Our results, using the PCR-SSP, showed that all donors were positive for the presence of a 2778 bp genomic segment obtained from the hybrid Rhesus box gene. The amplification of this segment implies that all donors have at least one RHD gene deletion allele, and another allele of the RHD gene may be a deletion or non-deletion allele. Therefore, the PCR-RFLP was used to check the status of the other allele for the presence or absence of RHD gene.
The results of PCR-RFLP showed that 198 (99%) of donors in both RH gene alleles had a hybrid
Rhesus box genomic segment, which means that these cases are homozygote for
RHD gene deletion. Analysis of exons 5, 7 and 10 using real-time PCR also showed that none of the three exons were amplified in these 198 donors, and the PCR-SSP and PCR-RFLP results were confirmed. Moreover, the PCR-RFLP results showed that 2 (1%) of the RhD-negative donors had a
RHD gene allele. The analysis of exons 5, 7 and 10 using real-time PCR showed that all three exons 5, 7 and 10 were amplified in one sample, but, in the other one, only exon 10 was amplified. In the first sample, further molecular analysis showed that the donor had a weak D allele type 11 that has not been detected by the weak D serological test. In some cases, the weak D phenotype may not be detected by the conventional serological tests (indirect anti-globulin test) [
17]. The 885 G>T mutation associated with the replacement of M295I results in lowered density of D antigen on red blood cells in the weak D type 11 [
18]. In the second sample that was positive only for exon 10, the results of further molecular analyses for exons 3, 4, 6 and 9 and introns 1 and 2 of
RHD gene indicated the presence of the
RHD-
CE (2-
9)-
D2 hybrid allele. Several studies in East Asia have shown that the most common mechanisms for the D-negative phenotype in this population are the
RHD gene deletion, the DEL allele (approximately 10–30%) and the
RHD-
CE-
D hybrid allele (approximately 10%) [
19,
20]. These genetic backgrounds of RhD-negative are different from the Iranian population located in the Middle East, while the frequency of our results is more similar to the European population [
21]. The frequency of D-negative phenotype with a non-deletion
RHD gene allele is approximately 0.6% in Caucasians, 10% in black Africans and 30% in Asians [
2,
22]. In our study, non-deletion
RHD gene allele was found in 2 (1%) D-negative donors, indicating more consistency with the Caucasian population. Genotyping of RhD-negative donors at the blood transfusion centers is very important because some alleles like DEL and weak D may not be detected by conventional serological tests and are considered as RhD-negative. Transfusion of these blood products into RhD-negative individuals, especially for girls and women with childbearing potential, may increase the risk of immunization with anti-D in these individuals. So, the screening of RhD-negative donors by genotyping for identification of
RHD gene alleles, especially in a population where these alleles are highly prevalent, can be very important [
17,
23]. Despite the high prevalence of DEL allele in East Asia (approximately 10–30% of the RhD-negative population), our results (high frequency of
RHD gene deletion) imply that the frequency of the DEL allele among the Iranian population should be very low and similar to the Caucasian population [
21,
24]. In our study, the
RHD gene deletion had the highest association with the D−C−E−c+e+ phenotype (92.92%) and 87.5% of the C+/E+ donors had
RHD gene deletion alleles. The frequency of D-negative phenotype with the presence of
RHD gene non-deletion alleles in association with C and E antigens has also been reported [
2,
11]. Both donors with non-deletion allele in this study were positive for C antigen.