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Validating the HPA-1 to -5 and -15 Detection by Homemade PCR-SSP, Real-Time PCR, and PCR-RFLP Methods.

Seyed Ghader Azizi^1,2, Shahram Samiee¹, Maryam Zadsar¹

¹Iranian Blood Transfusion Research Center, High Institute for Research & Education in Transfusion Medicine, Tehran, Iran.

Laboratory Medicine

|June 11, 2022

Summary

This study validated molecular methods for human platelet antigen (HPA) genotyping. Sequence-specific primer-polymerase chain reaction (PCR-SSP), real-time PCR, and PCR-RFLP showed 100% accuracy compared to sequencing.

Keywords:

HPAs PCRRFLP TaqMan real-time PCR human platelet antigens

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Area of Science:

Immunogenetics
Molecular Biology
Transfusion Medicine

Background:

Human platelet antigens (HPAs) are key determinants on platelet glycoproteins.
HPAs can trigger immune responses leading to platelet destruction.
Accurate HPA genotyping is crucial for transfusion compatibility and managing immune-related disorders.

Purpose of the Study:

To evaluate and validate molecular methods for HPA genotyping.
To assess the reliability of PCR-SSP, real-time PCR, and PCR-RFLP for HPA typing.
To compare these methods against sequencing as the gold standard.

Main Methods:

Implementation and validation of PCR-SSP, real-time PCR, and PCR-RFLP.
Genotyping of 10 blood donor samples at the Ardabil Blood Transfusion Center.
Validation against sequencing, a commercial DNA sample, and a commercial kit.

Main Results:

PCR-SSP, TaqMan Real-Time PCR, and PCR-RFLP demonstrated 100% concordance with sequencing.
Melting curve analysis (HPA-15) and PCR-RFLP (HPA-3) results were highly consistent.
All tested methods showed 100% repeatability with no false positives or negatives.

Conclusions:

The validated molecular methods (PCR-SSP, real-time PCR, PCR-RFLP) are reliable for HPA genotyping.
These techniques offer accurate and reproducible HPA typing.
The findings support the use of these methods in clinical and research settings.