RAPID FREEZING OF WHOLE-BLOOD OR BUFFY COAT SAMPLES FOR POLYMERASE CHAIN-REACTION AND CELL-CULTURE ANALYSIS - APPLICATION TO DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS IN BLOOD-DONOR AND RECIPIENT REPOSITORIES

Storage of lymphocytes for later use in prospective epidemiologic stud ies of blood donors and transfusion recipients has been limited by the cost of separating peripheral blood mononuclear cells (PBMCs). When t he Transfusion Safety Study began in 1985, it was decided to establish a cell repository of cryopreserved buffy coat (BC) samples, and thus far over 20,000 samples have been accumulated from enrolled subjects. To determine if these specimens could be used for polymerase chain rea ction, a simple thawing and pelleting technique for recovering hemoglo bin-free total white cells (WBCs) was developed. To validate the techn ique, parallel analysis was conducted of BCs, whole blood (WB), and PB MC samples from human immunodeficiency virus type 1 (HIV-1)-seropositi ve subjects. Immediate postthaw cell counts of 29 frozen-thawed (F-T) WB and BC samples averaged 90 percent of the prefreeze (input) values. Representative WBC populations were obtained by immediate pelleting. Amplification of HIV-1 gag sequences from F-T BCs and F-T WB was 94 an d 75 percent, respectively, which is as sensitive as that obtained wit h freshly separated PBMC lysates. Quantitative HIV-1 proviral load ana lysis by serial dilution of 23 F-T BCs and 8 WB lysates showed results comparable to those obtained with lysates of fresh PBMCs. Values for WBC differential and immunophenotyping could be applied to express vir al load relative to total WBCs, PBMCs, or CD4+ cells. These results es tablish the basis for simplified virologic analysis of cryopreserved B C or WB specimens.