Lymphocyte subset analysis on frozen whole blood

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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°C, in liquid nitrogen (LN2), and at −20°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°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/μl, 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. Cytometry 29:340–350, 1997.

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