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.

Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR).Aim: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. Methods: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/ KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. Results: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72).Conclusion: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.