High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis

Honghong He; Longhai Tang; Yiming Jin; Yujue Wang; Hongmei Wang; Shaohua Ding; Yezhou Chen; Jingjing Tian; Mingyuan Wang; Shengbao Duan

doi:10.1159/000530039

High-Throughput CD36 Phenotyping on Human Platelets Based on Sandwich ELISA and Mutant Gene Analysis

Transfus Med Hemother. 2023 Jun 27;51(1):32-40. doi: 10.1159/000530039. eCollection 2024 Feb.

Authors

Affiliations

¹ Suzhou Blood Center, Suzhou, China.
² Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China.

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.

Keywords: CD36 deficiency; Phenotyping; Sandwich ELISA; pCD36-negative phenotype database.

Grants and funding

This work was supported by a grant from the Weigao Research Fund Project of the Chinese Society of Blood Transfusion [Grant No. CSBT-WG-2020-06]; the Suzhou “National Mentor System” Training Program for Health Youth Backbone Talents [Grant No. Qngg2022033]; the Major Diseases Project of Suzhou [Grant No. GWZX202004]; the Key Medical Discipline Project of Suzhou [Grant No. SZXK202118]; the Jiangsu Provincial Science and Technology Plan Project [Grant No. BE2022743]; and the Suzhou Science and Technology Plan Project [Grant No. SJC2022013].