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

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.

Development and comparative evaluation of immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum

1990 ◽  
Vol 36 (12) ◽  
pp. 2053-2056 ◽  
Author(s):  
T Fonong ◽  
S M Evans ◽  
H A Homburger
Keyword(s):  
Comparative Evaluation ◽  
Clinical Laboratory ◽  
Connective Tissue Diseases ◽  
Positive Control ◽  
Analytical Sensitivity ◽  
Healthy Controls ◽  
Clinical Use ◽  
Immunoblot Assay ◽  
Negative Results ◽  
Positive Results

Abstract We developed rapid 24-h immunoblot assays for detecting autoantibodies to Scl 70 and Jo 1 antigens in serum. In comparative studies, we evaluated the analytical sensitivity of the immunoblot assays and commercial immunodiffusion assays for anti-Scl 70 and anti-Jo 1 autoantibodies with the use of positive control sera, and compared the frequencies of positive and negative results in a group of 116 sera, including specimens from 34 healthy controls and 82 patients with various connective-tissue diseases. The immunoblot assays were greater than 100-fold more sensitive than immunodiffusion for detecting both autoantibodies. Despite greater analytical sensitivity, there were no false-positive results by the immunoblot assay for anti-Scl 70 or anti-Jo 1 autoantibodies in sera from either the controls or the patients. The diagnostic sensitivity of the immunoblot assay for anti-Scl 70 autoantibodies in patients with scleroderma was greater than that of the immunodiffusion assay, 70% vs 20%, and was equivalent in patients with polymyositis, 43%. We conclude that rapid immunoblot assays for anti-Scl 70 and anti-Jo 1 autoantibodies are superior to immunodiffusion assays for clinical use and are suitable for routine use in the clinical laboratory.

scholarly journals Validation of Laboratory-Developed Molecular Assays for Infectious Diseases

2010 ◽  
Vol 23 (3) ◽  
pp. 550-576 ◽  
Author(s):  
Eileen M. Burd
Keyword(s):  
Infectious Diseases ◽  
Clinical Laboratory ◽  
Reference Interval ◽  
Attractive Alternative ◽  
Analytical Sensitivity ◽  
Additional Specimen ◽  
Molecular Assays ◽  
Clinical Laboratories ◽  
Regulatory Standards ◽  
Sensitivity Specificity

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.

Multi-site performance evaluation and Sigma metrics of 20 assays on the Atellica chemistry and immunoassay analyzers

2019 ◽  
Vol 58 (1) ◽  
pp. 59-68
Author(s):  
Tommaso Fasano ◽  
Jose Luis Bedini ◽  
Pierre-Antoine Fle ◽  
Malik Jlaiel ◽  
Karl Hubbert ◽  
...  
Keyword(s):  
Clinical Laboratory ◽  
Method Comparison ◽  
Analytical Performance ◽  
Medical Decision ◽  
Pooled Data ◽  
Performance Quality ◽  
Clinical Laboratories ◽  
Individual Site ◽  
Deming Regression ◽  
Allowable Error

Abstract Background The Atellica Solution comprises chemistry (CH) and immunoassay (IM) analyzers. Recently, six early adopter clinical laboratories across Europe evaluated the analytical performance of 20 CH and IM assays. To measure analytical performance quality, Sigma metrics were calculated for individual-site and pooled-site results. Methods Precision, detection capability, linearity, and method comparison studies were performed according to Clinical Laboratory Standards Institute protocols. Global Sigma metrics across sites were calculated from pooled data at the medical decision level using total allowable error (TEa) goals from CLIA for CH assays, and TEa goals from RiliBÄK for IM assays; and, the equation: Sigma metrics=%TEa–%bias/%CV. A pooled %CV was calculated by combining the imprecision obtained from individual sites. Bias calculations were performed against the ADVIA Chemistry system or ADVIA Centaur system using Deming regression analysis (Passing-Bablok regression for electrolytes) on the pooled-site data. The 103 individual-site Sigma metric calculations used individual-site imprecision and pooled-bias. Results The limits of blank and detection results agreed with the manufacturer’s claims. Most assays were linear across the assay range tested. Pooled Sigma metrics were good or better (>4 Sigma) for 18 of 20 assays; and, acceptable for urea nitrogen (3.1) and sodium (3.9), the latter values attributable to higher imprecision at one of five sites. Conclusions Sigma metrics for data generated across multiple real-world sites evaluating the Atellica Solution demonstrated good or better performance of greater than 4 Sigma for 18 of 20 assays tested. Overall, results verified the manufacturer’s claims that methods were fit for use in clinical laboratories.

scholarly journals Analytical performance evaluation of a commercial next generation sequencing liquid biopsy platform using plasma ctDNA, reference standards, and synthetic serial dilution samples derived from normal plasma

BMC Cancer ◽  
2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Suman Verma ◽  
Mathew W. Moore ◽  
Rebecca Ringler ◽  
Abhisek Ghosal ◽  
Kyle Horvath ◽  
...  
Keyword(s):  
Performance Evaluation ◽  
Next Generation Sequencing ◽  
Human Plasma ◽  
Allele Frequency ◽  
Liquid Biopsy ◽  
Clinical Laboratory ◽  
Reference Standards ◽  
Analytical Performance ◽  
Clinical Laboratories ◽  
Generation Sequencing

Abstract Background Circulating tumor (ct) DNA assays performed in clinical laboratories provide tumor biomarker testing support for biopharmaceutical clinical trials. Yet it is neither practical nor economically feasible for many of these clinical laboratories to internally develop their own liquid biopsy assay. Commercially available ctDNA kits are a potential solution for laboratories seeking to incorporate liquid biopsy into their test menus. However, the scarcity of characterized patient samples and cost of purchasing validation reference standards creates a barrier to entry. In the current study, we evaluated the analytical performance of the AVENIO ctDNA liquid biopsy platform (Roche Sequencing Solutions) for use in our clinical laboratory. Method Intra-laboratory performance evaluation of AVENIO ctDNA Targeted, Expanded, and Surveillance kits (Research Use Only) was performed according to College of American Pathologists (CAP) guidelines for the validation of targeted next generation sequencing assays using purchased reference standards, de-identified human plasma cell-free (cf) DNA samples, and contrived samples derived from commercially purchased normal and cancer human plasma. All samples were sequenced at read depths relevant to clinical settings using the NextSeq High Output kit (Illumina). Results At the clinically relevant read depth, Avenio ctDNA kits demonstrated 100% sensitivity in detecting single nucleotide variants (SNVs) at ≥0.5% allele frequency (AF) and 50% sensitivity in detecting SNVs at 0.1% AF using 20–40 ng sample input amount. The assay integrated seamlessly into our laboratory’s NGS workflow with input DNA mass, target allele frequency (TAF), multiplexing, and number of reads optimized to support a high-throughput assay appropriate for biopharmaceutical trials. Conclusions Our study demonstrates that AVENIO ctDNA liquid biopsy platform provides a viable alternative for efficient incorporation of liquid biopsy assays into the clinical laboratory for detecting somatic alterations as low as 0.5%. Accurate detection of variants lower than 0.5% could potentially be achieved by deeper sequencing when clinically indicated and economically feasible.

Lot variation and inter-device differences contribute to poor analytical performance of the DCA Vantage™ HbA1c POCT instrument in a true clinical setting

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Anders Abildgaard ◽  
Cindy Søndersø Knudsen ◽  
Lise Nørkjær Bjerg ◽  
Sten Lund ◽  
Julie Støy
Keyword(s):  
Clinical Setting ◽  
Clinical Laboratory ◽  
Point Of Care ◽  
Method Comparison ◽  
Glycated Haemoglobin ◽  
Analytical Performance ◽  
Point Of Care Testing ◽  
Performance Specifications ◽  
Ion Exchange Hplc ◽  
Analytical Imprecision

Abstract Objectives The glycated haemoglobin fraction A1c (HbA1c) is widely used in the management of diabetes mellitus, and the Siemens DCA Vantage™ point-of-care testing (POCT) instrument offers rapid HbA1c results even far from a clinical laboratory. However, the analytical performance has been questioned, and not much is known about effects of changing reagent lot, instrument and operator. We therefore compared the analytical performance of the DCA Vantage™ with established routine methods (Tosoh G8/G11 ion exchange HPLC) in a true clinical setting at two Danish hospitals. Methods We extracted all routine clinical HbA1c results incidentally drawn from the same patient within 48 h (n=960 pairs) and evaluated the effect of reagent lot, operator and instrument. We also performed a prospective method comparison in our diabetes out-patient clinic (n=97). Results The critical difference (CD) between two POCT results varied between 5.14 and 6.61 mmol/mol (0.47–0.55%), and the analytical imprecision of the DCA Vantage™ (CVA) was >3%. Significant effect of reagent lot and inter-instrument differences were found, whereas no effect of operator was seen. Conclusions The DCA Vantage™ HbA1c analysis does not fulfil the prevailing analytical performance specifications, but rigorous validation of new reagent lots and continuous recalibration of instruments may potentially improve the precision substantially. Our findings, therefore, clearly emphasise the necessity of a close collaboration between clinicians and laboratory professionals in the POCT field. Finally, POCT HbA1c results should always be interpreted together with other measures of glycaemic control to avoid inappropriate change of patient treatments due to measurement uncertainty.

scholarly journals Clinical laboratory: bigger is not always better

Diagnosis ◽  
2018 ◽  
Vol 5 (2) ◽  
pp. 41-46 ◽  
Author(s):  
Mario Plebani
Keyword(s):  
Quality Indicators ◽  
Clinical Laboratory ◽  
Diagnostic Errors ◽  
Test Volume ◽  
Laboratory Errors ◽  
Clinical Laboratories ◽  
Laboratory Services ◽  
Intermediate Outcomes ◽  
Performance Specifications ◽  
The Relationship

AbstractLaboratory services around the world are undergoing substantial consolidation and changes through mechanisms ranging from mergers, acquisitions and outsourcing, primarily based on expectations to improve efficiency, increasing volumes and reducing the cost per test. However, the relationship between volume and costs is not linear and numerous variables influence the end cost per test. In particular, the relationship between volumes and costs does not span the entire platter of clinical laboratories: high costs are associated with low volumes up to a threshold of 1 million test per year. Over this threshold, there is no linear association between volumes and costs, as laboratory organization rather than test volume more significantly affects the final costs. Currently, data on laboratory errors and associated diagnostic errors and risk for patient harm emphasize the need for a paradigmatic shift: from a focus on volumes and efficiency to a patient-centered vision restoring the nature of laboratory services as an integral part of the diagnostic and therapy process. Process and outcome quality indicators are effective tools to measure and improve laboratory services, by stimulating a competition based on intra- and extra-analytical performance specifications, intermediate outcomes and customer satisfaction. Rather than competing with economic value, clinical laboratories should adopt a strategy based on a set of harmonized quality indicators and performance specifications, active laboratory stewardship, and improved patient safety.

scholarly journals Spanish society of laboratory medicine external quality assurance programmes: evolution of the analytical performance of clinical laboratories over 30 years and comparison with other programmes

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
Carmen Perich ◽  
Carmen Ricós ◽  
Fernando Marqués ◽  
Joana Minchinela ◽  
Angel Salas ◽  
...  
Keyword(s):  
Quality Assurance ◽  
State Of The Art ◽  
Laboratory Medicine ◽  
Analytical Performance ◽  
External Quality Assurance ◽  
Biological Variability ◽  
External Quality ◽  
Spanish Society ◽  
Clinical Laboratories ◽  
Quality Specifications

AbstractThe purpose of this study is to understand the evolution of the analytical performance of the laboratories participating in the Spanish society of laboratory medicine (SEQCML) external quality assurance (EQA) programmes during its 30 years of operation and to compare it with the performance of other EQA programmes to establish whether the results are similar. The results obtained during this period are evaluated by applying the biological variability (BV) and state of the art-derived quality specifications. In addition, the results are compared with those obtained by other EQA programme organisations. It is noted that the laboratories participating in the EQA–SEQCML programmes have improved their performance over 30 years of experience and that the specifications derived from biological variation are achievable. It is difficult to compare EQA programmes, due to lack of accessibility and the differences in the design of these programmes (control materials, calculations used and analytical specifications established). The data from this study show that for some biological magnitudes the results obtained by the programmes are not yet harmonised, although efforts are being made to achieve this. Organisers of EQA programmes should also join the harmonisation effort by providing information on their results to enable comparison.

scholarly journals Diagnostic Challenges on the Laboratory Detection of Lupus Anticoagulant

Biomedicines ◽  
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.

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

2020 ◽  
Vol 58 (9) ◽  
pp. 1489-1497 ◽  
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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