Genetic typing of human platelet antigen 1 (HPA‐1) by oligonucleotide ligation assay in a specific and reliable semi‐automated system

Abstract

Genotyping of platelet alloantigens with the possibility of using any type of cellular material as a source of DNA has become a preferred procedure, particularly in thrombocytopenic patients when platelet counts are too low for phenotyping. Recently human platelet antigen 1 (HPA‐1) has been identified as an inherited risk factor for coronary thrombosis. The different detection methods currently used have disadvantages for large‐scale DNA diagnosis, including the need for electrophoresis (allele‐specific restriction enzyme analysis, amplification with sequence‐specific primers) or the potential risk of reduced specificity (allele‐specific oligonucleotide hybridization). In this report we describe the adaptation of an automated oligonucleotide ligation assay to genotype HPA‐1 in polymerase chain reaction (PCR)‐amplified DNA samples. HPA‐1a and HPA‐1b phenotypes corresponded to the results of the different genotyping assays. The genotypes determined with the ELISA‐based PCR‐oligonucleotide ligation assay were in 100% concordance with the results obtained by conventional allele‐specific restriction enzyme site analysis and PCR amplification with sequence‐specific primers. The automated oligonucleotide ligation assay provides a rapid, reliable, nonisotopic method to genotype human platelet antigens that can rapidly be applied to large population screening.

Oligonucleotide ligation assay: applications to molecular diagnosis of inherited disorders

“…The most time-consuming step in PCR-OLA has been the tedious sample preparation prior to PCR. Most of the PCR-OLA applications have been performed on DNA samples obtained from whole blood by the sodium-dodecyl-sulfate± proteinase K/phenol ± chloroform extraction method [20] or the fast puri® cation of DNA by commercially available kits [28]. To avoid tedious and time-consuming puri® cation of DNA, the dried blood spot (DBS) specimens were incorporated to the assay.…”

Rapid genotyping of human platelet antigen 1 (HPA‐1) with fluorophore‐labelled hybridization probes on the LightCyclerTM

“…The PCR‐OLA, although combining high sensitivity and specificity with the potential of automation, still requires several post‐amplification procedures, e.g. the ligation reaction and the subsequent ELISA‐based detection (Zotz et al , 1997).…”

Retrospective comparison of maternal vs. HPA‐matched donor platelets for treatment of fetal alloimmune thrombocytopenia

“…Platelets from both parents were phenotyped using a MAIPA. Both parents were genotyped using an oligonucleotide ligation assay as described previously [7]. A subject was classified as homozygous or heterozygous for HPA phenotype according to the reaction with antiserums specific for HPA-a and HPA-b epitopes.…”