Open AccessLymphocyte subset analysis on frozen whole blood
Author(s) -
Fiebig Eberhard W.,
Johnson Delene K.,
Hirschkorn Dale F.,
Knape Charlene C.,
Webster H. Kyle,
Lowder James,
Busch Michael P.
Publication year - 1997
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/(sici)1097-0320(19971201)29:4<340::aid-cyto11>3.0.co;2-u
Subject(s) - flow cytometry , microbiology and biotechnology , cytometry , cd3 , human immunodeficiency virus (hiv) , andrology , chemistry , biology , immunology , medicine , cd8 , antigen
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 (LN 2 ), 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 (r 2 = 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. © 1997 Wiley‐Liss, Inc.