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Purpose of the study The aim of this study was to investigate RHD alleles among Tunisian blood donors with D-negative phenotype and positive for C and/or E antigen. Patients and methods A total of 100 D-negative and C/E+ samples were analyzed by RHD genotyping using an initial test for RHD exon 10. In case of a positive reaction, further molecular investigations including real time quantitative PCR, allele specific PCR and nucleotide sequencing were done to elucidate the RHD involved mechanisms. Results Seventy-five percent of the studied samples lacked the RHD gene. Twenty-three percent carried the hybrid RHD-CE-D alleles (16 RHD-CE(3-7)-D , 5 RHD-CE(4-7)-D , 1 RHD-CE(4-8)-D , 1 RHD-CE(3-8)-D ) and 2% were weak D (1 weak D type 1 and 1 weak D type 5). Conclusion Our study proved the high frequency of RHD gene among serologically D-negative samples, positive for C and/or E antigen. Thus achieving systematically RHCE phenotyping in all transfusion centers on the Tunisian territory and considering blood donated from D-negative C/E+ persons as D-positive will be recommended to reduce anti-D allo-immunization.

SUMMARY Background Determination of the RHD zygosity is important for genetic counselling and risk evaluation of hemolytic disease of the newborn HDN in women with D iso-immunisation. Objectives We proposed to determine the genotype frequencies of the RHD locus using a PCR-SSP method and assignment of the most probable genotype (MPG) and analyse the concordance between the two methods. Methods The complete Rh phenotype and the frequencies of RH haplotypes were determined on 506 blood donors. RHD zygosity was determined by both assignment of the MPG and PCR-SSP specific for the hybrid Rhesus box. For RH:−1 samples, analysis of the RHD exon 10 was done to detect eventual RHD aberrant alleles. Results Among the 466 RH:1 samples, 54·08% were hemizygous and 45·92% homozygous by PCR-SSP, and 64·16% hemizygous and 35·84% homozygous by the MPG. The comparison between the methods showed discordant results in 135 RH:1 samples. For the 40 RH:−1 samples, hybrid Rhesus box was detected in all samples and RHD exon 10 was detected in three samples suggesting unequivocal alleles identified as one RHDψ, one (C)ces and one weak D type 4. Conclusion The PCR-SSP should replace the MPG. However, studying of aberrant RHD alleles and aberrant Rhesus boxes could confirm the accuracy of this method in Tunisian population.

International audience; BACKGROUND: The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD. MATERIALS AND METHODS: RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction - sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced. RESULTS: Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). DISCUSSION: RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD.

Determination of the RHD zygosity is important for genetic counselling and risk evaluation of hemolytic disease of the newborn HDN in women with D iso-immunisation.We proposed to determine the genotype frequencies of the RHD locus using a PCR-SSP method and assignment of the most probable genotype (MPG) and analyse the concordance between the two methods.The complete Rh phenotype and the frequencies of RH haplotypes were determined on 506 blood donors. RHD zygosity was determined by both assignment of the MPG and PCR-SSP specific for the hybrid Rhesus box. For RH:-1 samples, analysis of the RHD exon 10 was done to detect eventual RHD aberrant alleles.Among the 466 RH:1 samples, 54.08% were hemizygous and 45.92% homozygous by PCR-SSP, and 64.16% hemizygous and 35.84% homozygous by the MPG. The comparison between the methods showed discordant results in 135 RH:1 samples. For the 40 RH:-1 samples, hybrid Rhesus box was detected in all samples and RHD exon 10 was detected in three samples suggesting unequivocal alleles identified as one RHDψ, one (C)ce(s) and one weak D type 4.The PCR-SSP should replace the MPG. However, studying of aberrant RHD alleles and aberrant Rhesus boxes could confirm the accuracy of this method in Tunisian population.