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Required performance characteristics with suggested studies needed before implementation of FDA-approved/cleared tests and laboratory-developed testsa

Performance characteristic (reference[s]) and suggested studyRequirement(s) for:
FDA-approved/cleared testLaboratory-developed test
Reportable range (8), linearity study (for quantitative assays)5-7 concentrations across stated linear range, 2 replicates at each concn7-9 concentrations across anticipated measuring range (or 20-30% beyond to ascertain widest possible range); 2-3 replicates at each concn; polynomial regression analysis
Analytical sensitivity (14, 28, 33), limit-of-detection studyNot required by CLIA, but CAP requires LOD verification for quantitative assays; use 20 data points collected over 5 days60 data points (e.g., 12 replicates from 5 samples in the range of the expected detection limit); conduct the study over 5 days; probit regression analysis (or SD with confidence limits if LOB studies are used)
Precision (7, 13, 15, 40), replication experimentFor qualitative test, test 1 control/day for 20 days or duplicate controls for 10 days; for quantitative test, test 2 samples at each of 2 concentrations (4 samples) plus one control over 20 days or test 2 concentrations in triplicate over 5 daysFor qualitative test, minimum of 3 concentrations (LOD, 20% above LOD, 20% below LOD) and obtain 40 data points; for quantitative test, minimum of 3 concentrations (high, low, LOD) and test in duplicate 1-2 times/day over 20 days; calculate SD and/or CV within run, between run, day to day, total variation
Analytical specificity (28), interference studyNot required by CLIANo minimum no. of samples recommended; test sample-related interfering substances (hemolysis, lipemia, icterus, etc.) and genetically similar organisms or organisms found in same sample sites with same clinical presentation; spike with low concentration of analyte; paired-difference (t test) statistics
Accuracy (trueness) (13), comparison-of-methods study20 patient specimens within the measuring interval or reference materials at 2 concentrations (low and high) in duplicate over 2-5 runsTest in duplicate by both the comparative and test procedures over at least 5 operating days; typically 40 or more specimens; xy scatter plot with regression statistics; Bland-Altman difference plot with determination of bias; % agreement with kappa statistics
Reference interval (6)The reference interval stated by the manufacturer may be “transferred” if the stated reference interval is applicable to the population served by the clinical laboratory; if exptl verification is desired, test 20 specimens representative of the population; if the population is different, establish the reference interval by testing 60 (minimum, 40) specimensIf a nucleic acid target is always absent in a healthy individual and the tests is a qualitative test, the reference range is typically “negative” or “not detected” and reference interval studies do not need to be performed; for quantitative assays, the reference interval will be reported as below the LOD or LLOQ; for some analytes, the reference interval may be a clinical decision limit; if the intended use of the test is limited to patients known to be positive for the analyte being assayed, a reference interval may not be applicable