QUANTITATION OF WHITE CELL SUBPOPULATIONS BY POLYMERASE-CHAIN-REACTION USING FROZEN WHOLE-BLOOD SAMPLES

BACKGROUND: Previous methods for processing whole blood (WB) for nucle ic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative pol ymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and f rozen WE preparations were analyzed. The integrity of WBCs in the froz en WE samples was first assessed by flow cytometry using CD45 fluoresc ence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a dou ble-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentr ates. Y-chromosome-specific PCR was used to quantitate male WBCs in fi ve female WE samples spiked with serial dilutions of male WBCs and in serially collected frozen WE samples from four females transfused with male blood components. RESULTS: By Row cytometry, all major WBC subpo pulations in frozen-thawed WE were quantitatively recovered and immuno logically intact, although they were nonviable. HLA-DQ-A PCR quantitat ion of a dilution series from 8 to 16,700 per mt of WBCs spiked into d ouble-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r(2 )= 0.98, p<0.0001 for both), without significant difference between ob served and expected values (p>0.05). Y-chromosome-specific PCR results in female WE samples spiked with male WBCs were not significantly dif ferent in fresh and frozen preparations over a 3 log(10) range of male cells. The results of WBC survival studies on frozen WE samples were consistent with previous observations in fresh blood samples. CONCLUSI ON: Direct freezing of WE enables subsequent recovery of WBCs for quan titative PCR analyses, with results comparable to those of fresh prepa rations. This protocol should facilitate wider implementation of nucle ic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in trans fusion and transplant recipients.