Flow cytometric lymphocyte subset analysis using material from frozen whole blood

Area of Science:

• Immunology
• Cellular Biology
• Biotechnology

Background:

• Multicenter immune monitoring often requires cryopreserved peripheral blood mononuclear cells (PBMC).
• PBMC isolation from whole blood before freezing is standard but requires specific laboratory facilities.
• Field conditions may lack the infrastructure for PBMC separation, posing logistical challenges.

Purpose of the Study:

• To assess the feasibility of using whole blood (WB) frozen at -80°C as a source of viable lymphocytes.
• To compare lymphocyte subsets from frozen WB with those from cryopreserved PBMC.
• To determine the suitability of frozen WB for immunological studies, particularly T cell enumeration.

Main Methods:

• Whole blood (WB) samples from five healthy donors were frozen at -80°C.
• Peripheral blood mononuclear cells (PBMC) were isolated and cryopreserved using standard methods.
• Flow cytometry was used to analyze CD4 and CD8 T lymphocyte percentages and subsets in both frozen WB and cryopreserved PBMC.

Main Results:

• Lymphocyte analysis from WB frozen up to 120 days showed results comparable to PBMC frozen up to 10 days.
• CD4 and CD8 T cell counts and subset percentages were similar between the two sample types within the tested durations.
• The viability and integrity of lymphocytes in frozen WB were maintained sufficiently for T cell enumeration.

Conclusions:

• Contrary to initial assumptions, whole blood (WB) frozen at -80°C can serve as an appropriate source of viable lymphocytes.
• This method offers a practical alternative for immunological and epidemiological studies where PBMC isolation facilities are unavailable.
• Frozen WB simplifies sample handling and storage for immune monitoring, expanding research possibilities in field settings.