Workshop report on the genotyping of blood cell alloantigens

The immunization against alloantigens present on platelets, granulocytes and red blood cells (RBCs) is responsible for various clinical syndromes. Since the molecular basis of these antigens has become clear during the last decade, genotyping is nowadays used in several laboratories. However, many DNA-based techniques still have to be evaluated. We therefore organized a workshop on the genotyping of the most relevant alloantigens on platelets and granulocytes as well as on selected RBC alleles. DNA was isolated from peripheral blood lymphocytes or from B-lymphoblastoid cell lines (B-LCL). We distributed samples for the identification of platelet (n = 7), granulocyte (n = 6) and RBC (n = 4) polymorphisms, respectively. There were 33 institutions in Germany, Austria and Switzerland, which participated in at least one part of the workshop. Twenty-four laboratories reported results on HPA-1, and 23 laboratories on HPA-2, -3, and -5 typing. In addition, five laboratories typed for HPA-4 and -6. The HNA-1a/b (NA1/NA2) alleles were identified by eight laboratories, one of which also typed for HNA-1c (SH). The most frequent genes of the ABO (A1, B, O) and Rh (D, C, c, E, e) systems were typed by 12 participating laboratories, and an additional four laboratories restricted their RBC typing to the RHD gene. The typing technique mainly used for all three cell lineages was the polymerase chain reaction with sequence-specific primers. Other techniques were restriction fragment length analysis, oligonucleotide ligation assay, enzyme-linked mini-sequence assay or direct sequence analysis. The following typing errors were observed: HPA: 15/1442 (1.0%), HNA: 4/108 (3.7%), ABO: 5/96 (5.2%) RH 1/320 (0.3%). Our workshop demonstrated the existence of a number of reliable techniques for the genotyping of blood cell alloantigens and a high standard in the participating laboratories. In addition, we could show the usefulness of B-LCL as a source of reference DNA. However, the 5.2% rate of mistyping in the ABO system demonstrated that further efforts are needed to improve the precision of the genotyping techniques. Future workshops will have to challenge methods and participants with rare variants of RBC genes to guarantee reliable genotyping, e.g. in prenatal diagnosis of fetomaternal incompatibility.