Platelets from donors with consistently low HLA-B8, -B12, or -B35 expression do not undergo antibody-mediated internalization.

Patients refractory to platelet transfusions because of alloimmunization require HLA-matched platelets, which is only possible if a large HLA-typed donor pool is available. However, even then, patients with broad immunization or rare haplotypes may not have suitable donors. In these patients, transfusions with platelets showing low HLA class I expression may be an alternative to fully HLA-matched transfusions. In this study, we quantified the proportion of donors with consistently low HLA-B8, -B12, and -B35 expression on platelets using human monoclonal antibodies specific for these antigens. Furthermore, as model for in vivo clearance, antibody-mediated internalization of these platelets by macrophages was investigated. The expression of HLA-B8, -B12, or -B35 on platelets was extremely variable between individuals (coefficients of variation, 41.4% to 73.6%). For HLA-B8, but not for HLA-B12 or -B35, this variation was in part explained by zygosity. The variation was most pronounced in, but not exclusive to, platelets. Expression within one donor was consistent over time. Remarkably, 32% of 113 HLA-B8, 34% of 98 HLA-B12, and 9% of 66 HLA-B35 donors showed platelet antigen expression that was not or only minimally above background. Antibody-mediated internalization of platelets by macrophages correlated with antibody opsonization and antigen expression and was absent in platelets with low or minimal HLA expression. In conclusion, our findings indicate that a substantial proportion of donors have platelets with consistently low expression of specific HLA class I antigens. These platelets may be used to treat refractory patients with antibodies directed against these particular antigens, despite HLA mismatches.

A new preparation method for red blood cells for intrauterine transfusion enabling reduction of donor exposure.

BACKGROUND: Severe hemolytic anemia of the fetus, caused by maternal red blood cell (RBC) alloantibodies, is treated with intrauterine transfusion (IUT) of RBCs. Because IUT is associated with additional antibody formation, RBCs with the closest match between donor and mother are preferred. Because one fetus needs a median of three IUTs, finding such RBCs is complicated. Collection of repeated low-volume donations from one selected donor during the entire IUT treatment period would reduce donor exposure and possibly IUT-associated alloimmunization. STUDY DESIGN AND METHODS: Whole blood (WB) donations of 100 and 200 mL were diluted with saline, filtered, centrifuged, and separated to prepare experimental RBCs. Before and after gamma irradiation, the RBCs were sampled for comparison of in vitro quality with standard RBCs for IUT. An additional washing procedure was investigated to remove anti-A/-B. RESULTS: Experimental RBCs were leukoreduced to levels conforming with current guidelines and had final volumes of 44 (n = 12) and 84 (n = 8) mL with hematocrit levels between 0.80 and 0.88 L/L. Hemolysis was lower (0.12% vs. 0.42%), potassium leakage comparable, adenosine triphosphate levels lower (4.8 µmol/g Hb vs. 6.1 µmol/g Hb), and 2,3-diphosphoglycerate levels higher (10.3 µmol/g Hb vs 7.7 µmol/g Hb) at 6 hours after irradiation (product expiration time) compared to standard RBCs for IUT (n = 3). Anti-A/-B titers decreased substantially by the washing procedure. CONCLUSION: RBCs for IUT can be prepared from 100- or 200-mL WB donations, showing the potential of this new blood product to reduce donor exposure. A washing procedure is recommended to remove anti-A/-B.

Flow cytometric measurement of CD62P (P-selectin) expression on platelets: a multicenter optimization and standardization effort.

BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories. STUDY DESIGN AND METHODS: The effects of fixation, source and dilution of CD62P antibody, source of immunoglobulin G (IgG) isotypic antibody, and analysis of results were investigated. Once the optimal variables were defined, comparative studies were performed at five participating centers. In the final comparative study, eight split PLT concentrates were shipped to the centers, where samples were stained and fixed according to the uniform protocol. Analyses were performed using commercially available flow cytometers (BD Biosciences and Beckman Coulter). RESULTS: Uniformity between centers could be achieved by using a single clone for CD62P and IgG monoclonal antibody. A protocol was selected using fixation with 0.5 percent methanol-free formaldehyde. To increase conformity between flow cytometers, in the analysis of electronic data the thresholds of the isotypic control were set at 0.5 percent for the BD Biosciences and 2 percent for the Beckman Coulter flow cytometers. In the final comparative study, the 95 percent confidence intervals (CIs) for CD62P ranged between 8 and 21 percent in fresh and 20 to 40 percent in 8-day-old PLT concentrates. CONCLUSION: A uniform CD62P staining protocol and subsequent analysis can be used at multiple centers using different flow cytometers, yielding comparable results with acceptable 95 percent CIs.