The "Bioanalytical Method Validation" document lays down some criteria that may be applied to the validation of the most critical
assay parameters. It should be noted, however, that in some tests it may not be possible to reach the levels of accuracy and
precision specified in this document, while in others it may be possible to set closer limits. The criteria should be test-specific.9
Accuracy is determined by replicate analysis of samples containing known amounts of the analyte. It is recommended that at
least five determinations per concentration be done. A minimum of three different concentrations within the range of expected
concentration should be tested so that data comprise at least 15 analyses. The mean value should be within 15% of the actual
value, except at the lower limit of quantitation, where it should not deviate by more than 20%.
Precision should be measured using a minimum of five determinations on at least three different concentrations, testing multiple
aliquots of a single homogenous sample. The precision determined at each concentration should not exceed 15% of the coefficient
of variation (CV), except for the LLOQ, where it should not exceed 20%. The same criteria should be applied to each type of
measurement of precision (intra-assay, inter-assay).
The following criteria should be applied to the calibration or standard curve:
- Limit of detection should be at least three times the background or blank response
- Analyte response at the LLOQ should be at least five times the blank response
- Analyte (peak) response should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80%-120%
The simplest mathematical model that yields a useable relationship between concentration and response should be used.
In developing the standard curve, there must be <20% deviation of the LLOQ from the nominal concentration and <15% deviation
of other standards. No fewer than six points should be used to define the curve. At least four of six non-zero standards should
meet the above criteria, including the LLOQ and the calibration standard at the highest concentration. Considering the typical
asymptotic curve generated by most biological quantitative assays, the guidance recommends that more than six points should
be used to fit the curve. The concentration-response relationship is most often fitted to a four- or five-parameter logistic
model. Computer programs can perform this automatically, and some of these are capable of dealing with non-parallelism as
Acceptance criteria should be set for bioanalytical test validation before the series is run. The guidance offers the following
acceptance criteria: at least 67% of QC samples should be within 15% of their respective nominal value, and 33% of the QC
samples (not all replicates at the same concentration) may be outside the 15% limit. Some tests may require wider limits.
Some small points to consider in performing the replicate runs required to validate a bioassay, using the ubiquitous microplate
method, are as follows:
- Ensure that a certain number of microplates receive samples in reverse order ("front-to-back") or some other arrangement,
to limit potential effect of time or position on the plate. Positional effects may be very important. It has been reported
that multi-channel pipetting devices, which are often used for plate assays, may show variability between channels in the
- Perform identical validation runs using a sufficient number of lots of cells, media, standards, etc. to ensure that the test
is sufficiently robust to withstand lot-to-lot variation.
- Ensure that the SOP describing the assay is sufficiently detailed and that it gives unequivocal instructions for procedures
that may affect accuracy or precision of the test, e.g., mixing, washing steps.
- Be certain all reagents can withstand the exposure that occurs during plating out and further manipulation of the microplates
during the assay.
- Check that the plate reader has been adequately qualified for this assay.
- Record all data directly after reading, and have mathematical manipulations checked by another analyst, or, if they are normally
performed by the instrument's software, check this by manual re-calculation.
- If validation requires evaluation of a number of different test parameters, a "multi-dimensional" test plan can be used.