Apparent deviation from Mendelian rules of blood group inheritance is rarely observed. Blood group O parents with children expressing weak A subgroups have occasionally been described but not explained. A detailed serological investigation of such a family is described here. The ABO locus was analysed by PCR-ASP/restriction fragment length polymorphism genotyping and DNA sequencing. The propositus' RBCs were very weakly agglutinated with monoclonal anti-A but distinctly with polyclonal anti-A,B, i.e. typical for Ax. Serum anti-A1 (titre 4) and -B were present. Her parents' blood groups were both clearly O, with titres of serum anti-A1, and -A at 16 and 4, respectively. Adsorption/ elution studies demonstrated A antigen on the daughter's cells only. The ABO genotypes were: mother, AxO1; father, O1vO2; and propositus, AxO2. The Ax allele was an A1-O1v hybrid allele with a crossing-over breakpoint between positions 235 and 446 in intron 6 (Ax-4). Compared to the A1 glycosyltransferase, this allele predicts a protein with two amino acid substitutions (Phe216Ile and Met277Val) known to yield either weakly expressed or no A antigen on RBCs. This study suggests that the nature of the ABO allele in trans can influence A antigen expression, a phenomenon previously described as allelic enhancement (or reinforcement). Potential mechanisms for this are discussed.

BACKGROUND Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D negative by standard serology hence permitting incompatible transfusion to D negative recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D negative Polish donor population and to characterize its molecular background. STUDY DESIGN AND METHODS Plasma pools collected from 31,200 consecutive Polish donors typed as D negative were tested by real-time PCR for the presence of RHD specific markers located in the intron 4, exons 7 and 10. RHD positive individuals were characterized by PCR or cDNA sequencing and serology. RESULTS Plasma cross-pool strategy revealed 63 RHD positive donors harboring RHD*01N.03(n=17), RHD*15(n=12), RHD*11(n=7), RHD*DEL8(n=3), RHD*01W.2(n=3), RHD-CE(10)(n=3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, RHD(IVS1-29G>C) and two novel alleles: RHD*(767C>G)(n=3), RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen CONCLUSION 1/ Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2/ 0.2% of D negative Polish donors carry some fragments of the RHD gene; all of them were C or E positive. 3/ Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D negative recipient.

The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

Contrary to the mainstream blood group systems, P1PK continues to puzzle and generate controversies over its molecular background. The P1PK system comprises three glycosphingolipid antigens: Pk, P1 and NOR, all synthesised by a glycosyltransferase called Gb3/CD77 synthase. The Pk antigen is present in most individuals, whereas P1 frequency is lesser and varies regionally, thus underlying two common phenotypes: P1, if the P1 antigen is present, and P2, when P1 is absent. Null and NOR phenotypes are extremely rare. To date, several single nucleotide polymorphisms (SNPs) have been proposed to predict the P1/P2 status, but it has not been clear how important they are in general and in relation to each other, nor has it been clear how synthesis of NOR affects the P1 phenotype. Here, we quantitatively analysed the phenotypes and A4GALT transcription in relation to the previously proposed SNPs in a sample of 109 individuals, and addressed potential P1 antigen level confounders, most notably the red cell membrane cholesterol content. While all the SNPs were associated with the P1/P2 blood type and rs5751348 was the most reliable, we found large differences in P1 level within groups defined by their genotype and substantial intercohort overlaps, which shows that the P1PK blood group system still eludes full understanding.

Three kinds of non-ABO alloantibodies were detected, including anti-Dib described for the first time after HPCT. The rapid development of antibodies in the recipient despite intensive immunosuppression suggests their production by the donor cells primed by the RBC transfusion before HPC harvest. Their production by the residual host cells cannot be unequivocally excluded, however.