Analysis of reticulocyte chimaerism using flow cytometry

Purpose: After allogeneic bone marrow (BM) or peripheral blood progenitor cell (PBPC) transplantation, analysis of red blood cell (RBC) antigens using peripheral blood can be performed repeatedly to monitor transplant take, whereas the long presence of erythrocyte transfusions makes the exclusion of mixed chimaerism difficult. We characterized the blood group (BG) pattern of reticulocytes using flow cytometry (FC). Methods: EDTA anticoagulated washed erythrocytes were incubated one after the other with a primary IgG antibody detecting BG antigens (antiD, anti-C: Serologicals, UK ; anti-A, anti-c: eluat of anti-Ai/anti-c positive cord RBC), a secondary antibody (PE anti-human IgG F(ab)₂: Dianova) and thiazole orange. The optimum concentration of each used antibody was evaluated by titration. These double labelled preparations were analysed by FC employing a FACScan (Cellquest program, Becton Dickinson). Results: In order to estimate both the accuracy and the sensitivity of the method, artificially-made dual populations of human donor RBCs (BG 0 and Ai, CC and ce, D. and dd) were prepared according to following scheme: 100:0, 98:2. 95:5, 90:10. 50:50. 10:90, 5:95, 2:98. 0:100. Measured values of mixtures were very closely related to expected results. Repeated measurements (n=6) of one sample also displayed a high precision in one series and from day to day, respectively. From investigation of a series of BM or PBPC transplanted patients engraftment could be demonstrated early and rapidly by reticulocyte chimaerism analysis. The use of the method depends on a known BG difference between donor and recipient, whereas additional BG systems such as Duffy or Kidd could be employed. Conclusions: The proposed method provides appreciable help to follow engraftment after BM or PBPC transplantation. Thus the origin of RBCs produced of the regenerating marrow could be determined early and fast from the peripheral blood.