Journal Article

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

He, Ji, Zhu, Faming, Hong, Xiaozhen, Tao, Sudan, Chen, Shu

2019

Overview

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.

• Publisher: BioMed Central,BioMed Central Ltd,BMC
• Subject: Antibodies / Antibodies, Monoclonal - immunology / Antigen-antibody reactions / Antigens / Antigens, Human Platelet - analysis / Biomedical and Life Sciences / Biomedicine / Blood Platelet Disorders - immunology / Blood Platelets - cytology / Blood Platelets - immunology / Genotype / Glycoproteins / Health aspects / HLA antibodies / HLA antigens / HLA Antigens - analysis / HPA antibodies / Humans / Identification and classification / Immune System / Immunoassay - instrumentation / Immunoassay - methods / Isoantibodies / Luminex assay / MAIPA / Medical research / Medicine/Public Health / Microspheres / Monoclonal antibodies / Newborn infants / Novels / Purpura, Thrombocytopenic, Idiopathic - blood / Technology / Thrombocytopenia / Transitional care / Translational Genomics and Genetics / Workshops (Educational programs)