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Qualitative Assay Validation

Validation of a qualitative test differs from a quantitative test. Precision testing is required for qualitative tests that derive a qualitative result, such as negative or positive, from a quantitative value such as optical density. Precision testing is also required if the manufacturer's package insert provides precision specifications. Within run precision can be accomplished by running at least 20 replicates of negative control and 20 replicates of positive control in a single run. Between run precision involves running negative and positive control at least once per day for 20 days. Precision results are used to calculate the %CV for both the positive and negative controls. Precision is acceptable if the calculated CVs are less than or equal to the manufacturer's stated CV.

Accuracy of a new qualitative method is assessed by comparison to a method already in use in the laboratory or at a reference laboratory. Another possibility is to test samples with known values, such as proficiency test samples or commercial standards. A minimum of 10 samples for each expected result should be tested. For example, if a test gives results of positive or negative, accuracy studies must include 10 known positive and 10 known negative samples.

Accuracy can be assessed by calculating sensitivity and specificity using a contingency table.


Results from Comparison Study

Positive Negative
Positive # true positive (TP) # false positive (FP) TP+FP
Negative # false negative (FN) # true negative (TN) FN+TN
  1. Calculate the estimated Diagnostic Sensitivity(True positive rate) = 100 x [TP/(TP+FN)]
  2. Calculate the estimated Diagnostic Specificity(True negative rate) = 100 x [TN/(FP+TN)]
  3. Calculate the percent Positive Agreement (Positive Predictive Value)=100 x TP/(TP+FP)
  4. Calculate the percent Negative Agreement (Negative Predictive Value) =100 x TN/(TN+FN)
  5. Compare the results calculated above with the manufacturer’s stated claims for Sensitivity, Specificity and Agreement found in the test kit package insert.
  6. Results must be equal to, or greater than, the manufacturer’s claims for the method to be considered accurate.

Qualitative tests do not require linearity, AMR or reference range studies. For an FDA approved, unmodified method, the laboratory can accept the manufacturer's interference claims.

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