REGULATORY COMPLIANCE AND METHOD VALIDATION - Flow Cytometry in Drug Discovery and Development - page 244

Common anticoagulants for immunophenotyping and functional assessment are
ethylenediaminetetraacetic acid (EDTA), heparin, and acid citrate dextrose (ACD)
anticoagulants. Cyto-Chex BCT tubes (Streck, Inc.) are a newer product that can
allow extended stability; however, this product is not suitable for all antigens or for
functional studies as the product contains a mild fixative [43, 44]. Determination of
marker stability in appropriate anticoagulants during the assay development phase
will be beneficial for methods intended for use in clinical protocols. Sample stability
should be assessed in the collection container during shipment and storage, during
sample processing, and postfixation (if applicable). Understanding the limitations for
maintaining stable marker expression when collected in a particular additivewill help
define appropriate sample collection instructions for clinical protocols.
Whole blood is usually considered the first choice of sample matrix for flow
cytometry assays, as it closely mimics the functional status of cells in vivo.
Moreover, if a biomarker assay involves cell stimulation (e.g., phospho flow), the
readout is viewed as more appropriate in whole blood because the circulating
concentration of the drug is carried throughout the assay. The conundrum is that
whole blood is a perishable specimen; therefore, consideration should be given to
isolated cellular samples in lieu of a whole blood matrix. When cell counts are the
assay end point and the cell population under study is of low frequency, some
laboratories opt for a concentrated cellular sample to facilitate analysis of the
desired cell population.
If whole blood is not the matrix of choice, cell isolation procedures must be
optimized to safeguard against cell loss and to identify the most stable processing
conditions for the markers of interest. It is uncommon to hold isolated cells in buffer
or culture medium prior to phenotyping them because of the possibility that cells
will up- or downregulate their antigen profile. In the case that isolated cells cannot be
immediately stained, storage in liquid nitrogen is common. If the assay is to be
implemented in clinical studies using frozen PBMC, stability of markers after
cryopreservation is then an additional parameter to be investigated during assay
development before proceeding with validation of the assay.
12.3.1.6 Impact of PBMC Isolation Process on Cell Phenotype and Function
When using frozen PBMC as the sample matrix, optimization of immunopheno-
typing assays should include similar assessments to those made for peripheral
blood (i.e., appropriate anticoagulant, selection of reagents with high specificity
and staining intensity, etc.) with a few additional sample condition considerations:
(i) postcollection marker stability prior to PBMC isolation; (ii) impact of PBMC
isolation techniques, freezing conditions, storage temperature, and thawing method
on cell phenotype and function; and (iii) marker stability for the entire duration of
PBMC storage. Continuation of a stable environment is recommended for frozen
PBMC samples to preserve the integrity of markers, especially in functional
assays [45].
The process of PBMC isolation should be evaluated to optimize conditions for
analysis. Blood samples can either be directly collected in a cell preparation
tube containing anticoagulant and Ficoll gradient for PBMC separation through