In vitro Generated Megakaryocytes for the Detection of Human Platelet Antigen-Specific Alloantibodies

Abstract

Introduction: Serologic characterization of antihuman platelet antigen (HPA) alloantibodies is crucial in fetal neonatal alloimmune thrombocytopenia. The gold standard MAIPA assay requires fresh platelets from HPA-genotyped donors, which is challenging for some laboratories. Megakaryocytes express HPA epitopes and offer an alternative source for detecting anti-HPA antibodies. The objective of this study was to assess the efficacy of a novel assay called monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) for detecting anti-HPA antibodies.

Methods: CD34+ cells from buffy coats were differentiated into megakaryocytes in vitro. The performance of the MAIMA assay was evaluated using WHO reference reagents for HPA-1a, HPA-3a, and HPA-5b, along with sera samples from patients who had well-characterized anti-HPA antibodies.

Results: The WHO anti-HPA-1a reference reagent showed similar binding to megakaryocytes and platelets in MAIMA and MAIPA, respectively. On the other hand, optical density (OD) values for the WHO anti-HPA-3a reference reagent were lower in MAIMA than in MAIPA. Anti-HPA-5b antibodies were not detectable in MAIMA. Patients' sera containing anti-HPA-1a antibodies were successfully detected in MAIMA in all clinical samples. Moreover, OD values in MAIPA and MAIMA showed high correlation (r = 0.96, p < 0.001). MAIMA was reactive for samples with anti-HPA-3a as well as anti-HPA-3b; however, OD values were lower compared to MAIPA. Interestingly, all patient samples with anti-HPA-5b antibodies were tested negative in MAIMA.

Conclusion: In vitro generated megakaryocytes can be used to detect anti-HPA-1a alloantibodies. However, despite this potential, they may be less suitable for the detection of alloantibodies against other HPAs such as HPA-5b.

Keywords: Antibodies; Buffy coat; Fetal alloimmune thrombocytopenia; Platelet antigens.

Fig. 1.

Comparison of MAIMA and MAIPA for anti-HPA antibody detection. The optical density (OD) was compared in monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) assay and monoclonal antibody immobilization of platelet antigens (MAIPA) assay using WHO reference reagents for anti-HPA-1a, anti-HPA-3a, and anti-HPA-5b at different titrations. While OD values for anti-HPA-1a were similar between MAIMA and MAIPA for all tested titrations (a), they were significantly lower for anti-HPA-3a in MAIMA compared to MAIPA for most of the tested titrations (b). On the other hand, MAIMA failed to detect anti-HPA-5b antibodies (c). Platelets are represented with blue circles, and megakaryocytes with red circles. An OD of greater than 0.15 was considered positive (dashed line).

Fig. 2.

Reactivity of sera with anti-HPA-1a alloantibodies in MAIPA and MAIMA. The reactivity of sera with anti-HPA-1a and anti-HPA-1b alloantibodies was compared in monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) assay and in monoclonal antibody immobilization of platelet antigens (MAIPA) assay. The optical density (OD) values were similar between MAIMA and MAIPA for all tested sera in the HPA-1(a+b−) cells (a) for HPA-1(a+b+) cells (b). HPA-1(a−b+) megakaryocytes were negative with sera of HPA-1a (c). OD values of anti-HPA-1b alloantibodies were lower in MAIMA using HPA-1 (a−b+) or HPA-1 (a+b+) megakaryocytes (d). HPA-1(a+b−) megakaryocytes were negative with sera of HPA-1b (e). Platelets are represented with blue circles, and megakaryocytes with red circles. n refers to number of sera tested. An OD of greater than 0.15 was considered positive (dashed line).

Fig. 3.

Correlation between monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) assay and monoclonal antibody immobilization of platelet antigens (MAIPAs) assay for anti-HPA-1a alloantibody. The OD values in MAIPA and MAIMA showed a high correlation with HPA-1(a+b−) cells (a) as well as with HPA-1(a+b+) cells (b).

Fig. 4.

Reactivity of Sera with anti-HPA-3a and Anti-HPA-3b alloantibodies in MAIPA and MAIMA The reactivity of sera with anti-HPA-3a or anti-HPA-3b alloantibodies was compared in monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) assay and in monoclonal antibody immobilization of platelet antigens (MAIPA) assay. The optical density (OD) values were 33–50% lower in MAIMA then MAIPA for sera with HPA-3a alloantibody (a). Similarly, the OD values were 5–60% lower in MAIMA than those measured in MAIPA for sera with HPA-3b alloantibody (b). We investigated whether the variable reactivity of HPA-3a antibodies in MAIMA assay is due to the loss of sialic acid from platelet receptors on megakaryocytes during cultivation. To test our hypothesis, we treated megakaryocytes with oseltamivir, a neuraminidase inhibitor, and compared the reactivity of HPA-3a (c) and HPA-3b (d) patient sera in MAIMA assay with oseltamivir-treated and untreated megakaryocytes. n refers to number of sera tested. An OD of greater than 0.15 was considered positive (dashed line).

Fig. 5.

Reactivity of sera with anti-HPA-5b Alloantibodies in MAIPA and MAIMA. We compared the reactivity of sera with anti-HPA-5b alloantibodies in monoclonal antibody immobilization of megakaryocyte antigens (MAIMA) assay and in monoclonal antibody immobilization of platelet antigens (MAIPA) assay using HPA-5(a−b+) (a) and HPA-1(a+b+) (b) cells. MAIMA has failed to detect anti-HPA-5b alloantibodies in clinical samples. Platelets are represented with blue circles, and megakaryocytes represented with red circles. n refers to number of sera tested. An optical density (OD) of greater than 0.15 was considered positive (dashed line).

Fig. 6.

CD49b expression on in vitro generated megakaryocytes analyzed by flow cytometry. Expression of CD49b on in vitro generated megakaryocytes and peripheral blood platelets was analyzed using flow cytometry. Megakaryocytes were generated from CD34+ hematopoietic stem cells isolated from buffy coat using a standardized protocol, while platelets were isolated from peripheral blood. Cells were stained with antibodies against CD41, CD42a, and CD49b. Representative dot plots showing CD41 expression on the x-axis and CD42a or CD49b expression on the y-axis seen. Cells were stained with propidium iodide (PI), a DNA marker. The percentage of CD41/CD42a positive cells was similar between platelets and in vitro generated megakaryocytes. In comparison to peripheral blood platelets, in vitro generated megakaryocytes showed significantly lower levels of CD41/CD49b double positive cells.