Comparison of human platelet antigen (HPA)-1a typing by solid phase red cell adherence to HPA-1 allotypes determined by allelespecific restriction enzyme analysis

Abstract

Phenotype results for human platelet antigen (HPA)-1 by Capture-P(R), (Immucor, Inc., Norcross, GA) solid phase red cell adherence (SPRCA) were compared to results of allele-specific restriction enzyme analysis (ASRA) for the determination of HPA-1 allotype. Because the expression of HPA-1a and HPA-1b is determined by a single nucleotide substitution of thymine --> cytosine at position 196 of the gene encoding membrane glycoprotein (GP)-IIIa, it is possible to distinguish the alternate forms of the gene using ASRA. Primers (5'- GCTCCAATGTACGGGGTAAACTC-3' and 5'-CAGACCTCCACCTTGTGCTCTATG- 3') were designed to amplify the region of DNA that contains the polymorphism and a restriction enzyme (Nci I) was used to cleave the DNA in a predictable manner. Platelet-rich plasma for immunophenotying and anticoagulated whole blood for DNA extraction were obtained from 159 platepheresis donors. Of 159 SPRCA tests, 138 were valid and 21 were invalid due to positive autologous controls. For 135 HPA-1a-positive and 2 HPA-1a-negative phenotype tests the DNA typing results correlated: 135 positive samples were either HPA-1a/a or HPA-1a/b and 2 negative samples were HPA-1b/b. One donor that typed as HPA-1b/b by ASRA had a positive result of 2+ on SPRCA. This donor had been previously typed by SPRCA as HPA-1a-negative and DNA typed as HPA-1b/b by our laboratory. Based on these findings results of = 3+ by SPRCA are interpreted as HPA-1a-positive for donor screening purposes. SPRCA test results of = 2+ are considered equivocal and the HPA-1 allotype is determined by ASRA. HPA-1a-negative donors by SPRCA must be confirmed as HPA-1b/b by ASRA prior to issue for a patient that requires HPA-1anegative platelets.