Molecular testing for platelet and granulocyte antigens
Authors: Nogués N. 
Published: May 2011

Human platelet antigen (HPA) genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera, complementing the clinical history in the diagnosis of alloimmune platelet disorders such as neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet transfusion refractoriness (PR). Likewise, human neutrophil antigen (HNA) typing also plays a critical role in the investigation and diagnosis of some clinical conditions induced by granulocyte alloantibodies, like neonatal alloimmune neutropenia (NAIN) and transfusion‐related acute lung injury (TRALI).

Up to now, 27 HPA alloantigens have been described, including the 6 most clinically relevant biallelic systems and a significant number of low‐frequency or rare HPAs, which continues to increase with new reports. The genetic polymorphisms underlying these antigens have been characterized and in all cases but one, are single nucleotide polymorphisms (SNPs) in the coding sequence of the different membrane proteins where the HPA antigens are expressed. In contrast, the molecular basis of the HNA antigens are a little bit more heterogeneous. Within the 5 recognized HNA systems, the first alloantigens to be described, HNA‐1a and HNA‐1b differ in their coding sequence by 5 nucleotides, which result in 4 aminoacid changes in the protein sequence of the FcγRIIIb. Another alloantigen, the HNA‐2a, has been identified on the CD177 glycoprotein but cannot be genotyped, despite knowing the corresponding coding sequence. The HNA‐2a negative phenotype is due to splicing defects in the mRNA and no mutations have been detected in the gene sequence. The remaining HNA alloantigens result from single nucleotide changes, including the clinically relevant HNA‐3a, which has recently been characterized as the result of a single base change in the choline transporter‐like protein (CTL) 2 gene.

Currently, genotyping methods are routinely applied to HPA and HNA testing in all laboratories performing platelet and granulocyte immunohematology studies. Many different techniques have been developed for typing these polymorphisms, including PCR with sequence‐specific primers, which is still the most widely used, 5′‐nuclease assays and high‐throughput SNP typing platforms. These latter ones are based on specific hybridization to oligonucleotide probes immobilized in a solid suport, usually a glass slide or beads. Among these, the newest platform developed for HPA typing, ID‐HPA, is based on the xMAP® technology and the beads are analyzed with the Luminex 100/200™ System, which is a robust and convenient system, already implemented in most HLA laboratories. Interestingly, the same technology can recently be used for the detection of HPA and HNA antibodies, which offers the possibility of multiple applications with a unique platform. These features may foster the introduction of such flexible assay systems in the near future. In any case, already at present, different high‐throughput genotyping platforms analyzing HPA polymorphisms are already being used for large‐scale blood or platelet apheresis donor typing, and further automation will probably lead to their final implementation in centralized National Blood Centers, facilitating the provision of antigen‐negative platelets for alloimmunized patients.

Finally, it is worth to remark the novel strategies that have been recently developed for the non‐invasive fetal HPA‐1 typing from the maternal plasma DNA. These highly sensitive approaches may be applied in the future to other clinically significant HPA specificities, and will represent a major improvement in the management of pregnancies at risk of NAIT.