REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 250

criteria are important. The more practice and assay experience an analyst has
performing the assay, the less variability between analysts is expected. The data
generated between analysts should be within the inter-assay variability or
predetermined acceptance range (Table 12.1). Finally, the sample materials for
precision testing should mimic the expected samples in clinical trials, including
both samples from subjects (donors) and QC samples.
INTER - INSTRUMENT COMPARISON Differences in instrument performance can add
variability to an assay increasing the imprecision assessment. While a primary
cytometer may be used for the majority of validation tests and clinical sample
analysis for a given assay, a flow cytometry lab may have more than one instrument to
handle large numbers of samples from the clinical trials. The additional cytometer(s)
also serve as backup instruments when the primary cytometer is subject to
maintenance services. To ensure the cytometers are comparable within the same
lab or between different labs, the instrument-to-instrument comparison should be
performed at least every 6 months and after maintenance service. Clear
documentation of instrument maintenance and service is a good practice to obtain
high-quality data.
Accuracy The strict definition of accuracy is as the closeness of agreement between
the averages of the test results when compared to a well-defined official reference
standard. Accuracy assessment is not possible in flow cytometry assays in this regard,
as reference standards are not readily available. For those laboratories engaged in
proficiency testing services, comparability of results from one site against the group
can imply accuracy. In instances where an antigen is not included in the typical
proficiency testing panel, a biopharmaceutical company may elect to prepare samples
in-house and distribute them for analysis by as many flow cytometers and by as many
analysts as are available. Although this may be better described as an exercise in
robustness, it is quite important to to understand whether a given data set would agree
with another generated in a different time and place. The lack of accuracy standards do
not excuse those in the field from ignoring this important parameter during validation
exercises, and every effort should be made to speak to the accuracy of an assay despite
limitations of our field.
Specificity Cellular assays are often challenged by a complex matrix (i.e., whole
blood) presenting specificity and sensitivity issues. Reagents must be specific
enough to recognize the intended target in the presence of interfering substances
(i.e., compounds, proteins, etc.) and sensitive enough to measure low levels of
expression over the background signals. Antibody specificity and cross reactivity
within a complex biological matrix greatly influence assay sensitivity in flow
cytometric assays. Since both are related to proper reagent selection, specificity
and sensitivity are customarily discussed together.
Proper reagent selection is critical to the development of a flow cytometry assay.
The antibody clone must be specific for the intended target [5]. It is important to
understand assay specificity to determine the reliability of measurements in the