Development and Clinical Evaluation of Oligonucleotide Microarray for HLA-AB Genotyping

Abstract

Background: The antigens encoded by human leukocyte antigen (HLA) genes are primary antigens in immunological response of transplantation, and genotypes of HLA-A, B and DRB1 must be determined on donors and recipients before the transplantation is carried out. In this study, oligonucleotide microarray for HLA-AB genotyping was prepared and evaluated. Methods: Oligonucleotide probes were designed based on the sequence of the different genotypes of HLA-A and B and were fixed on silylated glass slides to form a microarray. Fluorescence-labeled target DNA was obtained by asymmetric polymerase chain reaction amplification of exon 2 and exon 3 of HLA-A and -B genes and hybridized to the microarray. The hybridized microarray was subsequently scanned and the result was analyzed by software in order to determine the genotypes of the tested sample. Results: The sensitivity of the microarray for HLA-AB genotyping was 10 ng/µl, with a coincidence rate of 100% with international reference, and a combined variation value of detected signal below 15%. Analysis of genotyping of 1295 cases of clinical samples showed that the general coincidence rate between the microarray method and conventional method (polymerase chain reaction-sequence-specific primer and flow cytometry reverse polymerase chain reaction sequence-specific oligonucleotide) was HLA-A: 99.61% and HLA-B: 98.30%, respectively. A total of five out of seven samples that had conflicting results of genotypes were proved to be microarray-assay reliable by DNA sequencing, suggesting a higher accuracy of the microarray method. Conclusion: The microarray for HLA-AB genotyping is satisfactory for clinical use in HLA-AB genotyping with its good specificity, sensitivity and reproducibility.

Keywords: :

• flow cytometry
• genotyping
• human leukocyte antigens
• oligonucleotide microarray
• polymerase chain reaction
• sequence-specific primers
• sequence-specific oligonucleotide probes

Acknowledgments

This project was supported by grant from the National Natural Sciences Foundation of China for Key Programme (39889001). We thank Prof. Wu Dalin and Li Dan for the supply of clinical samples.

Additional information

Funding

This project was supported by grant from the National Natural Sciences Foundation of China for Key Programme (39889001). We thank Prof. Wu Dalin and Li Dan for the supply of clinical samples.