Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads

Abstract

Background Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. Methods Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. Results The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. Conclusion Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

Platelet antibodies identification: comparison between two laboratory tests

“…Therefore, platelet serology is in constant development and we agree with Porcelijn et al, 2020 9 that there is variation in antigen recognition and that some challenging clinical issues need more than one assay. One example of this are the rare HPAs in the Chinese population for which beads could not cover the antibody detection 16 and it is crucial also for a mixed population, such as the Brazilian.…”

Report on the 19th International Society of Blood Transfusion Platelet Immunology Workshop 2018

“…When tested for the presence of anti-HPA-1, anti-HPA-2, anti-HPA-3, anti-HPA-5 and anti-HLA in 44 clinical samples, the results of the Luminex technology were in agreement with the MAIPA. Interestingly, the Luminex technology failed at detecting anti-HPA-15, until lately [10,11], thus further confirming that detection of anti-HPA-15 is challenging, likely as a result of the generally low and variable antigen expression level [12,13]. However, M€ ortberg et al (2016) found that the modified monoclonal antibody immobilization of platelet antigens assay (MAIPA) using biotinylated monoclonal antibodies, streptavidin-coated beads and detection by flow cytometry, allowed for improved HPA antibody detection with high sensitivity for all assessed antibodies (anti-HPA-1a, anti-HPA-3a, anti-HPA-5b and anti-HPA-15b).…”

“…They were able to optimize conditions to obtain good sensitivity, reproducibility and specificity. Tao and coworkers have used Luminex microbeads to simultaneously detect anti‐HPA‐1, anti‐HPA‐2, anti‐HPA‐3, anti‐HPA‐5 and anti‐HLA antibodies [10]. This assay shares similarities with the Pak Lx assay reported herein.…”