title: Lymphocyte subset analysis on frozen whole blood.
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.
authors.affiliation: Irwin Memorial Blood Centers, San Francisco, California 94118, USA.
authors.forename: E W
authors.initials: EW
authors.lastname: Fiebig
authors.forename: D K
authors.initials: DK
authors.lastname: Johnson
authors.forename: D F
authors.initials: DF
authors.lastname: Hirschkorn
authors.forename: C C
authors.initials: CC
authors.lastname: Knape
authors.forename: H K
authors.initials: HK
authors.lastname: Webster
authors.forename: J
authors.initials: J
authors.lastname: Lowder
authors.forename: M P
authors.initials: MP
authors.lastname: Busch
mesh_terms: D001793:Blood Preservation; D001800:Blood Specimen Collection; D002470:Cell Survival; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D018655:Lymphocyte Count; D016131:Lymphocyte Subsets / Q000166:cytology*; D008214:Lymphocytes