Rapid and reliable genotyping of human platelet antigen (HPA)-1, -2, -3, -4, and -5 a/b and Gov a/b by melting curve analysis.
BACKGROUND
The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)-1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA-4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence-specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems.


METHODS
A total of 105 healthy blood donors were genotyped for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b with new primers and probes designed for mutation detection by melting curve analysis (using LightCycler technology). Donor DNA was independently genotyped by an allele-specific assay, using SSPs, in a reference laboratory.


RESULTS
There was full concordance between the two genotyping methods, and genotype frequencies were comparable with previous studies in caucasians.


CONCLUSIONS
We present rapid and reliable detection systems for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b based on mutation detection of both alleles simultaneously by melting curve analysis. As the Gov system has been reported to have similar frequency of involvement in alloimmune thrombocytopenia as HPA-5, the opportunity for genotyping should aid the diagnosis of such patients.
Randen
Ingrid
I
Departments of Immunology and Transfusion Medicine, Ullevål University Hospital, Kirkeveien 166, 0407 Oslo, Norway. ingrid.randen@ioks.uio.no
Sørensen
Kirsten
K
Killie
Mette K
MK
Kjeldsen-Kragh
Jens
J
D016824:Antigens, Human Platelet / Q000235:genetics*; D001782:Blood Donors; D005260:Female; D005838:Genotype; D006801:Humans; D007231:Infant, Newborn; D039661:Integrin beta3; D007518:Isoantibodies / Q000276:immunology; D007519:Isoantigens / Q000276:immunology; D008431:Maternal-Fetal Exchange; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy; D011696:Purpura, Thrombocytopenic / Q000276:immunology
C115212:2a alloantigen, human; C071221:2b alloantigen, human; C044561:3a alloantigen, human; C083448:3b alloantigen, human; C057260:4a alloantigen, human; C510800:4b alloantigen, human; C071092:5a alloantigen, human; C055973:5b alloantigen, human; D016824:Antigens, Human Platelet; C061248:ITGB3 protein, human; D039661:Integrin beta3; D007518:Isoantibodies; D007519:Isoantigens; C083451:human platelet antigen 1b