Assessment of donor T-cell function in cellular blood components by the CD69 induction assay: effects of storage, gamma radiation, and photochemical treatment.

BACKGROUND: Functional donor T-lymphocytes in blood components may cause a variety of transfusion complications. A flow cytometric assay based on the measurement of induced CD69 expression may be an alternative to cell proliferation methods in determining the functional status of these cells in blood components. STUDY DESIGN AND METHODS: Seven units of whole blood, RBCs, and platelet concentrates (PCs) were stored under blood bank conditions. Half of 3 PCs each were gamma-radiated or treated with UVA+psoralen; the other half served as controls. Samples were analyzed for phorbolester-induced expression of CD69 as an indicator of cell responsiveness and for exclusion of propidium iodide as a measure of cell membrane integrity and viability. RESULTS: CD69 inducibility and propidium iodide exclusion decreased exponentially (half-life, 3. 3 and 8.1 days, respectively) during cold blood storage. Irradiation and UVA+psoralen treatment of PCs immediately reduced CD69 inducibility to 21 percent (controls, 82%; p = 0.004) and 12 percent (controls, 95%; p = 0.0008), respectively. The proportion of cells capable of propidium iodide exclusion was similar in treated samples and controls, but it declined faster in the treated samples during subsequent storage. CONCLUSION: Flow cytometric measurement of CD69 induction can be adapted to provide quantitative assessment of T-cell function in blood components. Results obtained by the CD69 assay are in general agreement with those previously reported by use of proliferation methods; the assay may be useful for special applications in transfusion medicine.

Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study.

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y-chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations.This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.

Lymphocyte subset analysis on frozen whole blood.

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.