{"title":"High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis.","authors":"Honghong He, Longhai Tang, Yiming Jin, Yujue Wang, Hongmei Wang, Shaohua Ding, Yezhou Chen, Jingjing Tian, Mingyuan Wang, Shengbao Duan","doi":"10.1159/000530039","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes.</p><p><strong>Study design and methods: </strong>A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT).</p><p><strong>Results: </strong>The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1-4.8%; inter-assay CV, 2.3-5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3-14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples.</p><p><strong>Conclusion: </strong>The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.</p>","PeriodicalId":23252,"journal":{"name":"Transfusion Medicine and Hemotherapy","volume":null,"pages":null},"PeriodicalIF":1.9000,"publicationDate":"2023-06-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10836864/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Transfusion Medicine and Hemotherapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1159/000530039","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/2/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"HEMATOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Background: CD36 deficiency is closely associated with fetal/neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness, and other hemorrhage disorders, particularly in Asian and African populations. There is a clinical need for rapid and high-throughput methods of platelet CD36 (pCD36) phenotyping to improve the availability of CD36 typing of donors and assist clinical blood transfusions for patients with anti-CD36 antibodies. Such methods can also support the establishment of databases of pCD36-negative phenotypes.
Study design and methods: A sandwich enzyme-linked immunosorbent assay (ELISA) for CD36 phenotyping of human platelets was developed using anti-CD36 monoclonal antibodies. The reliability of the assay was evaluated by calculating the intra-assay and inter-assay coefficients of variation (CV). A total of 1,691 anticoagulant whole blood samples from healthy blood donors were randomly selected. PCD36 expression was measured using a sandwich ELISA. PCD36 deficiency was confirmed by flow cytometry (FC). Mutations underlying pCD36 deficiency were identified using polymerase chain reaction sequence-based typing (PCR-SBT).
Results: The sandwich ELISA for pCD36 phenotyping had high reliability (intra-assay CV, 2.1-4.8%; inter-assay CV, 2.3-5.2%). The sandwich ELISA was used to screen for CD36 expression on platelets isolated from 1,691 healthy blood donors. Of these, 36 samples were pCD36-negative. FC demonstrated absence of CD36 expression on monocytes in three of the 36 cases. In the present study population, the frequency of CD36 deficiency was 2.13% (36/1,691), of which 0.18% (3/1,691) was type I deficiency and 1.95% (33/1,691) was type II deficiency. In addition, we used PCR-SBT to characterize the gene mutations in exons 3-14 of the CD36 gene in 27 cases of CD36 deficiency and discovered 10 types of mutations in 13 pCD36-negative samples.
Conclusion: The present study describes the development and characterization of a highly reliable sandwich ELISA for high-throughput screening for pCD36 expression. This novel method is feasible for clinical applications and provides a useful tool for the establishment of databases of pCD36-negative phenotype donors.
期刊介绍:
This journal is devoted to all areas of transfusion medicine. These include the quality and security of blood products, therapy with blood components and plasma derivatives, transfusion-related questions in transplantation, stem cell manipulation, therapeutic and diagnostic problems of homeostasis, immuno-hematological investigations, and legal aspects of the production of blood products as well as hemotherapy. Both comprehensive reviews and primary publications that detail the newest work in transfusion medicine and hemotherapy promote the international exchange of knowledge within these disciplines. Consistent with this goal, continuing clinical education is also specifically addressed.