REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 247

cell basis. There is a liability with the MFI readout, however, as the degree of reported
fluorescence intensity could change at different PMT settings on the same cytometer
and vary from instrument to instrument. For this reason, standardization methods
have been developed. Three useful approaches are to convert signal to molecules
of equivalent soluble fluorochrome (MESF), to report the antibody bound per cell
(ABC) using a standardized curve, and to calibrate the instrument using beads to
standardize signal output before sample analysis [50].
12.3.2 Method Validation (Prestudy Validation)
Optimized clinical trial methods and preclinical toxicology experiments require
validation. The key elements of an assay validation are an approved validation plan,
assay validation experiments, predetermined acceptance criteria of results, and
documentation of the validation. The stringency of the validation assessment depends
upon at which stage of drug development the assay is used and the intended
applicability of the assay output (Figure 12.1).
Because flow cytometry applications are varied (and include immunophenotyping,
pharmacokinetic, pharmacodynamic, biomarker, immunogenicity, and functional
assays), validation parameters will be somewhat different and depend on assay type.
Thankfully, general guidelines have been described in earlier reports [27, 51-57],
with additional advances along the way as these efforts mature. Although flow
cytometry assays to be used in discovery and early research phase need not be
validated as per GLP, they should be well characterized and reproducible using a
calibrated, properly functioning flow cytometer. If this assay is to be used for testing
clinical samples in early-phase clinical studies for exploratory biomarker purpose, the
assay needs to be validated using a fit-for-purpose approach at a minimum and
performed in a GLP-like environment [1]. Furthermore, if the assay is to be used in
later stage registrational studies, the need for validation is tangibly heightened. For a
diagnostic application of the assay, the validation of the assay is performed as per
CLIA guidelines. The validation of a flow cytometry assay for application in a clinical
setting is described in the following section, whereas validation of an assay for CLIA
certification is beyond the scope of this chapter (refer to Section 12.2).
12.3.2.1 Validation Plan The validation protocol is an essential tool used to guide
the validation process. It should describe all the elements of the validation process
including sample type and source, list of equipment, identification of scientists
performing validation experiments, approvers of the results of validation,
validation parameters, statistical methods, assay and result acceptability criteria
set prospectively, and assaymagnitude during validation (number of samples or plates
mimicking intended use of the assay). Validation study parameters to be considered in
the validation assessment include precision, sensitivity (limits of quantitation),
specificity, accuracy, stability, and reference ranges. Flow cytometric assay
validation plan considerations and suggested acceptance criteria are summarized
in Table 12.1. Prior to the start of a validation assessment, sufficient reagents should be
obtained to perform all experiments intended for validation, and the analyst should be