The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets.

Area of Science:

• Immunology
• Vaccinology
• Clinical Analysis

Background:

• Analyzing lymphocyte subsets is crucial for understanding immune responses, particularly in individuals experiencing adverse events post-vaccination.
• Delays between blood sample collection and processing can impact flow cytometry results, complicating accurate immunophenotyping.

Purpose of the Study:

• To propose and evaluate a protocol for analyzing blood samples from yellow fever 17DD vaccine recipients with serious adverse events.
• To determine if the time interval between blood sample collection and processing affects lymphocyte subset percentages.

Main Methods:

• Blood samples from nine healthy volunteers were analyzed immediately after collection and after 24 and 48-hour intervals.
• Two methods were compared: whole blood lysis and gradient sedimentation using Histopaque for peripheral blood mononuclear cell isolation.
• Lymphocyte subsets (CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B cells) were quantified using flow cytometry.

Main Results:

• The whole blood lysis method showed no significant changes in lymphocyte subset percentages across the time intervals.
• Histopaque gradient sedimentation resulted in significant variations in T lymphocyte subsets but not B cells over time.
• The whole blood lysis method demonstrated greater stability for immunophenotyping despite sample processing delays.

Conclusions:

• The whole blood lysis method is more reliable than Histopaque gradient sedimentation for immunophenotyping blood samples with potential transport delays.
• This finding is particularly relevant for analyzing samples from yellow fever vaccine recipients who develop serious adverse events.
• The proposed protocol using the whole blood lysis method offers a robust approach to minimize time-dependent variations in lymphocyte subset analysis.