Home⇛The Philippine Journal of Biochemistry and Molecular Biology (PJBMB)⇛vol. 1 no. 1 & 2 (2020)

Identification of Class I HLA Alleles in Anonymized Cell Therapy Specimens through Real-Time PCR with Melt- Curve Analysis

Joanne Jennifer E. Tan | Maria Teresa A. Barzaga | Francisco M. Heralde Iii

Discipline: Biological and Sport Sciences

 

Abstract:

Accurate human leukocyte antigen (HLA) typing is crucial for allogeneic dendritic cell anti-cancer vaccination, where at least a seven out of eight HLA match to the medium resolution allele level is required. Molecular methods can provide medium to high resolution typing but are expensive and time-consuming. A modified method to facilitate faster and more efficientmediumresolution identitymatching for some common alleles in Filipinoswas developed using real-time PCR withmelt-curve analysis.Themost commonFilipinoHLAClass Ialleles identified fromavailable databaseswere:A*02,A*24, B*15 andC*07.Primers specific to these alleleswere designed.DNAwere extracted from17 de-identified unstimulated stem cell specimens from the Molecular Diagnostics and Cellular Therapeutics Laboratory of the Lung Center of the Philippines (MDCTL-LCP). Comparison of the melt-curve profile was able to determine some degree of HLA allele identity. Distinct alleles from the samples that were identified through sequencing were A*03:01 and C*07:32. Higher resolution typing was not possible for some alleles due to highly similar sequences in the amplified region. The study thus explored the use of realtime PCR and melting curve analysis in describing some common HLA Class I alleles in Filipinos. Further replication and more specific primers will be needed to establish the melting curve profiles for HLA identification use.



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