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Published on: December 6, 2014
Lymphocyte subset analysis on frozen whole blood
E W Fiebig1, D K Johnson, D F Hirschkorn
1Irwin Memorial Blood Centers, San Francisco, California 94118, USA.
Insights
Analyzing frozen-thawed whole blood (F/T WB) for lymphocyte subsets provides comparable results to fresh samples. This method, using flow cytometry, is valuable for human immunodeficiency virus type 1 (HIV-1) research and clinical applications.
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.
Abstract:
An approach to perform lymphocyte subset analysis on frozen-thawed whole blood (F/T WB) is described. WB from 24 human immunodeficiency virus type 1 (HIV-1) seropositive individuals and 21 controls was analyzed fresh and after frozen storage (with or without dimethyl sulfoxide) at -80 degrees C, in liquid nitrogen (LN2), and at -20 degrees C. Analysis of F/T WB utilized 3-color flow cytometry with CD45 and right angle light scatter gating. Absolute cell counts were obtained for 30 samples by using staining tubes containing internal bead standards [TruCount, Becton Dickinson Immunocytometry Systems (BDIS), San Jose, CA]. The mean difference between CD3+4+ percentages for F/T (-80 degrees C storage for up to 1 year) and fresh WB was less than -0.2% (95% limits +/-3%, P = 0.5) with 39 of 45 (87%) results falling within 2% of the fresh values (P = 0.74). Absolute CD3+4+ cell counts for F/T WB were generally lower than corresponding results for fresh aliquots (median difference was 33 cells/microl, P < 0.0001), but the results were highly correlated (r2 = 0.975, P < 0.0001). Results were more variable, although still highly correlated, for CD3+8+ cells, and with other freezing and storage conditions. It is concluded that lymphocyte subset analysis using F/T WB yields comparable results to fresh samples, which should prove useful for a number of practical applications.
