Stability of immunophenotypic markers in fixed peripheral blood for extended analysis using flow cytometry.

The analysis of whole blood samples by flow cytometry for pharmacodynamic and biomarker assessments in clinical studies has been limited by the necessity to test these samples within a short time frame after blood collection. In most clinical studies, blood specimens are shipped to a centralized testing facility; it is critical to demonstrate specimen stability over a period of time which will encompass the time elapsed between specimen collection and testing. A possible solution to overcome this limitation is the use of a fixative to preserve the cell surface antigen stability in whole blood. We examined the stability of markers for T lymphocytes (CD3, CD4, CD8, CD45RA, and CD45RO), B lymphocytes (CD19), NK cells (CD16+CD56), activation (CD25 and HLA-DR), chemokine receptors (CCR5 and CXCR3) and skin homing (CLA) in fixed blood over 7 days and used this information to select the markers for global clinical studies. These assays with selected markers were further validated using fit-for-purpose approach (Lee et al., 2006) and to set the sample acceptability criteria for use in clinical sample testing. Most of the markers examined were stable when collected in Cyto-Chex® BCT for one week with the exception of the activation markers on T cells.