RhD-negative Blood Donors Research Articles

A Study of Key Factors Influencing the Willingness of RhD-negative Blood Donors to Donate Blood in Chongqing, China, 2017-2019

The importance of RhD-negative blood in clinical blood transfusion is becoming more and more prominent with the continuous advancement of medical technology, but the resources of blood donors are especially precious due to their rarity. As a densely populated area, the recruitment and retention of RhD-negative blood donors in Chongqing Municipality is crucial to ensure clinical blood supply. In this study, a questionnaire survey method was used to collect 1,200 valid questionnaires from RhD-negative blood donors in Chongqing Municipality during 2019-2021 by random sampling. The questionnaires covered the donors' knowledge of RhD-negative blood, knowledge of blood donation policy, motivation to donate blood, experience of blood donation and willingness to donate blood again. The data were statistically analyzed using SPSS software to explore the key factors affecting the willingness to donate blood through descriptive statistics and chi-square test. The results showed that factors such as donors' knowledge of RhD-negative blood, knowledge of blood donation policy, motivation and experience of blood donation significantly affected willingness to donate blood. Therefore, measures such as strengthening the publicity of RhD-negative blood knowledge, improving the blood donation policy, and upgrading the quality of blood donation services are recommended to promote the stable development of the RhD-negative blood donor workforce, to meet the urgent clinical demand for RhD-negative blood, and at the same time to enhance the satisfaction of the donors and the blood donation rate.

Prevalence of C, c, E, e, K, and k antigens in RhD-negative blood donors in and around Puducherry.

Prevalence of C, c, E, e, K, and k antigens in RhD-negative blood donors in and around Puducherry.

The Frequencies of ABO and Rh Phenotypes among Male Blood Donors in Northwestern Saudi Arabia.

Transfusion-dependent patients in Saudi Arabia are numerous because of the existence of life-threatening inherited diseases such as sickle cell anemia and thalassemia. Thus, analysis of the frequencies of the ABO and rhesus (Rh) phenotypes is vital. This study sought to evaluate the frequencies of the ABO and Rh phenotypes among male blood donors in Hail region. One hundred and twenty-six (126) blood samples were collected from male donors living in Hail region and were screened for ABO and Rh phenotypes. The collected data were statistically analyzed using GraphPad Prism (version 9.3.1). Among 126 blood donors, 43.6% were classified into the O blood group. Additionally, Rh antigen e was predominantly detected in this study (99.2%). A total of 103 blood donors exhibited D antigen, whereas 23 were negative for D antigen. The DCe/dce (R1r) phenotype was observed in 29.1% of RhD positive donors, while 73.9% of RhD negative blood donors expressed the dce/dce (rr) phenotype. The O phenotype and e antigen were most frequently observed in male blood donors from Hail province. RhD positive samples took advantage of total blood donor samples over RhD negative samples. CcDee (R1r) phenotype was commonly identified in the RhD positive population, whereas ccddee (rr) phenotype was consis¬tently detected in the RhD negative male donors.

Molecular basis of RhD-negative phenotype in North Indian blood donor population.

RHD gene typing is highly complex due to homology with RHCE genes. Molecular polymorphism of the RHCE and RHD genes have been characterized among various populations, but no studies have been undertaken among Indians. This study was undertaken to assess the genetic basis of RHD-negative phenotype in Indian blood donor population. Sample from a total of 200 phenotypically RhD-negative blood donors were analyzed for presence of RHD gene using polymerase chain reaction (PCR). RHD genotyping was done using three primer sets designed for exons 4 and 10 and one set for identification of pseudo (RHDΨ) gene between introns (int) 3 and 4. Amplified PCR products were analyzed by gel-electrophoresis (XY Loper, Uvitech, Cambridge) and confirmed by nucleotide sequencing (ABI 3730 xl 96 capillary system). No PCR product was found in 195/200 (97.5%) of study samples indicating homozygous gene deletion. Of the 5/200 (2.5%) showing RHD gene polymorphisms, 4/200 (2%) were positive for presence of exon 10 only (RHD-CE-D hybrid). RHDΨ gene was not detected in any of the samples tested. One sample showed presence of all three tested regions and was negative for RHDΨ gene. RHD gene deletion was found to be the most common cause of an RHD-negative phenotype while RHDΨ gene was, reported to be present in up to 39 per cent of various ethnic populations, but was not detected. RHD-CE-D hybrid gene (found in 2.5% individuals) is important for predicting the requirement of Rh prophylaxis during the antenatal period.

A practical and effective strategy in East Asia to prevent anti-D alloimmunization in patients by C/c phenotyping of serologic RhD-negative blood donors.

Serologic RhD-negative red cells can cause anti-D alloimmunization if they carry the Asian-type DEL or other DEL variants. RHD genotyping is a viable countermeasure if available, but inexpensive alternatives are worthy of consideration. RhD-negative blood donors in Japan were studied by anti-D adsorption-elution and RHD genotyping. We collated published case reports of RhD-negative red cell transfusions associated with inexplicable anti-D immunization. Of 2754 serologic RhD-negative donors, 378 were genotyped D/d. Anti-D adsorption-elution revealed 63.5% (240 of 378) to be DEL, of whom 96.7% (232 of 240) had the 1227G > A variant, diagnostic for the Asian-type DEL. All 240 donors also carried at least one C antigen; none had a cc phenotype. The chance of transfusing DEL red cells to genuinely RhD-negative Asian patients (based on a three-unit transfusion) ranges from 16.7% in Korea to 69.4% in Taiwan, versus 0.6% in Germany. Among 22 RhD-negative recipients of serologic RhD-negative red cells, who produced new or increased anti-D antibody titers, all 17 from East Asia were transfused with red cells with a C-positive phenotype or known to be Asian-type DEL or both. Serologic RhD-negative East Asians with a cc phenotype can be red cell donors for RhD-negative recipients, especially those of childbearing potential.

D variants in the population of D-negative blood donors in the north-eastern region of Croatia.

The aim of this study was to determine RHESUS D GENE (RHD) allelic variants among Croatian D-negative blood donors and compare our results with respective data from other European countries. Altered or reduced D antigen expression can result in D variants, which can be mistyped and can lead to the alloimmunisation of the blood recipient. RHD genotyping can distinguish D variants: weak D, partial D and DEL, thus preventing alloimmunisation. A total of 6523 samples obtained from D-negative Croatian donors were screened for the presence of RHD using the real-time polymerase chain reaction (PCR) method. PCR-SSP was performed for D variant genotyping by using commercial genotyping kits (Inno-Train, Kronberg, Germany). Genomic DNA sequencing for all 10 exons of the RHD was performed when the genotyping kits failed to assign a D variant. RHD molecular screening revealed 23 (0.35%) RHD-PCR positive samples, all C/E positive, in decreasing frequency: 11 hybrid RHD-CE (2-9) D-CE variants, 4 weak partial D type 11 and 2 weak D type 2. Six samples remained unresolved and were sequenced. For 12 of 23 samples (excluding large hybrids), an adsorption/elution of anti-D serum was performed, confirming that all 12 were RhD+. The calculated frequency of clinically significant D alleles in RhD-negative blood donors was 1:543 (0.18%) or 1:53 (1.89%) in C/E blood donors. Data on the significant frequency of D variants among serologically D-negative blood donors in the north-eastern region of Croatia could help in introducing RHD molecular screening of blood donors in a routine workflow.

RHD genotyping of serologic RhD-negative blood donors in a hospital-based blood donor center.

Serologic RhD-negative blood donors are tested by a method known to detect weak D antigen expression. Serology does not detect all red blood cells with RhD expression and RHD genotyping has been used to identify variant RHD alleles, which may lead to some RhD expression. The aim of this study was to determine the frequency of RHD variant alleles in serologic RhD-negative blood donors at a hospital-based donor center in Los Angeles. RHD genotyping of serologic RhD-negative blood donors over a 20-month period was performed using the Immucor RHD BeadChip assay. DNA sequencing was performed when the RHD BeadChip assay failed to assign a genotype. For RHD variants known or suspected to result in RhD expression, recipients of previous blood donations were investigated for alloimmunization. RHD genotyping was performed in 1174 RhD-negative blood donors, and 1122 were genotyped for RHCE variants. Eleven donors (0.94%) harbored mutations predicted to yield RhD expression. The predicted phenotypes were, in decreasing frequency, DEL, partial, and weak D phenotypes. Anti-D was not detected in 16 patients who had received blood from these donors after an average follow up of 182 days. Genotyping can be used to identify donors with the potential to sensitize RhD-negative recipients. In this limited study, 0.94% of serologic RhD-negative blood donors were found to have variant RHD alleles that might cause alloimmunization in RhD-negative recipients. To our knowledge, a study of this nature has not been reported in the United States.

Molecular typing of RhD-negative blood donors with C and/or E antigen

Background: Most people are either RhD positive or RhD negative, but there is also a number of persons with D antigen variants. The aim of this study was to prove, by using molecular diagnostic tests, whether the RHD gene and D antigen on the red cell membrane of the blood donors serologically typed as RhD-negative with RhD phenotype Ccddee and ccddEe, are so weak that they cannot be proven by serology techniques or the available anti-D test serums. Methods: Samples used are those of regular voluntary donors who were serotyped as RhD-negative, C and/or E positive. Samples were collected from voluntary donors at the Institute for Transfusion Medicine of the Republic of Srpska during the period from April 2016 to December 2018. Results: Among the serologically proven RhD-negative donors, 346 had C and/or E in their phenotype and those were subjected to molecular screening test. Conclusion: The first results of molecular typing match those published in literature, i.e. the RHD gene is present in some serologically RhD-negative forms, which was proven by molecular testing.

RHD Genotyping by Molecular Analysis of Hybrid Rhesus box in RhD-Negative Blood Donors from Iran

D antigen is the most important and immunogenic antigen of the Rh blood group. The RhD-negative phenotype has different genetic backgrounds with variable distribution in different populations. Hybrid Rhesus box, resulting from RHD gene deletion, is used in genotyping studies of the Rh blood group as a marker to identify the RHD gene deletion. This study for the first time identified genetic mechanisms for the occurrence of RhD-negative phenotype among the Iranian population. 200 RhD-negative blood donors were randomly selected from Tehran Blood Transfusion Center. The phenotype of D, C, Ε, e and c antigens was serologically identified, and DNA was extracted from buffy coat. The molecular analysis of hybrid Rhesus box was performed by PCR-SSP and PCR-RFLP. Moreover, the presence of different exons of RHD gene was investigated by real-time PCR on extracted DNA. Hybrid Rhesus box was detected in all samples, and PCR-RFLP confirmed that 198 (99%) were homozygous for an RHD gene deletion and 2 were heterozygous for hybrid Rhesus box in which one (0.5%) had a weak D type 11 and the other one (0.5%) had a RHD-CE (2-9)-D2 hybrid allele. Similar to Caucasians, the frequency of RHD gene deletion was high among the Iranian population studied in this investigation, so hybrid Rhesus box can be used as an efficient marker to detect RHD gene deletion in our population.

Polymorphism of RHD gene among RhD negative and D variant blood donors from Qingdao region

To study the molecular mechanism and polymorphism of D gene of RhD negative and D variants among voluntary blood donors from Qingdao region. For 220 D-negative phenotype cases and 5 D variant cases confirmed by serological test, exons 1 to 10 of the RHD gene were detected by a PCR-SSP method. The samples which contain all or part of the exons were sequenced. Among the 220 cases, 166 (75.45%) had complete absence of the RHD gene, while 54 (24.55%) had retained some or all of the 10 exons. Eight genotypes were identified, which included RHD 1227G>A in 28 cases (12.73%), RHD-CE- (2-9) -D in 19 cases (8.64%), RHD-CE- (3-7)-D in 1 case (0.45%), RHD 3G>A in 1 case (0.45%), RHD 711delC in 2 cases (0.91%), RHD 845G>A in 1 case (0.45%), RHD 1013T>C in 1 case (0.45%), and RHD 1227A/G in 1 case (0.45%). No mutation was found in all of the 10 exons. Two alleles were identified in the 5 cases of D variants, which included RHD 845G>A (4 cases) and RHD 697G>A (1 case). Absence of the whole RHD gene is common among RhD negative blood donors from Qingdao region, and there are rich genetic polymorphisms for this locus.

An effective diagnostic strategy for accurate detection of RhD variants including Asian DEL type in apparently RhD-negative blood donors in Korea.

The purpose of this study was to provide an effective RHD genotyping strategy for the East Asian blood donors. RhD phenotyping, weak D testing and RhCE phenotyping were performed on 110 samples from members of the RhD-negative club, private organization composed of RhD-negative blood donors, in the GwangJu-Chonnam region of Korea. The RHD promoter, intron 4, and exons 7 and 10 were analysed by real-time PCR. Two nucleotide changes (c.1227 G>A, and c.1222 T>C) in exon 9 were analysed by sequencing. Of 110 RhD-negative club members, 79 (71·8%) showed complete deletion of the RHD gene, 10 (9·1%) showed results consistent with RHD-CE-D hybrid, and 21 (19·1%) showed amplification of RHD promoter, intron 4, and exons 7 and 10. Of the latter group, 16 (14·5%) were in the DEL blood group including c.1227 G>A (N = 14) and c.1222 T>C (N = 2), 2 (1·8%) were weak D, 1(0·9%) was partial D, and 2 (1·8%) were undetermined. The RhD-negative phenotype samples consisted of 58 C-E-c+e+, 19 C-E+c+e+, 3 C-E+c+e-, 21 C+E-c+e-, 6 C+E-c+e+ and 3 C+E-c-e + . Notably, all 58 samples with the C-E-c+e+ phenotype were revealed to have complete deletion of the RHD gene. The C-E-c+e+ phenotype showed 100% positive predictive value for detecting D-negative cases. RHD genotyping is not required in half of D-negative cases. We suggest here an effective RHD genotyping strategy for accurate detection of RhD variants in apparently RhD-negative blood donors in East Asia.

Molecular mechanisms underlying DEL phenotype among ethnic Han individuals from Jiangsu

To explore the molecular mechanism underlying the DEL phenotype among RhD negative ethnic Han individuals from Jiangsu, China. The DEL phenotype was determined by an adsorption elution test among 57 RhD negative blood donors. The Rh C, c, E, and e phenotypes were detected by a tube method. PCR with sequence-specific primering (PCR-SSP) assay was used to determine the RHCE genotypes. The RHD gene of the DEL individuals were amplified with polymerase chain reaction and subjected to Sanger sequencing analysis. Among the 57 RhD negative donors, 10 (17.54%) were determined as having the DEL phenotype. The major RhCE phenotypes for DEL and RhD negative cases were RhCcee (80.0%) and Rhccee (61.7%), respectively. All RHD gene sequences of the 10 individuals have harbored a G>A mutation at position 1227 of exon 9. A proportion of RhD negative individuals determined by routine serological method are actually DEL with RHD gene mutations. RHD *1227A is the most prevalent DEL genotype among ethnic Han Chinese from Jiangsu. Further research on the phenotype and underlying molecular mechanism of DEL is important for blood transfusion.

Dried blood spots of pooled samples for RHD gene screening in blood donors of mixed ancestry.

In this study, we present a strategy for RHD gene screening based on real-time polymerase chain reaction (PCR) using dried blood spots of pooled samples. Molecular analysis of blood donors may be used to detect RHD variants among the presumed D-negative individuals. RHD genotyping using pooled samples is a strategy to test a large number of samples at a more reasonable cost. RHD gene detection based on real-time PCR using dried blood spots of pooled samples was standardised and used to evaluate 1550 Brazilian blood donors phenotyped as RhD-negative. Positive results were re-evaluated by retesting single samples using real-time PCR and conventional multiplex PCR to amplify five RHD-specific exons. PCR-sequence-specific primers was used to amplify RHDψ allele. We devised a strategy for RHD gene screening using dried blood spots of five pooled samples. Among 1550 serologically D-negative blood donors, 58 (3.74%) had the RHD gene. The non-functional RHDψ allele was detected in 47 samples (3.02%). The present method is a promising strategy to detect the RHD gene among presumed RhD-negative blood donors, particularly for populations with African ancestry.

Molecular RHD screening of RhD negative donors can replace standard serological testing for RhD negative donors

This work aims to assess the value of a generalized molecular RHD screening strategy which could replace routine serological screening of weak D by indirect antiglobulin test. Three independent studies were performed at the two Blood Transfusion Services Berne and Zurich. Donors investigated were 652 RhD negative, but RhC and/or RhE positive, 17,391 mainly Rhccee, and 8200 with normal RhCcEe phenotype distribution. In study I single samples, in studies II and III minipools of 24 and 20 donor samples were tested, respectively. Among 26,243 phenotypically RhD negative blood donors, 65 carriers of RHD alleles were identified. Thirty-one of them were redefined as RhD positive.

RhC Phenotyping, Adsorption/Elution Test, and SSP-PCR: The Combined Test for D-Elute Phenotype Screening in Thai RhD-Negative Blood Donors

The Rhesus (Rh) blood group is the most polymorphic human blood group and it is clinically significant in transfusion medicine. Especially, D antigen is the most important and highly immunogenic antigen. Due to anti-D, it is the cause of the hemolytic disease of the newborn and transfusion reaction. About 0.1%–0.5% of Asian people are RhD-negative, whereas in the Thai population, the RhD-negative blood type only occurs in 0.3%. Approximately 10%–30% of RhD-negative in Eastern Asian people actually were D-elute (DEL) phenotype, the very weak D antigen that cannot be detected by indirect antiglobulin test (IAT). There are many reports about anti-D immunization in RhD-negative recipients through the transfusion of red blood cells from individuals with DEL phenotype. D-elute phenotype screening in Thai RhD-negative blood donors was studied to distinguish true RhD-negative from DEL phenotype. A total of 254 Thai serologically RhD-negative blood donors were tested for RhCE phenotypes and anti-D adsorption/elution test. In addition, RhC(+) samples were tested for RHD 1227A allele by SSP-PCR technique. The RhD-negative phenotype samples consisted of 131 ccee, 4 ccEe, 1 ccEE, 101 Ccee, 16 CCee, and 1 CcEe. The 42 Ccee and 8 CCee phenotype samples were typed as DEL phenotype and 96% of DEL samples were positive for RHD 1227A allele. The incidence of RhC(+) was 46.4%, and 48 of the 118 RhC(+) samples were positive for both anti-D adsorption/elution test and SSP-PCR technique for RHD 1227A allele. The sensitivity and specificity were 96% and 100%, respectively, for RHD 1227A detection as compared with the adsorption/elution test. In conclusion, RhC(+) phenotype can combine with anti-D adsorption/elution test and RHD 1227A allele SSP-PCR technique for distinguishing true RhD-negative from DEL phenotype.

Detection of Weak D (Du) Phenotype among Rh-D Negative Males and Females in Kumasi, Ghana

Weak Rh D phenotypes are very frequent in Africans. They are capable of causing alloimmunization in Rh D-negative individuals. Some weak Ds may elude routine typing using direct agglutination techniques. This study aimed at determining the prevalence of weak D phenotype among Rhnegatives, using indirect antiglobulin technique. A total of 400 donors between the ages of 16 and 35 years who were grouped by the blood bank were randomly sampled over a period of 2 months. Three hundred and sixty nine (92.25%) were typed as Rh D-positive and 31 (7.75%) RhD-negative. Two (6.45%) of the Rh D-negative donors were weak D positive while 29 (93.55%) were weak D negative. Among the males 25 (9.43%) were Rh D-negative and 240 (90.57%) RhD-positive. Two (8%) of the 25 males were weak D positive. Among the females, 6 (4.44%) were Rh D-negative and 129 (95.56%) RhD- positive. This implies that, there are people in Kumasi with weak D phenotype which cannot be detected by the direct monoclonal anti-D agglutination. Consequently, indirect antiglobulin test may be indicated for such individuals typed Rh D-negative. This study has shown the need for a comprehensive policy on appropriate testing of donors and newborns, and management of Rh D-negative mothers in the Region. This should include weak D testing of all Rh Dnegative blood donors before transfusion in Rh D-negative patient. Keywords: Rhesus D, weak Rh D, indirect antiglobulin test, Kumasi-Ghana Journal of Science & Technology (Ghana) Vol. 28 (3) 2008: pp. 34-40

Prenatal genotyping for the RhD blood group antigen: considerations in developing an accurate test.

Experience performing prenatal genotyping for RHD has shown that consideration must be given to developing a molecular test capable of detecting recombination/gene conversion events involving the RHD and RHCE genes that can lead to erroneous results. Out of 50 prenatal RHD tests performed over the past 5 years, four samples were encountered that gave false-positive results. In only one of the tests, incorrect results were issued to the physician. In the other three instances, the erroneous nature of the test results was revealed through the analysis of multiple regions of the RHD gene and, more importantly, because the mother, and sometimes the father, were tested in parallel with the fetus. In an extension of the observations obtained from the prenatal testing program, a large panel of RhD-negative blood donors were subjected to molecular analysis of the RHD gene. Of 1,183 donors screened, 187 were found to phenotype as RhD negative. Of the 187 donors confirmed RhD negative serologically, 22 (11.8%) were found to retain remnants of the RHD gene that, depending upon the characteristics of the molecular assay performed, could lead to a false-positive result in a genotyping assay. On the basis of the experience presented here, it is recommended that any molecular RHD assay include an analysis of multiple areas of the RHD gene so as to allow for the detection of recombination/gene conversion events between the RHD and RHCE genes. Moreover, it is strongly recommended that the mother (at a minimum) and father be subjected to molecular analysis simultaneously with the fetus to confirm that the known phenotypes of the parent(s) are consistent with their respective genotypes.

Three unrelated Rh D gene polymorphisms identified among blood donors with Rhesus CCee (r'r') phenotypes

Human red blood cells are traditionally typed as Rhesus (Rh)-positive or -negative depending on the presence or absence of the Rh D antigen. A recent report demonstrated that the Rh D gene is completely absent in Rh D-negative individuals. In this study, Rh D-negative blood donors with ccee (n = 25) and CCee (n = 3) phenotypes were examined for the presence of absence of the D gene. Polymerase chain reaction (PCR) probes that hybridize to the 5' and 3' regions of the Rh CcEe gene and the closely related D gene were used in a Southern analysis. The D gene was absent in all ccee phenotypes examined. The CCee phenotypes showed three Rh D polymorphisms: one donor lacked the D gene, one donor had a partial deletion on one D gene at the 3' region, and the remaining donor appeared to have one normal D gene within the intron/exon regions examined. We conclude that, while the D gene may be absent in the majority of Rh D-negative phenotypes, rarer polymorphisms also occur that prevent expression of the D antigen resulting in the Rh D-negative phenotype.

Three Unrelated Rh D Gene Polymorphisms Identified Among Blood Donors With Rhesus CCee (r'r') Phenotypes

Human red blood cells are traditionally typed as Rhesus (Rh)-positive or -negative depending on the presence or absence of the Rh D antigen. A recent report demonstrated that the Rh D gene is completely absent in Rh D-negative individuals. In this study, Rh D-negative blood donors with ccee (n = 25) and CCee (n = 3) phenotypes were examined for the presence of absence of the D gene. Polymerase chain reaction (PCR) probes that hybridize to the 5' and 3' regions of the Rh CcEe gene and the closely related D gene were used in a Southern analysis. The D gene was absent in all ccee phenotypes examined. The CCee phenotypes showed three Rh D polymorphisms: one donor lacked the D gene, one donor had a partial deletion on one D gene at the 3' region, and the remaining donor appeared to have one normal D gene within the intron/exon regions examined. We conclude that, while the D gene may be absent in the majority of Rh D-negative phenotypes, rarer polymorphisms also occur that prevent expression of the D antigen resulting in the Rh D-negative phenotype.