Validation of Laboratory-Developed Molecular Assays for Infectious Diseases

SUMMARY Molecular technology has changed the way that clinical laboratories diagnose and manage many infectious diseases. Excellent sensitivity, specificity, and speed have made molecular assays an attractive alternative to culture or enzyme immunoassay methods. Many molecular assays are commercially available and FDA approved. Others, especially those that test for less common analytes, are often laboratory developed. Laboratories also often modify FDA-approved assays to include different extraction systems or additional specimen types. The Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards require clinical laboratories to establish and document their own performance specifications for laboratory-developed tests to ensure accurate and precise results prior to implementation of the test. The performance characteristics that must be established include accuracy, precision, reportable range, reference interval, analytical sensitivity, and analytical specificity. Clinical laboratories are challenged to understand the requirements and determine the types of experiments and analyses necessary to meet the requirements. A variety of protocols and guidelines are available in various texts and documents. Many of the guidelines are general and more appropriate for assays in chemistry sections of the laboratory but are applied in principle to molecular assays. This review presents information that laboratories may consider in their efforts to meet regulatory requirements.

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Challenges and Controversies to Testing for COVID-19

Journal of Clinical Microbiology ◽

10.1128/jcm.01695-20 ◽

2020 ◽

Vol 58 (11) ◽

Cited By ~ 2

Author(s):

Matthew J. Binnicker

Keyword(s):

Clinical Laboratory ◽

Threshold Value ◽

Current Debate ◽

Research Staff ◽

Viral Pathogen ◽

Patient Reports ◽

Molecular Assays ◽

Clinical Laboratories ◽

Laboratory Operation

ABSTRACT The coronavirus disease (COVID-19) pandemic has placed the clinical laboratory and testing for SARS-CoV-2 front and center in the worldwide discussion of how to end the outbreak. Clinical laboratories have responded by developing, validating, and implementing a variety of molecular and serologic assays to test for SARS-CoV-2 infection. This has played an essential role in identifying cases, informing isolation decisions, and helping to curb the spread of disease. However, as the demand for COVID-19 testing has increased, laboratory professionals have faced a growing list of challenges, uncertainties, and, in some situations, controversy, as they have attempted to balance the need for increasing test capacity with maintaining a high-quality laboratory operation. The emergence of this new viral pathogen has raised unique diagnostic questions for which there have not always been straightforward answers. In this commentary, the author addresses several areas of current debate, including (i) the role of molecular assays in defining the duration of isolation/quarantine, (ii) whether the PCR cycle threshold value should be included on patient reports, (iii) if specimen pooling and testing by research staff represent acceptable solutions to expand screening, and (iv) whether testing a large percentage of the population is feasible and represents a viable strategy to end the pandemic.

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Test Verification and Validation for Molecular Diagnostic Assays

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2011-0212-ed ◽

2012 ◽

Vol 136 (1) ◽

pp. 11-13 ◽

Cited By ~ 38

Author(s):

Kevin C Halling ◽

Iris Schrijver ◽

Diane L Persons

Keyword(s):

Clinical Laboratory ◽

Molecular Diagnostic ◽

Molecular Assay ◽

Validation And Verification ◽

Diagnostic Assays ◽

Molecular Assays ◽

Clinical Laboratories ◽

Hereditary Disorders ◽

Test Verification

With our ever-increasing understanding of the molecular basis of disease, clinical laboratories are implementing a variety of molecular diagnostic tests to aid in the diagnosis of hereditary disorders, detection and monitoring of cancer, determination of prognosis and guidance for cancer therapy, and detection and monitoring of infectious diseases. Before introducing any new test into the clinical laboratory, the performance characteristics of the assay must be “verified,” if it is a US Food and Drug Administration (FDA)–approved or FDA-cleared test, or “validated,” if it is a laboratory-developed test. Although guidelines exist for how validation and verification studies may be addressed for molecular assays, the specific details of the approach used by individual laboratories is rarely published. Many laboratories, especially those introducing new types of molecular assays, would welcome additional guidance, especially in the form of specific examples, on the process of preparing a new molecular assay for clinical use.

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Analytical Validation of qPCR-Based Multivariate Index Assays in a Clinical Laboratory: Practical Challenges and Limitations

The Journal of Applied Laboratory Medicine ◽

10.1373/jalm.2017.025924 ◽

2018 ◽

Vol 3 (2) ◽

pp. 267-281

Author(s):

Cheryl L Sesler ◽

Elena V Grigorenko

Keyword(s):

Clinical Laboratory ◽

Disease Status ◽

Analytical Performance ◽

Analytical Sensitivity ◽

Clinical Use ◽

Analytical Validation ◽

Clinical Laboratories ◽

Practical Strategy ◽

Regulatory Guidelines ◽

Performance Specifications

Abstract Background Multivariate index assays (MIAs) to evaluate disease status and/or therapeutic efficacy are increasingly being used in clinical laboratories as laboratory-developed tests (LDTs). Before clinical use, diagnostic and analytical performance specifications of LDTs must be established. Several regulatory guidelines have been published that address specific components of validation procedures, but the interpretation for the analytical validation of MIAs is ambiguous and creates confusion when implementing a novel MIA in the clinical laboratory. Content CLSI guidelines and published methods were evaluated to develop a validation strategy to establish analytical sensitivity, precision, specificity, and stability for qPCR-based MIAs. Limitations and challenges identified while evaluating guidelines and literature and implementing this strategy are discussed in this review, including sample sourcing and integrity, laboratory contamination, and sample throughput. Due to the diversity of qPCR-based MIAs, we discuss additional considerations for researchers intending to transfer MIAs to a clinical laboratory. Summary A practical strategy to assess the analytical performance characteristics for validation of qPCR-based MIAs was developed and tested before diagnostic clinical use. Several important limitations, challenges, and considerations were identified during development of the analytical validation procedures that are not addressed in regulatory guidelines or published literature. The described strategy can provide insight for future developers of MIAs and clinical laboratories implementing MIAs as LDTs.

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Measurement uncertainty for clinical laboratories – a revision of the concept

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2016-0311 ◽

2016 ◽

Vol 54 (8) ◽

Cited By ~ 3

Author(s):

Graham Ross Dallas Jones

Keyword(s):

Measurement Uncertainty ◽

Clinical Laboratory ◽

Reference Interval ◽

Clinical Decision ◽

Time Frame ◽

Specific Reference ◽

True Value ◽

Clinical Laboratories ◽

Decision Points ◽

Result Interpretation

AbstractThe uncertainty of a measurement result is a fundamental concept in metrology indicating the range within the “true” value of a measurement should lie. Although not commonly reported with results, the calculation of measurement uncertainty (MU) has become common in routine clinical laboratories. Interpretation of numerical pathology results is made by comparison with data from other measurements. As MU is aimed at assisting with result interpretation, it should be related to the specific comparison being made. There are three basic type of comparators: a previous result from the same patient, a population reference interval, or a clinical decision point. For each comparison, the “true” value is that which would have been obtained from the instrument used to make the comparator measurements if it was measured without uncertainty. The MU is the range of likely deviations from this true value due to the method used to produce the result under interpretation. For patient monitoring, if the two measurements were made on the same analyzer, the uncertainty is the imprecision of the assay over the relevant time frame. In comparing with a manufacturer-specific reference interval, the MU is deviation from the manufacturer’s master calibrator. For clinical decision points produced with the assays traceable to international references, the MU is related to deviation from that reference standard. For optimal use of MU in the clinical laboratory, it may be necessary to consider the use of the test result and the concept of a single MU for each result may need to be revised.

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Diagnostic deoxyribonucleic acid probes for infectious diseases.

Clinical Microbiology Reviews ◽

10.1128/cmr.1.1.82 ◽

1988 ◽

Vol 1 (1) ◽

pp. 82-101 ◽

Cited By ~ 141

Author(s):

F C Tenover

Keyword(s):

Infectious Diseases ◽

Ribonucleic Acid ◽

Deoxyribonucleic Acid ◽

Clinical Laboratory ◽

Brugia Malayi ◽

Nucleotide Sequences ◽

Sensitive Detection ◽

Predictive Values ◽

Detection Systems ◽

Sensitivity Specificity

Virtually all microorganisms contain some unique nucleotide sequences which can be the target of deoxyribonucleic acid probes. Probes have been used successfully to identify a wide variety of pathogens from the simple ribonucleic acid-containing polioviruses to the complex filarial worms Brugia malayi. Probe technology offers the clinical laboratory the potential both to extend the types of pathogens that can be readily identified and to reduce significantly the time associated with the identification of fastidious microorganisms. Over a dozen commercially prepared deoxyribonucleic acid probe tests are now available. This article explores the development of deoxyribonucleic acid probe tests and reviews the sensitivity, specificity, and predictive values of many of the diagnostic probes developed during the last several years. Prospects for newer, more sensitive detection systems for the products of hybridization reactions are also reviewed.

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Using Standards and Controls in Molecular Assays for Infectious Diseases

Molecular Diagnosis ◽

10.2165/00066982-200106040-00014 ◽

2001 ◽

Vol 6 (4) ◽

pp. 335-345 ◽

Cited By ~ 3

Author(s):

ROBERTA MADEJ

Keyword(s):

Infectious Diseases ◽

Molecular Assays

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Diagnostic Challenges on the Laboratory Detection of Lupus Anticoagulant

Biomedicines ◽

10.3390/biomedicines9070844 ◽

2021 ◽

Vol 9 (7) ◽

pp. 844

Author(s):

Armando Tripodi

Keyword(s):

Lupus Anticoagulant ◽

Clinical Laboratory ◽

External Quality Assessment ◽

State Of The Art ◽

Diagnostic Procedures ◽

External Quality ◽

Glycoprotein I ◽

Current State ◽

Clinical Laboratories

Lupus anticoagulant (LA) is one of the three laboratory parameters (the others being antibodies to either cardiolipin or β2-glycoprotein I) which defines the rare but potentially devastating condition known as antiphospholipid syndrome (APS). Testing for LA is a challenging task for the clinical laboratory because specific tests for its detection are not available. However, proper LA detection is paramount for patients’ management, as its persistent positivity in the presence of (previous or current) thrombotic events, candidate for long term anticoagulation. Guidelines for LA detection have been established and updated over the last two decades. Implementation of these guidelines across laboratories and participation to external quality assessment schemes are required to help standardize the diagnostic procedures and help clinicians for appropriate management of APS. This article aims to review the current state of the art and the challenges that clinical laboratories incur in the detection of LA.

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Gold nanoparticles in the clinical laboratory: principles of preparation and applications

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2011.732 ◽

2012 ◽

Vol 50 (2) ◽

Cited By ~ 34

Author(s):

Hassan M.E. Azzazy ◽

Mai M.H. Mansour ◽

Tamer M. Samir ◽

Ricardo Franco

Keyword(s):

Gold Nanoparticles ◽

Clinical Laboratory ◽

Sensitive Detection ◽

Detection Methods ◽

Practical Applications ◽

Molecular Assays ◽

Detection Of Biomarkers

AbstractIn order to meet the challenges of effective healthcare, the clinical laboratory is constantly striving to improve testing sensitivity while reducing the required time and cost. Gold nanoparticles (AuNPs) are proposed as one of the most promising tools to meet such goals. They have unique optophysical properties which enable sensitive detection of biomarkers, and are easily amenable to modification for use in different assay formats including immunoassays and molecular assays. Additionally, their preparation is relatively simple and their detection methods are quite versatile. AuNPs are showing substantial promise for effective practical applications and commercial utilization is already underway. This article covers the principles of preparation of AuNPs and their use for development of different diagnostic platforms.

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Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm-2019-1262 ◽

2020 ◽

Vol 58 (9) ◽

pp. 1489-1497 ◽

Cited By ~ 1

Author(s):

Lisa K. Peterson ◽

Anne E. Tebo ◽

Mark H. Wener ◽

Susan S. Copple ◽

Marvin J. Fritzler

Keyword(s):

Indirect Immunofluorescence ◽

Antinuclear Antibodies ◽

Current Practice ◽

Clinical Laboratory ◽

Immunofluorescence Assay ◽

Indirect Immunofluorescence Assay ◽

Medical Laboratory ◽

International Consensus ◽

Clinical Laboratories ◽

Reading Interpretation

AbstractBackgroundThe indirect immunofluorescence assay (IFA) using HEp-2 cell substrates is the preferred method by some for detecting antinuclear antibodies (ANA) as it demonstrates a number of characteristic staining patterns that reflect the cellular components bound as well as semi-quantitative results. Lack of harmonized nomenclature for HEp-2 IFA patterns, subjectivity in interpretation and variability in the number of patterns reported by different laboratories pose significant harmonization challenges. The main objectives of this study were to assess current practice in laboratory assessment of HEp-2 IFA, identify gaps and define strategies to improve reading, interpretation and reporting.MethodsWe developed and administered a 24-item survey based on four domains: educational and professional background of participants, current practice of HEp-2 IFA testing and training, gap assessment and the perceived value of International Consensus on Antinuclear Antibody Patterns (ICAP) and other factors in HEp-2 IFA assessment. The Association of Medical Laboratory Immunologists (AMLI) and American Society for Clinical Pathology administered the survey from April 1 to June 30, 2018, to members involved in ANA testing. This report summarizes the survey results and discussion from a dry workshop held during the 2019 AMLI annual meeting.ResultsOne hundred and seventy-nine (n = 179) responses were obtained where a significant number were clinical laboratory scientists (46%), laboratory directors (24%), supervisors (13%) or others (17%). A majority of respondents agreed on the need to standardize nomenclature and reporting of HEp-2 IFA results. About 55% were aware of the ICAP initiative; however, among those aware, a significant majority thought its guidance on HEp-2 IFA nomenclature and reporting is of value to clinical laboratories. To improve ICAP awareness and further enhance HEp-2 IFA assessment, increased collaboration between ICAP and the clinical laboratory community was suggested with emphasis on education and availability of reference materials.ConclusionsBased on these suggestions, future efforts to optimize HEp-2 IFA reading, interpretation and reporting would benefit from more hands-on training of laboratory personnel as well as continuous collaboration between professional organizations, in vitro diagnostic manufacturers and clinical laboratories.

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