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

Area of Science:

• Immunology
• Clinical Diagnostics
• Biomarker Analysis

Background:

• Flow cytometry analysis of whole blood for pharmacodynamic and biomarker assessments in clinical studies is limited by short sample turnaround times.
• Shipping blood specimens to centralized facilities necessitates demonstrating specimen stability over time, from collection to testing.

Purpose of the Study:

• To evaluate the stability of various cell surface markers in whole blood collected in a fixative over seven days.
• To identify stable markers for use in global clinical studies and establish sample acceptability criteria.

Main Methods:

• Whole blood samples were collected in Cyto-Chex® BCT and analyzed by flow cytometry.
• Stability of T lymphocyte markers (CD3, CD4, CD8, CD45RA, CD45RO), B lymphocyte markers (CD19), NK cell markers (CD16+CD56), activation markers (CD25, HLA-DR), chemokine receptors (CCR5, CXCR3), and skin homing markers (CLA) was assessed over seven days.
• Selected assays were validated using a fit-for-purpose approach.

Main Results:

• Most cell surface markers, including T and B lymphocyte populations, NK cells, chemokine receptors, and skin homing markers, remained stable for seven days in fixed blood.
• Activation markers (CD25, HLA-DR) on T cells showed instability over the seven-day period.
• The study successfully selected and validated stable markers for clinical use.

Conclusions:

• Cyto-Chex® BCT provides a viable solution for preserving whole blood samples for flow cytometry analysis for up to one week.
• This approach overcomes logistical challenges in clinical studies, allowing for delayed testing of most cell surface markers.
• Activation markers on T cells require careful consideration regarding sample handling and timing due to their instability.