REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 245

centrifugation (i.e., BD Vacutainer CPT tube containing heparin or citrate) or be
collected in an alternative anticoagulant for manual Ficoll gradient application and
cell separation. In both processes, marker stability should be evaluated prior to and
including PBMC isolation processing as any fluctuation in handling and storage can
affect marker instability and greatly impact result data [46]. Cell yield and viability
results will also vary depending on PBMC isolation conditions. Additional benefit
may be gained in obtaining PBMC from diseased donors (if available) to provide
insight into the expectation of marker expression in clinical samples and the effects of
isolation techniques on sensitive markers.
Reagents used during the PBMC isolation process may affect some markers of
interest and should be evaluated. Expression of some markers, such as CCR5, may be
affected by regents used for PBMC isolation (i.e., Ficoll) and may not be suitable for
sample collection in a particular flow cytometric method. In addition, the cell
preservation reagents, thawing media, and final cell suspension should be evaluated
for optimizing marker expression. One should not underemphasize the need to assess
stability of markers before and after PBMC isolation if this route is chosen.
A process of controlled freezing of isolated PBMC samples (e.g., using 1 C
cryogenic freezing containers at

70 C) followed by a prompt transfer to liquid
nitrogen storage is beneficial in maximizing PBMC recovery and marker stabi-
lity [47]. Mononuclear cells stored in liquid nitrogen are known to preserve the
majority of known surface markers and certain cell functionality aspects; however,
extended cryopreservation may be detrimental to certain phenotypes or functions
affecting intracellular immunophenotyping, functional, and tetramer assays. There-
fore, stability of marker expression should be determined over a period of extended
cryopreservation, if applicable [48]. If markers are stable after cryopreservation
during the testing period in the assay development stage, the method validation can be
initiated with greater confidence using batch frozen PBMC. In addition, ex vivo
stimulation methods can be implemented in batch processing using frozen PBMC
when long-term marker stability has been established.

12.3.1.7 Assay controls Inclusion of appropriate controls for reagents, samples,
instrument setup, and gating is critical for a flow cytometric assay. Requirement for
these controls is based on the assay type, the purpose of the experiment, and the
intended utility of the data generated. Assessment of relevant controls should occur
during the assay development phase and selection should be made before validation
begins. Awell-designed flow assay at a minimum must include controls for reagents
and method. Main categories of controls include (i) negative or autofluorescence
control, (ii) compensation or positive controls, (iii) specificity or gating controls, and
(iv) sample controls.

(i) Negative control is set up using cells (typically untreated, but can include
treated cells also) not stained with any antibodies to detect background/
autofluorescence of the cells. Autofluorescence tends to lower discrimination
between stained and unstained cells interfering with measurement of low-
level expression of bound fluorescent antibody. Autofluorescence is largely