spacer between the probe and the solid surface), probe length, and any
chemical modifications. The assay developer will design probes compatible with these fixed requirements
with consideration for the optimal probe length and guanine-cytosine (GC) content to allow for effective
hybridization. From a manufacturing point of view, the microarray feature is the area of each probe
location, which influences the probe density and, subsequently, the level of parallelism (ie, redundancy)
in the analysis. The available array area and feature size will determine the total number of probes that
can be applied to the microarray and its potential applications, such as individual sequence detection,
tiling, resequencing, etc.32 The physical arrangement of probes for slide-based platforms is best
distributed in space along the array surface, minimizing the potential effects of localized interferences,
such as small bubbles and precipitates. Incorporation of control probes is essential and may include
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exogenous sequence detection for spiked-in controls or endogenous sequence detection compatible with
the expected sample type.
5.3.2 Sequence Selection and Updating
The selection of microarray probes should be based on the most currently available sequence data.
However, the amount of publicly available sequence data is increasing exponentially and any design may
rapidly become outdated. Novel and unexpected genetic changes in infectious agents require the
microarray user to be aware of potential strain types that cannot be detected with a certain assay and to
understand its limitations. Updates to existing probes and assay designs should be undertaken judiciously
by microarray assay developers, particularly when new genetic variants are found to be circulating in the
population. In some jurisdictions, regulatory requirements for changing microarray assay design are
significant and manufacturers and laboratories should be aware of the considerable effort required before
changing assay designs. Laboratories should consider the limitations of each microarray assay for their
patient population, and either use an alternative assay or a reference laboratory and public health
resources for high-suspicion cases of infection with novel or emerging agents that may not be detected
with current platforms.
5.4 Preexamination Requirements
5.4.1 Collection and Handling
The collection and handling of specimens before examination should follow good laboratory practices as
described in CLSI document MM13.23 Furthermore, each laboratory performing these (and other)
molecular tests should have a written policy on acceptance and rejection criteria that has been agreed to
by those who order and/or procure the samples to be tested. The appropriate specimen type, collection
method, and transport matrix will be determined by the clinical presentation and type of infection for
which the assay is designed to detect. Modifications to IVD procedures, such as changes in the specimen
type analyzed or patient population, need to be validated by the performing laboratory. Both analytical
and clinical validation studies should be performed to validate the modification to the IVD procedure.
Laboratories should offer clinicians specific guidance and training in optimal patient sample collection
techniques. Some assays may require purified microbial isolates for typing and characterization while
others may examine direct patient specimens for detection of causative infectious agents. In particular,
direct patient specimens can have substantial variation in endogenous (eg, blood and mucus) and
exogenous (eg, drugs, metabolites, creams) substances that can affect assay performance. Transport
matrix containing known inhibitors of PCR, such as heparin, should be generally avoided. For blood
samples, EDTA and acid citrate dextrose are suitable alternative anticoagulants.33 For some types of
applications, tubes containing RNA stabilization reagents or viral inactivation reagents may be
preferred.34 Endogenous PCR inhibitors, such as heme and high levels of protein, can be present in
samples and should be shown not to interfere with assays if they are expected to be present in the
specimen type examined.35
5.5 Examination Requirements
5.5.1 Specimen Storage
After collection, specimen transport and storage conditions should be designed to optimize recovery of
nucleic acids for analysis. Samples may be transported and stored at room temperature, refrigerated or
frozen, and the measurand stability should be determined for expected laboratory operating conditions.
Recommendations for specific sample type storage can be found in CLSI document MM13.23
Laboratories performing microarray assays should verify appropriate transport and storage conditions,
particularly if they are different from those approved for a specific assay. Sample stability testing should
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also be performed to determine the acceptable ranges and conditions under which samples will retain
stability and can be used for the test (eg, time, temperature).
5.5.2 Automated Instrumentation
Microarray analyses are typically complex, multistep procedures requiring careful handling and attention
to detail. Robotic instrumentation, which is available for some processes, can reduce imprecision and
errors from manual pipetting and sample handling steps. Fully automated “hands-off” systems are in
development and will soon be available. When automated systems are used, laboratory personnel need a
detailed understanding of the robotic processes involved in order to adequately verify instrument
performance and troubleshoot potential errors or system failures. For example, investigation of potential
carryover contamination requires knowledge of the sample path and order taken in the automated
instrument.
5.5.3 Sample Nucleic Acid Extraction
The goal of the extraction process is to purify the nucleic acid of interest while removing contaminants
and inhibitors of downstream reactions. Several commercial reagents, kits, and instruments are available
for high-efficiency nucleic acid extraction. The clinical laboratory should verify the suitability of each
extraction protocol for the assay’s specimen type(s), collection device, and type of nucleic acid needed,
and microorganisms that will be detected by the assay. The extracted nucleic acid may consist of DNA
and/or RNA; may be derived from genomic/fragmented genomic material, episomes/plasmids, or
transcribed RNA; and may be derived from various organism types, including bacteria, mycobacteria,
virus particles, fungi, or parasites. Preincubation and lysis conditions will differ depending on input
material, which may consist of purified microbial isolates or direct clinical specimens (eg, blood;
respiratory; body fluid; frozen tissue; formalin-fixed, paraffin-embedded tissue). Difficult-to-extract
sample types, such as tissues, and some microorganisms, including gram-positive bacteria, mycobacteria,
or fungi with rigid cell walls may need enzymatic or mechanical pretreatment. The extraction efficiency
may be of prime consideration for assays aiming to detect low levels of pathogens in clinical samples,
because this is a major determinant to achieve clinically necessary limits of detection (LoDs). Carrier
material such as linear acrylamide or exogenous RNA can be added to increase extraction efficiency as
long as these materials do not interfere with downstream analysis.36
5.5.4 Amplification
Most microarray applications, particularly in infectious disease detection, will require amplification of
nucleic acid during analysis. Amplification techniques that will allow adequate detection in a background
of host and other microbial flora should be selected. Strategies for amplification include multiplex PCR,
DNA or complementary DNA (cDNA) targets generated through reverse transcription, or sequence-
independent techniques designed to amplify whole genomic material.37 While most current clinical
applications have focused on multiplexed specific amplification, it is difficult to amplify more than 20
targets using this approach, due to primer interference, preferential amplification of certain target
sequences, and spurious amplification products.38,39 Multiplex PCR requires careful primer design40 and
optimization of primer, buffer, template concentrations, and cycling temperatures in order to ensure
specific and uniform amplification.41 Alternative approaches such as spatial primer separation on solid
surfaces or within emulsions are possible,42,43 but may not be compatible with downstream microarray
analysis. Very broad pathogen detection and characterization assays may require more generalized
amplification methods. Several techniques can be used to generate relatively unbiased whole genome
amplification, such as fragmentation and adaptor ligation followed by PCR and random priming,37
multiply primed rolling circle amplification, and multiple displacement amplification.44 Regardless of the
amplification method used, it should be shown to amplify the pathogen targets of interest adequately for
array detection. The analytical LoD for each microarray target will vary by microarray platform and
hybridization/experimental conditions, but has been shown to be approximately 10 pg of DNA for several
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Escherichia coli genes.45 Specificity is somewhat less of a concern because downstream hybridization and
wash steps will remove nontarget amplicons, but large amounts of nonspecific amplification, such as with
primer dimers, can reduce amplification efficiency and interfere with hybridization kinetics. Therefore,
the amplification reaction(s) should be optimized to minimize off-target amplicons. Some microarray
methods may analyze part of the amplified material as QC for the assay, confirming the expected amount
and size distribution of amplicons before further analysis.
5.5.5 Labeling and Purification
Labeling of nucleic acid amplification products for microarray detection can take place during
amplification or as a separate step. Biotin and fluorescently labeled nucleotides are commonly used labels
and the choice of label largely depends upon the microarray testing platform and detection instrument.
Label incorporation should be shown to be quantitative and efficient and the amount of incorporated label
may be measured spectrophotometrically to ensure adequate amounts. Once amplified and labeled, the
target nucleic acid may have to be purified before applying for hybridization. Purification techniques
should be quantitative and efficient without substantial loss of amplified and labeled product.
5.5.6 Hybridization and Washing
The conditions for hybridization are based on the nature of the interaction with oligonucleotide probes on
solid surface or microsphere beads. Several variables need to be taken into account, including
temperature, probe, product lengths, GC content, product concentration, salt concentration, and addition
of other buffer agents such as denaturing reagents. Proper binding conditions can be predicted based on
knowledge of typical probe interactions and melting temperatures (Tm), but should be verified and
optimized experimentally using known positive samples for typical agents detected in the assay. 46,47 The
composition and temperature of wash buffer solutions is critical in order to efficiently remove any
nonspecific signals while maintaining true probe binding. During this step, care should be taken to ensure
that the signal label remains active (eg, by limiting the duration of light exposure for fluorescent labels).
Some fluorescent labels, notably Cy5 dye, are susceptible to ozone degradation, which could be a concern
at some testing locations.48-50 Leakage of the sample through gaskets or the presence of bubbles can
adversely affect the background signal and cause signal artifacts precluding proper analysis.
5.5.7 Detection
Signal detection is performed by specialized instruments that are able to measure the labeled nucleotide
signal for each probe on the array. Solid-phase microarrays use the probe location as a marker for the
sequence each spot is known to contain, while liquid-phase microbead arrays use a flow cytometer to
measure the signal intensity for each labeled bead population. The end user may have little control over
the instrument specifications, but should still ensure that proper maintenance is performed, because
changes in instrument calibration and alignment can have major detrimental effects on performance.
Solid-phase scans should be done at high enough resolution to differentiate probe spots from background
signals. If fluorescent light filter sets are used, the settings should minimize the amount of the signal in a
specific detection channel that falls into alternate detection channels so that a high positive signal is not
interpreted as positive for more than one signal moiety.
5.5.8 Controls
Inclusion of appropriate controls and quality indicators with each run is necessary to ensure adequate
assay performance.51 Controls can be added at various steps in the microarray test procedure and different
types can be used to investigate various steps of the entire assay. Controls can be internal to each sample
(spiked or endogenous) or external to patient samples as a check on assay performance. Extraction
controls are best designed to mimic the measurand being tested in patient samples and should go through
the entire testing process. Run controls may or may not be extracted, but are designed to ensure that the
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postextraction amplification, labeling, and detection are behaving as expected. Some microarray assays
may have additional controls, such as prelabeled nucleotides used for spot localization.52 Controls may be
derived from the assay manufacturer, third party suppliers, or laboratory-derived material, such as
cultured organism, cloned nucleic acid, or previously tested patient samples.
Microarray methods allow for multianalyte testing, and multiple controls can be used within a single
sample in order to verify adequate performance of different parts of the assay. Like other assays,
microarray controls should be used at concentrations reasonably near the LoD. If the control levels are too
high, partial inhibition of amplification may not cause failure, and erroneous patient results could be
reported. In some microarray tests, having multiple levels of control material could be useful to determine
the dynamic range, although currently most of these tests are qualitative rather than quantitative.
Laboratories should comply with minimum existing local and regional regulatory requirements in terms
of the composition, number (internal and external), and frequency of use or controls analyzed. However,
the director may decide to have additional controls based on the complexity and regulatory status of the
particular infectious disease assay being implemented. Additional controls may also be used for
troubleshooting of assays where false positives (ie, contamination) or false negatives (ie, reagent failures
or matrix inhibition effects) are suspected.
5.6 Postexamination Requirements
Appropriate data analysis and interpretation are key steps to generating test results that are meaningful
and useful for patient care. Microarray data can require complex manipulations to extract the result, and
the end user should be aware of the process and nature of the computational steps involved.
5.6.1 Preliminary Signal Analysis
Raw microarray data in the form of image files or fluorescent signal counts are transformed initially to
allow for meaningful analysis. Spot intensity refers to the overall target signal generated for each
microarray probe and is typically presented in relative units. The microarray instrument software will
present this data for further analysis using proprietary or open-source computing methods.
5.6.2 Data Normalization
Some types of microarray experiments will require substantial data normalization, although this typically
applies to gene expression experiments, which are out of the scope of this document. Data normalization
can be used to investigate relationships between related biological samples, and is most often used as a
research tool for comparative experiments.53 For more information on data normalization, see Section
10.2.5 and CLSI documents MM06,21 MM12,1 MM17,24 and MM19.25
5.6.3 Background Subtraction
Microarray background signal can arise from several sources including incomplete washing, array
features that bind dye or nucleotides, and imprecision in spot localization during image acquisition.54
Background subtraction can be applied to high-density microarrays to correct for nonspecific probe
binding. Color compensation can also be used in two-color array analyses in order to minimize signals
from spectral overlap. The nature of the sample type can affect the background signal on microarrays
greatly, and for clinical assays, it may be preferable to analyze specific probes rather than rely on
substantial background subtraction techniques.
5.6.4 Pattern Interpretation
The key to infectious disease microarray data analysis is in the interpretation of the signal based on probe
binding to identify the presence or absence of a particular microbe or genotype. The most straightforward
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type of analysis relates each probe signal to a predefined threshold value for positivity. Such analyses
may also involve an indeterminate range. The setting of these thresholds is of critical importance and is
based on extensive knowledge of the assay’s performance characteristics, including the signal dynamic
range, limit of blank (LoB), and nonspecific reactivity in various sample matrixes. Extensive studies are
performed by the manufacturer in the case of IVD devices, and by the laboratory in the case of LDTs, to
evaluate the sensitivity and LoD. These studies should define threshold values that allow reproducible
detection of low levels of organisms (ie, slightly above the test LoD) while maintaining high specificity
by not exhibiting a false-positive signal or resulting in various types of samples not containing the agent
or genotype of interest.
More complex methods of interpretation can be used to define the presence or absence of target
nucleotide sequences, particularly with high-density microarrays where hundreds to thousands of probe
sequences may be interrogated. In these types of assays, the presence of a particular organism may be
related to a certain pattern of probe signals, where similar strains may have overlapping, but not identical,
signal patterns. A tiling or resequencing microarray design can generate substantial microbial sequence
information with many nucleotide changes seen for each sample tested. In order to ensure the assay’s
ability to detect additional or novel changes, the analytical accuracy of these base pairs (bp) or genotyping
calls should be verified using a representative sample. Testing of individual microbial isolates for detailed
genotyping information will typically reveal only a single pattern for interpretation. However, if a mixed
culture is tested or the organism contains genetic rearrangements, the analysis may become more
complicated. When direct patient samples are tested, the interpretation should accommodate mixed flora
in the sample. Identification of a specific organism and known resistance markers for that organism
cannot always ensure that the two are related. For example, the mecA gene for methicillin resistance can
be present in both pathogenic Staphylococcus aureus and coagulase-negative staphylococci, which could
make it difficult to know which organism harbors the resistance element in a mixed sample without
knowing the strain-specific markers.55,56 For this reason, interpretation of multiplex genotyping results
from direct patient specimens should be performed with caution, especially from nonsterile sites.
5.6.5 Reporting
Patient reports from infectious disease microarray testing should be clear, concise, and contain actionable
information for diagnosis and treatment. Results from highly complex assays may require review and
summarization by a qualified provider or laboratory professional. Positive results should be prominently
displayed and pertinent negative results noted. As the scale and scope of infectious disease panels
increase, it will not be feasible or desirable to provide the entire list of genetic targets assayed; however,
that information should be readily available to medical providers. Laboratories providing these assays are
also advised to make their assay performance characteristics available to clinicians along with the clinical
significance of the results.
5.6.6 Novel or Unexpected Results
Genetic modifications in infectious organisms may lead to unexpected results for microarray assays.
Laboratories should be aware that these changes and modifications could lead to strains that will not be
detectable in their assay. Samples from patients with highly suspicious presentations for infection should
be subjected to alternate testing strategies, or sent to reference laboratories for further analysis.
In rare cases, microarray assays may detect organisms of public health significance that are suspected to
be highly infectious, pathogenic, or resistant to treatment. Atypical results demonstrating previously
unrecognized hybridization patterns could represent a new strain variant, such as occurred with pandemic
2009 H1N1 influenza.57 If these are truly novel or not previously seen in a certain geographic area, the
laboratory may have tested no similar species and may be unable to verify the identity or characteristics
of the agent in question. If these results are seen, laboratories should communicate with public health
officials and treating physicians regarding the nature of the suspected infectious agent and the limitations
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of the assay being performed. Based on these discussions and the clinical presentation, decisions may be
made to isolate the patient and/or treat empirically while samples are sent to referral public health
laboratories for further analysis and confirmation. Once the genomic sequence of the agent in question
has been determined, in silico analysis can be performed to investigate whether the results are consistent
with the predicted hybridization profile seen initially.58 The laboratory plays a key role in ensuring
effective communication among the team of providers and public health officials. Furthermore, the results
of confirmatory testing will indicate the accuracy of the microarray test method.
5.7 Contamination Control
Prevention of contamination in microarray analysis of infectious organisms presents some unique

challenges. Because clinically significant organisms can be present in the laboratory environment, the
laboratory should have both procedures for preventing contamination of the testing process and
procedures for recognizing contamination when it does occur (see CLSI document MM1925).59
Depending upon the type of organism being tested, contamination can result from:
 Previously tested patient samples
 Organisms (virus or bacteria) cultured from patient samples
 Control materials (eg, cultured reference strains, purified nucleic acid from reference strains,
synthetic nucleic acid specific to the test assay)
 Reagent and consumables contamination
 Vaccine materials handled by the clinic where samples are obtained59-61
 The laboratory worker handling the sample
Typically, the largest risk for contamination comes from DNA amplicons from previously amplified
material that are present at very high concentrations, whether of patient or control origin (see CLSI
document MM1925).62 The best defense against contamination is the development and rigorous
application of procedures that minimize contamination risk.63 These procedures will establish a
unidirectional workflow with designated preamplification and postamplification work areas. Ideally, the
preamplification area is in a physically separate room under positive pressure to avoid the introduction of
aerosols containing amplified material. Staff should avoid introducing contaminated material by wearing
appropriate clothing protectors, such as disposable gowns, hair covers, and booties, and by changing
gloves frequently between processing steps. Each sample processing step should be designed to avoid
contamination between specimens and among steps by using filter pipette tips and unique pipette sets
where necessary. Separate biosafety hoods or “dead-air” boxes can be used for each processing step,
including patient sample aliquotting, extraction, master mix preparation, and the addition of the extract to
the master mix before closing the tube and bringing to the postamplification area. Reagents should be
stored in small aliquots that can be disposed of if necessary and should be kept separately from patient
samples and control materials to avoid reagent contamination.
The postamplification work area should be kept clean and have surfaces that can be decontaminated
easily. Microarray tests often involve manipulation of amplified nucleic acid by opening tubes and adding
reagents for further incubations, which leads to the risk of cross-sample contamination. Some test
methods can include enzymatic reactions designed to reduce the risk of amplicon contamination (ie,
incorporation of a uracil-N-glycosylase nuclease step before amplification).64 To reduce the risk of cross-
sample contamination, careful sample handling is necessary, and the work area should be cleaned with
10% household bleach or commercially available DNA degradation reagents, usually followed by a
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deinonized water rinse and 70% ethanol to dry.65,66 UV light may be used to crosslink DNA on surfaces;
however, its potential damage to instruments and exposure to staff should be considered.67 Periodic
testing of work surfaces and items used in processing samples, such as pipettes and thermocyclers, should
be performed, and any detected contamination should be dealt with by thorough cleaning and retesting to
ensure decontamination effectiveness. As mentioned above, it may be useful to test laboratory workers
who have symptoms consistent with disease caused by the pathogen being detected. In the extreme, it
may be best to reassign such workers to other tasks until they test negative for the organism in question.
Contamination events can be detected in a variety of ways. A microarray assay result that is negative
when tested with standard reagents and a patient blank gives some confidence that the reagents are free of
contamination, but this is only as good as the sensitivity of the assay and will not pick up infrequent or
point source contamination. The completeness of the microarray analysis is also a strength in that some
forms of contamination, such as control material (eg, reference strains or synthetic nucleic acids) or
vaccine strains, may have characteristic signatures that are evident in the microarray data analysis. If
contamination is not ruled out by these methods, it may be necessary to amplify and sequence a portion of
the suspected contaminant. In this case, identical sequence between several different patient samples
would raise the suspicion that a contamination event had occurred, depending upon the amount of
variation found naturally in the organism. However, it must be emphasized that further manipulation of
the amplicon is inherently risky and should only be done as a last resort by highly trained personnel.
6 Platform Overview
Microarrays can be processed on a variety of platforms, including printed solid surface microarrays, in
situ–synthesized oligonucleotide microarrays, high-density silicon microarrays, electronic microarrays,
suspension liquid (bead) microarrays, and small volume PCR arrays. Details of major features of the
microarray technologies are summarized in Table 2.
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Table 2. Comparison of Major Features of the Microarray Technologies

Maximum
Number Oligonucleotide Binding
Section Technology Feature Size of Features Size Technology
Printed Solid Surface Covalent
6.1.1–6.1.3 20 100–150 nm Up to 30 000 25–80 base
Microarrays coupling
Parallel
6.2.2.2.1 In Situ Synthesis by Printing68 70–100 µm Up to 1 000 000 60 base
Synthesis
In Situ Parallel Synthesis Using
Parallel
6.2.2.2.2 Electrochemical < 100 µm Up to 1 000 000 35 base
Synthesis
Methodologies/Technologies69,70
Maskless Photogenerated Acid- Parallel
6.2.2.2.3 > 40 µm Up to 1 000 000 ≤ 100 base
Controlled In Situ Synthesis71-73 Synthesis
Mask-Directed In Situ Parallel
Parallel
6.2.2.2.4 Synthesis Using Photolabile < 25 µm Up to 1 000 000 25 base
Synthesis
Protecting Group74,75
Maskless In Situ Parallel Synthesis
Parallel
6.2.2.2.5 Using Photolabile Protecting < 25 µm > 1 000 000 ≤ 24 base
Synthesis
Group76
Spotting of Presynthesized Covalent
6.3.2.1 20 µm Up to 15 000 ≤ 75 base
Oligonucleotide Probes77-79 coupling
Van der Waal
6.3.2.2 Self-Assembling of Silica Beads80 3 µm > 1 000 000 40–60 base
forces
Streptavidin-
6.4 Electronic Microarrays81,82 50 µm 400 20–30 base
biotin
Suspension Liquid Bead Covalent
6.5 6–250 µm* Up to 500 15–20 base
Microarrays83 coupling
Customized 96- and 384-Well PCR 18–24 base† Small volume
6.6.1 1 µL volume 96–384 wells
Arrays84 PCR primer PCR reaction
18–24 base† Small volume
6.6.2 Microfluidic Card85,86 1 µL volume 384 wells
PCR reaction
Nested PCRs in Small Volume 18–24 base† Small volume
6.6.3 1 µL volume 102 wells
Arrays87 PCR reaction
3072 “through 18–24 base† Small volume
6.6.4 Nanoliter Volume PCR Arrays88,89 33 nL volume
holes” PCR reaction
*
Diameter of microbead used in this assay rather than printed probe.
†
Length of PCR primer rather than probe size.
Abbreviation: PCR, polymerase chain reaction.
6.1 Printed Solid Surface Microarrays
Printed solid surface microarrays were originally developed by printing multiple nucleic acid probes on
filter paper membranes and using standard Southern blotting chemicals and techniques.90,91 As the
potential for microarray technology was realized, further printing techniques were developed using
standard glass microscope slides and silicon.92,93 These materials provide a great improvement over the
standard filter paper (including linear array),92-94 because it is stable through the most stringent of washes,
is temperature resistant, and has only minimal background fluorescence levels.95
6.1.1 Probes Used
Nucleic acid probes for printed solid surface microarrays can include double-stranded DNA (dsDNA),
PCR amplicons from shotgun library clones, or cDNA, and are generally 200 to 800 bp in length.20 A
second type of nucleic acid probe is an oligomer, which consists of single-stranded DNA (ssDNA). These
are synthesized by commercial companies in lengths between 25 to 80 bases.20
6.1.2 Probe Printing
There are two common printing methods for fabricating printed solid surface microarrays—a noncontact
method and a contact method. The noncontact method uses an inkjet style of printing and the contact
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method involves the capillary actions of small quill pins to dispense the suspended probes in one of the
various binding buffers currently available.
The printed spots are typically between 100 and 150 nm in diameter and the microarrays are available
with densities ranging from 40 features up to 30 000 features.20
6.1.3 Oligomer Binding
For dsDNA probes, early studies determined that the positive charge of the silica in glass slides provides
a good binding media for the negatively charged backbone of DNA.96 Later studies determined that the
coating of the glass slides by amines and UV crosslinking enhances the binding. This enhancement is due
to the covalent bonding of the nucleic acid oligomers, via the probe’s thymidine bases, to the amine
coating on the slides.95 The ssDNA oligomer probes, with their smaller size, require UV crosslinking to
coated slides that provide an aldehyde or epoxy functional group.20 Technology has continued to improve,
and several companies have begun to manufacture their own coated slides, resulting in more consistent
spot printing and morphology. One major improvement of manufactured slides is a hydrophobically
patterned surface that prevents irregular spotting by controlling the location of the probes printed in
hydrophilic buffer (see Figures 1a and 1b).
Abbreviations: PCR, polymerase chain reaction; RNA, ribonucleic acid.

Figure 1a. Workflow Summary of Printed Microarrays. Probes are synthesized or PCR amplified and
spotted onto coated microarray slides. In this figure, nucleic acids are extracted and fluorescently labeled
before being hybridized to printed probes. Hybridization is detected by a fluorescent reader, providing
data for analysis. (Adapted with kind permission from Springer Science and Business Media, from: Ehrenreich A. DNA
microarray technology for the microbiologist: an overview. Appl Microbiol Biotechnol. 2006;73(2):255-273.)96
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Abbreviations: LoC, “lab-on-chip”; PCR, polymerase chain reaction.

Figure 1b. Low-Density Oligonucleotide Printed Microarray. Shown here is an LoC that integrates
both the PCR amplification and the microarray detection. Technological details of LoC: (a1) scanning
electronic microscopy vertical section of PCR silicon buried channel (× 150); (a2) scanning electronic
microscopy images of LoC outlet (× 70); (a3) integrated sensors composed by Al/Si/Cu metal layers.
(Adapted with kind permission from De Gruyter, from: Foglieni B, Brisci A, San Biagio F, et al. Integrated PCR amplification
and detection processes on a Lab-on-Chip platform: a new advanced solution for molecular diagnostics. Clin Chem Lab Med.
2010;48(3):329-336.)92,93
6.2 In Situ–Synthesized Oligonucleotide Microarrays
6.2.1 Introduction
Probes in this class of microarray consist of oligonucleotides (generally 25 to 75 bp long) that are
synthesized in situ on the surface of the solid supports.
Compared to long DNA (eg, cDNA) probes, DNA probes based on oligonucleotides offer several
advantages such as:
 Better sequence reproducibility during production, because they are prepared by well-controlled
automated chemical synthesis processes
 Reduction of the risk of cross-hybridization to nontarget sequences due to sufficiently long

overlapping regions that forms stable duplexes between a probe and a target
 Greater ability to pick sequence elements with favorable hybridization properties (length, GC content,
sequence variation)
The in situ methodology allows high parallel synthesis of oligonucleotides so that it is possible to obtain
oligonucleotide microarrays with improved design in a short amount of time and at a low cost.
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This class of microarray is used for applications related to specific gene profiling and is also available for
DNA analysis applications, such as copy number analysis, drug metabolism analysis, genome-wide
genotyping, molecular cytogenetics, resequencing analysis, and targeted genotyping analysis.
This section provides an overview of the main technologies and methods currently used for in situ–
synthesized oligonucleotide microarrays.
6.2.2 Methods and Technologies Overview
6.2.2.1 Phosphoramidite Chemistry
The in situ–synthesized oligonucleotide microarrays are developed via parallel synthesis based on
protection-deprotection chemistry. Among the several methods developed for the in situ oligonucleotide
synthesis, the phosphoramidite chemistry97 is one that is regularly employed and routinely achieves better
than 98.5% stepwise yield on a DNA synthesizer. Conventional synthesis is carried out on a variety of
solid supports (see CLSI document MM121), and involves multiple cycles of four main reaction steps:
1. Chemical deprotection of solid support-linked nucleotide to release the terminal hydroxyl (OH) group
(HO5ʹ-Rx)
2. Coupling of the terminal OH group with 5ʹ-O-4,4ʹ dimethoxytrytil (DMT)–protected

nucleophosphoradimite monomer (DMT-O5ʹ-Ry) to form the DMT-O5ʹ-Rx O5ʹ-Ry derivative
3. Capping of the free OH-deprotected group that fails to couple
4. Phosphite internucleotide oxidation of DMT-O5ʹ-Rx O5ʹ-Ry dimer bond to the final nucleotide
phosphate tri-ester
At the end of the synthesis, the protecting groups of both nucleobases and phosphotriester are chemically
removed by appropriate treatment.98,99
The key advantages of this approach are the large number of parallel reactions conducted in a small area
(eg, less than 1 cm2) with automated and standardized reaction processes that allow array miniaturization,
regularity in spot alignment and geometry, and high uniformity.
To optimize the array manufacturing quality, it is important that the probe be covalently linked to the
surface and extended by sufficient length from the surface to minimize steric hindrance.100 This is
achieved by the incorporation of two specific moieties into the probe chain: the spacers and linkers.
Spacers allow the probe to extend from the surface while linkers permit covalent bonding of the probe
with the surface. They can also be employed to modulate the probe surface density and modify the surface
properties, such as charge, adhesion, autofluorescence, and hydrophilicity/hydrophobicity, which are key
factors for obtaining uniform hybridization signals.101-109
6.2.2.2 Methods to Prepare In Situ–Synthesized Oligonucleotide Microarrays
There are several methods that have been reported to prepare in-situ–synthesized oligonucleotide
microarrays. These are illustrated in the following sections.
6.2.2.2.1 In Situ Synthesis by Printing
This method employs printing technologies (inkjet or other) to deliver the chemical reagents/solutions for
the oligonucleotide synthesis directly to the spot onto the microarray solid surface. It uses the
phosphoradimite chemistry (see Section 6.2.2.1) and the reaction is gated through delivery of adenine (A),
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cytosine (C), guanine (G), and thymine (T) phosphoradimite to specific sites (see Figure 2).110-113
This method enables the synthesis of oligonucleotides of various lengths and is flexible in chip design. It
has been used successfully to synthesize a 20 000 spot microarray on 2.5 × 7.5 cm2 glass slides with 60-
base oligonucleotides and spot features of about 100 µm.
Figure 2. In Situ Inkjet-Printed Long Oligonucleotide Microarray. (A) Noncontact in situ inkjet
printing technology deposits a small volume (picoliters) of nucleotides to the first layer on the microarray
surface. (B) Subsequent rounds of base-by-base printing extend the length of specific oligonucleotide
probes. (C) Close-up of a nucleotide base being added to growing oligonucleotide chain. (D) The final
product is a 60-base in situ–synthesized probe as a feature on a microarray containing thousands of
specifically synthesized probes. (Adapted with kind permission from the American Society for Microbiology, from: Miller
MB, Tang YW. Basic concepts of microarrays and potential applications in clinical microbiology. Clin Microbiol Rev.
2009;22(4):611-633.)20
6.2.2.2.2 In Situ Parallel Synthesis Using Electrochemical Methodologies/Technologies
This methodology is based on an array composed of individually addressable microelectrodes that, on

activation, generate acid precisely at specific locations. The generated acid deprotects the acid-labile
protecting group (DMT) during oligonucleotide synthesis. DNA microarrays of 35-base oligonucleotides
with a feature size of < 100 µm can be achieved using this method.69
6.2.2.2.3 Maskless Photogenerated Acid-Controlled In Situ Synthesis
This technique employs digital photolithography to generate acid locally from a photogenerated acid
(PGA) precursor. It is possible to project a specific light patterning to the microarray solid surface to
generate acid (H+) locally via a PGA precursor. The acid is then used to deprotect the DMT-protected
surface, covalently linked nucleotide (see Section 6.2.2.1). The reaction gates through single nucleotide
cycles. There are a large number of PGA compounds available that are also compatible with
microelectronic processing.114-116
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A digital optical module is employed for light patterning projection onto the reaction surface. This optical
module is software driven, thereby achieving synthesis of a large number of different molecules on the
same reaction surface.
Considered the most advanced technology, the projection device can be based on a digital micromirror
device (DMD),117-119 which is a reflective display device consisting of electromechanically controlled
micromirrors that allow projecting images with resolutions of a few microns per pixel. This method
allows oligonucleotide synthesis of various lengths, up to 100 base with feature size > 40 µm.
Commercial technologies are available with microarrays having densities up to 4 million features.
6.2.2.2.4 Mask-Directed In Situ Parallel Synthesis Using Photolabile Protecting Group
This method uses a mask to cleave a photolabile protecting group (PLPG) that was previously introduced
to the surface oligonucleotide chain (see Figure 3). This generates free OH groups that subsequently react
via single nucleotide cycles according to the phosphoramidite chemistry described in Section
6.2.2.1.120,121
At the beginning of the synthesis, the surface reactive OH groups are blocked by a PLPG moiety (such as
α-methyl-[2-nitropiperony-1] oxycarbonyl). Photomasks selectively expose light in predetermined areas
of the solid surface, thus cleaving the PLPGs from the terminus of the growing chain to produce free OH
groups. The free OH can then react with PLPG phosphoramidite monomers to add a new base at the 5ʹ
end of the oligonucleotide. In the nonilluminated surface areas, PLPGs remain to block the terminal OH
group. These steps are repeated sequentially every cycle, adding a different nucleotide monomer
according to the sequences to be synthesized. This process permits the synthesis of high-density
oligonucleotide arrays with feature size < 25 µm and lengths up to 25 nucleotides.
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Abbreviations: A, adenine; C, cytosine; G, guanine; T, thymine; UV, ultraviolet.

Figure 3. Photomask-Assisted In Situ–Synthesized Short Oligonucleotide Microarray. (Top)
Photolithography—A lithographic mask is used to transmit or block UV light from the chemically
protected microarray surface. The order of sequence synthesis is determined by sequential application of
specific lithographic masks. (Bottom) Chemical synthesis—Protecting groups (circles) are removed by
UV light, allowing addition of a single protected nucleotide. Sequential cycles of light deprotection,
changes in masks and filtering patterns, and addition of single nucleotides form microarray features with
specific oligonucleotide probes. (Figure 3 provided by Sherry Dunbar.)
6.2.2.2.5 Maskless In Situ Parallel Synthesis Using Photolabile Protecting Group
This methodology represents an advance on the one described in Section 6.2.2.2.4. It uses digital
photolithography instead of a mask to photocleave a PLPG. Using this method has several advantages,
primarily the significant “speed up” of the synthesis process with subsequent reduction of both time and
manufacturing cost without losing other advantages, such as high density or small feature synthesis
(potentially millions of oligonucleotides on a 1-cm2 surface). Thus, customized microarray design can be
easily manufactured (see Figure 4).76,117
To overcome the limitations of the PLPG chemistry (see Section 6.2.2.2.4), different PLPG groups
belonging to the family of 2-(2-nitrophenyl) propoxycarbonyl have been employed. These PLPGs can
enhance the efficiency of the photolytic deprotection reaction by more than 10% compared to that of the
standard 5ʹ-protection chemistry.121,122 This technology is now used to synthesize short oligonucleotide
(24-base DNA) high-density microarrays with feature size < 25 µm.
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Abbreviations: A, adenine; C, cytosine; DMD, digital micromirror device; G, guanine; T, thymine.

Figure 4. Digital Micromirror Synthesized Maskless Oligonucleotide Microarray. Maskless array
synthesizer technology is depicted in this figure. This technology uses a DMD to create virtual masks.
During photomediated synthesis, the DMD forms the pattern of UV light needed to direct the specific
nucleic acid addition. UV light removes the PLPG (circles) from the microarray surface, allowing the
addition of a single protected nucleotide to the growing oligonucleotide chain. The cycling of DMD
filtering, light deprotection, and nucleotide addition creates oligonucleotide features 60 to 100 bp in
length on the surface of the microarray. (Adapted with kind permission from the American Society for Microbiology,
from: Miller MB, Tang YW. Basic concepts of microarrays and potential applications in clinical microbiology. Clin Microbiol
Rev. 2009;22(4):611-633.)20
6.3 High-Density Silicon Microarrays
6.3.1 Introduction
This class of microarray contains a large number of oligonucleotide probes (25 to 75 bp long) attached to
silicon-based substrates. The advantages of using oligonucleotide probes have already been discussed in
Section 6.2.1. The methodologies described in this section couple the flexibility of using oligonucleotide
lengths up to 75 bp with high-quality synthesis.
This section provides an overview of the main technologies and methods currently used for high-density
silicon microarrays.
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6.3.2 Methods and Technologies Overview
The high-density silicon microarrays are made by following one of the two main methods:
 Deposition of presynthesized oligonucleotide probes on silicon-based surfaces by using spotting

robots followed by a covalent immobilization of oligonucleotide-DNA via chemical reaction of the
end-tagged oligonucleotides with surface functional groups.123-125
 Random self-assembling of silica beads (few µm in size) in silicon microwells. Each bead is covered
with hundreds of thousands of copies of a specific oligonucleotide that act as the capture sequences
(probe) in the assays.80
6.3.2.1 Spotting of Presynthesized Oligonucleotide Probes
Inkjet printing and microspotting methods are used to fabricate custom microarrays by direct spotting of
the oligonucleotide onto the silicon-based substrates. Microspotting methods typically use noncontact or
contact printing technologies, such as those by robotic pin or piezoelectric inkjet instruments, to deposit
presynthesized oligonucleotides on a surface according to a predetermined pattern (layout) containing one
oligonucleotide per site. Various types of spotters are employed to fabricate microarrays.77
The probe-substrate attachment methods are based on covalent bond formation between the
presynthesized 5-amino probe and the appropriate surface chemical moiety bound to the solid support
(derivatized substrate).77 The surface chemical groups on the derivatized substrate determine how the
oligonucleotide will be immobilized on the surface and the type of interactions that dominate the
attachment process. Silane coupling agents are commonly used to activate silicon surfaces for subsequent
immobilization of oligonucleotides with a variety of chemical modification of the silane chain.78 Among
these, a common silane coupling agent is 3-glycidoxypropyltrimethoxysilane. This agent is often selected
in preference to other coupling agents because of the reproducibility and higher density of oligonucleotide
probe immobilization it allows.79
This method enables the generation of DNA microarrays with various length of oligonucleotide (25 to 60
base) and with feature size of 70 µm.
6.3.2.2 Self-Assembling of Silica Beads
These arrays are made by random self-assembling of oligonucleotide-covered silica beads (about 3 µm
size) that fit into silicon microwells. These chips are built using either fiber optic bundles or planar silica
slides, and the silicon microwells are made by the plasma etching process.
Each bead is covered with hundreds of thousands of copies of a specific full-length oligonucleotide (40 to
60 base) that act as the capture sequences probe in the assays. The oligonucleotides are coupled to the
beads through an amine linkage. Individual bead lots (with each lot having only one oligonucleotide
sequence bound) are fabricated with specific fluorescent signatures that allow them to be differentiated
when they are pooled together with other lots. When pooled together, they form assay panels for 96 to 1
million or more targets.
Once the beads are randomly assembled on the arrays, they are uniformly spaced about 6 µm apart, and
initially, the location of a particular probe is unknown and a decoding process is used to find the location
of each bead. The decoding process is carried out by performing several hybridization/stripping cycles.
For example, in the case where there are just 16 different bead types, the array is hybridized to 16
“decoder oligonucleotides,” each one of which is a match for one of the oligonucleotide sequences (bead
types) on the beads in the array. After the decoder oligonucleotides are labeled with four different
fluorescent dyes, the array is then imaged and stripped. A second hybridization occurs when the decoder
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oligonucleotides are labeled with a different dye than in the first hybridization. Following the second
round of hybridization and imaging, it is possible to identify the exact bead type precisely at each position
on the array (see Figure 5).
Figure 5. Self-Assembling Microbead Array. The microbead array consists of 96 1.4-mm fiber-optic
bundles (bottom left). Each bundle is an individual array consisting of 50 000 5-µm fiber-optic strands,
each of which is chemically etched to create a microwell for a single bead (top left). The platform can
assay one to 16 samples at a time on a silicon slide (bottom right) that has been processed to provide
microwells for individual beads (top right). Both platforms rely on 3-µm silica beads that randomly self-
assemble (center). (Adapted with kind permission from Fan JB, Hu S, Craumer Q, Barker D. BeadArray-based solutions for
enabling the promise of pharmacogenomics. Biotechniques. 2005;39(10 Suppl):S583-8.)126
6.4 Electronic Microarrays
Electronic microarrays use active hybridization through electric fields to control nucleic acid transport.
Microelectronic cartridges use complementary metal oxide semiconductor technology for electronically
addressing nucleic acids.127 A typical electronic microarray cartridge has 12 connectors that control 400
individual test sites.20,128 The microarray is generated by transport of negatively charged nucleic acids to
specific sites when an electric field is applied to one or more test sites on the microarray.129,130 The surface
of the microarray contains streptavidin. Biotinylated probes reach these locations through the application
of a specific electrical field, so that the streptavidin-biotin binding occurs and the probes are then
immobilized onto the surface. The electric field is then removed from the active features, and new test
sites can be activated.20,128
Once the probes have been hybridized at discrete test sites, the microarray is ready for the application of
fluorescently labeled target DNA. Target DNA passively hybridizes with the immobilized probes on the
microarray or is concentrated electronically.131,132 Electronically addressing the capture probe first is the
most commonly used assay format, but amplicon first and sandwich assays have also been described.133 If
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hybridization occurs between the probe and the target DNA, fluorescent reporters will be present at the
positive test site(s) and will be detected when the electronic microarray is scanned and analyzed by an
imaging instrument using a charge-coupled device (CCD) camera with data collection and analysis
software (see Figure 6).20,134,135
Negatively charged
DNA
Figure 6. Electronic Microarray. (A) A positive electric current is applied to test sites, facilitating the
active movement and concentration of negatively charged DNA probes to the activated locations. (B)
Once the first probe is bound to its targeted location(s) by streptavidin-biotin bonds, the test site(s) can be
deactivated, and the current can be applied to a different test site. This process is repeated until all the
probes are arrayed. (Adapted with kind permission from the American Society for Microbiology, from: Miller MB, Tang
YW. Basic concepts of microarrays and potential applications in clinical microbiology. Clin Microbiol Rev. 2009;22(4):611-
633.)20
6.5 Suspension Liquid Bead Microarrays
Suspension liquid bead microarray technologies are three dimensional arrays that use microparticles
(microspheres or beads) as the solid support for the binding reaction and flow cytometry or imaging for
detection of the bead and associated target (see Figure 7).136-140 Several platforms are available in various
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configurations and generally allow multiplexing through unique features of the particle subsets. The
particles can be distinguished into subsets by different classification features, such as internally absorbed
fluorophores, size or diameter, and surface or composition characteristics. A specific capture moiety
(oligonucleotide probe) is attached to the surface of the beads through covalent coupling or adsorption.
The target nucleic acid is captured by hybridization to the complementary probe sequence, and labeled
with a reporter molecule (ie, fluorescent dye) to allow detection and quantitation of the specific binding
that has occurred at the particle surface. Multiple readings are made per particle set or target, providing a
mean, median, or cumulative output signal of the result.136-140
Figure 7. Suspension Bead Arrays. (A) A red dye and an infrared dye are added to microspheres in
different relative concentrations to create 100 beads, each with a unique spectral identity. (B) Biotinylated
primers are used to amplify nucleic acid targets and the amplicons are then denatured and hybridized to
microspheres tagged with target-specific sequence probes. Probe-target hybridization is measured using a
streptavidin-bound fluorophore. (C) Flow cytometry technology is used to analyze the microsphere
suspension. The probe is analyzed by a red laser that is used to determine the spectral identity of the bead.
The reporter fluorochrome is excited by a green laser, which quantifies the probe-target reaction on the
microsphere surface. (Adapted from Clin Chim Acta, Vol. 363 / No. 1-2, Dunbar SA, Applications of Luminex xMAP
technology for rapid, high-throughput multiplexed nucleic acid detection, pp. 71-82, Copyright © 2006, with permission from
Elsevier.)136
6.5.1 Synthesis of Beads
To demonstrate an example of the synthesis of beads, one particle-based detection platform uses
polystyrene microspheres that are internally dyed with two or three spectrally distinct fluorochromes.
Using precise amounts of each of these fluorochromes, an array is created consisting of different bead sets
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with specific spectral addresses. An additional fluorochrome coupled to a reporter molecule quantifies the
biomolecular interaction (hybridization event) that has occurred at the bead surface. After completion of
assay incubations with a target nucleic acid sample and a detectable reporter reagent, the reactions are
analyzed within a reader, classifying the beads based on the spectral address (ie, bead identity) and
quantifying the bound fluorophore in the reporter detection channel. Microsphere reagents precoupled
with unique capture oligonucleotides are commercially available from some manufacturers for nucleic
acid microarray assay development. These reagents incorporate the use of universal oligonucleotides that
are chosen to have identical hybridization parameters, but minimal crossreactivity.136-140
6.5.2 Reading of Beads
Microsphere analysis platforms are generally flow based or employ the use of CCD imaging. In one type
of flow analysis instrument, the beads are introduced into a rapidly flowing fluid stream and, through
hydrodynamic focusing, are interrogated individually as they pass by two separate lasers. A 635-nm, 10-
milliwatt red diode laser excites the fluorochromes contained within the microspheres and a 532-nm, 13-
milliwatt yttrium aluminum garnet laser excites the reporter fluorochrome bound to the bead surface.
High-speed digital signal processing classifies the microsphere based on its spectral address and
quantifies the amount of probe on the microsphere surface. In a second instrument format, the
microspheres are magnetic and the reacted beads are passed through an imaging flow cell where a
magnetic actuator pulls the beads out of suspension and holds them in place for optical analysis. Red
light-emitting diodes (630 nm) are used to excite the fluorescent dyes contained within the microspheres
and green light-emitting diodes (515 to 521 nm) are used to excite the reporter fluorochrome bound to the
bead surface. A CCD imager identifies the bead region and quantifies the bound reporter.20,130,134
6.5.3 Suspension Bead Chemistries
A variety of assay chemistries have been used for the development of suspension bead microarrays,
including direct capture, competitive hybridization, and universal array capture sequences in combination
with solution-based enzymatic reaction.136 Direct hybridization of a labeled PCR-amplified target DNA to
bead sets bearing specific oligonucleotide capture probes is the simplest assay chemistry that can provide
single nucleotide discrimination (see Figure 7B). It takes advantage of the fact that the Tm for
hybridization of a perfectly matched template compared to one with a single base mismatch can differ by
several degrees for capture oligonucleotides approximately 15 to 20 nucleotides in length. Typically, for
single nucleotide discrimination, capture probes are designed to be matched in length at approximately 20
nucleotides. The probes are complementary in sequence to the labeled strand of the PCR product and the
polymorphic nucleotide is located at or near the center of the probe. Probes are modified to provide a
spacer between the hybridizing sequence and microsphere surface, and a terminal group for covalent
attachment to the functional group on the bead (eg, a primary amine for carboxylated microspheres). PCR
primers are typically designed to amplify 50- to 300-bp regions of target sequence with one primer of
each pair biotinylated at the 5ʹ end for labeling the target strand of the amplicon. Using a small target
DNA minimizes the potential for steric hindrance to affect hybridization efficiency adversely at the bead
surface.138-140
6.5.3.1 Competitive Hybridization
Competitive hybridization is similar to direct hybridization in probe and target design. However, in the
competitive assay format, unlabeled double-stranded PCR-amplified targets compete with labeled single-
stranded oligonucleotide targets for annealing to the sequence-specific capture probes on the
microspheres.
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6.5.3.2 Enzymatic Reactions
Another approach is to use a sequence-specific enzymatic reaction in solution to determine the target
sequence, followed by capture onto the bead surface for detection. This format involves the incorporation
of a specific capture sequence during the enzymatic step that allows hybridization to a complementary
universal “tag” sequence on the bead surface. Commonly used enzymatic methods for sequence
determination rely on the discriminating ability of DNA polymerases and DNA ligases, and include
allele-specific primer extension, target-specific primer extension (TSPE), oligonucleotide ligation assay
(OLA), single-base chain extension (SBCE), and target-specific PCR (TS-PCR) (see Figure 8). This
approach takes advantage of the speed of solution-phase hybridizations and permits the universal tag bead
sets to be used in many different assays where new sequences can be targeted by adding the appropriate
capture sequence to the target-specific oligonucleotide used in the enzymatic step.136
Abbreviations: OLA, oligonucleotide ligation assay; PCR, polymerase chain reaction; SBCE, single-base chain extension; TS-
PCR, target-specific polymerase chain reaction; TSPE, target-specific primer extension.
Figure 8. Chemistries Used for Universal Tag Microsphere Capture Assays. (A) TSPE—Sample
DNA is amplified and annealed to target-specific primers in a reaction containing a DNA polymerase and
deoxynucleotide triphosphates (one with a biotin label). Targets are extended and extension products are
captured onto complementary bead sets and labeled with streptavidin-R-phycoerythrin. (B) OLA—
Sample DNA is amplified and annealed to target-specific primers in a reaction containing a DNA ligase
and biotinylated reporter probe. Ligation products are captured onto complementary beads sets, and
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labeled with streptavidin-R-phycoerythrin. (C) SBCE—Sample DNA is amplified and annealed to target-
specific primers in a reaction containing a DNA polymerase and a biotinylated dideoxynucleotide
triphosphate (in separate reactions for each nucleotide). Targets are extended and extension products are
captured onto complementary beads sets, and labeled with streptavidin-R-phycoerythrin. (D) TS-PCR—
Sample DNA is amplified using target-specific upstream primers paired with downstream biotinylated
primers. PCR products are simultaneously hybridized to complementary bead sets and labeled with
streptavidin-R-phycoerythrin. This example depicts a spacer between address and target-specific primer
sequences to prevent amplification of the capture sequence. (Adapted with kind permission from Springer Science
and Business Media, from: Dunbar SA. Bead-based suspension arrays for the detection and identification of respiratory viruses.
In: Tang YW and Stratton CW, eds. Advanced Techniques in Diagnostic Microbiology. 2nd ed. New York, NY: Springer
Science; 2013:813-834.)135
6.6 Small Volume Polymerase Chain Reaction Arrays
Over the last decade, several platforms have been developed that allow small volume PCR reactions to
occur in an array format containing hundreds to thousands of individual reactions. While this allows one
sample to be interrogated for many different target nucleic acid sequences at one time, it usually comes at
the cost of performing the individual reactions on a small amount of the original sample (tens of µL down
to tens of nL). As a practical matter, these volumes are sufficient to detect a single copy gene target in the
human genome. In some cases, the volume of original sample interrogated is sufficient to test for an
infectious disease agent with sensitivity comparable to more conventional real-time PCR methods. This is
due in part to a function of the degree of concentration of nucleic acid through the sample preparation
step.
In principle, pathogen nucleic acid targets of interest can be pre-enriched (eg, by whole genome or a
specific PCR preamplification) before being amplified in the PCR array. Nested PCR for pathogens has
been described by several groups141,142 and this can be applied to some of the PCR array platforms
described in the following sections.
6.6.1 Customized 96- and 384-Well Polymerase Chain Reaction Arrays
Primers dried down into the wells of a 96- or 384-well PCR plate constitute a form of array in which the
user mixes the sample with a real-time PCR master mix (without primers) and aliquots this into the wells
of the array. This has been used to monitor gene expression for different panels of genes, including those
that change in response to viral infection.84 Real-time PCR is performed with a dsDNA binding dye as the
fluorescent readout of the reaction. Microplates can be customized with different primer sets including
those specific to viruses and bacteria.
6.6.2 Microfluidic Card
A customizable, 384-well microfluidic card enables a laboratory to perform hundreds of real-time PCR
reactions simultaneously (see Figure 9). The card is manufactured with oligonucleotide primers and
probes dried down into each of the 1-µL volume reaction chambers. Eight sample-loading ports are
connected to 48 of the reaction wells. Each sample is mixed with the enzymes and buffers for PCR, added
to a sample-loading port, and briefly centrifuged. The centrifugation distributes the sample into each of
the reaction chambers connected to the sample loading port via microfluidic channels. The channels are
then permanently sealed and the card is placed into a real-time PCR instrument. Amplification curve data
are acquired using the appropriate excitation/emission filters for the fluorophores used in the probes.
In the format that has been used for the detection of respiratory pathogens, one sample port is reserved for
a negative control and another one can be used for a positive control. Hydrolysis probe assays for viruses,
bacteria, and three control reactions are spotted into duplicate wells of the array. The sample makes up
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20% of the mix centrifuged into the wells of the array so that a total of 0.2 µL of sample (from the
replicate reactions) is interrogated for each individual assay.
Figure 9. Microfluidic Cards. This low-density array card is a 384-well microfluidic card that permits
384 simultaneous real-time PCR reactions. The card allows one to eight samples to be amplified in
parallel and hybridized against 12 to 384 probes (one probe per well) that are preloaded into the wells on
the card. (Adapted with kind permission from Springer Science and Business Media, from: Keys DN, Au-Young JK, Fekete
RA. TaqMan array cards in pharmaceutical research. Methods Mol Biol. 2010; 632:87-97.)143
6.6.3 Nested Polymerase Chain Reactions in Small Volume Arrays
Small volume PCR arrays have also been implemented as the last stage of a multiplex nested reaction.
Raw sample is injected into a disposable pouch and nucleic acid is purified. It is then subjected to a first
stage multiplex reverse transcription PCR. This first stage amplification is diluted 225-fold and mixed
with a second PCR master mix before being spread across the second stage PCR array. This array
contains 102 wells of 1-µL volume, which has been spotted with PCR primers nested inside the
amplicons from the first stage PCR. A Peltier device adjacent to one side of the array controls thermal
cycling while a light-emitting diode and camera on the other side enable detection of fluorescence
generated by a dsDNA binding dye present in the master mix. Amplicons are detected and identified
through the analysis of postamplification DNA melt curves (see Figure 10).
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Abbreviations: DNA, deoxyribonucleic acid; PCR, polymerase chain reaction; RNA, ribonucleic acid.
Figure 10. Example of a Nested PCR in a Small Volume Array. (A, top) Disposable reagent pouch
used for nucleic acid purification and nested multiplex PCR. Colored liquid in the top of the pouch
indicates the location of the lyophilized chemicals and enzymes used to carry out the chemistry. (B, top)
Schematic of the pouch showing the different stages of the chemical and enzymatic steps. PCR II
indicates the second stage inner PCR, which occurs in an array of 102 one-microliter wells. (A, bottom)
Schematic of the array. Primers for different PCR assays are spotted in triplicate across the array. Two
control assays (a process control [yeast] and a control for the inner PCR [PCR 2]) and two organism-
specific assays are indicated. (B, bottom) To start the nested PCR reactions in the array, diluted amplicon
from the first stage PCR is mixed with additional DNA polymerase, nucleotides, and a fluorescent
dsDNA binding dye. This mixture is flooded over the top of the array and into the wells through holes in
the top of each well. After the wells fill, a clear plastic bladder in the instrument inflates to seal the wells
shut. Unique sets of primers in each well allow amplification of specific targets if they are present in the
first stage amplification. (Adapted with kind permission from Poritz MA, Blaschke AJ, Byington CL, et al. FilmArray, an
automated nested multiplex PCR system for multi-pathogen detection: development and application to respiratory tract infection.
PLoS One. 2011;6(10):e26047.)87
6.6.4 Nanoliter Volume Polymerase Chain Reaction Arrays
Thousands of PCR reactions can be performed in a microarray format that uses “through-holes” in which
the PCR primers bind to the sides of the holes in the array.89 The volume of each reaction is 33 nL and
both probe and dsDNA binding dye formats are available. Custom arrays with user-chosen formats and
primers can be ordered from the manufacturer. The array contains 3072 “through-holes” configured as 48
subarrays of 64 holes that can be injected with 12 to 48 different samples. The amplification reaction
occurs in a specialized real-time PCR instrument, but the output is similar to that of a standard real-time
PCR instrument.
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7 Clinical Applications
7.1 Overview
Rapid and accurate detection of life-threatening infectious agents is pivotal to the optimization of medical
treatment of patients with infectious diseases, the control of potential infectious disease outbreaks, and to
ensuring the protection of the public’s health. Diagnostic methodologies, which are capable of detecting
multiple agents, are important, particularly in early diagnosis, to assist in the treatment of disease
conditions and to address the continuous challenges in detecting existing and emerging infectious diseases
rapidly.
Nucleic acid testing technology is constantly being revisited by research scientists and clinicians for the
development of a variety of sophisticated research tools, such as real-time PCR,144 whole genome
expression,145 sequence analysis,146 and microarrays.147 These technologies are largely used in laboratory
research and, in recent years, also have demonstrated their potential in clinical diagnostic
applications.148-150 Research initiatives in the past two decades, especially recent advances in molecular
biology and bioinformatics, have allowed multigene detection and identification by microarray
technology.20 Also referred to as DNA arrays, DNA chips, bio-chips, or gene-chips, DNA microarray-
based identification of pathogens has attracted much attention from clinical researchers because of its
potential efficiency and high-throughput capabilities.151
Unlike classical nucleic acid detection assays, DNA microarrays can offer the possibility to interrogate
the expression levels of a large number of selected sequence targets in a single specimen simultaneously.
However, despite having shown promising outcome in clinical laboratory settings,152-157 widespread
application of the microarray technology for detection and identification of pathogenic microorganisms
has yet to be established.
Various types of microarrays have been developed and reported by commercial manufacturers and
research investigators for genotyping infectious pathogens and detection of antimicrobial drug resistance.
This section broadly covers the implication of the microarray technology for the diagnosis and
identification of pathogens and discusses its significance and limitations over conventional testing
methodologies. Efforts are focused primarily on the clinical potential to:
 Detect pathogens in clinical samples.

 Detect antimicrobial resistance genes.
 Rapidly identify potential bioterrorism pathogens.
The challenges presented by the wealth of information derived from a microarray experiment are
discussed in the following particular examples.
7.2 Detection of Pathogens in Clinical Samples
7.2.1 Detection of Pathogens in Respiratory Tract Specimens
Applications of microarrays to detect and characterize respiratory viruses began with solid arrays. The
first respiratory pan-viral microarray system that covered a variety of viral genome sequences was
described in 2002.158-161 Subsequently, resequencing microarrays were developed to use short
oligonucleotides for the simultaneous identification of respiratory pathogens at both the species and the
strain level. Solid microarray systems for the detection and identification of a panel of respiratory viruses
are commercially available.162-165 Liquid microarray combines flow cytometry, fluorescent microspheres,
and matrix hybridization into a flexible, open-architecture, multianalyte profiling system. Rapid
hybridization kinetics, flexibility in assay design and format, and relatively low costs has made liquid
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microarray the most practical microarray platform for clinical diagnostic applications.166,167 Numerous
studies, including those on several commercially available systems, have reported the use of liquid
microarray technology for the detection and differentiation of a panel of common respiratory viral
pathogens (eg, influenza, respiratory syncytial virus, parainfluenza).139,168,169 More recently, a sample-to-
answer, nested-multiplex PCR system has been developed with a second-stage small volume PCR array
that has assays to detect 20 viruses and bacteria (eg, Bordetella pertussis, Chlamydophila pneumoniae,
and Mycoplasma pneumoniae).87 The system has been compared to LDTs and to other commercially
available IVD devices.163,170-173 Specificity of the assays is very similar. Sensitivity for most organisms
tested is comparable with the selected exceptions, reflecting the most genetically diverse organisms. The
diagnostic yield of the system has also been tested on respiratory samples from immunocompromised
patients.172,174 It is still too early to determine the effect on patient outcomes of these multiplex systems.
7.2.2 Detection of Pathogens in Blood
Although detection of sepsis by the classical methods of direct blood culture and identification using the
bacterial growth characteristics is a well-established and robust procedure, there is growing interest in the
use of microarrays to supplement or replace this technique. Eventually, microarrays will supplement
bacterial culture by nucleic acid analysis of the culture or of clinical isolates.175,176 Much effort is focused
on completely replacing blood culture with direct detection of bacteria in blood using some form of
microarray technology to distinguish among the large number of potential pathogens 177; however, this
effort is technically challenging given the low level of organisms found in the blood stream. These efforts
will also benefit blood donor screening. It may be possible to qualify the deferred donors for reentry
based on approved microarray technologies.178-183
Emerging or unknown infectious agents may quickly be introduced from one region to another due to
frequent travel to and from endemic areas, thus complicating the actual diagnosis of infections with
similar clinical symptoms. In addition, changes in global climate are affecting the prevalence and
distribution of existing and emerging pathogens (eg, malaria, dengue, yellow fever), making these
pathogens more common or even endemic in areas where they were once rare or nonexistent. In the
absence of IVD devices to precisely detect or identify these pathogens, inaccurate diagnosis based on
nonspecific clinical symptoms may prevent physicians from treating patients with the appropriate drugs.
Additionally, rapid emergence of new viral strains of HIV, hepatitis B virus (HBV), or hepatitis C virus
(HCV)184-188 and recombinant 189-192 or drug-resistant viral mutants193-195 pose a serious threat to the public
health.
7.2.3 Detection of Pathogens in the Gastrointestinal Tract
Microarray technologies have been applied to the detection and differentiation of gastrointestinal
pathogens, first in LDTs and, more recently, in commercially available devices. In addition to detection of
foodborne pathogenic bacteria, viruses, and parasites, microarray technologies can be used to further
characterize microorganisms by providing information for specific identification of isolates, to understand
the pathogenesis based on the presence of virulence genes, and to indicate how new pathogenic strains
evolved epidemiologically and phylogenetically.196 Glass slide microarrays have been used to identify
bacteria and to study gene expression in complex microbial populations, such as those found in food and
gastrointestinal tracts, to provide data to potentially improve food safety.197 A spotted microarray
comprising 489 probes was developed for detection of 40 bacterial pathogens of medical, veterinary, and
zoonotic importance; associated genes that encode for antimicrobial and metal resistance; and DNA
elements important for horizontal gene transfer among bacteria.198 The method was validated using
pathogenic bacteria derived from pure culture and following inoculation in complex sample matrixes,
such as soil and manure.
Gastrointestinal viruses, such as noroviruses, rotaviruses, adenoviruses, and astroviruses have been
detected and successfully genotyped using microarray techniques.199-202
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Parallel PCR in microarray format (up to 384 singleplex real-time PCR reactions) was developed and
applied to simultaneous detection of 19 enteric pathogens.203
Suspension microarrays using direct capture of PCR amplified targets have been developed as LDTs and
used for detection of enteric bacteria from culture and stool.204,205 Suspension microarrays for
gastrointestinal pathogens using a TS-PCR chemistry have been used as LDTs, have become
commercially available as IVD devices for use in the clinical diagnostic laboratory, and have been applied
to outbreak investigations and evaluation of tissues for agents of gastroenteritis.206-208
7.3 Pathogen Genotyping
Various microarray platforms have been developed by commercial manufacturers and clinical
laboratories for genotyping infectious pathogens. These assays typically use either target amplification
followed by reverse probe hybridization (eg, linear arrays and line probe assays), microfluidic
amplification followed by solid-phase electrochemical detection, or amplification followed by flow
cytometric detection of target sequences hybridized to probes immobilized on microspheres suspended in
liquid. Linear arrays or line probe assays are commercially available in some countries for genotyping of
HBV,209,210 HCV,211,212 and human papilloma virus (HPV).94,213,214 Laboratory-developed, dot-blot solid-
surface microarrays have been described for genotyping Chlamydia trachomatis,215,216 Chlamydophila
psittaci,217 Vibrio cholera,218 HBV,219-221 HCV,222,223 HPV,224-226 measles virus,227 and noroviruses.200,201 A
genotyping assay for HCV based on PCR amplification of the 5ʹ untranslated and core regions of the viral
genome followed by hybridization to signal probe and electrochemical detection of captured target
cDNA-signal probe complex in a microfluidic chamber228 is also commercially available in some
countries. Bead-based microarrays for genotyping HCV229 and HPV230-232 have been developed by various
investigators. Detailed recommendations and specific guidelines for infectious disease genotyping can be
found in CLSI document MM10.233
7.4 Detection of Antimicrobial Resistance Genes
With the rise of nosocomial infections caused by multidrug-resistant bacteria, various investigators have
also developed microarray methods to rapidly amplify and detect genotypic antibacterial drug resistance
mutations. These methods, which are mainly of the printed solid-phase microarray type, have been
described for detecting antimicrobial drug resistance to extended-spectrum β-lactams, carbapenems, and
aminoglycosides in Enterobacteriaceae.234-237
Detection of antimicrobial drug resistance in infectious pathogens has been the focus of several diagnostic
manufacturers. Different methods have been used for detection of antibiotic resistance markers, with the
most common method being detection of gene mutations that confer resistance to a specific drug.
Amplifying and sequencing the genes of interest to detect known genotypic mutations has been the main
molecular method of detecting genotypic drug resistance in these pathogens. However, microarray
methods to detect known genotypic drug resistance mutations have been described for Staphylococcus
aureus, enterococci, HBV,221 and HIV.238 Most, if not all, of these methods are based on printed solid-
phase microarrays.
Efforts to detect antibiotic resistance genes in bacteria have resulted in several assays that are used in
clinical laboratories. One uses bacteriophage for detection of methicillin resistance in S. aureus. The other
two assays use molecular methods for the detection of the mecA gene sequences in S. aureus. One applies
real-time PCR for amplification of the mecA target sequence from S. aureus, while the other assay does
not use a target amplification method to detect mecA sequence directly from positive blood culture
specimens. The latter takes advantage of the natural amplification of the pathogen in blood cultures and
uses hybridization of the target nucleic acid (mecA gene sequence) to a complementary sequence (capture
probe) fixed on a spot of a microarray.239 Captured mecA targets are also hybridized to a second
complementary oligonucleotide (detector probe) that carries a gold nanoparticle. The signal is amplified
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by the addition of silver.239 When light is passed through the microarray, it will be scattered at the
locations of the spots where the mecA gene and detector probe have bound.240 The spot is read by the
reader, which will indicate a positive result for the presence of the mecA target gene in the pathogen. The
test turnaround time for this assay is about 2.5 hours, with five to 10 minutes of hands-on time.
Recently, a multitude of microarray-based devices have been developed for the detection of mutations
(eg, Mycobacterium tuberculosis) associated with resistance to rifampin and isoniazid.241-245 Among them,
one DNA microarray that incorporates specific nucleotides at a given position of the rpoB, inhA, and katG
gene has been developed to detect multidrug resistance–related mutations in different kinds of clinical
specimens.242-244,246 The same format has been used to detect drug resistance in HBV.247,248
7.5 Detection of Bioterrorism Pathogens
Microarrays will also have a role to play in the related problem of detection of biothreat agents 86;
however, the challenge here is to identify one of a large number of pathogens that may have been
deliberately disseminated into a population. The largest barrier to the use of microarrays in the detection
of biothreat agents is the logistics of maintaining a supply of validated arrays available for the rare
outbreak. Several applications of microarray technologies to the detection of biothreat agents have been
described and are being evaluated for their potential utility in biothreat monitoring systems in public
spaces.249,250
7.6 Other Uses of Microarray Technology
The overall trend for molecular testing has been toward less expensive and more streamlined processes,
which have made it an increasingly viable option in clinical testing. The potential for molecular testing,
and microarray in particular, is enhanced by the increases in sequences uploaded to nucleotide databases.
With mass sequencing becoming less expensive, the amount of genomic information will continue to
increase.
Microarray testing generates large amounts of data and continued work is necessary to develop computer
software to accommodate this large amount of data that requires accurate analysis. As microarray
technology continues to become more established, and the accuracy of analysis continues to increase,
there will likely be a continuing number of microarray platforms that meet government-required
guidelines for clinical testing. The ability of microarray testing to test for high numbers of targets in
parallel will greatly enhance diagnostic testing by providing comprehensive data to health care workers
about multiple types of diseases. As this “lab-on-a-chip” technology becomes more commonly accepted,
there will be an overall reduction in the cost of health care and potentially an increase in health overall.
8 Assay Performance Verification and Validation
8.1 Assay Verification and Validation
8.1.1 Overview
Assay performance verification and validation processes have been well described for molecular
diagnostics tests in many publications and CLSI documents. In particular, CLSI document MM1724 is a
key resource for microarray users. This section summarizes the areas that are unique to microarrays and
includes references for the user to consult for further general information. As stated in CLSI document
MM17,24 verification is defined as the process of confirming performance specifications previously
established by the manufacturer and validation is defined as the process of establishing the performance
characteristics of a new assay or an assay with a new or modified method, specimen type, clinical
indication, or patient population, which would have been typically established by the manufacturer. The
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scope of verification and validation studies will depend on the regulatory status of the test being
implemented as well as the complexity of the method and clinical use of the test. As a result, each
laboratory will need to develop its own particular verification and validation protocols.251 Users can also
refer to available guidance documents for more detailed discussion and recommendations for test
verification and validation (see CLSI documents MM06,21 MM03,26 EP28,252 EP05,253 EP06,254 EP09,255
EP12,256 EP15,257 and EP17258). Table 3 provides detailed recommendations for specific studies that
should be conducted when evaluating the performance characteristics of microarray infectious disease
tests.
8.1.2 In Vitro Diagnostic Devices, Modified In Vitro Diagnostic Devices, and

Laboratory-Developed Tests
As stated in CLSI document MM17,24 a three-tiered categorization approach can be taken to describe the
necessary validation process for microarray diagnostic tests. IVD devices are those approved or certified
by regulatory agencies (eg, US Food and Drug Administration [FDA]–cleared or –approved, CE-marked)
where the test manufacturer has performed acceptable validation studies. Modified IVD devices are tests
performed using changes to cleared, approved, or CE-marked devices, such as changes in specimen types,
patient population, instrumentation, software, or intended use. LDTs are unapproved test methods that are
developed for clinical diagnosis in an individual laboratory where that laboratory is responsible for
establishing the performance characteristics of the test.
8.1.2.1 In Vitro Diagnostic Devices
In comparison to full validation, as described in Sections 8.1.2.2 and 8.1.2.3, verification is usually less
complex and needs less hands-on time, fewer samples, and fewer resources. Each laboratory is
responsible for determining its verification protocol and several guidelines are available for use that
describe the number of specimens to be tested and analysis to perform. It is important that the samples
used in verification are in the matrixes stated in the test system’s instructions for use. If multiple specimen
types are listed as acceptable in the test system package insert, samples that are used for assay
performance verification should include all the specimen types that the clinical laboratory will encounter.
For some microarray tests, laboratories may not have access to the samples necessary to verify assay
performance for all targets present (eg, rare respiratory viruses or organism subtypes). The laboratory may
choose not to report results for that target. However, if a patient is later determined to have one of these
organisms, the laboratory may confirm the result at a reference laboratory and include this as part of its
assay verification. Alternatively, if the laboratory confirms an appropriately high specificity and negative
predictive value for the organism/subtype target in its patient population, it may report out negative
results, but it must also confirm any positives at a reference laboratory and use this data to verify the
assay performance for this rare target. Note that if detection of a particular organism or subtype will lead
to specific therapeutic changes (eg, vancomycin for methicillin-resistant S. aureus or oseltamivir for
influenza A), laboratories should verify assay performance before reporting negative results for these
targets.
Although more complex than single-measurand molecular diagnostic devices, multiplex microarray
device verification protocols can be used to maximize sample pooling to ensure all potential organisms
are tested. For example, a test panel containing 16 different targets could be verified using pools of two,
four, or eight target organisms. In addition, protocols for determining the performance characteristics can
be combined to maximize testing efficiency.
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Table 3. Required Performance Characteristics With Recommended Studies Before Implementation of Infectious Disease Microarray
Tests as IVD Devices, Modified IVD Devices, or LDTs
Assay Performance
Characteristic IVD Recommendations Modified IVD Recommendations LDT Recommendations
Accuracy (trueness) (see CLSI Laboratories need to confirm the Laboratories need to establish the Laboratories need to establish the assay
documents EP12256 and EP15257) manufacturer’s specifications by testing performance of the assay, including performance, demonstrating an acceptable
specimens known to be positive by another the test modification. This can be lower 95% confidence interval boundary.
Comparison-of-methods study: method. While the total number of samples completed by showing comparable FDA and CLSI recommend at least 35
The laboratory director must to be tested will depend on the number of performance between the modified patient specimens positive for each
determine the appropriate microarray targets, a minimum of 20 and unmodified assay results or by microarray measurand for validation‡ (see
number of positive and negative specimens positive for at least one establishing the performance de novo CLSI document MM1724).260
specimens needed to verify or microarray measurand should be included. If (as in LDT recommendations).
validate assay accuracy. Data available, 10 specimens per measurand type Recommendations for use of alternate
should be analyzed for the entire should be tested.* specimen types (modified IVD)
microarray panel, groups of include testing at least 15 positive and
related organisms, and each Higher density arrays may group similar 15 negative samples.259
organism type/subtype types of target measurands together for
separately. analysis to avoid the need for testing very
large numbers of samples for accuracy
verification.†
©
Clinical and Laboratory Standards Institute. All rights reserved.
Precision (see CLSI documents Testing of pooled samples positive for Testing should include positive Testing should include negative samples
MM03,26 EP05,253 EP12,256 and multiple measurands is recommended to samples near the LoD and negative and two levels of positive samples (high
EP15257)261 reduce the number of replicates.* Test samples using the modified procedure positive and near the LoD). Test controls
controls in singlicate over 10 days for a total or specimen matrix. Test controls in in singlicate or duplicate over at least 20
Replication study: of 10 data points per measurand. singlicate over 20 days or in duplicate days for a total of at least 20 data points
The laboratory needs to establish over 10 days for a total of 20 data per measurand.
overall assay variability points per measurand.
including all relevant variables
(eg, run, day, operator, reagent
lot), and intra-assay variability
(if applicable).
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Table 3. (Continued)
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Assay Performance Characteristic IVD Recommendations Modified IVD Recommendations LDT Recommendations
Analytical sensitivity (see CLSI Not required by regulatory agencies, but Laboratories need to establish the LoD, Laboratories need to establish the LoD
document EP17258)262,263 may be confirmed at the laboratory including the test modification. This can using quantified material to
director’s discretion. be done by showing comparable demonstrate absolute detection limits.
LoD study: performance between the modified and Perform LoD study using quantified
The laboratory needs to establish the unmodified assay results or by source material with probit regression
LoD for modified IVD and LDT establishing performance de novo (as in analysis (see CLSI document
assays. LDT recommendations). EP17258).
Example: Validation of a nonapproved
specimen matrix can consist of a
confirmation of similar or clinically
acceptable detection levels in a dilution
series of simulated patient samples. An
adequate number of replicates near the
LoD are recommended, with LoD at the
lowest dilution with 95% detection (see
CLSI document EP17258).
Analytical specificity (see CLSI Not required by regulatory agencies, but Laboratories need to establish the assay specificity in the appropriate specimen
document MM0326)262 may be confirmed at the laboratory matrix. Potentially crossreacting organisms, including pathogens and normal flora
director’s discretion. that may cause a false-positive reaction, should be investigated. Testing of pooled
Specificity study: controls spiked with potentially reacting substances would be an acceptable
The laboratory needs to determine method.
the effects of potentially
crossreacting organisms for modified
IVD and LDT assays.
Interference (see CLSI document Not required by regulatory agencies, but Laboratories need to establish the effect of potentially interfering substances on
EP07264) may be confirmed at the laboratory the assay as used in the specified format. This may include endogenous substances
director’s discretion. found in the test sample matrix, as well as exogenous substances seen in the
Interference study: patient population. The laboratory should include positive interference (ie,
The laboratory needs to determine crossreacting organisms) and negative interference (eg, hemoglobin, heparin).
the effects of potentially interfering Testing of pooled controls spiked with potentially interfering substances would be
substances for modified IVD and an acceptable method.
LDT assays.
Reportable range (see CLSI
document EP06254)
Not applicable to qualitative microarray assays.
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Linearity study (for quantitative
assays)
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Table 3. (Continued)
Number 3
Assay Performance
Characteristic IVD Recommendations Modified IVD Recommendations LDT Recommendations
Reference range (see CLSI
document EP28252)
For measurands typically not detected in normal (uninfected) populations, verification of reference interval not required.
Reference range study
*
More limited verification can be considered for rare organism subtypes where clinical material is not readily available. In these cases, testing of simulated patient samples may be
acceptable before clinical use. Simulated patient specimens consist of measurand spiked into individual patient specimen matrix known to be negative or a pool of patient specimens
known to be negative for the specific measurand. Replicate testing of samples can be used to increase the number of results available for analysis when sample availability is limited.
Multiplex testing verification protocols in areas other than infectious disease have included reference material for each measurand and a total of 20 patient samples covering the
microarray targets.265
†
More limited verification can be considered for measurands in which detection has limited impact on patient treatment or alternative diagnostic tests are used in patient management.
Organisms may be grouped together for analysis based on species relatedness and those that can be expected to react similarly with the microarray analysis procedure. Accuracy studies
should include known locally circulating strains to ensure adequate detection of these organism types. Comprehensive verification and validation recommendations should be followed
for each analyte where incorrect results could lead to failure to provide a diagnosis and incorrect patient management decisions (ie, detection of methicillin-resistant S. aureus and/or M.
tuberculosis) and for organism subtypes that lead to different clinical management decisions.
‡
When performing the validation, laboratories should establish an acceptable lower boundary of the 95% confidence interval for accuracy. For example, if 20 patient samples are
concordant between the microarray assay and reference method for each measurand, the lower bound of the 95% confidence interval is 83%. If 35 patient samples are concordant for
each measurand, the lower bound of the 95% confidence interval is 90% (see CLSI document MM1724).
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Abbreviations: FDA, US Food and Drug Administration; IVD, in vitro diagnostic; LDT, laboratory-developed test; LoD, limit of detection.
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8.1.2.2 Modified In Vitro Diagnostic Devices
When a laboratory modifies an IVD test in order to meet its specific testing needs, the validation plan is
usually more extensive than verification, depending on the modifications. Some of the modifications may
be minor; others can be extensive. Examples of modifications include, but are not limited to:
 Applying the test to a different patient population

 Using additional or alternative specimen types
 Using different sample preparation methods, or changing steps in the assay procedure
 Using different instrument or software versions
 Applying different cutoffs
With any modifications, the analytical and clinical specifications of the original IVD test may no longer
be appropriate for the test’s intended uses. Therefore, the validation of the modified IVD test should first
assess and redefine any test parameters and specifications related to the modifications introduced and
ensure that these will meet the intended use of the test. For example, if bronchoalveolar lavage fluid is
used for respiratory viral detection in an IVD test that was validated for nasopharyngeal swabs or
aspirates, parameters such as the analytical sensitivity, clinical sensitivity, and specificity will need to be
redefined.
8.1.2.3 Laboratory-Developed Tests
The validation of LDTs is perhaps the most involved process because there are no predefined and
validated performance requirements and specifications. Refer to CLSI document MM1724 for a
complete description of the validation planning and testing process.
If the number of available patient specimens known to contain particular measurands is not sufficient, the
test material may be created by “spiking,” or the addition of an organism into a pool of patient specimens
known to be negative for the specific measurand.
An element of validation that is sometimes overlooked is the assessment of the vendors that will supply
the key critical raw materials of the developed assay. The concept of caveat emptor (“let the buyer
beware”) should be realized by establishing a good vendor qualification process. There are many
examples of commercial reagents being contaminated with nucleic acids and more contamination
problems are being discovered with more sensitive and broad-based detection schemes, such as
microarrays.266-268 This is particularly important when using an LDT for which a laboratory is reliant upon
the quality of the critical raw reagent materials. In this process, a vendor should be evaluated to determine
whether it could provide the necessary reagents to the standards that the purchasing medical laboratory
requires. Clear written standards or specifications should be developed for each critical material
purchased. The technical basis for these standards should be defined during assay development, and again
for all critical materials and reagents, as well as consumables. For example, many microarray tests use
oligonucleotide as primers for PCR. Specifications for primers should be defined in terms of sequence,
purity, and scale before ordering. Depending on the assay, one or all of these factors could have an impact
on the quality of LDT results. Other critical components of LDTs include:
 Materials for nucleic acid purification

 Fluorescent, chemiluminescent, or colorimetric dyes
 Polymerases
 Deoxynucleotide triphosphates
 Buffers
 Storage tubes
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Laboratories should be aware that manufacturers do not always notify customers when they make process
changes to reagent manufacture. Because process changes may affect reagent performance, it is best to
develop quality indicator tests for raw materials that ensure that critical performance criteria are met
before incorporating them into an LDT. Testing of multiple lots of test reagents is advisable early on in
the process. Best quality practices include submitting a copy of the agreed-upon specification to the
vendor with each material order. Vendors should also be assessed based on their skills in material
preparation and evaluation of the quality management systems they have in place. Finally, it is
recommended that a primary and backup vendor be identified for critical materials to ensure an
uninterrupted supply. A similar strategy for vendor qualification can be applied to selection of reference
laboratory services to be used as the gold standard comparator method.
Because of the inherent multiplex nature of microarray assays, which can easily provide output data on
tens of thousands of genetic targets (probes) simultaneously, the analytical and clinical verification and
validation of this type of test can be particularly challenging. The traditional approach of determining all
assay characteristics for each probe individually may not be feasible, as the number of replicate assays
needed increases exponentially with the number of target probes included on the array. Additionally,
control material for each individual probe may not be available. Even if such material could be
manufactured (eg, synthesized or cloned DNA targets), this would not reflect the actual nucleic acid being
detected from the organism.
Large efforts to generate analytical performance data using artificial targets would likely not inform the
laboratory about the true limitations of the microarray test on patient samples, which is a major goal of
test verification and validation. However, because actual clinical specimens containing the entire diversity
of target organisms may be difficult or impossible to obtain, verification and validation protocols may
include a mixture of patient samples, cultured organisms, and artificial nucleic acid targets to obtain a
reasonably comprehensive analysis of the microarray assay performance in individual laboratories.
Suboptimal amounts of nucleic acid, particularly excessive amounts, can cause incorrect calls to a greater
degree in multiplex, microarray assays than in single individual assays, and some test systems are more
sensitive to nucleic acid quantity than others are.
Depending on the assay, the laboratory may not quantify each specimen DNA before performing the
assays, and thus, it is important to determine the range of nucleic acid output from the extraction method
to be used and to ensure that this range is compatible with the requirements of the assay. Studies should
also ensure that different organism subtypes and/or strains are correctly identified or detected, which is
essential when there are geographic and population variations.
More complex microarray assays containing large numbers of probes or complex mathematical
algorithms for data analysis need additional validation studies before patient use. The concept of signature
classifier, prediction rule, or score result because of a global output based on a number of probe
results/calls increases the validation burden, because the algorithm must be shown to be accurate in the
face of potential background signals, and the output interpretation is meaningful clinically. Most
infectious disease microarray applications will call a positive result as the identification of an organism in
a patient sample, or will subtype a cultured isolate. Therefore, the sensitivity and specificity of the entire
algorithm for accurate identification in patient isolates needs to be determined relative to a gold standard
method.
The fact that different targets may have different sensitivity thresholds should also be considered in the
validation. Because the prevalence of different targets will affect the positive predictive values of the
result, interpretation of positive results for targets with different prevalence will also present a significant
challenge. Because of these factors, the prospect of full validation of microarray tests is an arduous task
for most of today’s laboratories. Yet, validation of the test is required to ascertain a safe use of the assay
for public health. The verification of existing assays that are validated, cleared, approved, or marked by a
government regulatory body is a much more feasible alternative to the average clinical laboratory.
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8.1.3 Clinical Validation
Clinical validation of a new diagnostic microarray test (ie, a test for which clinical utility has not been
sufficiently established) should demonstrate the safety and effectiveness of the test through the validation
of clinical test performance characteristics. Refer to CLSI document MM1724 for detailed information on
the key steps of clinical validation and the essential clinical validation parameters.
8.1.3.1 Specimen Types
A critical factor of a successful clinical validation is a clinical sample set, which should include an
adequate number, type, and variety of samples to ensure that test results can be adequately interpreted and
that the limitations of the testing and test results are known.
When selecting samples, the following factors should be considered:
 The prevalence of the organism, mutations, or variants being evaluated

– Samples should be appropriate for establishing test performance specifications and defining
limitations.
 Inclusion of sample types that represent the real-life expectations of the use of the assay (eg,
nasopharyngeal swabs/aspirates, CSF, cultured organisms, fresh or frozen tissue, paraffin-embedded
tissue)
 Inclusion of samples that represent each of the possible reportable results, where possible
– For a multiplex microarray test, all the strains and variants or mutations to be detected should be
included in the performance establishment. In certain situations, naturally occurring samples that
contain target genotypes are difficult to obtain for rare mutations and variants; in these instances,
the alternative control samples and alternative control procedures that will be used should be
included in the establishment of performance specifications.
 Inclusion of samples at microbial concentrations that cover the range of concentrations likely to be
present in the infected individual
 Performance specifications to be established
 Control materials, calibration materials, and other reference materials needed for the test procedures
Although prospective samples are preferred for clinical validation, under specific circumstances, well-
characterized banked or archived samples that have been tested using validated methods can be used,
especially if the clinical utility and validity has been established in the literature. Special consideration
should be given to the evaluation of the performance of the test in fresh vs banked specimens to make
sure the samples will be representative of the actual use of the test.
Sample size should have sufficient statistical power to detect differences of clinical importance for each
organism/genotype. An appropriate number of samples from uninfected and infected individuals, or
cultured organisms with specific genotypes, should be included. Consultation with a statistician is
recommended to determine the appropriate power calculations to consider many variables affecting test
validity, such as the infection prevalence, asymptomatic carriage rate, and the acceptable sensitivity and
specificity. An appropriate number of samples for each specimen type should also be included. If an
organism or genotype has a very low prevalence or very low frequency, alternately constructed samples
may be considered. When samples representing rare markers or mutations cannot be obtained or
constructed, lack of these samples may be acceptable, as long as strong and fully verified performance to
more common mutations is demonstrated using clinical specimens. When specific detection of certain
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organisms or subtypes will lead to therapeutic changes (eg, vancomycin for methicillin-resistant S. aureus
or specific antituberculosis therapy for genetically defined M. tuberculosis strains), the test validation
should include these organisms.
Preexamination factors, such as specimen collection and handling methods, transportation and storage
conditions (eg, type of anticoagulants and fresh vs frozen), extraction methods, and input DNA and RNA
specifications (eg, integrity and purity) should be specified, documented, and validated. Sample stability
testing should also be performed to determine the acceptable ranges and conditions under which samples
will retain stability and can be used for the test (eg, time, temperature).
To ensure compliance with local and national regulations, the need for an internal review and informed
consent should be assessed for the use of the clinical specimens in the clinical validation study, including
the use of banked or archived samples.
8.1.3.2 Results Evaluation
The analytical gold standard or “clinical truth” for which the test will be validated should be well defined.
The analytical gold standard may include reference laboratory test results on split specimens, preferably
from clinical laboratories. When clinical laboratory tests are not available, the gold standard may be PCR
and sequencing for the genome region covering the microarray probe target(s). For new organism or
genotype detections, the clinical validation includes analysis of clinical outcomes as the “clinical truth.”
For example, clinical validation of a new respiratory virus may include evaluation of patient respiratory
symptoms and the need for intubation and intensive care unit admission. Test results are compared to
these analytical or clinical gold standards to determine the appropriate test characteristics.
Analysis for microarray measurands should be performed individually, globally (ie, for all detected
organisms/subtypes), and grouped for similar organism types. Grouped analysis can allow for greater
statistical power and confidence in the determined assay performance characteristics when using a limited
number of samples. This can also provide test performance statistics that are more easily understood and
adopted by the practicing clinician.
When using a standard reference method, the reference test results are considered the “truth.” The test
performance will be calculated and reported as clinical sensitivity and clinical specificity. The diagnostic
accuracy of the new method is determined by the extent of agreement it demonstrates when compared to
the reference method. Likelihood ratios for positive and negative results or receiver operating
characteristic curves (if a numerical output is measured) may also be calculated.269
When a standard reference method is not available or is impractical for some rare
conditions/mutations/genotypes, a comparison to a well-characterized method that has the same or similar
intended use(s) may be employed as part of clinical validation. In this case, the comparator results may or
may not be the “truth.” Therefore, test performance should be assessed in terms of positive and negative
percent agreements. Analysis of discrepant results may be performed to inform the analysis of sensitivity
and specificity, particularly when a new microarray method is expected to be more sensitive than existing
clinical laboratory tests.
8.1.3.3 Limitations Arising From Validation
Any study may have its limitations; test validation studies are no exception. It is important to document
these limitations in order to ensure correct interpretations of the results. Some usual limitations may
include, but are not limited to, sample selection (eg, number of samples available, organisms/genotypes
assessed in the validation vs organisms/genotypes included in the assay), and reference/comparison
method selection.
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Limitations of the test, revealed through the validation or based on published literature, should also be
analyzed and documented.
Any limitations and contraindications for use of the test, including factors that adversely affect accuracy
of test performance and any technical limitations of the assay, should be identified. Assay limitations can
severely affect the analysis of any assay. Knowledge of assay technology, including locations of primers
and microarray probes, can allow the user to predict where limitations may occur and focus validation
studies to define these limitations clearly.
Limitations can be inherent to the assay and may include:
 Methodology
 Assay components
 Primer location and sequence
 Amplification efficiency/reaction kinetics
 LoD
 Limitations related to type of sample and sample storage
 Effects of sample processing
 Method used to purify nucleic acids
 Technical limitations that lead to incorrect results
Limitations can also exist as a result of the patient or the sample and may include:
 Availability of clinical samples

 Characteristics of patient population
 Mutations in primer/probe binding sites
 Unknown or unexpected alleles
 Unknown deletions or polymorphisms
 Inhibitors present in the samples
 Compromised samples
 Insufficient DNA or cells in the sample
Some of the limitation issues are discussed in further detail in CLSI documents MM03,26 MM05,270 and
MM17.24
8.1.3.4 Ongoing Validation
The acceptance of the test verification/validation data and the implementation of the new test in patient
testing should not be considered the end of the monitoring and evaluation of the test’s performance.
Ongoing validation can be considered as continual and incremental improvements that are made to an
examination. The performance specifications derived from the validation should be used to assess the
“validity” of each test run and this information should be added to the validation file at appropriate
intervals. In many cases, the accumulation of data over time is an important additional component of the
initial validation, which can be used to continually improve the assessment of test accuracy and quality.
Several aspects to ongoing validation of a test that need to be considered by the laboratory include:
 Continual documentation of information on clinical validity and usefulness

– Because the accumulation of clinical validity information on infectious diseases is a continuous
process, laboratories should stay current as new data become available.
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 Monitoring and staying current with genome databases of mutations and variants that relate to the
microarray tests performed in the laboratory
 Monitoring and evaluating results of internal QC, external quality assessment, and nonconformities
related to the test or technique as appropriate
 Performing proficiency testing (PT) and carefully investigating any deficiencies observed
 Testing new component reagent lots to ensure that they will work in the assay
– Testing of new lots of critical reagents may involve a small-scale QC study (eg, a comparison
between the new and old lots of material) and/or a precision study. The extent of this ongoing
validation will depend upon the criticality of the reagent. After a new reagent lot is approved,
samples of older reagent lots should be maintained to assist in troubleshooting.
A test will need to be revalidated if changes are made to the test. The extent of revalidation should be
commensurate with the extent of the change. For example, if there is a significant change to the assay (eg,
new amplification primers or microarray probe designs), a full revalidation of the test may be necessary.
A risk analysis (eg, a failure modes and effects analysis) is helpful to identify potential failure modes of
the changes introduced and provide information about the additional validation that may be required.
Minor changes to the assay (eg, a change in the supplier of MgCl2 or buffer) may require minimal
revalidation. Alternate suppliers of reagents or disposables need to be qualified.
Expansion of the intended use of the test, such as the addition of a new sample type (eg, adding CSF as a
sample type for a blood test) or the addition of a new organism mutant (eg, a new strain of influenza) to
be tested, should be associated with a validation of the test with the new sample type or mutation.
8.2 Considerations Related to Microarrays
8.2.1 Validation of Test System vs Target Validation
A full performance evaluation for multianalyte systems for every target (eg, accuracy, precision, LoD,
stability, interference) may be beyond the scope of many accredited laboratories. Precision analysis alone
may be cost prohibitive to perform for every target. While IVD devices do not require full evaluation,
many laboratories have a clinical need to perform tests using alternate specimen types, which would
require extensive validation experiments.
An alternative approach would be to perform full validation experiments using representative targets and
more limited experiments to evaluate additional target organisms. The analytical test system must
undergo full verification/validation (as appropriate), but there is no reason to think that organism types
with nucleic acid sequence similarity that would predict similar annealing and hybridization efficiencies
would perform significantly differently on the test system. Each organism type that may interact
differently with the system should undergo full validation. For example, a respiratory virus panel could be
validated using RNA virus (influenza) and DNA virus (adenovirus) with more limited validation data
obtained for other viruses detected. Addition of a bacterium to the assay would require full validation for
this change in the target organism. Microarray targets for specific organisms leading to clinically
actionable results, such as detection of resistance markers, would require more extensive validation as a
resistant strain may not be detected with the same primers/probes as the parent strain. In addition, some
organisms with inherent genomic instability due to error-prone polymerases would require more extensive
validation whereas strain characterization for organisms and strains without known clinical significance
could undergo limited validation.
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8.2.2 Validation of Rare Organisms/Hard to Find Samples
Laboratories may find it difficult to obtain representative cultures of all organisms detected in a panel and
clinical samples verified as samples containing the organism may be extremely rare. However, these may
be important pathogens with seasonal or geographic restrictions or emerging infectious threats.
Microarrays allow these infrequent target organisms to be detected, and the public health benefit through
additional detection in accredited laboratories is likely to outweigh the risk of false positives or failure to
detect these because of the absence of available tests.
Laboratories may approach this using an ongoing validation approach that recognizes the limitations of
their microarray assay for detection of organisms for which their laboratory has been unable to verify
performance characteristics. During the initial microarray test validation, an adequate number of samples
should be assayed and shown to be negative for the rare organism to ensure a high level of specificity.
Potential crossreactions should be taken into account both in silico and through interference studies.
After the assay is put into clinical use, a rare patient sample may test positive for the organism in
question. The laboratory then has the responsibility to verify that the organism is truly present in that
sample through additional testing at reference or public health laboratories while maintaining
communication channels with the physician(s) and infection control providers to ensure appropriate
treatment (ie, understanding the limitations of an initially unverified result) and public health precautions,
where necessary.
Among many other important considerations in designing a microarray intended for detection of
infectious diseases, the most critical initial parameter is the selection and generation of the desired probe
sequences for the respective pathogens, including their mutational variant strains, by careful analysis of
the nucleotide databases.
The identified nucleotide sequences are usually synthesized in situ unless the DNA fragments are
generated by other methods, such as plasmid constructs, PCR amplicons, or purified restriction digestion
fragments. The type of chemistry for nucleotide synthesis or other alternative methodologies used to
generate the DNA fragments, including the labeling procedures, should be optimized for steady
production. Subsequent steps also need to be properly validated to maintain consistency during the course
of the entire manufacturing process. A set of in-house probes printed on miniaturized array formats may
be considered to establish parameters for process validation. The use of an additional (or preferably more)
intra-array platform consisting of the identical set of probes generated from three or more independent
production lots could assist the generation of product specifications, especially in the development of a
new device. An average of at least triplicate determinations within the acceptable CV from the individual
platforms, normalized in terms of a standardized unit rather than arbitrary signal intensities derived from
each array formats, may be potentially suitable for establishing proper specifications. In addition, values
expressed in terms of standard units, yielding comparative data from various microarray platforms, may
be virtually adapted for unified data analysis and interpretation of results, irrespective of the type or
design of the microarray platforms.
High clinical sensitivity and specificity of an IVD device are crucial for accurate diagnosis of the
infectious diseases. A DNA microarray consisting of probe sequences highly specific to each pathogen
can be validated for its optimized performance using analytical reagents, normal specimens negative for
the infectious diseases, and known positive clinical specimens from the respective pathogen-specific
disease conditions. Such extensive validation may be labor intensive and highly time consuming;
however, once properly validated, these steps could potentially maximize the sensitivity of a microarray
assay system and minimize the nonspecific signals by adjusting the standardized assay cutoff threshold
value accordingly. A number of recently published studies have shown repeatability and reproducibility
of results from microarrays.271-275 Therefore, issues concerning irreproducibility or often-contradictory
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results must be carefully addressed for the improved performance before this technology can be
successfully introduced to clinical diagnostics.
In the United States, the MicroArray Quality Control project276-278 was initiated by a group of
scientists at the FDA and the US Environmental Protection Agency in an attempt to resolve
the above issues and other concerns related to the accuracy of data analysis
(http://www.fda.gov/ScienceResearch/BioinformaticsTools/MicroarrayQualityControlProject/default.htm).
These scientists closely evaluated key issues surrounding the reliability of DNA microarray data and have
generated a number of publications to facilitate scientific progress in various disciplines such as drug
development, clinical diagnostics, and the management of risk-benefit assessment. Other resources may
also be useful.279-282
Test results from licensed in vitro blood screening assays intended for detecting infectious diseases are
used to prevent potentially tainted blood (or plasma) units from being transfused or used in further
manufacturing of blood products. In addition, reliable results from an approved diagnostic test are pivotal
determining factors for clinicians in making decisions to treat patients with appropriate therapeutics.
Therefore, it is imperative that robust preclinical and clinical validation studies for microarray tests are
performed to establish safe and effective performance of the IVD devices. Development of miniaturized
microarrays comprising highly standardized nucleotide probes derived only from various genomic targets
specific to each of the transfusion-transmitted–related pathogens, including different subtypes,
recombinant strains, and up-to-date mutational variants, may potentially limit the issue of sample DNA
crossreactivity with the thousands of human gene sequence probes that are part of other large genomic
microarray platforms. Carefully prepared assay validation study protocols, detailed proposals for
precision, preclinical and clinical studies, and methods for data analysis and interpretation of results,
performed before or during the early stages of product development, may assist manufacturers in
performing effective process validation of microarray technologies intended for clinical purposes.
8.2.3 Instrumentation and Analysis Software
Any instrumentation or software used in the assay system should be evaluated separately for performance.
Equipment evaluation can be straightforward, such as verification of pipette accuracy and precision. More
sophisticated equipment and software may require installation qualification (IQ), operational qualification
(OQ), and performance qualification (PQ). Many manufacturers of life science or research use only
systems often supply validation packages for an additional cost. However, manufacturer-supplied IQ, OQ,
and PQ validation packages require that end-user assay developers (eg, those who develop LDTs) carry
out an additional PQ specific to the assay system to ensure that the instrument settings are appropriate for
the particular test performance. For some genetic testing methods, this can be quite extensive. An
example of a verification in which instrument/software PQ is determined for a molecular genetics test is
illustrated by van der Stoep et al.283 In the United States, for FDA-approved instrumentation,
documentation of the instrument validation is required and, therefore, repeat verification of the
instrumentation by the medical laboratory is not.
8.2.4 Characterization and Handling of Reagent Components
For molecular tests that use RNA and/or amplification enzymes, it is critical that the components be well
characterized and appropriately handled. DNA/RNA–based controls, nucleic acid extraction reagents,
buffers, and PCR enzymes can all be sources of contamination or other failure modes. The critical reagent
components should be identified during the risk assessment of the test design, and should be characterized
and controlled appropriately.
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8.2.5 Data Evaluation
Determination of whether a change in a test value is significant depends on the precision of the overall
system and the shape of the distribution of values about the mean. If the values follow a normal
distribution, parametric tests such as Student’s t-test are used for data analysis. If values do not follow a
normal distribution, nonparametric tests should be applied or the data should be transformed to comply
with parametric criteria. Datasets can be examined for normality using methods such as the Kolmogorov-
Smirnov test. In cases of non-normally distributed data, the user needs to decide if nonparametric tests are
appropriate, or if the data should be log10 transformed, and then analyzed using parametric methods.
Consideration needs to be given to the type of data being analyzed (eg, within-run precision or
comparison of methods) to determine the proper statistical methods to apply. If, for example, Student’s
t-test for dependent variables is appropriate for normal data, a comparable nonparametric method such as
the Wilcoxon matched pairs test needs to be used. If non-normal data are transformed, the appropriate
transformation function must be used. An example is log10 transformation of values for measurands
(analytes) having large potential measuring intervals. Sample size also needs to be considered, because
molecular diagnostic testing costs compared to the costs of other tests (eg, those of clinical chemistry
assays) often limit the number of samples that are run. With small sample sizes (eg, < 20 replicates), it is
difficult to determine whether data conform to a normal distribution, potentially leading to erroneous
results if parametric methods are used for analysis. Nonparametric methods are well suited for small
sample sizes and may be more applicable. Formulas for the statistical computation of within-run
imprecision and for comparison with manufacturers’ claims or other performance criteria can be found in
CLSI documents EP05,253 EP09,255 and EP15.257
9 Quality Control and Quality Assurance
9.1 Laboratory Quality Assurance and Quality Indicator Considerations
There are several important QA and quality improvement issues to be considered when microarrays are
used for clinical diagnostics. QA includes routine QC, PT, technical staff competency, instrument
calibration, and clinical correlation. QC measures and validates the correct performance of a test. QA is a
test process to include correct test results for all phases of the testing process (ie, preexamination,
examination, and postexamination). Regardless of assays used or reagent source, all clinical laboratories
must adopt practices and procedures to minimize the risk of contamination causing false-positive results,
along with controlling for false-positive results due to inhibition (refer to CLSI document MM0326). Due
to the complexity and diversity of microarray technology, an attempt to determine a “one-size-fits-all”
policy for all of them would be unsuccessful. However, there are standard guidelines that have been
applied to clinical tests in the past that can be applied successfully to arrays that are to be used for clinical
diagnostic use.
PCR arrays (described in Section 6.6) are available in “open” and “closed” formats. Most array platforms
are “open” format with regard to the ability of the end user to choose the capture oligonucleotides and
probes. However, there are an increasing number of arrays that are in a “closed” format, meaning the user
relies upon the manufacturer to provide a set of controls and the appropriate data interpretation.87 These
“closed” platforms are becoming more abundant as IVD device manufacturers seek to develop systems
that are easy to use, but also have FDA clearance or CE registration. In such systems, the end user is not
able to modify the assays to include custom controls. Internal or process controls are included in the
system and the instrument software will report an invalid result based on failure of the internal controls;
however, the end user does not necessarily have access to the data analysis that resulted in the invalid call.
The internal process controls demonstrate that one or more of the steps of sample preparation, reverse
transcription of cDNA into RNA, and PCR amplification of the target work satisfactorily. This addresses
the overall performance of the system. However, it does not demonstrate that the analyte-specific
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components of the array are functioning properly. Use of analyte-specific positive and negative controls is
necessary to demonstrate satisfactory function of each analyte-specific component of the microarray.
For example, the following may be performed at least once each day that patient specimens are
assayed284:
 For each qualitative procedure, include a negative and positive control.
 For each test system that has an extraction phase, include two control materials, including one that is
capable of detecting errors in the extraction process.
 For each molecular amplification procedure, include two controls, and if reaction inhibition is a
significant source of false-negative results, include a control material capable of detecting the
inhibition (refer to CLSI document MM0326).
However, as mentioned above, for systems with internal and/or procedural controls that monitor the entire
analytical process, less reliance may be placed on the use of external controls, especially closed systems
that adequately control reagent integrity and sample processing adequacy, and that incorporate internal
controls to monitor for sample interference and/or inhibition.
9.1.1 Positive and Negative Controls
Good laboratory practice recommends the use of positive and negative controls to ensure integrity of the
assay reagents and proper performance of the assay procedure. These controls are intended to monitor
substantial reagent failure at each step of the assay protocol (ie, sample preparation, amplification,
hybridization, and detection).
9.1.1.1 Negative Controls
Negative controls include DNA polymerase-free and RNA polymerase-free water, which can be included
as extraction negative control, PCR negative control, and hybridization/detection negative control.
Multiple negative controls are recommended for each assay run, and they can be interspersed throughout
the run (eg, first, mid, and last positions).
9.1.1.2 Positive Controls
Positive controls, or known positive samples containing the targeted sequences (external positive
controls), should be included with each assay run of patient specimens. Such controls should be prepared,
extracted, and tested in the same manner as patient specimens. Results from external controls should be
examined before the results from patient specimens. If the positive or negative control for a given
measurand does not perform as expected, all results for that measurand in the batch of samples should be
examined to determine if repeat testing is required.
Probes specific for certain nucleotide sequences may be used with internal (positive) controls in some
arrays. The internal control is added to each clinical specimen before extraction of nucleic acids, and it
allows the user to ascertain whether the assay is functioning properly. Failure to generate a positive result
for the internal control indicates a failure at either the extraction step, and/or the amplification step, and
may be indicative of the presence of amplification inhibitors, which can lead to false-negative results.
In PCR arrays of both the open and closed formats, testing of the whole system with positive and negative
controls becomes the primary method for verifying the assay performance in clinical laboratories.
Samples used for this purpose can be derived from clinical samples (ie, identified using a different test
method) or contrived samples that contain each of the target pathogens or sequences with the appropriate
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general operating instructions. For organisms that can be grown in culture, well-characterized organisms
or culture supernatants can be purchased from commercial vendors and be tested either neat or diluted
into specimen transport media. However, some pathogens are not available for purchase commercially,
either because of restrictions due to intellectual property rights or because they cannot be grown in
culture. In such cases, well-characterized clinical specimens can serve as useful surrogates (see CLSI
document MM1724).
In multiplex assay systems, normal positive control material from clinical or cultured material can serve
as both a positive control (for the organism identified in previous testing) and a negative control (for all
organisms not detected in prior testing). If the assay platform is sufficiently sensitive or the control
material is of sufficiently high concentration, then the control material can be pooled together to save time
and cost. Limitations of the sample pooling approach occur when similar organisms are tested in the same
array panel. Assays that detect two different nucleotide sequences within a given organism cannot be
tested with a panel of organisms that together contain both sequences. For this reason, assay
manufacturers can recommend combinations of organisms that can be tested together with their assays,
but clinical laboratories performing the assay validation must understand the assay details well enough to
avoid testing unsuitable combinations of organisms.
For some arrays, nucleic acid controls can be purchased from manufacturers who specialize in
commercial production of such products. If these controls contain synthetic nucleic acids, they must be
certified for testing on the assay platforms of interest so that they contain the appropriate nucleic acid
sequences to be amplified in the PCR array and avoid generating false-negative test results.
QC is more difficult to perform for microarrays than single-target assays. Ideally, a positive control
should be included for each nucleic acid target in each run of the assay. In reality, this is neither practical
nor cost effective for arrays capable of detecting a large number of targets. Positive controls for all targets
in such assays can be tested on a rotation schedule, using different representative controls on different
assay runs. Alternatively, multiple sequence targets in the appropriate matrix can be used to conserve
cost. The approach for an open test system should typically include an internal control, a negative control,
and one or more positive controls, with all positive controls included at a user-defined test frequency. In a
closed test system, the user should adhere to the assay manufacturer’s recommendations for QC.
9.1.1.3 Assay Reagents
QC of microarray assays include testing new reagents or kits of new lots and shipment, using necessary
controls and parallel testing of at least one negative clinical sample and one positive clinical sample
previously tested with current reagents or kits.
Laboratories should include documentation and guidance related to QA practices in the molecular assay
standard operating procedure. This documentation should include precise directions (ie, frequency and
techniques), criteria for acceptability, and guidance for reagent or kit implementation. Reagent or test kit
documentation should always include information on the manufacturer; type of reagent; safety data sheet;
lot number with known stability (ie, expiration date) and acceptable shelf life, storage, and handling
conditions; and lot-to-lot variation data for trending purposes.
9.1.2 Other Considerations
Key elements of the QA procedure for molecular assay development, including laboratory design and
practices, unidirectional workflow, and practices within the laboratory to prevent contamination, are
discussed in detail in CLSI document MM03.26 Also, QA during development of molecular diagnostic
tests is also described in detail in CLSI document MM03.26
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9.1.3 Proficiency Testing
Assurance of good-quality results generated from routine testing of patient specimens requires PT (see
CLSI documents GP27285 and GP29286). Periodic challenges from testing samples from external
laboratory accreditation agencies, such as the College of American Pathologists or alternate PT programs,
are the typical source of PT samples. Using interlaboratory programs can be acceptable if no external
systems are available for the specific measurands. For example, split specimens with documented criteria
from the standard operating procedure could be used and are typically directed by the QA department of
the clinical laboratory. Unacceptable PT challenges should be investigated and finalized by corrective
action. PT leads to quality improvement, which is the cornerstone of QA.
9.1.4 Technical Staff Competency
Laboratory personnel must be competent in microarray test performance (see CLSI document QMS03287).
QA requires monitoring the preexamination, examination, and postexamination phases of testing.
Operator competency assessment includes observation and documentation of the technologist performing
the test on a scheduled basis. This should include validation of staff adherence to the standard operating
procedure exactly as stated, biosafety, patient confidentiality, result interpretation, reporting, and QC
documentation. In addition, laboratory personnel should have documented evidence of continuing and
relevant education.
9.1.5 Instrument Quality Assurance
The quality program must be well documented and organized (see CLSI document QMS13288). The
documentation for all instruments must include cleaning, function, calibration, maintenance, repairs, and
service. For further details and guidance, refer to the relevant specialty checklists (eg, microbiology and
molecular pathology) from the Laboratory Accreditation Program of the College of American
Pathologists (http://www.cap.org/apps/cap.portal?_nfpb=true&_pageLabel=accreditation) or from ISO.10
Instrument calibration should be performed on a semiannual basis or as the manufacturer recommends. If

multiple instruments of the sample type are in use, the process consists of validating the instruments
against each other with an adequate number of representative patient test samples.
10 Data Analysis and Interpretation
10.1 Challenges of Adapting Microarray Technologies to Detection of Pathogens
The application of microarray technology to the detection and identification of infectious agents is still
new enough that the advantages and limitations compared to existing nucleic acid detection and
serological testing approaches are still being worked out. Complex statistical and bioinformatics
challenges, in addition to adequate assessment of accuracy, specificity, reproducibility, and reliability of
data or data analysis, constitute some of the many intrinsic challenges with microarrays.289 Nevertheless,
customized microarrays for the detection and identification of pathogens, including their genetic variants,
may potentially be of great utility to diagnostic laboratories for the development of specific panels or
disease-specific applications. The following sections address analysis methods and interpretation of
microarray data. Detailed recommendations for the reporting of results can be found in CLSI document
MM03.26
10.1.1 Variable Sensitivity of Measurand Detection
The major challenge underlying the molecular diagnostics of many viruses, especially influenza, HIV,
HCV, or even some bacteria and emerging parasites, is their extreme genetic mutational variability.
Nucleotide sequences of pathogens of the same species or even the same serotype may be significantly
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heterogeneous. These difficult challenges are generally taken into consideration, requiring careful
analysis of the most recent databases and phylogenetic trees, when designing the primer sequences for
PCR amplification and target probe segments for their subsequent detection steps.
Microarrays and the multiplex results they generate highlight this problem because they may show
excellent sensitivity for most of the analytes they are designed to detect, but lower sensitivity for a few of
the analytes.172 The manufacturer of such a system can report the lower sensitivity in the product insert;
however, this may not meet the needs of the clinicians using the test. Because it may take a year or more
for an IVD device manufacturer to revise the microarray, perform the necessary analytical and clinical
studies, and receive regulatory agency clearance or approval for the revised panel, it may be necessary for
the laboratory to bring on a secondary test for particular measurands in the interim.
10.1.2 Microarray Signatures From Novel Pathogens
The last decade saw the emergence of several novel human pathogens, such as severe acute respiratory
syndrome, the pandemic 2009 influenza H1N1, and limited cases of avian influenza H5N1infection, into
the human population. As microarrays become more common in the diagnostic laboratory, they will
become part of the “early warning” system for the detection of these novel pathogens. In many of these
cases, the novel pathogen will be detected by some, but not all, of the tests on the microarray. Influenza
provides the best example of this because there are assays on most microarray platforms for sequence
elements conserved across all strains of influenza, including avian and ovine, and there are subtype-
specific assays that recognize the H1 and H3 serotypes of the hemagglutinin (HA) gene. A novel
influenza may not be detected at all or it may be detected as “HA untypeable.” In the right context, this is
highly informative.290
In the last four years, massively parallel sequencing has made even greater advances in microbial
surveillance and outbreak monitoring than microarray technology. In the future, any novel pathogen,
identified by an outbreak of “negatives” using conventional testing or microarray technology, is likely to
be rapidly sequenced, and then conventional singleplex PCR assays will be developed to detect the
presence of the organism.291,292 In such cases, microarray diagnostics will play a supportive role to the
singleplex assay by providing a presumptive diagnosis for an emerging agent that has become prevalent
in the community, or by identifying patients with co-infections to the novel pathogen than may need
additional treatment.
10.2 Microarray Data Analysis Steps
10.2.1 Low-level Analysis
Microarray analysis technology involves a complex electrical-optical-chemical process that spans detector
array fabrication, target preparation, fluorescence dye labeling, hybridization, washing, fluorophore
excitation, imaging-using optics, slide scanning, analog to digital conversion using either CCDs or
photomultiplier tubes, and, finally, image storage and archiving.293
Low-level analysis is a term widely used in the microarray field; it refers to all of the steps from
hybridization imaging (pixel-level data) to the final quantification of genetic targets (DNA or RNA).294
Issues of primary importance and challenges are image analysis, assay QC, background correction, and
normalization.
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10.2.2 Image Analysis
10.2.2.1 Spot Identification and Grid Alignment
The problem of gridding (sometimes denoted as addressing or spot finding) is to assign an image
coordinate to every element of the spot array. If the array is on a membrane, there is frequently nonlinear
warping of the matrix, which means that the observed spot array will not have the strict regularity of an
array printed to glass. A significant rotation of the overall spot array can sometimes be observed.
Segmentation allows the classification of pixels belonging to a spot as signal (ie, corresponding to a spot
of interest) or background. Fixed circle segmentation fits a circle with a constant diameter to all spots in
the image. This method works when all the spots in the array are circular and of the same size, which is
rarely the case. In adaptive circle segmentation, the circle’s diameter is estimated separately for each spot.
Though the adaptive circle segmentation is more flexible than the former method, the spots are rarely
perfectly circular in practice and can exhibit oval and “doughnut” shapes. Two frequently used methods
in microarray image analysis for adaptive segmentation are watersheds and seeded region growing. The
histogram segmentation uses a target mask that is chosen to be larger than any spot in the image. Signal
and background intensity estimates are determined from the histogram of pixel values for pixels within
the masked area for a spot.295
10.2.2.2 Determination of Spot Intensity
Quantification deals with the measuring of the spot signal and background values. Histogram
segmentation techniques measure these values directly. For other segmentation methods, it is necessary to
calculate signal and background intensities based on the segmentation, and possibly including spot quality
measures. Most microarray analysis approaches define the signal intensity as the mean, median, or
another percentile of pixel values within the segmented spot mask.
10.2.3 Assay Quality Control
10.2.3.1 Internal Controls
The presence of exogenous or endogenous sample controls is important to ensure that the multiple steps
of microarray analysis (ie, extraction, amplification, hybridization, and detection) have occurred as
expected, ensuring that negative results are not due to assay failure. Ideally, the appropriate internal
control will be present at a level near the LoD for the assay, and closely mimic the nature of the organism
being sought. Multiple internal controls can be incorporated to help determine the precise step of assay
failure. Internal controls should be shown not to interfere with assay sensitivity and QC metrics, such as
signal levels for the internal control, can help to evaluate assay performance over time.
10.2.3.2 Amplification Quality Control
The presence of adequate target nucleic acid and incorporation of fluorescent dye can be evaluated using
spectrophotometric parameters, such as the optical density at 260 and 280 nm and measuring the
fluorescence incorporation at the appropriate wavelength. The size distribution of amplified products can
be evaluated through gel electrophoresis or column methods. Other methods of amplification QC, such as
PCR or quantitative PCR for specific targets, can be used depending on the need for highly precise
amounts of input nucleic acid for hybridization.
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10.2.3.3 Hybridization Quality Control
For hybridization, the quality of a microarray is usually assessed by factors such as:
 Spatial effect (uneven slide washing, nonuniform background)

 Chip effect (manufacturing defects)
 Procedural artifacts (high background, foreground signal saturation, dust, contamination)
 Multichip normalization scaling factor
Image-based spot QA (or grid screening) is used to identify grid cells that contain valid spots and to
eliminate invalid spots from further analysis. In order to detect invalid or defective spots, spot validity
criteria (metrics) must be defined. In general, criteria for evaluating spot validity can be divided into two
classes. The first class of spot validity criteria is for assessing foreground and background intensities. It
includes assessing absolute background and foreground levels, background variation, foreground
saturation, and foreground-to-background intensity ratio (or signal-to-noise ratio). The second class is for
evaluating morphological properties of foreground, such as spot shape and size irregularities, or spot
location (position offset).296
The determined metrics can be compared across the array probes to determine that the hybridization
meets QC criteria. Microarrays should have acceptable levels of background intensity, background
variation, and foreground saturation. Inclusion of positive and negative control probes across the surface
of the microarray can be useful in evaluating adequate hybridization conditions.
10.2.4 Background Correction
Background represents a value of the measured signal intensity that is presumed to be due to nonspecific
binding of target to the probe, and it must be removed from the signal intensity measurement in order to
quantify the amount of target nucleic acid present in the sample accurately. Spot intensity signals obtained
from scanning a microarray do not only originate from fluorescent molecules attached to hybridized
DNA, but also from other sources referred to as background. For example, the source of the background
signal could be fluorescence from the coating of the glass or contamination from the hybridization and
washing procedures. The scanner device can also be a major source of background due to varying filter
bandwidths, optics, or photomultiplier tubes. Background is assumed to be additive to the true spot signal
such that the measured spot intensity equals true intensity plus background intensity.297
Background variations occur due to microarray slide preparation (eg, hybridization and spotting errors)
and inappropriate acquisition procedures (eg, presence of dust or dirt) can be detected by microarray QA.
The variation due to image acquisition instruments (eg, nonlinearity of imaging components) cannot be
removed by a user. Thus, many image-processing algorithms compensate for background variations by
modeling their probability distribution function. The most frequent model is the gaussian or normal
probability distribution function. Other statistical models to consider would be a uniform or a functional
probability distribution function, depending on the observed properties of acquired images.
The term background correction, also referred to as signal adjustment, describes a wide variety of
methods. More specifically, a background correction method should perform some or all of the following:
 Corrects for background noise and processing effects
 Adjusts for crosshybridization, which is the binding of nonspecific DNA (ie, noncomplementary
binding) to the array
 Adjusts expression estimates so that they fall on the proper scale, or are linearly related to
concentration
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It is important to note that this definition is somewhat broader than is often used in the wider community.
Often, only methods dealing with the first problem have been referred to as background correction
methods. The methods of background correction will vary depending on the specific array technology
used. Some array systems, such as spotted cDNA microarrays, can use pixels surrounding a spot to
compute the local background adjustment. Other technologies with very densely spaced probes require
that the probe intensities themselves be used to determine any background correction adjustment.
10.2.4.1 Determination of Background Intensity
The standard method for estimating the background of a spot is to assume that the background level is the
same as the intensity in the proximity of the spot and excludes other spots. Two major approaches exist
for estimating this intensity—local background and global background. Local background is determined
for an area near each spot, and after identifying the so-called background pixels within this area, the
background estimate of the spot is then taken as the sample median (or mean) of these pixels. Global
background is determined from the signal of negative control spots on the array.
10.2.4.2 Global Background Correction
The global background intensity is typically calculated as the mean or median value of the negative
control probes (ie, those probes for which there is not a corresponding target in the same type examined).
This method will subtract the mean/median negative probe signal from all probes on the array to bring the
average signal for negative probes close to zero. Some probes may have small negative values following
background subtraction, and can be assumed to have spot intensities of zero for further analysis.
10.2.4.3 Local Background Correction
The signal derived from pixels surrounding a spot can be used to calculate the background intensity for
each probe in the array, which can then be subtracted from the measured spot intensity. This method is
typically used when there are variations in the array surface that can lead to nonspecific binding of
labeled target, such as with spotted cDNA microarrays. Other commercial array platforms have less
nonspecific binding effect, and local correction may not be necessary for these.
10.2.5 Normalization
Data quantification (or spot feature extraction) refers to extracting descriptive values of foreground and
background pixels for each spot. Ideally, extracted descriptors (also called features or attributes) should
be directly proportional to the nucleic acid quantity in the solution that was deposited in a spot, and
should represent the deposited target gene level. However, fluorescent intensity measurements in each
channel might be scaled or distorted differently according to some linear or nonlinear functions during
data preparation steps. Thus, normalization of extracted spot descriptors can be desirable.
The motivation for normalizing microarray images and/or extracted descriptors comes from the fact that
one would like to compare results obtained from multiple slides, scanners, or laboratories, and multiple
microarray techniques. The difficulty of performing meaningful comparisons arises from different slide
preparations (eg, amounts of amplified nucleic acid), scanner settings, microarray protocols, or labeling
specifics. Normalization will identify and remove the effects of systematic variation in the measured
fluorescence intensities other than differential expression, such as different labeling efficiencies of the
dyes; different scanning parameters; and print-tip, spatial, or plate effects.
The purpose of normalization is to adjust for these variations, primarily for label efficiency and
hybridization efficiency, so that signal intensity can be compared between different array preparations.
Therefore, before multiple microarray measurements can be integrated into a single analysis, the reported
measurements need to be normalized, or modified (possibly corrected), to make them comparable. When
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microarrays are used to collect gene expression data in an experiment, normalization might simply be a
matter of adjusting the overall brightness of each scanned microarray image. This approach assumes that
all of the arrays use homogeneous populations of similar cells, the same microarray technology, and the
same quantity of RNA. Other normalization methods include using expression levels of “housekeeping”
genes, using assumptions that most genes do not change across experiments, using spline mathematics, or
other nonlinear techniques.298
In general, the approaches to normalization can be divided into:
 Methods using statistical descriptors, such as the median and the mean
 Techniques using control spots, such as housekeeping genes or spiked-in controls
 Correlation (regression) analyses
For arrays with a big number of probes (eg, thousands to tens of thousands), a global normalization is
generally used. The aim of the normalization is to make all of the arrays have the same
mean/median/quantiles. There is also the major assumption that most of the genes do not vary among
arrays (ie, even between normal vs disease cases), so that the total fluorescence signal is forced to be
constant. For small arrays (eg, < 100 probes) the normalization can be based on housekeeping genes or
spiked-in controls, because the overall expression of the arrays cannot be assumed to be constant.
10.2.5.1 Normalization Using Control Probes
This technique requires inserting spots of known intensities or genes of known expression level (eg,
spiked internal controls) into a microarray slide or use genes that are known to be constant across
specimens (eg, housekeeping genes used as endogenous controls). By using spiked internal control spots,
one can normalize all other spots with respect to the reference intensities defined by the control spots. In
this case, it is recommended to scatter the control spots across an entire slide so that local variations can
be normalized accurately.
10.2.5.2 Global Normalization
This normalization method uses statistical descriptors such as sample mean, median, mode, or percentile
of intensity distribution. This particular normalization can be performed by either division or subtraction
of statistical descriptors. For example, one could apply Z-transformation to the problem that consists of
subtracting the sample mean from all intensities and dividing their values by the SD of the microarray
spot intensities.
10.2.5.3 Normalization Using Regression Analysis
Regression ratios are quite often used as part of fluorescence channel normalization or normalization
across multiple arrays. While the majority of analyses assume linear dependency, it is also possible to
assume nonlinear models, such as piecewise linear, polynomial, or curve dependency models. Approaches
to model nonlinear fluorescence channel dependencies are based on:
 Introducing higher order models (eg, locally weighted polynomial regression [Lowess] or exponential
model)
 Dividing an intensity range into segments where linearity can be assumed (piece-wise linear model)
 Combining the two previous strategies
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10.2.5.4 Visualization of Normalization Impact
The impact of normalization procedures on expression data may be visually examined using box plots,
pair-wise scatter/correlation plots, and data variability vs mean (MA) plots. For box plots, multiple arrays
are plotted side by side with each box plot representing one array. After the normalization, it is expected
that the signal distributions from all the arrays are similar. Particularly, in each box plot, the range, first
quartile, median, and the third quartile are shown. For the scatter plots and the MA plots, it is expected
that all of the data points for each array spot fall along the straight line.
10.3 Threshold Value Determination
10.3.1 Limit of Blank
Following low-level analysis, the resulting background-adjusted, normalized data must be examined to
identify which probes are most likely to yield insight into the biological process being evaluated; in this
case, the detection or genetic characterization of infectious pathogens. Due to the nature of the assay (eg,
detection of the fluorescence signal), all of the spots will give non-negative results, some of which are due
to true hybridization, and some are simply noise. Most of the background-corrected signals are still non-
negative; this means that some of the signals cannot be treated as reliable quantifications of DNA amount.
The upper limit of the background signal can be determined by calculating a percentile of the upper limit
of the signals measured from those known nonspecific probes (eg, scrambled sequences or pathogen
sequences known to be absent in the samples analyzed). This measure is generally referred to as the LoB.
10.3.1.1 Samples to Be Examined
LoB should be determined using samples that are representative of the matrix to be tested. For organism
detection tests, LoB would require clinical samples, while genotyping or subtyping arrays would use
cultured organisms or clinical samples, depending on the sample type analyzed. Because large-scale
microarray probes can interact differently with different specimen blanks, it is preferable to test individual
negative samples rather than replicates of known blank samples. The number of samples/replicates used
should be at least 20 (see CLSI document EP17258).
10.3.1.2 Data Analysis
CLSI document EP17258 has an extensive description of LoB determination, which depends largely on the
accepted values for α error (ie, Type I or false-positive error). While the most common approach uses α =
0.05, this may not be appropriate for large-scale microarray testing, where several hundred or thousand
individual probe-level measurements are made. In particular, the risk of a Type I error for a large probe
set would be very high, and it may be more appropriate to use α = 0.01 or even 0.001, depending on how
many positive probes are required for a positive result.
Once an appropriate α value has been chosen, the LoB can be determined using parametric or
nonparametric methods. If the data follow a gaussian distribution, parametric analysis can be applied, and
LoB determined as the mean plus a multiple of the SD. For example, if α = 0.01, the LoB is at the 99th
percentile above the mean, and LoB = mean + 2.58 • SD. If the data do not follow a gaussian distribution, a
nonparametric method can be used to determine the 99th percentile of ranked background spot intensities,
and this value is taken as the LoB.
LoB should be calculated for each probe on the microarray, as well as across all the probes. The result can
be calculated using a nonparametric approach or parametric approach, if the distribution of microarray
probe signals is within a normal distribution. The highest level for LoB can be applied for the entire
microarray, and probes with significantly elevated LoB are ideally excluded from analysis due to their
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nonspecific signal. Thus, the primary goal of LoB studies for microarrays is to ensure that there are no
poorly acting or crosshybridizing probes that could yield potential false-positive results.
10.3.2 Limit of Detection
Any signal value greater than LoB (eg, LoB + δ) can be claimed with high confidence that it is not a
measurement from a blank sample. However, due to the assay variation, the repeated measures of a low
sample with true concentration of LoB + δ may very likely have values below LoB. Therefore, the
confidence on the decision of whether a signal is noise or a true measure of the gene expression is
associated with two possible errors—false-positive error (Type I error) and false-negative error (Type II
error). LoB only specifies a limit that should be exceeded for a result to be declared significantly higher
than a blank measurement; it does not, however, address the lowest value that can be distinguished from a
blank value with reasonable assurance. Actually, repeated measurements of a sample with a true value
exactly equal to the limit of statistical significance yield a distribution with 50% of values below and 50%
above the limit because of random measurement error. LoD is the lowest value that significantly exceeds
the measurements of a blank sample. It is the minimum sample concentration (ie, amount of nucleic acid
for microarrays) that provides measured concentration values exceeding the LoB with a specified
probability. If the Type II error level is set as β = 0.95, 95% of the measurements of a sample with a
concentration that equals LoD should exceed the LoB. All the probes with signal greater than LoD are
consequently deemed detectable, and thus, the target is determined to be present in the tested sample.
Each probe should have its own LoB and LoD, where feasible. LoD and LoB can be determined by a
variety of statistical methods using repeated measures of a single low sample (eg, a blank sample) or
repeated measures of multiple low samples (see CLSI document EP17258).
10.3.2.1 Specimens to Be Analyzed
Positive samples containing representative organism genotypes should be included in an LoD study.
While it may not be feasible to analyze all genotypes, representative organisms should be chosen to
encompass the range of detection/subtyping probes used on the microarray. Once LoD has been
determined for a certain organism genotype, additional types can be assessed using verification testing.
The initial determination of LoD should encompass at least five dilutions for each starting sample.
Typically, a tenfold dilution series will be analyzed. At least five replicates for each sample should be
performed, at which point the lowest dilution with 100% detection (ie, signal value greater than LoB) will
guide further LoD determination. At least three dilutions around this point should be analyzed with at
least 10 replicates and at least one dilution showing 100% detection.
10.3.2.2 Data Analysis
Probit analysis is typically used in determining LoD for infectious disease targets, which is described in
CLSI document EP17.258 The level corresponding to 95% detection rate is taken as LoD. If the probit
analysis does not show a linear response, testing of additional replicates or dilutions may be necessary.
Alternatively, using the protocol described above, LoD can be estimated conservatively as the lowest
level with 100% detection. This approach can be useful when analyzing large probe sets, where it may be
difficult to obtain a linear probit analysis for each probe. Probe-level LoD should be reported for each
individual genotype, or for a class of organisms using the highest applicable LoD.
LoDs for each probe on the microarray can be used to develop data analysis sets, or algorithms, to classify
samples as having particular organisms or genotypes detected or not detected. Therefore, it is not essential
that each probe have the same LoD, but that each minimal probe set used to define an organism type as
detected have reasonable comparable LoD. If a particular probe containing sequence known to be present
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in a positive sample has a much higher LoD, the probe is not sufficiently sensitive for detection and
should be excluded from the analysis.
10.4 Classifier Development (Supervised Learning)
10.4.1 Manual Classification Algorithms
For smaller probe sets, the classification for each target on the array can represent a single organism or a
set of organism types that has been determined manually at the probe design step. Multiple and/or
degenerate probes can be designed for each target sequence in order to detect mutant organisms. The
validation of the manual classification algorithm will be approached at both the probe design stage (see
Section 5.3.2) and evaluation with a set of organisms with known subtypes/phenotypes (see Section
10.4.2). An example of this approach would be a microarray to detect influenza A, using known
conserved regions as universal detection probes, along with subtyping probes for each strain to be
detected by the array. When using multiple probes for the same target sequence, the LoD will likely vary
among the different probes, and a classification scheme will have to be developed to resolve discrepant
calls at the probe level. The algorithm used should be based on the biology and known sequence
variability of the organism being detected, and will have to be validated using known organisms (see
Section 8).
10.4.2 Classification/Prediction Algorithm Development
After the probe detection limits have been identified, a statistical prediction/classification algorithm can
be developed using these probes and the known phenotype classes. Some of the general statistical
approaches include:
 Linear or quadratic discriminant analysis

 Logistic regression
 k-nearest neighbors
 Classification and regression trees
 Support vector machines
 Naïve Bayes classifier
In developing a prediction rule, the results for multiple samples from each of the classes/phenotypes for
each organism/genotype must be known through a reference method, because this is the gold standard for
model development and performance evaluation. For infectious disease microarrays, these would
typically consist of a set of samples or isolates with known subtype/serotype/resistance profiles. Most
class prediction methods do not work well when there are a large number of independent variables (ie,
genes/spots/probe sets) in comparison to the number of samples, because this typically results in
overfitting of the model. The implications of overfitting are that the model works well for the data at
hand, but may perform poorly when applied to an independent dataset. Therefore, filtering the set of
spots/probe sets to reduce the dimensionality is essential. Once a set of “important” probes has been
identified, the multivariate function producing the prediction rule must be defined. Examples of
multivariate methods include weighted voting, support vector machines, compound covariate prediction,
k-nearest neighbors, and others as mentioned in this section. It is essential that once the prediction rule
has been defined, an unbiased estimate of the true error rate must be calculated.
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10.4.3 Evaluate Model Performance
10.4.3.1 Use of Training Dataset and Independent Test Dataset
Applying the prediction rule to an independent dataset and k-fold cross-validation are two commonly used
methods that provide a measure of the quality of a prediction algorithm. In a setting where many
observations are available, it is recommended that the dataset be randomly divided into two parts,
representing a training and test dataset. The prediction algorithm is built using the training dataset, and
once a final model has been developed, the prediction rule is applied to the test dataset to estimate the
generalization error. In the clinical diagnostic setting, it is recognized that often a laboratory may be
constrained by the number of samples, so that withholding a large portion of the data for validation
purposes may limit the ability of developing a prediction rule. Therefore, cross-validation has proven
useful for small datasets.
10.4.3.2 Use of Cross-Validation From a Single Dataset
K-fold cross-validation requires one to split the dataset randomly into k equally sized groups. Thereafter,
the model is fit to k − 1 parts of the data and the generalization error is calculated using the kth remaining
part of the data. This procedure is repeated so that the generalization error is estimated for each of the k
parts of the data, providing an overall estimate of the generalization error and its associated standard
error. With a sample size of N  200, five- or 10-fold cross-validation will provide an estimate of the
generalization error that is approximately unbiased. With smaller sample sizes, five- or 10-fold cross-
validation tends to overestimate the generalization error. Therefore, in small sample settings, N-fold
cross-validation, or “leave-one-out,” is recommended as it provides an unbiased estimate of the
generalization error.
A permutation-based p-value for the cross-validation rate may be used to assess the strength of the
multivariate predictor. The procedure is as follows: for each random permutation of class labels, the entire
cross-validation procedure is repeated to determine the cross-validated misclassification rate obtained
from developing a multivariate predictor with two random classes. The final p-value is the proportion of
the random permutations that give as small a cross-validated misclassification rate as was obtained in the
observed data. In summary, N-fold cross-validation is likely to be the most practical method for assessing
the generalization error of a prediction algorithm when modeling microarray data, and the probability of
the cross-validation error being as extreme or more extreme than the one observed may be calculated
using permutation testing.
10.4.3.3 Evaluation Methods
The developed prediction algorithm is applied to the test or cross-validation dataset and its results are
compared with the known status of each sample from the reference method. The test characteristics using
the developed prediction algorithm can then be calculated based on the reference method. Comparison to
the reference method can be done in a 2 × 2 table format, with calculations of test sensitivity, specificity,
accuracy, and other relevant parameters. Discrepancies may arise due to failures of the microarray method
or reference method, particularly when a less sensitive reference method is used. Analysis using
additional assays can be used to resolve discrepant results in order to more accurately determine the test
characteristics of the microarray method (see CLSI documents EP12256 and EP24299).
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251
Burd EM. Validation of laboratory-developed molecular assays for infectious diseases. Clin Microbiol Rev. 2010;23(3):550-576.
252
CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved Guideline—Third Edition. CLSI
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253
CLSI. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—Second Edition. CLSI document
EP05-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2004.
254
CLSI. Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved Guideline. CLSI document
EP06-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2003.
255
CLSI. Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved Guideline—Third Edition. CLSI
document EP09-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2013.
256
CLSI. User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline—Second Edition. CLSI document EP12-A2.
257
CLSI. User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition. CLSI document EP15-A2.
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CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved Guideline—Second Edition. CLSI
document EP17-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2012.
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Association for Public Health Laboratories/STD Steering Committee. Example verification protocol for Chlamydia trachomatis and
Neisseria gonorrhoeae: nucleic acid amplification testing of rectal swabs, October 15, 2009.
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CLSI. Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition. CLSI document EP07-A2. Wayne, PA: Clinical
and Laboratory Standards Institute; 2005.
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Lacbawan FL, Weck KE, Kant JA, Feldman GL, Schrijver I; Biological and Molecular Genetic Resource Committee of the College of
American Pathologists. Verification of performance specifications of a molecular test: cystic fibrosis carrier testing using the Luminex
liquid bead array. Arch Pathol Lab Med. 2012:136(1):14-19.
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Zheng H, Jia H, Shankar A, Heneine W, Switzer WM. Detection of murine leukemia virus or mouse DNA in commercial RT-PCR reagents
and human DNAs. PloS One. 2011;6(12):e29050.
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Tilburg JJ, Nabuurs-Fransssen MH, Van Hannen EJ, Horrevorts AM, Melchers WJ, Klaassen CH. Contamination of commercial PCR
master mix with DNA from Coxiella burnetii. J Clin Microbiol. 2010;48(12):4634-4635.
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in-house genotypic HIV drug resistance tests. Antivir Ther. 2010;15(1):121-126.
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Second Edition. CLSI document EP24-A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2011.
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Number 3 MM22-A
The Quality Management System Approach

Clinical and Laboratory Standards Institute (CLSI) subscribes to a quality management system approach in the
development of standards and guidelines, which facilitates project management; defines a document structure via a
template; and provides a process to identify needed documents. The quality management system approach applies a
core set of “quality system essentials” (QSEs), basic to any organization, to all operations in any health care
service’s path of workflow (ie, operational aspects that define how a particular product or service is provided). The
QSEs provide the framework for delivery of any type of product or service, serving as a manager’s guide. The QSEs
are as follows:
Organization Personnel Process Management Nonconforming Event Management

Customer Focus Purchasing and Inventory Documents and Records Assessments
Facilities and Safety Equipment Information Management Continual Improvement
MM22-A addresses the QSE indicated by an “X.” For a description of the other documents listed in the grid, please
refer to the Related CLSI Reference Materials section, beginning on page 82.
Event Management
Customer Focus
Nonconforming
Documents and
Purchasing and
Improvement
Facilities and
Organization
Management
Management
Assessments
Information
Equipment
Personnel
Continual
Inventory
Records
Process
Safety
X
EP05
EP06
EP07
EP09
EP12
EP15
EP17
EP24
EP28
GP27 GP27 GP27
GP29 GP29
M29
MM03
MM05 MM05
MM06
MM09
MM10
MM12
MM13
MM17
MM19 MM19 MM19 MM19 MM19 MM19 MM19 MM19 MM19 MM19 MM19 MM19
QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01
QMS03
QMS13
©
Volume 34 MM22-A
Path of Workflow
A path of workflow is the description of the necessary processes to deliver the particular product or service that the
organization or entity provides. A laboratory path of workflow consists of the sequential processes: preexamination,
examination, and postexamination and their respective sequential subprocesses. All laboratories follow these
processes to deliver the laboratory’s services, namely quality laboratory information.
MM22-A addresses the clinical laboratory path of workflow processes indicated by an “X.” For a description of the
other documents listed in the grid, please refer to the Related CLSI Reference Materials section on the following
page.
Preexamination Examination Postexamination
Sample management
Results review and
receipt/processing
Sample collection
Results reporting
Sample transport
and archiving
Interpretation
Examination
Examination
follow-up
ordering
Sample
X X X X X
MM03 MM03 MM03 MM03 MM03 MM03
MM05 MM05 MM05 MM05 MM05 MM05 MM05
MM06 MM06 MM06 MM06 MM06 MM06 MM06 MM06
MM09 MM09 MM09 MM09 MM09 MM09 MM09 MM09
MM10 MM10 MM10 MM10
MM12 MM12 MM12 MM12 MM12 MM12 MM12
MM13 MM13 MM13 MM13
MM19 MM19 MM19 MM19 MM19 MM19
QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01 QMS01
©
Number 3 MM22-A
Related CLSI Reference Materials

EP05-A2 Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline—
Second Edition (2004). This document provides guidance for designing an experiment to evaluate the
precision performance of quantitative measurement methods; recommendations on comparing the resulting
precision estimates with manufacturers’ precision performance claims and determining when such
comparisons are valid; as well as manufacturers’ guidelines for establishing claims.
EP06-A Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach;

Approved Guideline (2003). This document provides guidance for characterizing the linearity of a method
during a method evaluation; for checking linearity as part of routine quality assurance; and for determining
and stating a manufacturer’s claim for linear range.
EP07-A2 Interference Testing in Clinical Chemistry; Approved Guideline—Second Edition (2005). This document
provides background information, guidance, and experimental procedures for investigating, identifying, and
characterizing the effects of interfering substances on clinical chemistry test results.
EP09-A3 Measurement Procedure Comparison and Bias Estimation Using Patient Samples; Approved
Guideline—Third Edition (2013). This document addresses the design of measurement procedure
comparison experiments using patient samples and subsequent data analysis techniques used to determine the
bias between two in vitro diagnostic measurement procedures.
EP12-A2 User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline—Second Edition
(2008). This document provides a consistent approach for protocol design and data analysis when evaluating
qualitative diagnostic tests. Guidance is provided for both precision and method-comparison studies.
EP15-A2 User Verification of Performance for Precision and Trueness; Approved Guideline—Second Edition
(2006). This document describes the demonstration of method precision and trueness for clinical laboratory
quantitative methods utilizing a protocol designed to be completed within five working days or less.
EP17-A2 Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved
Guideline—Second Edition (2012). This document provides guidance for evaluation and documentation of
the detection capability of clinical laboratory measurement procedures (ie, limits of blank, detection, and
quantitation), for verification of manufacturers’ detection capability claims, and for the proper use and
interpretation of different detection capability estimates.
EP24-A2 Assessment of the Diagnostic Accuracy of Laboratory Tests Using Receiver Operating Characteristic
Curves; Approved Guideline—Second Edition (2011). This document provides a protocol for evaluating
the accuracy of a test to discriminate between two subclasses of subjects when there is some clinically relevant
reason to separate them. In addition to the use of receiver operating characteristic curves and the comparison
of two curves, the document emphasizes the importance of defining the question, selecting the sample group,
and determining the “true” clinical state.
EP28-A3c Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory; Approved
Guideline—Third Edition (2010). This document contains guidelines for determining reference values and
reference intervals for quantitative clinical laboratory tests. A CLSI-IFCC joint project.
GP27-A2 Using Proficiency Testing to Improve the Clinical Laboratory; Approved Guideline—Second Edition
(2007). This guideline provides assistance to laboratories in using proficiency testing as a quality
improvement tool.
GP29-A2 Assessment of Laboratory Tests When Proficiency Testing Is Not Available; Approved Guideline—
Second Edition (2008). This document offers methods to assess test performance when proficiency testing
(PT) is not available; these methods include examples with statistical analyses. This document is intended for
use by laboratory managers and testing personnel in traditional clinical laboratories as well as in point-of-care
and bedside testing environments.

CLSI documents are continually reviewed and revised through the CLSI consensus process; therefore, readers should refer to
the most current editions.
©
Volume 34 MM22-A
Related CLSI Reference Materials (Continued)

M29-A3 Protection of Laboratory Workers From Occupationally Acquired Infections; Approved Guideline—
Third Edition (2005). Based on US regulations, this document provides guidance on the risk of transmission
of infectious agents by aerosols, droplets, blood, and body substances in a laboratory setting; specific
precautions for preventing the laboratory transmission of microbial infection from laboratory instruments and
materials; and recommendations for the management of exposure to infectious agents.
MM03-A2 Molecular Diagnostic Methods for Infectious Diseases; Approved Guideline—Second Edition (2006).
This guideline addresses topics relating to clinical applications, amplified and nonamplified nucleic acid
methods, selection and qualification of nucleic acid sequences, establishment and evaluation of test
performance characteristics, inhibitors, and interfering substances, controlling false-positive reactions,
reporting and interpretation of results, quality assurance, regulatory issues, and recommendations for
manufacturers and clinical laboratories.
MM05-A2 Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline—Second
Edition (2012). This guideline addresses the performance and application of assays for gene rearrangement
and translocations by both polymerase chain reaction (PCR) and reverse-transcriptase PCR techniques, and
includes information on specimen collection, sample preparation, test reporting, test validation, and quality
assurance.
MM06-A2 Quantitative Molecular Methods for Infectious Diseases; Approved Guideline—Second Edition (2010).
This document provides guidance for the development and use of quantitative molecular methods, such as
nucleic acid probes and nucleic acid amplification techniques of the target sequences specific to particular
microorganisms. It also presents recommendations for quality assurance, proficiency testing, and
interpretation of results.
MM09-A Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine; Approved Guideline (2004). This
document addresses automated, PCR-based, dideoxy-terminator, and primer extension sequencing done on
gel- or capillary-based sequencers. Topics covered include specimen collection and handling; isolation of
nucleic acid; amplification and sequencing of nucleic acids; interpretation and reporting of results; and quality
control/assessment considerations as appropriate.
MM10-A Genotyping for Infectious Diseases: Identification and Characterization; Approved Guideline (2006).
This guideline describes currently used analytical approaches and methodologies applied to identify the
clinically important genetic characteristics responsible for disease manifestation, outcome, and response to
therapy in the infectious disease setting. It also provides guidance on the criteria to be considered for design,
validation, and determination of clinical utility of such testing.
MM12-A Diagnostic Nucleic Acid Microarrays; Approved Guideline (2006). This guideline provides
recommendations for many aspects of the array process including: a method overview; nucleic acid extraction;
the preparation, handling, and assessment of genetic material; quality control; analytic validation; and
interpretation and reporting of results. A CLSI-IFCC joint project.
MM13-A Collection, Transport, Preparation, and Storage of Specimens for Molecular Methods; Approved
Guideline (2005). This document provides guidance related to proper and safe biological specimen collection
and nucleic acid isolation and purification. These topics include methods of collection, recommended storage
and transport conditions, and available nucleic acid purification technologies for each specimen/nucleic acid
type. A CLSI-IFCC joint project.
MM17-A Verification and Validation of Multiplex Nucleic Acid Assays; Approved Guideline (2008). This guideline
provides recommendations for analytic verification and validation of multiplex assays, as well as a review of
different types of biologic and synthetic reference materials.
MM19-A Establishing Molecular Testing in Clinical Laboratory Environments; Approved Guideline (2011). This
guideline provides comprehensive guidance for planning and implementation of molecular diagnostic testing,
including strategic planning, regulatory requirements, implementation, quality management, and special
considerations for the subspecialties of molecular genetics, infectious diseases, oncology, and
pharmacogenetics.
©
Number 3 MM22-A
Related CLSI Reference Materials (Continued)

QMS01-A4 Quality Management System: A Model for Laboratory Services; Approved Guideline—Fourth Edition
(2011). This document provides a model for medical laboratories that will assist with implementation and
maintenance of an effective quality management system.
QMS03-A3 Training and Competence Assessment; Approved Guideline—Third Edition (2009). This document
provides background information and recommended processes for the development of training and
competence assessment programs that meet quality and regulatory objectives.
QMS13-A Quality Management System: Equipment; Approved Guideline (2011). This guideline provides
recommendations for establishing equipment management processes from selection through decommission of
equipment used in the provision of laboratory services.
©
Active Membership
(As of 1 February 2014)
Industry and Large Commercial Ventana Medical Systems Inc. (AZ) Atlanticare Regional Medical Center (NJ) Capital Health Regional Medical Center (NJ)
Laboratories Verinata Health, Inc. (CA) Atrium Medical Center (OH) Capital Region Medical Center (MO)
Viracor-IBT Reference Laboratory (MO) Audie L. Murphy VA Hospital (TX) Cardinal Hill Rehabilitation Hospital (KY)
Abbott Laboratories (IL) Wellstat Diagnostics, LLC (MD) Augusta Health (VA) Carl R. Darnall Army Medical Center
Accelerate Diagnostics Inc. (AZ) XDx, Inc. (CA) Aultman Hospital (OH) Department of Pathology (TX)
AdvaMed (DC) Austin Diagnostic Clinic (TX) Carle Foundation Hospital (IL)
Ariosa Diagnostics (CA) Health Care Professions/Government Austin Health (Australia) Carolinas Healthcare System (NC)
ARUP Laboratories (UT) Austin Regional Clinic, P.A. (TX) Carolinas Hospital System (SC)
Astellas Pharma (IL) 436 Medical Group - Dover Air Force Base Austin State Hospital (TX) Caromont Regional Medical Center (NC)
AstraZeneca Pharmaceuticals (MA) (DE) Avera McKennan Laboratory (SD) Carpermor S.A. de C.V. (Mexico)
Astute Medical, Inc. (CA) 51 MDSS/ Laboratory (AP) AZ Sint-Lucas Hospital (Belgium) Carroll Hospital Center (MD)
Axis-Shield PoC AS (United Kingdom [GB]) 59th MDW/859th MDTS/MTL (TX) Azienda Ospedale Di Lecco (Italy) Carteret General Hospital (NC)
Bayer Healthcare, LLC Diagnostic Division Aberdeen Royal Infirmary (United Kingdom Azienda Ospedaliera Verona Cary Medical Center (ME)
(KS) [GB]) B.B.A.G. Ve U. AS., Duzen Laboratories Cass County Memorial Hospital (IA)
BD (NJ) Academisch Ziekenhuis-VUB (Belgium) (Turkey) Castle Medical Center (HI)
Beckman Coulter, Inc. (PA) ACG (Colombia) Banyan Biomarkers (CA) Catholic Health Initiatives (KY)
Bioanalyse, Ltd. (Turkey) ACL Laboratories (IL) Baptist Health Medical Center (FL) Catholic Medical Center (NH)
Biohit Oyj. (Finland) ACL Laboratories (WI) Baptist Health Medical Center-Little Rock Cayuga Medical Center At Ithaca (NY)
bioMeríeux, Inc. (MO) Adams Memorial Hospital (IN) (AR) CD Diagnostics, Inc. (PA)
Bio-Rad Laboratories, Inc. (CA) ADNOC Medical Center (United Arab Baptist Health System (TX) CDC - Nigeria (Nigeria)
Canon U.S. Life Sciences, Inc. (MD) Emirates) Baptist Hospital Laboratory (FL) Cedars-Sinai Medical Center (CA)
Cempra Pharmaceuticals, Inc. (NC) Advanced Laboratory Services (PA) Baptist Hospital of Miami (FL) Cedimat Medical Center (FL)
Cepheid (CA) Adventist Health System (FL) Baptist Memorial Health Care Corporation - Cellnetix Pathology & Laboratories (WA)
Cerexa, Inc. (CA) Adventist Medical Center (OR) Hospital Laboratories Works (TN) Center for Disease Detection (TX)
Clinical Reference Laboratory (MO) Affiliated Laboratory, Inc. (ME) Barnes-Jewish Hospital (VT) Centers for Disease Control and Prevention
Cubist Pharmaceuticals, Inc. (MA) AFRIMS (Thailand) Bassett Healthcare (NY) (GA)
Diagnostica Stago (NJ) Aga Khan University Hospital (Pakistan) Basurto Hospital (Spain) Centers for Disease Control and Prevention -
DX Assays Pte Ltd. (Malaysia) AHS Morristown (NJ) Baxter Regional Medical Center (AR) Ethiopia (Ethiopia)
Eiken Chemical Company, Ltd. (Japan) Akron Children’s Hospital (OH) Bay Area Hospital (OR) Centers for Disease Control and Prevention -
Elanco Animal Health (IN) Akron General Medical Center (OH) Bay Medical Center (FL) Tanzania (Tanzania)
Enzo Clinical Labs (NY) Al Hada Armed Forces Hospital/TAIF/KSA BayCare Health System (FL) Centers for Medicare & Medicaid Services
Eurofins Medinet (VA) (Saudi Arabia) Bayhealth Medical Center-Kent General (MD)
Exosome Diagnostics, Inc. (MN) Al Noor Hospital (United Arab Emirates) Hospital (DE) Centers for Medicare & Medicaid
GlaxoSmithKline (NJ) Alamance Regional Medical Center (NC) Baylor Health Care System (TX) Services/CLIA Program (TX)
Greiner Bio-One GmbH (Austria) Alameda County Medical Center (CA) Bayou Pathology, APMC (LA) Central Baptist Hospital (KY)
Greiner Bio-One Inc. (NC) Alaska Native Medical Center (AK) Baystate Medical Center (MA) Central Maine Medical Center (ME)
Himedia Labs Ltd (India) Alaska Regional Hospital (AK) BC Centre for Disease Control (Canada) Central Newfoundland Regional Health
Hologic, Inc. (MA) Albany College of Pharmacy & Health Beaufort Delta Health and Social Services Center (Canada)
Icon Laboratories, Inc. (NY) Sciences (NY) Authority (Canada) Central Ohio Primary Care Physicians (OH)
Insmed Incorporated (NJ) Albany Medical Center Hospital (NY) Beaver Dam Reference Lab (WI) Central Pennsylvania Alliance Laboratory
Instrumentation Laboratory (MA) Albemarle Hospital (NC) Beebe Medical Center (DE) (PA)
Intuity Medical (CA) Albert Einstein Medical Center (PA) Beloit Memorial Hospital (WI) Central Vermont Medical Center (VT)
ITC Corp (NJ) Alberta Health Services (Canada) Berkshire Medical Center (MA) Central Washington Hospital (WA)
Johnson & Johnson Pharmaceutical Research Alexandra Health Pte Ltd (Singapore) Berlin Memorial Hospital (WI) Centre Hospitalier Anna-Laberge (Canada)
& Develop., L.L.C. (NJ) Alfred I. du Pont Hospital for Children (DE) Beth Goldstein Consultant (PA) Centre Hospitalier Lyon SUD (France)
Kaiser Permanente (CA) All Children’s Hospital (FL) Beth Israel Deaconess Medical Center (MA) Centro Medico Imbanaco (Colombia)
Laboratory Corporation of America (VA) Alliance Community Hospital (OH) Beth Israel Medical Center (NY) Ceylon Hospitals Limited (Sri Lanka)
Laboratory Specialists, Inc. (OH) Allina Labs - 13201 (MN) Biodesign Institute at ASU (AZ) CGH Medical Center (IL)
Life Laboratories (MA) Alpena Regional Medical Center (MI) Bio-Reference Laboratories (NJ) Chaleur Regional Hospital (Canada)
LifeLabs (Canada) Alta Bates Summit Medical Center (CA) Blanchard Valley Hospital (OH) Chambersburg Hospital (PA)
LifeLabs Medical Laboratory Services Altru Health Systems (ND) Blount Memorial Hospital (TN) Champlain Valley Physicians Hospital (NY)
(Canada) Alvarado Hospital Medical Center Laboratory Blue Mountain Health System (PA) Chang Gung Memorial Hospital (Taiwan)
LipoScience, Inc. (NC) (CA) Blue Ridge Regional Hospital (NC) Charleston Area Medical Center (WV)
Mbio Diagnostics, Inc. (CO) Alverno Clinical Laboratories, Inc. (IN) Bon Secours Health Partners (VA) Chatham - Kent Health Alliance (Canada)
Merck & Company, Inc. (NJ) American Association for Clinical Chemistry Bon Secours Hospital (Ireland) Chesapeake General Hospital (VA)
Merial Limited & Newport Laboratories (MO) (DC) Bozeman Deaconess Laboratory (MT) Chester County Hospital (PA)
Microbiologics (MN) American Association for Laboratory Braintree Rehabilitation Hospital (MA) Cheyenne Regional Medical Center (WY)
Micromyx, LLC (MI) Accreditation (MD) Brandywine Hospital (PA) Chi Solutions, Inc. (MI)
Micropoint Bioscience, Inc. (CA) American Hospital Dubai (United Arab Brant Community Healthcare System/Brant Chia-Yi Chang Gung Memorial Hospital
Nihon Kohden Corporation (Japan) Emirates) General Hospital (Canada) (Taiwan)
Nissui Pharmaceutical Co., Ltd. (Japan) American Medical Laboratories (Israel) Brazosport Regional Health System (TX) Chickasaw Nation Division of Health -
Nova Biomedical Corporation (MA) American Medical Technologists (VA) Breathitt Veterinary Center, Murray State Chickasaw Nation Medical Center (OK)
NovaBiotics (United Kingdom [GB]) American Society for Clinical Pathology (IL) University (KY) Children’s Healthcare of Atlanta (GA)
Novartis Institutes for Biomedical Research American Society for Microbiology (DC) Brian All Good Community Hospital/121 Childrens Hosp.- Kings Daughters (VA)
(CA) American Society of Phlebotomy Technicians Combat (CA) Children’s Hospital (AL)
Optimer Pharmaceuticals, Inc. (CA) (SC) Bridgeport Hospital (CT) Children’s Hospital & Medical Center (NE)
Ortho-Clinical Diagnostics, Inc. (NY) American Type Culture Collection (VA) Bristol Hospital (CT) Children’s Hospital & Research Center At
Oxyrase, Inc. (OH) Ampath (South Africa) British Columbia Institute of Technology Oakland (CA)
PathCare Pathology Laboratory (South Africa) Ann & Robert H. Lurie Children’s Hospital of (Canada) Children’s Hospital Boston (MA)
PerkinElmer (Finland) Chicago (IL) Brockville General Hospital (Canada) Childrens Hospital Los Angeles (CA)
PerkinElmer Genetics, Inc. (PA) Anna Jaques Hospital (MA) Bronson Methodist Hospital (MI) Children’s Hospital of Central California (CA)
Pfizer Inc (PA) Anne Arundel Medical Center (MD) Broward General Medical Center (FL) Children’s Hospital of Philadelphia (PA)
Phadia AB (Sweden) Anson General Hospital (Canada) Brownwood Regional Medical Center (TX) Childrens Hospital of Wisconsin (WI)
Philips Healthcare Incubator (Netherlands) Appalachian Regional Healthcare System Bryan Medical Center (NE) Children’s Hospitals and Clinics (MN)
QML Pathology (Australia) (NC) BSA Health System (TX) Children’s Medical Center (TX)
Quest Diagnostics Nichols Institute (CA) Arhus Universitets Hospital (Denmark) Buena Vista Regional Medical Center (IA) Chilton Memorial Hospital (NJ)
Quotient Bioresearch Ltd. (United Kingdom Arizona State Health Laboratory (AZ) Bumrungrad Hospital (Thailand) Chinese Committee for Clinical Laboratory
[GB]) Arkansas Children’s Hospital (AR) C. Gregory Bowling, MD APMC (LA) Standards (China)
Roche Diagnostics, Inc. (IN) Arkansas Dept of Health (AR) Cadham Provincial Laboratory-MB Health Chino Valley Medical Center (CA)
Sanofi Pasteur (PA) Armed Forces Health Surveillance Center (Canada) Christiana Care Health Services (DE)
Sarstedt, Inc. (NC) (AFHSC) (MD) California Department of Public Health (CA) Christus Santa Rosa-Westover Hills (TX)
Sekisui Diagnostics (MA) Arnot Ogden Medical Center Laboratory (NY) California Pacific Medical Center (CA) Christus St. Patrick Hospital (LA)
Seventh Sense Biosystems (MA) Arrowhead Regional Medical Center (CA) Cambridge Health Alliance (MA) CHU Sainte-Justine: Department of
Siemens Healthcare Diagnostics Inc. (CA) Asan Medical Center (Korea, Republic of) Cambridge Life Science (United Kingdom Microbiology and Immunology (Canada)
Sonic Healthcare USA (TX) Asante Health System (OR) [GB]) CHUM Hospital Saint-Luc (Canada)
SRL Limited (India) Ashe Memorial Hospital (NC) Camden Clark Memorial Hospital (WV) CHW-St. Mary’s Medical Center (CA)
Streck Laboratories, Inc. (NE) Asiri Group of Hospitals Ltd. (Sri Lanka) Campbellford Memorial Hospital (Canada) Cibola General Hospital (NM)
Sysmex America, Inc. (IL) Aspen Valley Hospital (CO) Canadian Science Center for Human and Cincinnati Children’s Hospital Medical Center
Tetraphase Pharmaceuticals (MA) ASPETAR (Qatar Orthopedic and Sports Animal Health (Canada) (OH)
The Medicines Company (Canada) Medicine Hospital) (Qatar) Canadian Society for Medical Laboratory Citizens Memorial Hospital (MO)
Theranos (CA) Aspirus Wausau Hospital (WI) Science (Canada) City of Hope National Medical Center (CA)
Theravance Inc. (CA) Associacao Das Pioneiras Sociais (Brazil) Canberra Hospital (Australia) City of Milwaukee Health Department (WI)
Thermo Fisher Scientific (CA) Association of Public Health Laboratories Cape Cod Hospital (MA) Clara Maass Medical Center (NJ)
Thermo Scientific Microbiology Sdn Bhd (MD) Cape Fear Valley Medical Center Laboratory Cleveland Clinic (OH)
(Malaysia) Atlantic Diagnostics Laboratories (PA) (NC)

Clifton Fine Hospital (NY) Eastern Gateway Community College (OH) Hardy Diagnostics (CA) Jackson Health System (FL)
Clinica Alemana De Santiago (Chile) Eastern Health - Health Sciences Centre Harford Memorial Hospital (MD) Jackson Hospital & Clinic, Inc. (AL)
Clinica Hospital San Fernando (Panama) (Canada) Harris Methodist HEB Hospital (TX) Jackson Purchase Medical Center (KY)
Clinical and Laboratory Standards Institute Eastern Health Pathology (Australia) Harris Methodist Hospital Southwest (TX) Jameson Memorial Hospital (PA)
(PA) Eastern Ontario Regional Laboratory Hartford Hospital (CT) Japan Assn. of Clinical Reagents Industries
Clinical Hospital Merkur (Croatia/Hrvatska) Association (EORLA) (Canada) Harvard Vanguard Medical Associates (Japan)
Clinique St. Luc (Belgium) Easton Hospital (PA) (MA) Jeanetics Laboratory Consulting, LLc (CA)
CLMA (IL) Edgerton Hospital & Health Services (WI) Hawaii Pathologists Laboratory (HI) Jefferson Memorial Hospital (WV)
CML HealthCare (Canada) Edmonds Community College (WA) Hawaii State Hospital (HI) Jefferson Regional Medical Center (PA)
COLA (MD) Edward Hospital (IL) Healdsburg District Hospital (CA) Jennings American Legion Hospital (LA)
College of American Pathologists (IL) Effingham Hospital (GA) Health Canada (Canada) Jersey Shore University Medical Center
College of Physicians and Surgeons of El Camino Hospital (CA) Health Network Lab (PA) (NJ)
Alberta (Canada) Emerson Hospital Laboratory (MA) Health Waikato (New Zealand) Jessa Ziekenhuis VZW (Belgium)
College of Physicians and Surgeons of Emory University Hospital (GA) Healthscope Pathology (Australia) Jiao Tong University School of Medicine -
Saskatchewan (Canada) Emory University School of Medicine (GA) Heartland Health (MO) Shanghai No. 3 People’s Hospital (China)
College of the North Atlantic (Canada) Empire College (CA) Helen Hayes Hospital (NY) John C. Lincoln Hospital - N.MT. (AZ)
College of Veterinary Medicine, Auburn Ephrata Community Hospital (PA) Hendrick Regional Laboratory (TX) John D. Archbold Hospital (GA)
University (AL) Erie County Medical Center Corporation Hendricks Regional Health (IN) John F. Kennedy Medical Center (NJ)
Collingwood General & Marine Hospital (NY) Henrico Doctors’ Hospital - Parham (VA) John H. Stroger, Jr. Hospital of Cook
(Canada) Erlanger Health Systems (TN) Henry Ford Hospital (MI) County (IL)
Columbia Memorial Hospital (NY) ESCMID (Switzerland) Henry M. Jackson Foundation for the John Hopkins APL (MD)
Columbia Memorial Hospital (OR) Estes Park Medical Center (CO) Advancement of Military Medicine-MD John Muir Health (CA)
Columbus Regional Healthcare System Ethiopian Health and Nutrition Research (MD) Johns Hopkins Medical Institutions (MD)
(NC) Institute (Ethiopia) Henry M. Jackson Foundation-Brook Army Johnson City Medical Center Hospital (TN)
Commonwealth of Kentucky (KY) Evangelical Community Hospital (PA) Medical Ctr (BAMC) (TX) Jonathan M. Wainwright Memorial Veterans
Commonwealth of Virginia (DCLS) (VA) Evans Army Community Hospital (CO) Hi-Desert Medical Center (CA) Affairs Medical Center (WA)
Community College of Rhode Island- Evanston Hospital, NorthShore University Highlands Medical Center (AL) Jones Memorial Hospital (NY)
Flanagan Campus (RI) HealthSystem (IL) Hillcrest Medical Center (OK) Jordan Valley Community Health Center
Community Hospital (IN) Exempla - Saint Joseph Hospital (CO) Hinsdale Pathology Associates (IL) (MO)
Community Hospital of the Monterey Exempla Lutheran Medical Center (CO) Hoag Memorial Hospital Presbyterian (CA) JPS Health Network (TX)
Peninsula (CA) Fairfax County Health Department (VA) Holstebro Hospital (Denmark) Jupiter Medical Center (FL)
Community Hospitals of Williams County Farrer Park Hospital (Singapore) Holy Name Hospital (NJ) Kaiser Medical Laboratory (HI)
(OH) Fayette County Memorial Hospital (OH) Holy Redeemer Hospital & Medical Center Kaiser Permanente (GA)
Community Medical Center (MT) FDA Ctr. for Devices/Rad. Health (CDRH) (PA) Kaiser Permanente (MD)
Community Medical Center (NJ) (MD) Holy Spirit Hospital (PA) Kaiser Permanente Colorado (CO)
Complexe Hospitalier de la Sagamie Federal Medical Center (MN) Holzer Health System (OH) Kaiser Permanente Medical Care (CA)
(Canada) Firelands Regional Medical Center (OH) Hong Kong Accreditation Service Kaiser Permanente San Francisco (CA)
CompuNet Clinical Laboratories (OH) Fisher-Titus Memorial Hospital (OH) Innovation and Technology Commission Kaleida Health Center for Laboratory
Coney Island Hospital (NY) Flagler Hospital Inc. (FL) (Hong Kong) Medicine (NY)
Consultants Laboratory of WI LLC (WI) Fletcher Allen Health Care (VT) Hong Kong Sanatorium & Hospital (Hong Kalispell Regional Medical Center (MT)
Contra Costa Regional Medical Center (CA) Fleury S.A. (Brazil) Kong) Kansas State University (KS)
Conway Medical Center (SC) Florida Department of Health (FL) Hopital Charles Lemoyne (Canada) Kaohsiun Chang Gung Memorial Hospital
Cook Children’s Medical Center (TX) Forrest General Hospital (MS) Hopital Cite de La Sante De Laval (Canada) (Taiwan)
Cookeville Regional Medical Center (TN) Forsyth Medical Center (NC) Hopital de Granby-CSSS Haute-Yamaska Karmanos Cancer Institute (MI)
Countess of Chester Hospital (United Fort Defiance Indian Hospital (AZ) (Canada) KCHL St. Elisabeth Hospital (Netherlands)
Kingdom [GB]) Fort Loudoun Medical Center (TN) Hopital Maisonneuve-Rosemont (Canada) Keck Hospital of USC (CA)
Counties Manukau District Health Board, Fox Chase Cancer Center (PA) Hopital Santa Cabrini Ospedale (Canada) Keck School of Medicine-USC (CA)
Middlemore Hospital (New Zealand) Franklin Memorial Hospital (ME) Hopkins County Memorial Hospital (TX) Keelung Chang Gung Memorial Hospital
Covance CLS (IN) Fresno Community Hospital & Medical Horizon Health Network (Canada) (Taiwan)
Covenant Medical Center (TX) Center (CA) Hospital Albert Einstein (Brazil) Keller Army Community Hospital (NY)
Crozer-Chester Medical Center (PA) Ft. Belvoir Community Hospital (VA) Hospital de Tjongerschans (Netherlands) Kennedy Health System (NJ)
CSSS Alphonse-Desjardins (Canada) Fundacao Faculdade de Medicina (Brazil) Hospital Italiano Laboratorio Central Kenora-Rainy River Reg. Lab. Program
CSSS St-Jerome (Canada) Fundacion Mexicana Para la Salud Capitulo (Argentina) (Canada)
Cyruss Tsurgeon (LA) Peninsular A.C (Mexico) Hospital Sacre-Coeur de Montreal (Canada) Kindred Healthcare (KY)
Dameron Hospital Association (CA) Gamma-Dynacare Laboratories (Canada) Hotel Dieu Grace Hospital Library (Canada) King Abdulaziz Hospital (Saudi Arabia)
Danbury Hospital (CT) Garden City Hospital (MI) Houston Medical Center (GA) King Abdulaziz Medical City-
Darwin Health Library, NT Dept. of Health Gateway Regional Medical Center (IL) Hunt Regional Healthcare (TX) NGHA/DPLM-Riyadh (Saudi Arabia)
(Australia) Geary Community Hospital (KS) Hunterdon Medical Center (NJ) King Fahad Specialist Hospital-Dammam,
Daviess Community Hospital (IN) Geisinger Medical Center (PA) Huntington Memorial Hospital (CA) K.S.A. (Saudi Arabia)
Dayton Children’s Medical Center (OH) Genesis Healthcare System (OH) Hutchinson Clinic, P.A. (KS) King Faisal Specialist Hospital & Research
Deaconess Hospital Laboratory (IN) Genesis Medical Center (IL) Hutt Valley Health District Health Board Center (Saudi Arabia)
Dean Medical Center (WI) Genome DX (Canada) (New Zealand) King Faisal Specialist Hospital & Research
Delano Regional Medical Center/Laboratory George Mason University (VA) IDEXX Reference Laboratories (Canada) Center (Saudi Arabia)
(CA) Ghent University Hospital (Belgium) Imelda Hospital (Belgium) King Hussein Cancer Center (Jordan)
Delaware Public Health Laboratory (DE) Glasgow Royal Infirmary (United Kingdom Indiana University - Chlamydia Laboratory Kingsbrook Jewish Medical Center (NY)
Delnor Community Hospital (IL) [GB]) (IN) Kingston General Hospital (Canada)
Delta Regional Medical Center (MS) Golden Valley Memorial Hospital (MO) Indiana University Health Bloomington KK Women’s & Children’s Hospital
Denver Health Medical Center (CO) Golwilkar Metropolis (India) Hospital (IN) (Singapore)
Department of Veterans Affairs (DC) Good Samaritan Hospital (IN) Indiana University Health Care - Pathology Kuakini Health System (HI)
DHHS NC State Lab of Public Health (NC) Good Samaritan Hospital Medical Center Laboratory (IN) Kuwait Cancer Control Center (Kuwait)
Diagnostic Accreditation Program (Canada) (NY) Industrial Technology Research Institute La Rabida Childrens Hospital (IL)
Diagnostic Center for Population & Animal Good Shepherd Medical Center (TX) (ITRI) (Taiwan) Lab Express (AZ)
Health (MI) Gottlieb Memorial Hospital (IL) INEI-ANLIS "Dr. C. G. Malbrán" Lab Médico Santa Luzia LTDA (Brazil)
Diagnostic Laboratory Medicine, Inc. (MA) Grady Memorial Hospital (GA) (Argentina) Labor Stein + Kollegen (Germany)
Diagnostic Laboratory Services, Inc. (HI) Grana S.A. (TX) Ingalls Hospital (IL) Laboratorio Bueso Arias (Honduras)
Diagnostic Medicine Services (Iceland) Grand River Hospital (Canada) Inova Central Laboratory (VA) Laboratorio Clinico Amadita P. de Gonzales
Diagnostic Services of Manitoba (Canada) Grays Harbor Community Hospital (WA) Institut National de Sante Publique du S.A. (FL)
Dialysis Clinic, Inc. Laboratory (TN) Great Plains Regional Med. Ctr. (NE) Quebec (Canada) Laboratorio Médico De Referencia
Dimensions Healthcare System Prince Great River Medical Center (IA) Institute Health Laboratories (PR) (Colombia)
George’s Hospital Center (MD) Greater Lowell Pediatrics (MA) Institute of Public Health (Slovenia) Laboratory Alliance of Central New York
DMC University Laboratories (MI) Greenville Memorial Medical Campus (SC) Institute of Tropical Medicine Dept. of (NY)
Docro, Inc. (CT) Grey Bruce Regional Health Center Clinical Sciences (Belgium) Laboratory for Medical Microbiology and
Doctor’s Data, Inc. (IL) (Canada) Institute of Veterinary Bacteriology Infectious Diseases (Netherlands)
DoctorsManagement (TN) Gritman Medical Center (ID) (Switzerland) Laboratory Medicin Dalarna (Sweden)
Donalsonville Hospital (GA) Group Health Cooperative (WA) Integrated BioBank (Luxembourg) Laboratory of Clinical Biology Ziekenhuis
Door County Medical Center (WI) Grove City Medical Center (PA) Integrated Diagnostics (WA) Oost-Limburg (ZOL) (Belgium)
Dr Sulaiman Al Habib Medical Group Guelph General Hospital (Canada) Integrated Regional Laboratories (HCA) Laboratory of Veterinary Medicine
(Saudi Arabia) Gulf Medical College Hospital & Research (FL) (Luxembourg)
Drug Scan Inc. (PA) Centre (United Arab Emirates) Interim LSU Hospital/Med. Center of La LabPlus Auckland District Health Board
DuBois Regional Medical Center (PA) Gundersen Lutheran Medical Center (WI) (LA) (New Zealand)
DUHS Clinical Laboratories (NC) Gunnison Valley Hospital (CO) Interior Health (Canada) LAC/USC Medical Center (CA)
Duke University Medical Center (NC) Guthrie Clinic Laboratories (PA) International Accreditation New Zealand Lafayette General Medical Center (LA)
Dynacare Laboratory (WI) Gwinnett Medical Center (GA) (New Zealand) Lahey Clinic (MA)
Dynacare NW, Inc - Seattle (WA) Halton Healthcare Services (Canada) International Federation of Clinical Lake Charles Memorial Hospital (LA)
DynaLIFE (Canada) Hamad Medical Corp-DLMP LAB QM Chemistry (Italy) Lake Norman Regional Medical Center
E. A. Conway Medical Center (LA) (Qatar) International Health Management (NC)
East Houston Regional Medical Center (TX) Hamilton Hospital (TX) Associates, Inc. (IL) Lakeland Regional Laboratories (MI)
East Texas Medical Center - Tyler (TX) Hamilton Regional Laboratory Medicine Istituto Cantonale Di Microbiologia Lakeland Regional Medical Center (FL)
East Texas Medical Center (ETMC) Program - St. Joseph’s (Canada) (Switzerland) Lakeridge Health Corporation - Oshawa Site
Henderson (TX) Hanover General Hospital (PA) Jackson County Memorial Hospital (OK) (Canada)
East Texas Medical Center-Pittsburg (TX) Harbor - UCLA Medical Center (CA)

Lakeview Medical Center (WI) Medlab Central (New Zealand) National Institutes of Health Department of Ontario Medical Association Quality
Lamb Healthcare Center (TX) Medlab Ghana Ltd. (Ghana) Lab Medicine (MD) Management Program-Laboratory Service
Lancaster General Hospital (PA) Medstar Health (DC) National Jewish Health (CO) (Canada)
Landstuhl Regional Medical Center (AE) Memorial Health System (CO) National Pathology Accreditation Advisory Onze Lieve Vrouwziekenhuis (Belgium)
Lane Regional Medical Center (LA) Memorial Hermann Healthcare System (TX) Council (Australia) Orange County Community College (NY)
Lawrence and Memorial Hospitals (CT) Memorial Hospital at Gulfport (MS) National Society for Histotechnology, Inc. Orange Park Medical Center (FL)
LeBonheur Children’s Hospital (TN) Memorial Hospital of Carbondale (IL) (MD) Ordre Professionnel Des Technologistes
Lee Memorial Hospital (FL) Memorial Hospital of Rhode Island (RI) National University Hospital (Singapore) Médicaux Du Québec (Canada)
Legacy Laboratory Services (OR) Memorial Hospital of Texas County (OK) Pte Ltd (Singapore) Orebro University Hospital (Sweden)
Leiden University Medical Center Memorial Medical Center (IL) National University of Ireland, Galway Oregon Health and Science University (OR)
(Netherlands) Memorial Medical Center (PA) (NUIG) (Ireland) Oregon Public Health Laboratory (OR)
LewisGale Hospital Montgomery (VA) Memorial Medical Center (TX) National Veterinary Institute (Sweden) Orillia Soldiers Memorial Hospital (Canada)
Lewis-Gale Medical Center (VA) Memorial Regional Hospital (FL) Nationwide Children’s Hospital (OH) Orlando Health (FL)
Lexington Medical Center (SC) Memorial Sloan Kettering Cancer Center Naval Hospital Lemoore (CA) OSF - Saint Anthony Medical Center (IL)
L’Hotel-Dieu de Quebec (Canada) (NY) Naval Hospital Oak Harbor (WA) OSF St. Elizabeth Medical Center (IL)
Licking Memorial Hospital (OH) Menonnite General Hospital (PR) Naval Medical Center San Diego (CA) OSU Veterinary Diagnostic Laboratory
LifeBridge Health Sinai Hospital (MD) Mercy Franciscan Mt. Airy (OH) NB Department of Health (Canada) (OR)
LifeCare Medical Center (MN) Mercy Health Center (MO) Nebraska LabLine (NE) OU Medical Center (OK)
Lithuanian Society of Laboratory Medicine Mercy Hospital (IA) Nellis Air Force Base (NV) Our Lady of the Lake Regional Medical
(Lithuania) Mercy Hospital (MN) Netlab SA (Ecuador) Center/FMOL Health System (LA)
Little Company of Mary Hospital (IL) Mercy Hospital Jefferson (MO) New Brunswick Community College Our Lady’s Hospital For Sick Children
Littleton Regional Healthcare (NH) Mercy Hospital St. Louis (MO) (Canada) (Ireland)
Lodi Memorial Hospital (CA) Mercy Integrated Laboratories / Mercy St. New Brunswick Provincial Veterinary Overlake Hospital Medical Center (WA)
Loma Linda University Medical Center Vincent (OH) Laboratory (Canada) Ozarks Medical Center (MO)
(LLUMC) (CA) Mercy Medical Center (CA) New Dar Al Shifa Hospital - Kuwait PA Veterinary Laboratory (PA)
Lompoc Valley Medical Center (CA) Mercy Medical Center (IA) (Kuwait) Pacific Diagnostic Laboratories (CA)
London Health Sciences Center (Canada) Mercy Medical Center (MD) New Hampshire Public Health Labs. (NH) Palmer Lutheran Health Center (IA)
Long Beach Memorial Medical Center- Mercy Medical Center (OH) New Hanover Regional Medical Center Palmetto Baptist Medical Center (SC)
LBMMC (CA) Mercy Regional Medical Center (OH) (NC) Palmetto Health Baptist Easley (SC)
Long Island Jewish Medical Center (NY) Meritus Medical Laboratory (MD) New Lexington Clinic (KY) Palo Alto Medical Foundation (CA)
Longmont United Hospital (CO) Methodist Dallas Medical Center (TX) New London Hospital (NH) Pamela Youde Nethersole Eastern Hospital
Longview Regional Medical Center (TX) Methodist Healthcare (TN) New Medical Centre Hospital (United Arab (Hong Kong East Cluster) (Hong Kong)
Louisiana Office of Public Health Methodist Hospital (TX) Emirates) Paris Community Hospital (IL)
Laboratory (LA) Methodist Hospital Pathology (NE) New York City Department of Health and Park Nicollet Methodist Hospital (MN)
Louisiana State University Medical Ctr. Methodist Medical Center (TN) Mental Hygiene (NY) Parkview Adventist Medical Center (ME)
(LA) Methodist Sugarland Hospital (TX) New York Eye and Ear Infirmary (NY) Parkview Health Laboratories (IN)
Lower Mainland Laboratories (Canada) MetroHealth Medical Center (OH) New York Presbyterian Hospital (NY) Parkwest Medical Center (TN)
Luke Thiboutot (MA) Metropolitan Hospital Center (NY) New York State Dept. of Health (NY) Parrish Medical Center (FL)
Luminex Corporation (TX) Miami Children’s Hospital (FL) New York University Medical Center (NY) Pathgroup (TN)
Lummi Tribal Health Center (WA) Michigan Dept. of Community Health (MI) New Zealand Blood Service (New Zealand) Pathlab (IA)
Lutheran Hospital of Indiana Inc. (IN) Michigan State University (MI) Newark Beth Israel Medical Center (NJ) Pathology Associates Medical Lab. (WA)
Lynchburg General (VA) Microbial Research, Inc. (CO) Newborn Metabolic Screening Program/ PathWest Laboratory Medicine WA
Lyndon B. Johnson General Hospital (TX) Microbiology Specialists, Inc. (TX) Alberta Health Services (Canada) (Australia)
Lyster Army Health Clinic (AL) Mid America Clinical Laboratories (IN) Newman Regional Health (KS) Pavia Hospital Santurce (PR)
MA Dept. of Public Health Laboratories Mid Coast Hospital (ME) Niagara Health System (Canada) PeaceHealth Laboratories (OR)
(MA) Middelheim General Hospital (Belgium) Ninewells Hospital and Medical School Peninsula Regional Medical Center (MD)
Mackenzie Health (Canada) Middlesex Hospital (CT) (United Kingdom [GB]) Penn State Hershey Medical Center (PA)
Mafraq Hospital (United Arab Emirates) Midland Memorial Hospital (TX) Noble’s Hospital (United Kingdom [GB]) Pennsylvania Dept. of Health (PA)
Magnolia Regional Health Center (MS) Midwestern Regional Medical Center (IL) NorDx - Scarborough Campus (ME) Pennsylvania Hospital (PA)
Main Line Clinical Laboratories, Inc. Mile Bluff Medical Center / Hess Memorial Norman Regional Hospital (OK) Peoria Tazewell Pathology Group, P.C. (IL)
Lankenau Hospital (PA) Hospital (WI) North Bay Regional Health Center (Canada) PEPFAR Tanzania (PA)
Maine General Medical Center (ME) Milford Regional Hospital (MA) North Carolina Baptist Hospital (NC) PerkinElmer Health Sciences, Inc. (SC)
Mammoth Hospital Laboratory (CA) Ministry of Health - Zambia (Zambia) North District Hospital (China) Peterborough Regional Health Centre
Margaret R. Pardee Memorial Hospital (NC) Ministry of Health and Social Welfare - North Kansas City Hospital (MO) (Canada)
Maria Parham Medical Center (NC) Tanzania (Tanzania) North Oaks Medical Center (LA) Peterson Regional Medical Center (TX)
Marietta Memorial Hospital (OH) Minneapolis Community and Technical North Philadelphia Health System-St. PHIA Project, NER (CO)
Marin General Hospital (CA) College (MN) Joseph’s Hospital (PA) Phoebe Putney Memorial Hospital (GA)
Marion County Public Health Department Minneapolis Medical Research Foundation North Shore Hospital Laboratory (New Phoenix Children’s Hospital (AZ)
(IN) (MN) Zealand) Phoenixville Hospital (PA)
Marquette General Hospital (MI) Minnesota Department of Health (MN) North Shore Medical Center (MA) Physicians Choice Laboratory Services (NC)
Marshfield Clinic (WI) MiraVista Diagnostics (IN) North Shore-Long Island Jewish Health Physicians Laboratory & SouthEast
Martha Jefferson Hospital (VA) Mission Hospitals Laboratory (NC) System Laboratories (NY) Community College (NE)
Martin Luther King, Jr./Drew Medical Mississippi Baptist Medical Center (MS) North York General Hospital (Canada) Physicians Preferred Laboratory (TX)
Center (CA) Mississippi Public Health Lab (MS) Northcrest Medical Center (TN) Piedmont Atlanta Hospital (GA)
Martin Memorial Health Systems (FL) Missouri State Public Health Laboratory Northeast Georgia Health System (GA) Piedmont Henry Hospital (GA)
Mary Black Memorial Hospital (SC) (MO) Northeastern Vermont Regional Hospital Pioneers Memorial Health Care District
Mary Greeley Medical Center (IA) Mobile Infirmary Association (AL) (VT) (CA)
Mary Hitchcock Memorial Hospital (NH) Modesto Memorial Hospital (CA) Northfield Hospital & Clinics (MN) Placer County Public Health Laboratory
Mary Washington Hospital (VA) MolecularMD Corp. (OR) Northridge Hospital Medical Center (CA) (CA)
Massachusetts General Hospital (MA) Monadnock Community Hospital (NH) Northside Hospital (GA) Pocono Medical Center School of Medical
Massasoit Community College (MA) Mongolian Agency for Standardization and Northside Medical Center (OH) Technology (PA)
Mater Health Services - Pathology Metrology (Mongolia) Northumberland Hills Hospital (Canada) Portneuf Medical Center (ID)
(Australia) Monongahela Valley Hospital (PA) Northwest Arkansas Pathology Associates Poudre Valley Hospital (CO)
Maury Regional Hospital (TN) Monongalia General Hospital (WV) (AR) Prairie Lakes Hospital (SD)
Mayo Clinic (MN) Montana Department of Public Health and Northwestern Medical Center, Inc. (VT) Presbyterian Hospital - Laboratory (NC)
Mayo Clinic Health Systems in Waycross Human Services (MT) Northwestern Memorial Hospital (IL) Presbyterian/St. Luke’s Medical Center
(GA) Montefiore Medical Center (NY) Norton Healthcare (KY) (CO)
Mayo Clinic Scottsdale (AZ) Morehead Memorial Hospital (NC) Norwalk Hospital (CT) Preventive Medicine Foundation (Taiwan)
McAlester Regional Health Center (OK) Morristown Hamblen Hospital (TN) Notre Dame Hospital (Canada) Prince George Regional Hospital (Canada)
McAllen Medical Center (TX) Mount Nittany Medical Center (PA) Nova Scotia Association of Clinical Princess Margaret Hospital (Hong Kong)
McCullough-Hyde Memorial Hospital (OH) Mt. Auburn Hospital (MA) Laboratory Managers (Canada) Proasecal LTD (Colombia)
MCG Health (GA) Mt. Sinai Hospital (Canada) Nova Scotia Community College (Canada) ProMedica Laboratory (OH)
McLaren Northern Michigan (MI) Mt. Sinai Hospital - New York (NY) Novus Path Labs (India) Prometheus Laboratories Inc. (CA)
MCN Healthcare (CO) Mt. Sinai Hospital Medical Center (IL) NSW Health Pathology (Australia) Providence Alaska Medical Center (AK)
Meadows Regional Medical Center (GA) MultiCare Health Systems (WA) NTD Laboratories Inc (NY) Providence Everett Medical Center (WA)
Meadville Medical Center (PA) Munson Medical Center (MI) NW Physicians Lab (WA) Providence Hospital (AL)
Med Health Services Laboratory (PA) Muskoka Algonquin Healthcare (Canada) Oakton Community College (IL) Providence St. Joseph Medical Center (CA)
Med. Laboratories Duesseldorf (Germany) Nacogdoches Memorial Hospital (TX) Ocean County Medical Laboratories (NJ) Providence St. Mary Medical Center (WA)
Medecin Microbiologiste (Canada) Nanticoke Memorial Hospital (DE) Ochsner Clinic Foundation (LA) Provista Diagnostics (AZ)
Media Lab, Inc. (GA) Nash General Hospital / Laboratory (NC) Oconee Memorial Hospital (SC) Public Health Ontario (Canada)
Medical Center Enterprise (AL) Nassau County Medical Center (NY) Octapharma Plasma (NC) Puget Sound Blood Center (WA)
Medical Center Hospital (TX) National Cancer Institute, CCR, LP (MD) Odense University Hospital (Denmark) Pullman Regional Hospital (WA)
Medical Center of Central Georgia (GA) National Cancer Institute, CDP, NIH (MD) Office of Medical Services Laboratory (DC) Queen Elizabeth Hospital (Canada)
Medical Centre Ljubljana (Slovenia) National Food Institute Technical University Ohio Health Laboratory Services (OH) Queen Elizabeth Hospital (China)
Medical College of Virginia Hospital (VA) of Denmark (Denmark) Ohio State University Hospitals (OH) Queensland Health Pathology Services
Medical Laboratories of Windsor, LTD National Health Laboratory Service C/O Ohio Valley Medical Center (WV) (Australia)
(Canada) F&M Import & Export Services (South Oklahoma Heart Hospital, LLC (OK) Quest - A Society for Adult Support and
Medical Laboratory Sciences Council of Africa) Oklahoma State University: Center for Rehabilitation (Canada)
Nigeria (Nigeria) National Institute of Health (Thailand) Health Sciences (OK) Quinte Healthcare Corp. - Belleville General
Medical University Hospital Authority (SC) National Institute of Health-Maputo, Olive View-UCLA Medical Center (CA) Site (Canada)
Medical, Laboratory & Technology Mozambique (Mozambique) Oneida Healthcare Center (NY) Quintiles Laboratories, Ltd. (GA)
Consultants, LLC (DC)

Radiometer Medical A/S (Denmark) Seoul National University Hospital (Korea, St. Olavs Hospital (Norway) The University of Tokyo (Japan)
Ramathibodi Hospital (Thailand) Republic of) St. Peter’s Bender Laboratory (NY) Thomas Jefferson University Hospital, Inc.
Randers Regional Hospital (Denmark) Seton Healthcare Network (TX) St. Peter’s Hospital (MT) (PA)
Range Regional Health Services (MN) Seton Medical Center (CA) St. Rita’s Medical Center (OH) Thunder Bay Regional Health Sciences
RCPA Quality Assurance Programs Pty Shands Jacksonville (FL) St. Tammany Parish Hospital (LA) Centre (Canada)
Limited (Australia) Shared Hospital Laboratory (Canada) St. Thomas Hospital (TN) Torrance Memorial Medical Center (CA)
Reading Hospital (PA) Sharon Regional Health System (PA) St. Thomas-Elgin General Hospital Touro Infirmary (LA)
Redlands Community Hospital (CA) Sharp Health Care Laboratory Services (Canada) Tri-Cities Laboratory (WA)
Regina Qu’Appelle Health Region (Canada) (CA) St. Vincent Hospital (NM) TriCore Reference Laboratories (NM)
Regional Laboratory of Public Health Shiel Medical Laboratory Inc. (NY) St. Vincent’s Medical Center (FL) Trident Medical Center (SC)
(Netherlands) Shore Memorial Hospital (NJ) Stanford Hospital and Clinics (CA) Trillium Health Partners Credit Valley
Regional Medical Laboratory, Inc. (OK) Shriners Hospitals for Children (OH) Stanton Territorial Health Authority Hospital (Canada)
Regions Hospital (MN) Silliman Medical Center (Philippines) (Canada) Trinity Health Systems (OH)
Reid Hospital & Health Care Services (IN) Silverton Health (OR) Stat Veterinary Lab (CA) Trinity Hospital of Augusta (GA)
Renown Regional Medical Center (NV) SIMeL (Italy) State of Alabama (AL) Trinity Medical Center (AL)
Research Institute of Tropical Medicine Singapore General Hospital (Singapore) State of Ohio Corrections Medical Center Trinity Muscatine (IA)
(Philippines) Singulex (CA) Laboratory (OH) Tripler Army Medical Center (HI)
Rhode Island Dept. of Health Labs (RI) Sky Lakes Medical Center (OR) State of Washington Public Health Labs Trumbull Memorial Hospital (OH)
Rhode Island Hospital (RI) Slidell Memorial Hospital (LA) (WA) Tucson Medical Center (AZ)
Rice Memorial Hospital (MN) SMDC Clinical Laboratory (MN) State of Wyoming Public Health Laboratory Tuen Mun Hospital, Hospital Authority
Ridgeview Medical Center (MN) Sociedad Espanola de Bioquimica Clinica y (WY) (Hong Kong)
Riverside Health System (VA) Patologia Molec. (Spain) Statens Serum Institut (Denmark) Tufts Medical Center (MA)
Riverton Memorial Hospital (WY) Sociedade Brasileira de Analises Clinicas Steward Norwood Hospital (MA) Tulane Medical Center Hospital & Clinic
Riverview Healthcare Assoc. (MN) (Brazil) Stillwater Medical Center (OK) (LA)
Riyadh Armed Forces Hospital, Sulaymainia Sociedade Brasileira de Patologia Clinica Stony Brook University Hospital (NY) Tulane University Health Sciences Center
(Saudi Arabia) (Brazil) Stormont-Vail Regional Medical Ctr. (KS) (LA)
RMIT University (Australia) South Bay Hospital (FL) Sturgis Hospital (MI) Twin Lakes Regional Medical Center (KY)
Robert E. Bush Naval Hospital (CA) South Bend Medical Foundation (IN) Summa Health System (OH) U.S. Medical Ctr. for Federal Prisoners
Robert Wood Johnson University Hospital South Bruce Grey Health Centre (Canada) Sunnybrook Health Sciences Centre (MO)
(NJ) South County Hospital (RI) (Canada) U.S. Naval Hospital, Yokosuka, Japan (AP)
Robert Wood Johnson University Hospital South Dakota State Health Laboratory (SD) Sunrise Hospital and Medical Center (NV) UC Davis Medical Center Department of
Rahway (NJ) South Eastern Area Laboratory Services SUNY Downstate Medical Center (NY) Pathology & Laboratory Medicine (CA)
Rockford Memorial Hospital (IL) (Australia) Susan B. Allen Hospital (KS) UC San Diego Health System Clinical
Roger Williams Medical Center (RI) South Miami Hospital (FL) Susquehanna Health System (PA) Laboratories (CA)
Roosevelt General Hospital (NM) South Peninsula Hospital (AK) Sutter Health (CA) UCI Medical Center (CA)
Roper St. Francis Healthcare (SC) South Texas Laboratory (TX) Sutter Health Sacramento Sierra Region UCLA Medical Center (CA)
Ross University School of Veterinary South West Medical Center (KS) Laboratories (CA) UCONN Health Center (CT)
Medicine (Saint Kitts and Nevis) Southeast Alabama Medical Center (AL) SV Biosystems (CA) UCSF Medical Center China Basin (CA)
Roswell Park Cancer Institute (NY) SouthEast Alaska Regional Health Swedish American Health System (IL) UMass Memorial Medical Center (MA)
Rouge Valley Health System (Canada) Consortium (SEARHC) (AK) Swedish Medical Center (CO) UMC of El Paso- Laboratory (TX)
Royal Children’s Hospital (Australia) Southern Health Care Network (Australia) Sydney South West Pathology Service UMC of Southern Nevada (NV)
Royal Hobart Hospital (Australia) Southern Hills Medical Center (TN) Liverpool Hospital (Australia) Umea University Hospital (Sweden)
Royal Victoria Hospital (Canada) Southern Maryland Hospital (MD) Tahoe Forest Hospital (CA) UNC Hospitals (NC)
Rush Copley Medical Center (IL) Southern Pathology Services, Inc. (PR) Taichung Veterans General Hospital Union Clinical Laboratory (Taiwan)
Rush Health Systems (MS) Southwest General Health Center (OH) (Taiwan) United Christian Hospital (Hong Kong)
Rush University Medical Center (IL) Southwestern Regional Medical Center Taiwan Society of Laboratory Medicine United Clinical Laboratories (IA)
Russellville Hospital (AL) (OK) (Taiwan) United Health Services Hospital / Wilson
SA Pathology (Australia) Sparrow Hospital (MI) Tallaght Hospital (Ireland) Hospital Lab (NY)
SAAD Specialist Hospital (Saudi Arabia) Spaulding Hospital Cambridge (MA) Tampa General Hospital (FL) United Memorial Med Center (NY)
Sacred Heart Hospital (FL) Speare Memorial Hospital (NH) Taranaki Medlab (New Zealand) United States Air Force School of
Sacred Heart Hospital (WI) Spectra East (NJ) Tartu University Clinics (Estonia) Aerospace Medicine / PHE (OH)
Saddleback Memorial Medical Center (CA) St Elizabeth Hospital (WI) Tataa Biocenter (Sweden) United States Coast Guard (NJ)
Sahlgrenska Universitetssjukhuset (Sweden) St Rose Dominican Hospital (AZ) Taylor Regional Hospital (KY) Universidad de Guadalajara (Mexico)
Saint Francis Hospital & Medical Center St. Agnes Healthcare (MD) Temple Community Hospital (CA) Universidade Federal Do Rio de Janeiro
(CT) St. Anthony Hospital (OK) Temple University Hospital - Parkinson (Brazil)
Saint Francis Medical Center (IL) St. Antonius Ziekenhuis (Netherlands) Pavilion (PA) Universitaet Zuerich (Switzerland)
Saint Mary’s Regional Medical Center (NV) St. Barnabas Medical Center (NJ) Tenet Healthcare (PA) Universitair Ziekenhuis Antwerpen
Salem Hospital (OR) St. Charles Medical Center-Bend (OR) Tennessee Department of Health (TN) (Belgium)
Salisbury University (MD) St. Charles Parish Hospital (LA) Tewksbury Hospital (MA) University College Hospital (Ireland)
Samaritan Health Services (OR) St. Clair Hospital (PA) Texas A & M University (TX) University General Hospital (TX)
Samaritan Regional Health System (OH) St. David’s South Austin Hospital (TX) Texas Children’s Hospital (TX) University Health Network Laboratory
Samkwang Medical Laboratory (Korea, St. Elizabeth Community Hospital (CA) Texas Department of State Health Services Medicine Program (Canada)
Republic of) St. Elizabeth’s Medical Center (NY) (TX) University Hospital (TX)
Sampson Regional Medical Center (NC) St. Francis Health Center (KS) Texas Health Harris Methodist Hospital University Hospital Center Sherbrooke
Samsung Medical Center (Korea, Republic St. Francis Hospital (SC) Cleburne (TX) (CHUS) (Canada)
of) St. Francis Hospital & Health Centers (NY) Texas Health Harris Methodist Hospital Fort University Hospitals of Cleveland (OH)
San Angelo Community Medical Center St. John Hospital and Medical Center (MI) Worth (TX) University Medical Center at Princeton (NJ)
(TX) St. John Medical Center (OH) Texas Health Presbyterian Hospital Dallas University Medical Center of El Paso (TX)
San Francisco General Hospital-University St. John’s Hospital (IL) (TX) University Medical Center Utrecht
of California San Francisco (CA) St. John’s Hospital (WY) Texas Scottish Rite Hospital for Children (Netherlands)
San Jose State University (CA) St. John’s Hospital & Health Center (CA) (TX) University of Alabama Hospital Lab (AL)
San Juan Regional Medical Group (NM) St. John’s Regional Health Center (MO) The Broad Institute (MA) University of Alberta - Medical Genetics
Sanford Health (ND) St. Joseph Health Center (MO) The Charlotte Hungerford Hospital (CT) (Canada)
Sanford USD Medical Center (SD) St. Joseph Hospital (CA) The Cheshire Medical Center (NH) University of Arizona Medical Center (AZ)
Santa Clara Valley Health & Hospital St. Joseph Hospital (NH) The Children’s Mercy Hospital (MO) University of Arkansas for Medical Sciences
Systems (CA) St. Joseph Medical Center (TX) The City Hospital Dubai UAE (United Arab (AR)
Santa Rosa Medical Center (FL) St. Joseph Regional Health Center (TX) Emirates) University of Bonn (Germany)
Santiam Memorial Hospital (OR) St. Joseph’s Health Centre (Canada) The Clinical Microbiology Institute (OR) University of British Columbia (Canada)
Sarasota Memorial Hospital (FL) St. Joseph’s Hospital & Medical Center The Cooley Dickinson Hospital, Inc. (MA) University of California Veterinary Medical
Saratoga Hospital (NY) (AZ) The Doctor’s Clinic (OR) Teaching Hospital (CA)
SARL Laboratoire Caron (France) St. Jude Children’s Research Hospital (TN) The First Hospital of China Medical University of Chicago Hospitals
Saskatchewan Disease Control Laboratory St. Jude Medical Center (CA) University (China) Laboratories (IL)
(Canada) St. Luke’s Episcopal Hospital (TX) The Good Samaritan Hospital (PA) University of Cincinnati Medical Center
Saskatoon Health Region (Canada) St. Luke’s Hospital (IA) The Hospital for Sick Children (Canada) (OH)
Saudi Aramco Medical (TX) St. Luke’s Hospital (MN) The Joint Commission (IL) University of Cologne Medical Center
SC Department of Health and St. Luke’s Hospital (MO) The Joint Pathology Center (MD) (Germany)
Environmental Control (SC) St. Luke’s Hospital (PA) The Korean Society for Laboratory University of Colorado Health Sciences
Schneck Medical Center (IN) St. Luke’s Hospital at The Vintage (TX) Medicine (Korea, Republic of) Center (CO)
Schuyler Hospital (NY) St. Luke’s Medical Center (AZ) The Michener Inst. for Applied Health University of Colorado Hospital (CO)
Scientific Institute of Public Health St. Luke’s Regional Medical Center (ID) Sciences (Canada) University of Delaware (DE)
(Belgium) St. Mark’s Hospital (UT) The Nathan S. Kline Institute (NY) University of Guelph (Canada)
Scott & White Memorial Hospital (TX) St. Mary Medical Center (CA) The Naval Hospital of Jacksonville (FL) University of Hong Kong (Hong Kong)
Scripps Health (CA) St. Mary Medical Center (PA) The Nebraska Medical Center (NE) University of Illinois Medical Center (IL)
Scuola Di Specializzaaione- University St. Mary’s Good Samaritan (IL) The Norwegian Institute of Biomedical University of Iowa Hospitals and Clinics
Milano Bicocca (Italy) St. Mary’s Health Center (MO) Science (Norway) (IA)
Seattle Cancer Care Alliance (WA) St. Mary’s Hospital (CO) The Ohio State University-Vet Hospital University of Iowa, Hygienic Lab (IA)
Seattle Children’s Hospital/Children’s St. Mary’s Hospital (NJ) (OH) University of Kentucky Medical Center
Hospital and Regional Medical Center St. Mary’s Hospital (NY) The Permanente Medical Group, Inc. (CA) Hospital (KY)
(WA) St. Mary’s Hospital (WI) The University of Texas Medical Branch University of Ljubljana Faculty of Medicine
Seminole Hospital District (TX) St. Mary’s Medical Center (IN) (TX) (Slovenia)
Sentara Healthcare (VA) St. Mary’s Medical Center (WV) The University of the West Indies, Trinidad University of Louisville Hospital (KY)
Sentinel CH SpA (Italy) St. Michael’s Hospital (WI) Campus (Trinidad and Tobago)
St. Nicholas Hospital (WI)

University of Maryland Medical System West Georgia Health Systems (GA) Mintrude Charles-Young (Canada) Brian Lubbers (KS)
(MD) West Penn Allegheny Health System- Pauline Cyr (Canada) Darrell Lundrigan (Canada)
University of Miami (FL) Allegheny General Hospital (PA) Redintor Dagos (Philippines) Dr. Raquel Yahyaoui Macias (Spain)
University of Miami - Clinical Genetics West Shore Medical Center (MI) Dr. Jeff Dahlen PhD (CA) Dr. Roberta Madej (CA)
Labs (FL) West Valley Medical Center Laboratory Imelda Daniel (CA) Randolph D. Maloney (MA)
University of Michigan, Department of (ID) Saffiatou Darboe (Gambia) Mr. David Manalan F(ASQ), CSQE, CBA
Pathology (MI) West Virginia Bureau for Public Health Ms. Arlene Darmanie MS (Trinidad and (MA)
University of Minnesota Medical Center- (WV) Tobago) Linda M Mann (CA)
Fairview (MN) West Virginia Univ. Hospitals (WV) Dr. Trivikram Dasu PhD (WI) Kristin M Marckel (MN)
University of Missouri Hospital (MO) Westchester Medical Center (NY) Ms. Diana R. DeHoyos MS, MT(ASCP) Barbara Masten (NM)
University of MS Medical Center (MS) Western Baptist Hospital (KY) (TX) Christine McRoberts (Canada)
University of New Mexico (NM) Western Healthcare Corporation (Canada) Dr. Maria del Pilar Aguinaga PhD, Dr. Piet Meijer PhD (Netherlands)
University of North Carolina - Health Western Maryland Regional Medical Center CLDir(NCA) (TN) Jacques F Meis (Netherlands)
Services (NC) (MD) Anne Delaney (AZ) James J. Miller (KY)
University of Oregon (OR) Western Missouri Medical Center (MO) Dr. Francoi Depasse PharmD, MSc (France) Laura Miller (CA)
University of Pennsylvania (PA) Western State Hospital (VA) Narendra Desai (CA) Kathryne Miskavige (ND)
University of Pennsylvania Health System Whangarei Hospital (New Zealand) Dr. Edward P. Desmond PhD (CA) Ms. Barbara Mitchell (KS)
(PA) Wheaton Franciscan Laboratories At St. Patricia Devine (MA) Ian Morrissey (Switzerland)
University of Pittsburgh Medical Center Francis (WI) Ms. Diana L. Dickson MS, RAC (PA) Mohamed Hanafy Morsy (Saudi Arabia)
(PA) Wheeling Hospital (WV) Pinar Eker (Turkey) Anna Murphy (NJ)
University of Queensland (Australia) Whitehorse General Hospital (Canada) Omer Eltoum (Qatar) Nombuso Ndlovu (South Africa)
University of Rochester Medical Center Whitman Hospital & Medical Center (WA) Sahar Gamil EL-Wakil (Saudi Arabia) Joseph Oduor Ochieng (Kenya)
(NY) Wickenburg Community Hospital (AZ) Mike Ero (CA) Melanie O’Keefe (Australia)
University of South Alabama Medical William Beaumont Army Medical Center Mr. German Esparza BSc (Colombia) Jeffrey O’Kelley (GA)
Center (AL) (TX) Galen Eversole (NV) Olajumoke Oladipo (NY)
University of Tasmania (Australia) William Beaumont Hospital (MI) Dr. William Fales (MO) Ms. Margaret Ordonez Smith de Danies
University of Tennessee, College of William Osler Health Centre (Canada) Ms. Sue Forrest (Australia) (Colombia)
Veterinary Medicine (TN) Williamson Medical Center (TN) Dr. Timothy S. Frana DVM, MS, MPH, Samir Osman (Qatar)
University of Texas Health Center (Tyler) Wilson Medical Center (NC) PhD (IA) Mr. Jan Ostrup (Finland)
(TX) Winchester Hospital (MA) Dr. Jeff Fuller PhD, FCCM, ABMM Dr. Elizabeth Palavecino MD (NC)
University of Texas Health Science Center Winn Army Community Hospital (GA) (Canada) Dr. Mark G. Papich DVM, MS (NC)
(TX) Winter Haven Hospital, Inc. (FL) Mary Lou Gantzer (DE) A. K. Peer (South Africa)
University of Texas Southwestern Medical Wisconsin State Laboratory of Hygiene Patricia Garrett (ME) Armando Perez-Cardona (FL)
Center (TX) (WI) Dr. Valerio M. Genta MD (VA) David S Perlin (NJ)
University of Utah Hospital & Clinics (UT) Wishard Health Sciences (IN) Marc Goldford (IN) Linda Perryman (GA)
University of Virginia Medical Center (VA) Womack Army Medical Center (NC) Carlos Gonzalez (TX) C. Anne Pontius (TN)
University of Washington Medical Center Women & Infants Hospital (RI) Merran Govendir (Australia) Aida Porras (Colombia)
(WA) Womens and Childrens Hospital (LA) Tanya Graham (SD) Philip A Poston, PhD (VA)
University of Wisconsin Health (WI) Women’s Health Care Group of PA (PA) Neil Greenberg (NY) Dr. Mair Powell MD, FRCP, FRCPath
University of Wisconsin Medical Woods Memorial Hospital (TN) David Grier (NC) (United Kingdom [GB])
Foundation (WI) Woodside Health Center (Canada) Ann M. Gronowski (MO) Pam Prescott (GA)
UPMC Bedford Memorial (PA) WuXi AppTec Co., Ltd. (China) Jason Gruver (IA) Dr. Mathew Putzi (TX)
Urology of Virginia, PLLC (VA) Wyckoff Heights Medical Center (NY) Dr. Tibor Gyorfi (GA) Albert Rabinovitch (CA)
USA MEDDAC-Japan Wyoming County Community Hospital John F Halsey (SC) Tawni Reller (MN)
Uvalde Memorial Hospital (TX) (NY) Dr. W. Harry Hannon PhD (GA) Ms. Allison Remensperger (CA)
VA (Asheville) Medical Center (NC) Yale New Haven Hospital (CT) Dr. Muain Haseeb (Saudi Arabia) Lisa Reninger (IL)
VA (Bay Pines) Medical Center (FL) York General Health Care Services (NE) Judy Horton (MD) Dr. Robert P. Rennie PhD (Canada)
VA (Castle Point) Hudson Valley Health York Hospital (PA) B. Y. Hsieh (Taiwan) Mary Rice (CO)
Care System (NY) Yukon-Kuskokwim Delta Regional Hospital Po-Ren Hsueh (Taiwan) Jennifer Rogers (MI)
VA (Central Texas) Veterans Health Care (AK) Mr. Darren C. Hudach (OH) Dr. Markus Rose DVM, PhD (Germany)
System (TX) Yuma Regional Medical Center (AZ) Clark B Inderlied (CA) Daniel Ryan (CA)
VA (Dayton) Medical Center (OH) T. S Isbell (MO) Dr. Linoj Samuel PhD, D(ABMM) (MI)
VA (Indianapolis) Medical Center (IN) Individuals Dr. Megan E. Jacob PhD (NC) Rana Samuel (NY)
VA (Miami) Medical Center (FL) Ellis Jacobs (NJ) Caroline Satyadi (CA)
VA (Tampa) Hospital (FL) Mohamed O Abdelhalim (Oman) Carlos A. Javier (FL) Theresa Schnellman (SC)
VA (Tuscaloosa) Medical Center (AL) Franklin Adkinson (MD) Benjamin B John (MA) Kathleen Selover (NY)
Vail Valley Medical Center (CO) Park Ae Ja (Korea, Republic of) Judith Johnston (CA) Nilesh Shah (CA)
Valley Health / Winchester Medical Center Shana Ahmad (NY) Sumy Joseph (NC) Dinah Shore Myers (NC)
(VA) Lawal Akeem (Nigeria) Stephen Kahn (IL) Dr. Venkatakrishna Shyamala PhD (MD)
Valley Medical Center (WA) Ahmed M. Albarrag (Saudi Arabia) Jiesheng Kang (MA) Abdullah Mohd. Siddiqi (Saudi Arabia)
Vancouver Island Health Authority (SI) Erika B Ammirati (CA) Mr. Bob Kaplanis PBT, MT(ASCP) (AZ) Jane L. Smith (TN)
(Canada) Stephen Apfelroth (NY) Dr. Steven C. Kazmierczak PhD, DABCC, Judi Smith (MD)
Vanderbilt University Medical Center (TN) Chris Aug (CT) FACB (OR) David Soloy (TX)
Vejle Hospital (Denmark) Ahmed M Azaybi (Saudi Arabia) Harvey Ronald Kennedy, MD (NJ) Anna V. Sombong (Philippines)
Vermont Department of Health (VT) Cary Baird (OH) Natalie J. Kennel (CA) Steffini Stalos (TX)
Vernon Memorial Hospital (WI) Susan Barber (NC) Mr. Klaus M. Kjoller MSc (Denmark) Len Tanaka (HI)
Veterans Memorial Hospital (IA) Nancy Behling (AZ) William F. Koch (MD) Michelle Vanderpool (CA)
Via Christi Regional Medical Center (KS) Lynn Bell (IN) Jo Anne Koch-Owens (FL) Suresh H Vazirani (India)
Virginia Mason Medical Center (WA) Steven Bellistri (PA) Denise Kramer (OH) Lenin Villalta (Ecuador)
Virginia Physicians, Inc. (VA) Melissa Bennett (Canada) Mr. Narayan Krishnaswami MS, MBA Kim Walker (CA)
Virginia Regional Medical Center (MN) Dr. Lynette Y. Berkeley PhD (MD) (MO) Megan Waller (MD)
Virtua - West Jersey Hospital (NJ) Bhaskar Bhattacharya (India) Martin Kroll (NJ) Dr. Hui Wang PhD (China)
WakeMed (NC) Elma Kamari Bidkorpeh (CA) Jan Krouwer (MA) Jayesh Warade (India)
Warren Hospital (NJ) Deborah Bishop (WV) Kristi Kuper (TX) Peter Warn (United Kingdom [GB])
Waterbury Hospital (CT) Abbejane Blair (MA) Jennifer Kwon (NY) Mr. Niels Wartenberg (MN)
Watson Clinic (FL) Elizabeth Brown (PA) Dr. Patrick B. Kyle PhD (MS) Mr. Marlon A. Webb (MD)
Wayne Memorial Hospital (GA) Steven Brown (OR) Michael LaFleur (MA) Dr L.A. Nilika Wijeratne (Australia)
Weber State University (UT) Vanessa Buchan (New Zealand) Debra Larsen (TX) Matthew A Wikler (NJ)
Weed Army Community Hospital Donald R Callihan (MD) Professor Szu-Hee Lee MD, PhD (Australia) Bernadette Wildemore (GA)
Laboratory (CA) Ms. Natalie Campbell RT (Canada) Dr. Thomas J. Lenk PhD (CA) Dr. Emily S. Winn-Deen PhD (CA)
Weeneebayko General Hospital (Canada) Sheldon Campbell (CT) Sarah B Leppanen (CA) Mr. Dennis Winsten (AZ)
Weirton Medical Center (WV) Alan T. Cariski (CA) Andrew Leung (CA) Ms. Sheila M. Woodcock ART, MBA,
Wellington Regional Medical Center (FL) A. Bjoern Carle (ME) Jacob B Levine (NY) FCSMLS(D) (Canada)
Wellstar Douglas Hospital Laboratory (GA) Dr. Maria Paz Carlos DVM, PhD, MBA Ernst Lindhout (Netherlands) Ginger Wooster (WI)
Wellstar Health Systems (GA) (MD) Kristian Linnet (Denmark) Dr. Shangwei Wu PhD (China)
WellStar Paulding Hospital (GA) Eileen Carreiro-Lewandowski (MA) Yuqing Liu (China) Stanley Wu (GA)
Wenatchee Valley Medical Center (WA) Dr. Alexis Carter MD, FCAP, FASCP (GA) Philip Lively (PA) Michelle L. Zaharik (Canada)
Wesley Medical Center (KS) Dr. Jose B. Casals (Denmark) Mark Loch (MN) Jing Zhang (CA)
Ning Cegielski (WA) Moushumi Lodh (India) Dr. Marcia L. Zucker PhD (NJ)
Tony Chan (China) Stefano A. Lollai (Italy)

Number 3 MM22-A
NOTES
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February 2014

This document provides guidance for the laboratory development

Licensed to: Kuzya Kuzmich

For additional information on committee participation or to submit comments, contact CLSI.

Clinical and Laboratory Standards Institute

Licensed to: Kuzya Kuzmich

ISBN 1-56238-951-3 (Print)

Consensus Committee on Molecular Methods

Document Development Committee on Microarrays for Diagnosis and Monitoring of Infectious

Yi-Wei Tang, MD, PhD, Subhash Dhawan, PhD Staff

Sridar V. Chittur, RPh, PhD

Sabrina Conoci, PhD

Shuguang Huang, PhD

Teresa J. Raich, PhD, MBA

Wanda C. Reygaert, PhD

Hossein Salimnia, PhD, D(ABMM)

Microarrays for Diagnosis and Monitoring of Infectious Diseases; Approved

4.1 A Note on Terminology

CLSI, as a global leader in standardization, is firmly committed to achieving global harmonization

allele-specific primer extension – a solution-based, sequence-specific enzymatic reaction technology

assay – a quantitative determination or measurement of the amount, activity, or potency of a constituent

clinical laboratory – laboratory for the biological, microbiological, immunological, chemical,

deletion – loss of one or more nucleotides from a nucleic acid sequence.

imprecision – dispersion of independent results of measurements obtained under specified conditions;

in silico – an analysis performed on a computer or via computer simulation.

measurand – quantity intended to be measured (JCGM 200:2012)6; NOTE: See analyte.

measurement procedure – detailed description of a measurement according to one or more measurement

medical laboratory – see clinical laboratory.

oligonucleotide ligation assay (OLA) – a solution-based, sequence-specific enzymatic reaction

phenotype – the observed biochemical, physiological, and/or morphological characteristics of an

precision (measurement) – closeness of agreement between indications or measured quantity values

primer – an oligonucleotide usually of 18 to 25 bases which, when hybridized to a complementary strand

proficiency testing (PT) – determination of laboratory testing performance by means of interlaboratory

real-time polymerase chain reaction (PCR)//homogeneous, kinetic polymerase chain

repeatability (measurement) – measurement precision under a set of repeatability conditions of

reproducibility (measurement) – measurement precision under reproducibility conditions of

target-specific primer extension (TSPE) – a solution-based, sequence-specific enzymatic reaction

4.3 Abbreviations and Acronyms

TS-PCR target-specific polymerase chain reaction

5 General Considerations in Developing Microarray Assays

5.1 Microarray Test Workflow

 Nucleic acid extraction/purification

Table 1. CLSI Documents Pertaining to Microarray Analysis Workflow

5.2 Assay Design Considerations

5.2.1 Target Selection

5.2.2 Primer Assessment

5.3 Probe Design and Update

5.3.1 Physical Probe Arrangement

5.3.2 Sequence Selection and Updating

5.4 Preexamination Requirements

5.4.1 Collection and Handling

5.5 Examination Requirements

5.5.1 Specimen Storage

5.5.2 Automated Instrumentation

5.5.3 Sample Nucleic Acid Extraction

5.5.5 Labeling and Purification

5.5.6 Hybridization and Washing

5.6 Postexamination Requirements

5.6.1 Preliminary Signal Analysis

5.6.2 Data Normalization

5.6.3 Background Subtraction

5.6.4 Pattern Interpretation

5.6.6 Novel or Unexpected Results

5.7 Contamination Control