Towards a Regional Registry of Extended Typed Blood Donors: Molecular Typing for Blood Group, Platelet and Granulocyte Antigens

Abstract

Background: The provision of compatible blood products to patients is the most essential task of transfusion medicine. Besides ABO and Rh, a number of additional blood group antigens often have to be considered for the blood supply of immunized or chronically transfused patients. It also applies for platelet antigens (HPA) and neutrophil antigens (HNA) for patients receiving platelet or granulocyte concentrates. Here, we describe the molecular screening for a number of blood group, HPA, and HNA alleles. Based on the screening results we are building up a regional blood donor registry to provide extended matched blood products on demand.

Methods: We developed and validated TaqMan™ PCR and PCR-SSP methods for genetic markers defining 37 clinically relevant blood group antigens (beyond ABO and Rh), 10 HPA, and 11 HNA. Furthermore, we describe a feasible method for fast molecular screening of the HNA-2^null phenotype. All data were statistically evaluated regarding genotype distribution. Allele frequencies were compared to ExAC data from non-Finnish Europeans.

Results: Up to now more than 2,000 non-selected regular blood donors in south-west Germany have been screened for blood group, HPA, and HNA alleles. The screening results were confirmed by serology and PCR-SSP methods for selected numbers of samples. The allele frequencies were similar to non-finnish Europeans in the ExAC database except for the alleles encoding the S, HPA-3b and HNA-4b antigens, with significantly lower prevalence in our cohort, as well as the LU14 and the HNA-3b antigens, with a higher frequency compared to the ExAC data. We identified 71 donors with rare blood groups such as Lu(a+b-), Kp(a+b-), Fy(a-b-) and Vel-, and 169 donors with less prevalent HPA or HNA types.

Conclusion: Molecular screening for blood group alleles by using TaqMan™ PCR is an effective and reliable high-throughput method for establishing a rare donor registry.

Keywords: Blood groups; Genotyping; Granulocytes; High- and low-prevalence antigens; Molecular blood typing; Platelets; Polymerase chain reaction.

Fig. 1

Representative result of TaqMan™ PCR-based typing of 96 samples for the c.787A>T mutation in CD177 and CD177P1. Primers were designed to amplify a 88 bp fragment from both genes CD177 and CD177P1. The FAM-labeled probe detects the wild-type allele and the VIC-labeled probe is specific for the null haplotype including the c.787A>T mutation. FAM signals below the threshold (dashed line) indicate absence of the wild type allele in both genes and, therefore, indicate the HNA-2^null phenotype. Control: donor sample with confirmed homozygosity for the c.787A>T mutation by CD177 genomic sequencing and confirmed HNA-2^null phenotype by flow cytometry. In addition to the control three donors revealed absence of the wild type allele, i.e. homozygosity for the c.787A>T mutation. As expected, the HNA-2^null phenotype was confirmed for these samples.