REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 251

presence of the biologic matrix or interfering substances. Where possible, assays
intended for use in clinical trials should evaluate possible drug and metabolite
interferences prior to implementation. Generally, laboratories rely on cluster of
differentiation (CD) designations or the antibody vendor to characterize reagent
specificity. It is not difficult to find those in our field that are uncomfortable with this
approach, but there is no solution to this issue other than performing specificity
experiments in-house. In some cases, testing a variety of antibody clones (when
available) can lend credence to the reproducibility of the result; in other cases,
Western blots on cell lysates have been commonly used.
Sensitivity Sensitivity is the measure of assay performance with known negative
samples used in determining the lowest limit of measurement in known positive
samples. GLP guidelines define the lowest limit of quantitation as the lowest level at
which a method can accurately measure, whereas CLIA defines sensitivity as the
lowest value able to be reported [6, 8]. Reagent sensitivity is important in developing a
flow cytometric assay to find the minimum staining intensity above nonspecific
staining able to be detected in an assay. Sensitivity assessment should determine both
the maximum fluorescence intensity of a positive population and the maximum
separation between positive and negative populations. These results can be used to
determine the appropriate amount of reagent in a particular assay to be most
sensitive [58]. Even though commercial reagents are usually provided with
recommended amounts for use in staining protocols, the reagents should be
titered to confirm saturating conditions in a particular protocol.
Stability Sample handling and storage conditions can greatly impact the assay-
specific phenotype and/or function being monitored. Therefore, stability for a
biological marker must be validated from the time of sample collection to the
time of analysis. Validation may include assessment of physiological expression,
effect of matrix, storage and shipping effects, sample handling, processing conditions,
and effects of reagents. Early determination of stability limitations will provide
valuable information for assay transition to clinical trials.
It is common practice to begin assay validation with intra-assay and inter-assay
precision experiments; however, evaluation of sample stability limitations first may
provide valuable insight into how to manage clinical protocol sample collection,
handling, and shipping conditions. Consider the alternative scenario where post-
collection sample stability is validated first, and 48 h postcollection is determined to
be the maximum limit for marker stability. Subsequent precision validation experi-
ments can then be performed with samples that mimic the 48 h postcollection
conditions of acceptable clinical samples. The use of samples that are less than
optimal (not freshly collected) in validation will provide a more realistic estimation of
assay performance in clinical trials.
Storage, Handling, and ShippingConditions Sample stability should be validated in
the collection container to determine marker stability during shipment and storage.
Many cell surface markers are stablewhen samples are stored at ambient temperature;