Lymphocyte subset analysis on frozen whole blood

Area of Science:

• Immunology
• Flow Cytometry
• Virology

Background:

• Lymphocyte subset analysis is crucial for monitoring immune status, particularly in conditions like HIV-1 infection.
• Preserving whole blood (WB) samples through freezing is desirable for logistical and storage efficiency.
• Evaluating the reliability of frozen-thawed WB for accurate lymphocyte subset enumeration is essential.

Purpose of the Study:

• To assess the feasibility and accuracy of performing lymphocyte subset analysis on frozen-thawed whole blood (F/T WB).
• To compare lymphocyte subset percentages and absolute counts from F/T WB with those from fresh WB.
• To determine the impact of various freezing and storage conditions on sample integrity.

Main Methods:

• Whole blood samples from HIV-1 seropositive individuals and controls were analyzed fresh and after storage at -80°C, liquid nitrogen, and -20°C.
• Three-color flow cytometry was employed, utilizing CD45 and right-angle light scatter gating for analysis.
• Absolute cell counts were determined using internal bead standards (TruCount tubes).

Main Results:

• CD3+4+ percentages in F/T WB (stored at -80°C for up to 1 year) showed minimal difference compared to fresh WB (mean difference < -0.2%, P=0.5).
• High correlation (r²=0.975, P<0.0001) was observed between absolute CD3+4+ cell counts of F/T WB and fresh WB, though F/T counts were generally lower.
• CD3+8+ cell results were more variable but still highly correlated; other freezing conditions yielded more variability.

Conclusions:

• Lymphocyte subset analysis on frozen-thawed whole blood yields comparable results to fresh samples, supporting its practical utility.
• The described flow cytometry approach with F/T WB is a viable method for immunological assessments.
• This technique offers a practical solution for sample management in HIV-1 research and clinical settings.