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Common I RHD i variants and zygosity were assessed by sequence-specific primer PCR (PCR-SSP), as previously reported.4 DNA sequencing of I RHD i exons 1-10 and flanking intron regions was performed using in-house primers. Moreover, the I c.1228-1G>C i mutation from the proband was then passed to the daughter, who typed an I RHD c.1228-1G>C i allele and a normal I RHD i gene. Currently, 336 I RHD i alleles have been identified according to ISBT allele nomenclature version v5.0 (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-groug-terminology). [Extracted from the article]

Abstract: The RhD mRNA was analyzed in both Rh-positive and Del phenotypes with RHD 1227A allele through sequencing. As a result, five and six transcripts were detected in Rh-positive and Del, respectively. Four of them have identical sequences between Rh-positive and Del. Those are the transcripts with exon 9, exons 8 and 9, exons 7 and 9, and exons 7–9 spliced out compared normal RhD mRNA that was detected in Rh-positive but not in Del individuals. Unexpectedly, two additional transcripts were found in Del individuals. Its exon 9 or exons 8–9 were spliced out, but interestingly both possess a 170 bp segment of sequence from intron 7 of RHD. We may conclude that a normal RhD protein does not exist in a Del individual with RHD 1227A allele since the exon 9 was always spliced out due to the silent mutation at the end of exon 9. The transcripts without exon 9 was also detected in the normal Rh-positive individual and further confirmed by a specific reverse-transcription PCR (RT-PCR) system. [Copyright &y& Elsevier]

Objective: To explore the feasibility of RhCcEe blood group antigen mixed visual field identification in patients with regular blood transfusion, to follow up and evaluate the efficacy of matched transfusion and its clinical significance., Methods: RhCcEe genotyping for 142 patients with regular transfusion in our hospital was carried out by PCR-SSP method. According to the results of genotyping, 48 patients voluntarily selected the continuous transfusion of RhCcEe matched red blood cells, 46 patients received random blood transfusion (RhCcEe mismatched transfusion), 42 patients received partial RhCcEe matched transfusion (unable to provide fully matched RhCcEe donors each time), and 6 patients' blood transfusion data were lost. After 3-6 months of the RhCcEe matched transfusion, all patients were tested by RhCcEe microcolumn gel card and compared with the results before RhCcEe matched transfusion. The positive rates of alloantibodies, DAT and the percentage of red blood cell invalid transfusion were followed up and evaluated for the above-mentsioned 3 types of regular transfusion patients in the past 5 years., Results: Out of the 48 patients who underwent conti-nuous RhCcEe matched transfusion, only 1 case showed stratification, the remaining 47 cases had clear gel card results without stratification, suggesting that PCR-SSP genotyping was feasible. In addition, another 42 patients who could not receive RhCcEe matched transfusion each time and 46 patients with random blood transfusion were found to have a mixed vision phenomenon again. but the results was still difficult to confirm the results. For the transfusion results in the past 5 years, follow-up analysis showed that there were 1 case alloantibody (anti-Jka) (1/48) , 1 case of DAT positive (1/48) and 2 cases of invalid transfusion (2/48) in the RhCcEe matched transfusion group; 7 cases of alloantibodies (3 anti-E, 1 anti-E+anti-c, 1 anti-C, 1 anti-M, 1 anti-Fya) (7/46), 6 case of DAT positive (6/46) and 9 case of invalid transfusion (9/46) in the random transfusion group; 6 cases of alloantibodies (1 anti-E, 1 anti-E+autoantibody, 1 anti-C, 1 anti-c, 1 anti-M and 1 other antibody) (6/42) and 7 case of DAT positive (7/42) and 8 case of invalid transfusion (8/42) in the partial RhCcEe matched transfusion group. The statistical analysis showed that the positive rate of alloantibodies and the invalid infusion rate of RBC in each group were significant differences between RhCcEe matched transfusion group and the random transfusion group as well as betwen Rhce fe matched transfusion group and the partial matched transfusion group（P＜0.05）, but there was no statistical difference between the random transfusion group and the partial matched transfusion group（P>0.05）., Conclusion: PCR-SSP genotyping technique can be used to detect RhCcEe mixed vision in patients with regular blood transfusion. Continuous RhCcEe matched transfusion can effectively prevent the occurrence of alloimmunization, and improve the clinical transfusion efficacy and safety of the patients with regular blood transfusion, which has very important clinical significance.

Objective: To investigate the feasibilily of screening and identifying the red blood cell type alloantibodies by means of surface plasman resonance(SPR) technique so as to provide a new method for detecting the transfusion compatibility of red blood cells., Methods: The RBC antigens for screening the alloantibody were fixed on the SPR chip surface by means of amino coupling method; the analysis conditions of SPR chip were optimized and then the control serum with RBC blood group antibody positive was detected; the performance of SPR chip for detection of serum was analysed; the consistance of rusults detected by SPR technique and microcolum agglutination for clinieal samples of 129 thalasstmia patients with history of lone-term blood transfusion were compared; at the same time, the blood group amtibodies in 7 patients with blood group antibody positive were identified before blood transfusion by using SPR chip so as to select the RBC antigen compatible blood for transfusion; and the efficacy of RBC transfusion was followed up and evaluated., Results: The repeatability, sensitivity and specificity of SPR chip technique for detecting the blood group alloantibodies all were better. The SPR technique and microcolumn agglutination method were not significant different for screening blood group alloantibodies (χ2 = 0.333, P＞0.05), and the overall consistency was 97.2%; the results of SPR technique in 7 patients with positive blood group antibodies were as follows: 3 cases with anti-E, 1 case anti-M, 1 case anti-C, 1 case anti-Jka and 1 case autoantibody, which were consistent with the results of microcolumn agglutination tests, and the compatible red blood cells were selected for transfusion, of which the infusion of 6 cases was effective. In only 1 case the infusion was ineffective because of autoantibody., Conclusion: For screening and identification of blood group alloantibodies, the performance of SPR chip technique is equivalent to the micro-column agglutination, but the procedure of SPR technique is simpler, faster and high-throughput and label-free, which can meet the basic requirements for rapid screening and identification of blood group alloantibodies before transfusion of red blood cells.

Objective: To investigate the irregular antibody production and its relationship with Rh factor genotypes and the loci of thalassemia gene mutations for the β-thalassemic children with long-term transfusion, so as provide experimental basis for clinical safe and effective transfusions for thalassemic children., Methods: The peripheral blood from 246 children with β-thalassemia was collected in our hospital; the extraction of genomic DNA and Rh factor (C/c, E/e) genotypes were assayed by PCR-SSP method, the irregular antibodies were screened and identified by serological method, the genotypes for thalassemia and gene mutations were analysed by PCR-RD method., Results: The genotypes of Rh factors classified by PCR- SSP in the 246 cases of β-thalassemia children were as follws: Ce/Ce (143/246, 58.1%), CE/ce (59/246, 24%), cE/cE (14/24, 5.7%), Ce/ce (12/246, 4.9%); The positive rate of irregular antibody was 7.7% (19/246), including anti-E (7/19), anti-c (5/19), anti-C (2/19), anti-E and anti-c (2/19), anti-e (1/19), anti-D (2/19); Of the 19 cases with positive irregular antibody, the genotypings of Rh factor were: Ce/Ce (11/19), CE/ce (2/19), cE/cE (2/19), Ce/ce (2/19), cE/ce (2/19); the gene mutations location of thalassemia for 19 cases with positive irregular antibody: CD41-42M (13/19), CD71-72M (2/19), IVS-II-654M (3/19), -28M (1/19)., Conclusion: The irregular antibody production for β-thalassemic children with long-term transfusion may have some relevance with Rh factor genotypes and thalassemia genetic mutations. This study possesses a certain significance for effective prevention of RBC alloimmune response of β-thalassemia children and improvement of efficacy and safety of clinical transfasion blood.

Background: This study is a comprehensive analysis of RHD in D-negative phenotypes in saline, in Xi'an, Shanxi province, central China., Material and Methods: DCcEe in saline was measured for each blood sample from every donor between January 2008 and June 2012 in the Xi'an Blood Centre, China. D-negative results were confirmed by an indirect antiglobulin test and further investigated by adsorption-elution as required. The initial step of molecular analysis was RHD zygosity testing. Then RHD was detected by a sequence-specific polymerase chain reaction system for RHD(1227G>A), weak D type 15, and RHD(711delC) alleles for the samples carrying at least one RHD. For the remaining non-identified samples, ten RHD exons were amplified using a previously widely used RHD coding region sequencing method. Some RHD/RHCE conversion alleles were identified while those remaining were submitted to direct sequencing., Results: Overall, 2,493 D-negative samples in saline were detected in a total of 890,403 donors (D-negative rate, 0.28%). Among the D-negative individuals, RHD deletion (d/d) was assessed in 1685 donors (67.59%). Non-functional RHD alleles were detected in 184 donors (7.38%), the most common being the RHD-CE(2-9)-RHD and RHD(711delC) alleles. Two new alleles were observed and family investigations were performed; RHD(1227G>A) DEL was detected in 516 individuals (20.70%), and weak D or partial D variants were identified in 108 donors (4.33%). The most common alleles were weak D type 15, D(VI) type 3 and D(V) type 2. Four new weak D alleles were noted, and two cases of RHD(1227G>A)/weak D type 15 heterozygosity were confirmed., Conclusions: Currently, it seems to be difficult to observe any new RHD alleles in the Han Chinese population. D prediction in this population is easier because popular alleles are dominant, accounting for about 99.80% of alleles in D-negative people. Weak D types and partial D variants are rare and occur in approximately 0.01% of the population.

Background: Despite the introduction of anti-D prophylaxis into clinical practice, RhD alloimmunisation remains a problem, particularly in the context of transfusions and pregnancy-induced alloimmunisation. The incidence of RhD alloimmunisation among phenotypically RhD-negative individuals is unknown in most countries. We investigated RhD alloimmmunisation in RhD-negative pregnant women and transfusion recipients in south-east China in order to optimise the prevention of this phenomenon., Methods: We analysed the RhD alloimmunisation status of RhD-negative pregnant women and transfusion recipients in south-east China. The RhD blood types of the study population were identified by standard serological methods. The D antigen was further tested with the indirect antiglobulin test to exclude or confirm weak D or partial D types. RhC, c, E and e antigens were typed in all subjects. If anti-D antibody screening was positive, the specificity and titre of the antibody were determined. The Del phenotype was investigated by the polymerase chain reaction sequence-specific primer method., Results: An anti-D antibody was found in 61 of 416 RhD-negative pregnant women (14.66%), and in 11 of 227 RhD-negative transfusion recipients (4.85%). None of the 72 RhD-negative pregnant women or transfusion recipients with anti-D had the Del phenotype. Anti-D antibodies were not detected among Del phenotype individuals and Del phenotypes were not found in anti-D antibody producing individuals., Discussion: Our study suggests that the risk of alloimmunity-induced neonatal haemolysis increases in true RhD-negative multipara. Perinatal protection would be necessary in these patients, while antenatal anti-D testing and Rh immune globulin prophylaxis would be unnecessary for RhDel pregnant women. Pregnant women and transfusion recipients with the Del type seldom produce anti-D antibody. RhD-negative recipients are not at risk of alloimmunisation after transfusion with Del red blood cells.

Background: The characteristics of the D antigen are important as they influence the immunogenicity of D variant cells. Several studies on antigenic sites have been reported in normal D positive, weak D and partial D cases, including a comprehensive analysis of DEL types in Caucasians. The aim of this study was to assess D antigen density and epitopes on the erythrocyte surface of Asian type DEL phenotypic individuals carrying the RHD1227A allele in the Chinese population., Materials and Methods: A total of 154 DEL phenotypic individuals carrying the RHD1227A allele were identified through adsorption and elution tests and polymerase chain reaction analysis with sequence-specific primers in the Chinese population. D antigen density on the erythrocyte surface of these individuals was detected using a flow cytometric method. An erythrocyte sample with known D antigen density was used as a standard. Blood samples from D-negative and D-positive individuals were used as controls. In addition, D antigen epitopes on the erythrocyte surface of DEL individuals carrying the RHD1227A allele were investigated with 18 monoclonal anti-D antibodies specific for different D antigen epitopes., Results: The means of the median fluorescence intensity of D antigen on the erythrocyte membrane surface of D-negative, D-positive and DEL individuals were 2.14±0.25, 193.61±11.43 and 2.45±0.82, respectively. The DEL samples were estimated to have approximately 22 D antigens per cell. The samples from all 154 DEL individuals reacted positively with 18 monoclonal anti-D antibodies specific for different D antigen epitopes., Discussion: In this study, D antigen density on the erythrocyte surface of DEL individuals carrying the RHD1227A allele was extremely low, there being only very few antigenic molecules per cell, but the D antigen epitopes were grossly complete.

This study was aimed to investigate 1 case of rare RHD845A/1227A genotype pedigree and analyse their characters. The D antigen was determined by saline method and indirect anti-globulin test (IAT), the RHD1227A, RHD845A alleles and RHD zygosity were detected by PCR-SSP assay, the RHD coding region was analysed by gene sequencing. The results showed that the serological result of RH(D) antigen was found to be negative in one sample by saline assay, but positive by IAT. The analysis of RHD gene sequence indicated that RHD genes in the 845th and 1227th location were G/A base heterozygosis, it was speculated that the individual genotype may be RHD845A/1227A. Family investigation demonstrated the proband's father was RhD negative, his mother was RhD positive, the results of PCR-SSP assay showed that his father carried the RHD1227A alleles, whose genotype was RHD1227A/RHD(-), however, his mother carried RHD845A alleles, her genotype was RHD845A/RHD(+), which proved that the proband's genotype was RHD845A/1227A, inheriting the RHD1227A and RHD845A alleles from his father and mother respectively. It is concluded that 1 case of rare RHD845A/1227A genotype is found, further study proved that this rare heterozygosis come from the hereditary of RHD845A and RHD1227A alleles, rather than the formation of individual gene mutation.

This study was aimed to detect the level of the peripheral blood Breg and CD4(+) T cell subgroups in patients with chronic idiopathic thrombocytopenic purpura (CITP) before and after therapy, and to analyse the charge of related cytokines and their correlation, to explore their roles in the pathogenesis of CITP. A total of 35 CITP cases were taken as the research group and 35 healthy persons were served as the control group. The peripheral blood mononuclear cells (PBMNC) were separated, the percentages of Th1, Th17, Th22 and Breg cells were detected by flow cytometry before and after treatment of glucocorticoid, and the IFN-γ, IL-17, IL-22 and IL-10 levels from PBMNC culture supernatant also were determined by ELISA. The results showed that there was significant difference as compared with the healthy controls, the proportion of peripheral blood Th1, Th17, Th22 cell subgroups all increased in CITP patients before treatment with glucocorticoid, the regulatory B cells (Breg) ratio was reduced, the differences had statistical significance (P < 0.05), but the differences were no statistically significant after treatment with glucocorticoid (P > 0.05). The levels of IFN-γ, IL-17, IL-22 from culture supernatant all increased in CITP patients before treatment, the level of IL-10 was lower than that of the healthy control, the difference was statistically significant (P < 0.05), but the there were no statistically significant differences after treatment (P > 0.05). There were positive correlation between the Breg cells and IL-10 expression in CITP patients (P < 0.05), the Breg cells and Th1, Th17, Th22 cells showed a negative correlation, IL-10 and IFN-γ, IL-17, IL-22 levels also showed a negative correlation. It is concluded that the down-regulation of regulatory B cells proportion and the IL-10 level may participate in the mechanism of CD4(+) T cell immunity disorder in CITP, which can provide new targets and ideas for the clinical immune regulation therapy.

Flow cytometry was previously applied for analysis of Rh(D) antigen density, therefore it was suggested that the flow cytometry may be used for routine detection of weak D positive phenotypes. This study was purposed to evaluate its practicability. Six weak D positive and 7 DEL individuals were detected by using saline, IAT and absorption/elution test from 2010 to 2011 years. By RHD genotyping, zygosity analysis and sequencing, 3 cases of weak D type 15, 3 cases of partial D type DVI-III and 7 cases of DEL carrying RHD1227A alleles were identified. Taking 2 normal Rh(D)-positive and 2 D-negative samples as controls, all the samples were tested by using flow cytometry, and the median fluorescence intensities were observed as well. The results indicated that all weak D type 15 and partial D type DVI samples were detected to be positive by flow cytometry, as compared with 2 Rh(D)-negative samples (P < 0.05). Seven 7 DEL samples were tested to be negative (P > 0.05), although one of 7 DEL was tested as "±" in IAT and strong positive in absorption/elution. The RHD zygosity analysis showed this DEL individual as RHD(+)/RHD(+) homozygote. It is concluded that the sensitivity of detecting D antigen by flow cytometry is similar to that of IAT, but lower than absorption/elution test. As for detecting weak D or partial D antigens, IAT is easier than flow cytometry; as for identifying DEL, the flow cytometry is not sensitive enough.

RHD gene has different alternative transcripts. This study was aimed to construct expression vector of normal mRNA, DEL9 and DEL89 transcripts from RHD gene. Total RNA was extracted from Rh(D) positive umbilical blood cells of newborn. Intact RhD cDNA, DEL9 and DEL89 transcripts were obtained by one-step and two-step RT-PCR, respectively. The obtained products were cloned into pCR4 TOPO sequencing vector for choosing the right transcript. RHD gene was amplified again from the sequencing plasmid DNA, and then subcloned into pcDNA3.1/V5-His TOPO expression vector; DEL9 and DEL89 were cloned into the expression vector directly. Gene sequence and direction were identified by sequencing. The results showed that the sequence and direction of target genes were right, thus these 3 different expression vectors were correctly constructed. It is concluded that expression vector is constructed from different transcripts of RHD gene, which lays a foundation for further exploring the membranous protein expression of Rh(D) antigen.

Objective: To investigate the RHD zygosity of Rh(D)-positive Chinese Hans in order to study the mother-fetus Rh isoimmunization prophylaxis., Methods: Rh(D) blood group of 31 115 donors were serotyped, and the RHD zygosities were analyzed, or determined through a PCR method for 3628 donors of Rh(D)-positive individuals., Results: Among the 31 115 donors, 99 were tested Rh(D)-negative by indirect antiglobulin test (IAT) (0.318%). The d frequency was 0.056 41, D was 0.943 59, and Dd heterozygosity was 0.106 45 (10.6%). However the rate was 0.090 32 (about 9.0%) after excluding DEL (IAT-negative). For the 3628 PCR tested donors, 3383 were DD (93.2%), 245 were Dd (6.8%). After excluding nonfunctional RHD alleles, 7.4% of the donors were carrying one functional RHD. It showed that an Rh(D)-negative Chinese Han woman gives an Rh(D)-negative child at a rate of 3.7%-4.5% when her husband is Rh(D)-positive., Conclusion: Fetus Rh(D) genotyping may be unnecessary for Chinese Hans if invasive operation was needed for prenatal diagnosis. The Rh prophylaxis could be chosen assuming an Rh(D)-positive fetus.

An unknown HLA-B allele which was similar to HLA-B*270401 was detected by FLOW-SSOPCR-SSP and heterozygous sequence-based typing (SBT) in Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. Amplifying exon 2-5(include intron 2-4) of the HLA-B*27 allele separately by using allele-specific primers and sequencing in both directions. Identifying the difference between the novel B*27 allele and B*270401. The sequence of novel B*27 from exon 2 to partial exon 5 is 1 815 bp. There are 10 nt changes from B*270401 in exon 3-4, at nt634where A-->C(codon130 AGC-->CGC, 130 S-->R); nt670 where A-->T (codon142 ACC-->TCC, 142 T-->S); nt683 where G-->T (codon146 TGG-->TTG, 146 W-->L); nt698 where A-->T (codon151 GAG-->GTG, 151 E-->V); nt774 where G-->C (codon176 GAG-->GAC, 176 E-->D); nt776 where C-->A (codon177 ACG-->AAG, 177 T-->K); nt781 where C-->G (codon179 CAG-->GAG, 179Q-->E); nt789 where G-->T (codon181 GCG-->GCT) resulting no coding change; nt1438 where C-->T (codon206 GGC-->GGT) resulting no coding change; nt1449 where G-->C (codon210 GGG-->GCG, 210G-->A). In IMGT/HLA database, only three alleles (B*270502/2706/2732) have sequences of introns. The same sequence in intron 2 showed homology between the novel HLA-B*27 allele and B*2706, but their homology could not be supported in intron 3-4. Comparing the sequence of the novel B*27 allele in intron 3 and 4 with B*27 group, it showed there are three mutations at nt106 C-->G, nt179 G-->A, nt536 G-->A and one deletion at nt168 in intron 3 and one mutations at nt82 T-->C in intron 4, but the sequence of the novel B*27 allele in intron 3 and 4 was all the same to B*070201. The sequence was submitted to Gen-Bank and the accession number was DQ915176. The allele has been confirmed as an extension of B*2736 by the WHO Nomenclature committee in November 2006.

Objective: To identify HLA novel allele in Chinese Han individual., Methods: An unknown HLA-B allele which was similar to HLA-B*5610 was detected by polymerase chain reaction-sequence specific oligonucleotide probes(PCR-SSOP), PCR-sequence specific primer(PCR-SSP) and heterozygous sequence-based typing (SBT) in a Chinese Han individual. Its anomalous patterns suggested the possible presence of new allele. The HLA-B*56 allele was amplified separately by using allele-specific primers and sequencing exons 2-4 in both directions. The differences between the novel B*56 allele and B 5610 were identified., Results: There were 4nt changes from B*5610 in exon 3, at nt379 where C>G (codon 127 CTG>GTG, 127 Leu>Val); nt412 where A>G (codon 138 AAC>GAC, 138 Asn>Asp), nt419 where T>C and nt420 where A>C (codon 140 TTA>TCC, 140 Leu>Ser). The sequence was submitted to Genbank and the accession number was EF016753., Conclusion: This allele is a novel HLA-B allele, and has been officially named HLA-B*5618 by the WHO Nomenclature Committee in September 2006.

To identify HLA novel allele in Chinese Han individuals, an unknown HLA-A allele was detected by PCR-SSP and FLOW-SSO in Chinese Han individuals. Heterozygous sequence-based typing (SBT) showed that there were 3 differences compared with database in exon 2. Its anomalous patterns suggested the possible presence of either a novel A * 30 or a novel A * 24. To separate the two alleles and to determine whether the allele is novel, the HLA-A * 30 and HLA-A * 24 alleles were amplified separately by using a commercial kit for the single allele-specific sequencing strategy, and both alleles for exons 2 - 4 were sequenced according to the manufacturer' protocol. To prepare B-lymphoblastoid cell line of the novel HLA allele by using Epstein-Barr virus-infected B-lymphoblastoid cells in the peripheral blood. The results indicated that the sequencing results showed HLA-A alleles of the sample to be HLA-A * 240201 and a new A * 30 allele. The sequences of the new A*30 were identical to those of HLA-A * 300101 except for three nucleotide changes in exon 2: at nt 121 (A-->C), nt 123 (T-->C) and nt 126 (A-->G), resulting in an amino acid residue substitution from S (AGT) to R (CGC) at codon 17 and a synonymous substitution from G (GGA) to G (GGG) at codon 18. Immortalized B-lymphoblastoid cell line of the novel HLA-A * 3018 allele was achieved, the sequence of HLA-A * 3018 allele was submitted to GenBank and its accession number was DQ872509. In conclusion, the HLA-A * 3018 is a novel HLA-A allele and has been officially named HLA-A * 3018 by the WHO Nomenclature committee in August 2006 (HWS10004039).

Objective: To observe the effect platelet antigen modification by mPEG-SPA with different molecular masses., Methods: Platelet CD42a was modified by 5 kD and 20 kD mPEG-SPA, respectively, and the fluorescence intensity of CD42a was detect by flow cytometry and the three-dimensional structure of CD42a simulated to analyze the distribution of lysine in CD42a molecule., Results: After platelet CD42a modification by 5 kD and 20 kD mPEG-SPA, the fluorescence intensity of CD42a decreased sharply by 85.54% and 88.65%, respectively, and multiple lysine regions were identified on the surface of CD42a molecule., Conclusion: Both 5 kD and 20 kD mPEG-SPA allow useful modification of platelet CD42a, but 20 kD mPEG-SPA is more advantageous than 5 kD mPEG-SPA.

Objective: To study the genetic feature of weak D type 15 allele (RHD845A) in a Chinese family., Methods: Rh D, C, c, E and e phenotypes of 4 members in a weak D type 15 family were tested by serological and polymerase chain reaction (PCR), D antigen was proven by indirect antiglobulin test. A pair of primers specific for RHD845A were designed, and a sequence specific primer-PCR (PCR-SSP) method was established to detect RHD845A allele in all family members. Subsequently the dual-tube PCR method was used to determine the RHD zygosity of 4 members., Results: The RHD845A allele existed in all 4 family members and the RHD zygosity test showed that all members were RHD +/RHD + homozygous. The parents and nephew possessed one normal RHD gene as RHD845A allele carriers, which caused RhD positive. The proband and his old-sister took two RHD845A alleles, which caused weak D phenotype., Conclusion: The proband is the weak D type 15 allele homozygous. The weak D type 15 gene is an ancestral allele, but not a mutation.