Real-time PCR genotyping of human platelet alloantigens HPA-1, HPA-2, HPA-3 and HPA-5 is superior to the standard PCR-SSP method.
Genotyping of the human platelet alloantigens (HPA) is useful for the diagnosis and therapy of the patients with alloimmune thrombocytopenic syndromes, such as post-transfusion refractoriness to platelets, post-transfusion thrombocytopenic purpura and foetomaternal alloimmune thrombocytopenia. We have developed, optimized and validated a new method for simultaneous genotyping of HPAs - HPA-1, HPA-2, HPA-3 and HPA-5 - by using the real-time polymerase chain reaction (PCR) based on TaqMan technology. Its performances were compared to those of the standard PCR-sequence-specific primers (SSP) method by testing 120 DNA samples. Several discrepancies between the two methods have been observed, especially in the HPA-3 genotyping. Evidently, the PCR-SSP method produced several false positive results due to its technical drawbacks. Based on our comparison, we believe that the new real-time TaqMan PCR assay for the HPA-1, HPA-2, HPA-3 and HPA-5 genotyping is faster, more reliable and reproducible, compared to the standard PCR-SSP.
Ficko
T
T
Department of Immunohaematology, Blood Transfusion Centre of Slovenia, Slajmerjeva 6, SI-1000 Ljubljana, Slovenia.
Galvani
V
V
Rupreht
R
R
Dovc
T
T
Rozman
P
P
D016824:Antigens, Human Platelet / Q000235:genetics*; D005838:Genotype; D006801:Humans; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011696:Purpura, Thrombocytopenic / Q000175:diagnosis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D016824:Antigens, Human Platelet