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Background: Molecular typing for RHCE blood group alleles has been established in many countries for patients and blood donors. In the Chinese literature nearly 80% of transfused patients with alloimmunization have antibodies specific for antigens of the Rh blood group system. We investigated if it is feasible to match packed red blood cells (RBCs) for Chinese β-thalassemia patients by RHCE genotyping. Methods: In this study, 481 patients with β-thalassemia were enrolled. They were genotyped for RHCE alleles by a simple PCR method with sequence-specific primers (PCR-SSP). Among these patients, 203 continuously received RBCs of the identical Rh subgroups according to the genotyping results for at least 3 months. Subsequently, their phenotypes were tested through a micro-column gel card method. For validation purposes, 400 donors were serologically typed with the same technology, of which 164 were genotyped too. Finally, the C, c, E, and e frequencies and the feasibility of the simple genotyping method were analyzed. Results: All patients showed mixed-field agglutination in the Rh subgroup gel cards before the same Rh subgroups in blood donors were selected for blood transfusion. The results, however, lacked mixed-field agglutination in all 203 cases after transfusion with RBC concentrates selected for the patient's C, c, E, and e antigens for at least 3 months. The genotyping results of 164 donors were all consistent with the serological results. Whole coding regions of RHCE were sequenced in 7 individuals with weak c, E, or e antigens. In only one sample we observed a 1059G>A nucleotide mutation coding for a truncated RhCE polypeptide (GenBank KT957625), in the other 6 samples no sequence variant was found. Both patients and donors were predominantly CcEe and CCee, with a prevalence of 55.3% and 24.9% for patients or 49.3% and 31.3% for donors, respectively. It revealed that about 80% of Chinese could receive Rh-matched RBCs easily. Conclusion: A simple RHCE genotyping technique is safe enough for Rh-matched transfusion of β-thalassemia patients in Chinese Han.

C*12:02:36 differs from C*12:02:02:01 by one nucleotide change at nucleotide 438 in exon 3 from T to C.

A*11:333 differs from A*11:01:01 by one nucleotide change at nucleotide 865 in exon 4 from G to A.

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.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.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（P0.05）.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.定期输血患者RhCcEe混合视野鉴定及配型输注效果分析.探讨定期输血患者RhCcEe血型抗原混合视野鉴定的可行性，跟踪评价其配型输注效果及其临床意义.采用PCR-SSP方法对我院142例定期输血患者进行RhCcEe基因分型；根据基因分型结果，其中48例患者自愿持续进行RhCcEe同型输血，46例按临床输血规范进行RhCcEe随机输血，42例进行部分RhCcEe同型输血，6例患者流失了输血资料；在RhCcEe配型输注3-6个月后对所有患者采用RhCcEe微柱凝胶卡复查，并与他们配型输血前结果比较，跟踪评价近5年3类定期输血患者输血后同种抗体发生率、直接抗人球蛋白试验（DAT）阳性率及红细胞无效输注率.在48例持续RhCcEe同型输血患者中仅1例出现分层现象，其余47例凝胶卡结果清晰，此结果提示PCR-SSP基因分型的可行性；另外42例部分同型输血患者及46例随机输血患者凝胶卡检测仍然出现混合视野，结果较难确认。跟踪分析近5年输血效果显示，RhCcEe同型输血组发生同种抗体1例（抗-Jka）（1/48），DAT阳性1例（1/48），输注无效2例（2/48）；随机输血组同种抗体7例（抗-E 3例、抗-E+抗-c 1例、抗-C 1例、抗M 1例、抗-Fya 1例）(7/46)，DAT阳性6例(6/46)，输注无效9例（9/46）；部分RhCcEe配型输血组同种抗体6例（抗-E1例、抗-E合并自身抗体1例、抗-C 1例、抗-c 1例、抗-M 1例、其它1例）(6/42)，DAT阳性7例（7/42），输注无效8例（8/42）；统计学分析各组同种抗体阳性率与红细胞无效输注率显示，配型输血组与随机输血组、部分配型输注组间均有统计学差异（P＜0.05），而随机输血组与部分配型输血组间无统计学差异（P0.05）.PCR-SSP基因分型技术可应用于定期输血患者RhCcEe血型混合视野检测，持续RhCcEe配型输血可有效预防同种免疫产生，并提升定期输血患者临床输血效果与输血安全性，显示出具有一定的临床意义.

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.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.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.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.SPR技术在红细胞血型同种抗体筛选与鉴定中的应用研究.探讨利用表面等离子体共振（Surface plasman resonance，SPR）技术对红细胞血型同种抗体筛选与鉴定的可行性，为红细胞输注相容性检测提供一种新方法.采用氨基偶联法在SPR芯片表面固定红细胞抗原谱，优化芯片分析条件，检测红细胞血型抗体阳性对照血清，分析SPR芯片技术的检测性能；对比研究SPR芯片技术与微柱凝集法检测129例长期输血的地中海贫血患者临床样本的一致性；同时运用SPR芯片技术对7例血型抗体阳性患者输血前进行血型抗体鉴定，选择红细胞抗原相容性血液输注；跟踪评价红细胞输注效果.SPR芯片技术检测血型同种抗体的重复性、敏感性与特异性均较好；SPR技术与微柱凝集法对129例临床样本血型同种抗体筛查结果显示，2种方法无显著性差异（χ2=0.333, P＞0.05），总体一致性为97.2%；SPR技术对7例血型同种抗体阳性患者鉴定结果为：抗-E 3例、抗-M 1例、抗-C 1例、抗-Jka 1例、自身抗体1例，与微柱凝集法鉴定结果基本一致；SPR技术选择配合性输血后，6例患者红细胞输注有效，1例患者存在自身抗体输注无效.SPR芯片技术筛检与鉴定红细胞血型抗体的性能与微柱凝集法总体一致，而SPR技术操作更简便快速、高通量且非标记，可满足批量红细胞输注前血型同种抗体快速筛查和鉴定的基本要求.

Background This study was aimed to establish a novel strategy based on the surface plasmon resonance (SPR) technology for platelet compatibility testing. Methods A novel surface matrix was prepared based on poly (OEGMA-co-HEMA) via surface-initiated polymerization as a biosensor surface platform. Type O universal platelets and donor platelets were immobilized on these novel matrices via amine-coupling reaction and worked as a capturing ligand for binding the platelet antibody. Antibodies binding to platelets were monitored in real time by injecting the samples into a microfluidic channel. Clinical serum samples (n = 186) with multiple platelet transfusions were assayed for platelet antibodies using the SPR technology and monoclonal antibody-immobilized platelet antigen (MAIPA) assay. Results The novel biosensor surface achieved nonfouling background and high immobilization capacity and showed good repeatability and stability after regeneration. The limit of detection of the SPR biosensor for platelet antibody was estimated to be 50 ng/mL. The sensitivity and specificity were 92% and 98.7%. It could detect the platelet antibody directly in serum samples, and the results were similar to MAIPA assay. Conclusions A novel strategy to facilitate the sensitive and reliable detection of platelet compatibility for developing an SPR-based biosensor was established in this study. The SPR-based biosensor combined with novel surface chemistry is a promising method for platelet compatibility testing.

Background As an emerging metropolis with population expansion from 2 million to 10 million from 1993 to 2012, the clinical demand for blood in Shenzhen has increased 20 times. To deal with this big challenge, Shenzhen utilized voluntary nonremunerated blood donation (VNRBD) in 1993 for the first time in China. After two decades of efforts, Shenzhen has achieved self-sufficiency in its blood supply and guaranteed its blood security by nonpaid blood donation. Study Design and Methods We summarized the strategies to achieve self-sufficiency and security in the blood supply in Shenzhen during two decades, including the legal construction of VNRBDs and the continuously improving strategies to recruit and retain nonpaid donors. The collection data of whole blood (WB) and apheresis platelet (PLT) donations were retrieved, and donor demographic and donation characteristics were analyzed. Results From 1993 to 1998, paid and nonpaid blood donations coexisted in Shenzhen. From the year 1999, all WB for clinical use came from VNRBDs. From 1999 to 2012, the donors who chose to donate 400 mL each time and repeat and regular donors increased sharply to meet the fast growth of clinical demand. From the year 2005, the clinical demand for PLTs was entirely satisfied by nonpaid donations. Conclusions After two decades of practice, we believe that the legal regime of VNRBD is fundamental guarantee for long-term self-sufficiency and security in the blood supply. In addition, strengthening the publicity to improve the public's awareness and improving donation services and measures to recruit more nonpaid donors and retain repeat and regular donors are very important.

Background Generally Rh-negative patients need to be transfused with Rh-negative red blood cells. For pregnant women carrying Rh-positive fetus, the antenatal anti-D detection and Rh immunoglobulin prophylaxis are required worldwide. In East Asia, a RhD variant, termed “Asia type” DEL, was found in approximately 30% of apparent Rh-negative individuals. The antigenic and molecular properties of the DEL were previously defined. Few data discuss whether DEL could be immunized by D antigen clinically although DEL was reported arousing alloimmunization to true Rh-negative patients. Study design and methods To determine whether the DEL variant can be immunized to the D antigen, we retrospectively evaluated 104 Rh-negative pregnancies with allo-anti-D antibodies, and we also tracked 199 consecutive apparent Rh-negative pregnant women, with a history of gestations or parturitions but not subject to anti-D gamma-globulin prophylaxis, for evidence of allo-anti-D. The DEL variant was first excluded by ccee phenotypes and then identified through PCR analysis or sequencing. Results In the retrospective study, we expected to find 30 DEL variants, yet none of the anti-D alloimmunized women were DEL -positive. And in the second group, none of 44 DEL -positive women versus 38 of 155 (24.5%) true Rh-negative women (those excluding DEL) formed allo-anti-D. Conclusion The data indicate that the “Asia type” DEL variant does not appear at risk of alloimmunization to the D antigen. It strongly suggests that the antenatal Rh immune globulin prophylaxis is unnecessary for DEL women. Furthermore, it implicates that the “Asia type” DEL may be deemed Rh-positive safely for clinical transfusion therapy.

Background Severe acute respiratory syndrome (SARS) is a newly recognized infectious disease that caused an outbreak in south China in 2003. The cause of SARS was identified as a novel coronavirus (CoV). The existence of asymptomatic seroconvertors and the detection of the SARS-CoV RNA in plasma during the course of infection all suggest that SARS could, as least theoretically, be transmitted by transfusion. An estimate of the risk of SARS transmission through blood transfusion will contribute to decisions concerning blood safety monitoring and may be useful in the design of strategies to decrease the risk of transfusion-transmitted infections. Study design and methods Case onset dates from the 2003 Shenzhen SARS epidemic and investigational results from Taiwan on viremia in humans are used to estimate the number of cases that were viremic throughout the epidemic. Estimates of the asymptomatic-to-clinically confirmed SARS-CoV infection ratio, the proportion of asymptomatic infections reported in a seroprevalence survey in Hongkong, and the population size of Shenzhen are used to infer the SARS-CoV transfusion–transmission risk. Statistical resampling methods are used. Results Based on data from Shenzhen, Hongkong and Taiwan, the maximum and mean risk (per million) of SARS-CoV transmission from donors in Shenzhen were estimated as 23.57 (95% CI: 6.83–47.69) and 14.11 (95% CI: 11.00–17.22), respectively. The estimated risk peaked on April 02, 2003. Conclusions Although there are currently no confirmed reports of the transmission of SARS-CoV from asymptomatic individuals, recent research data indicate that transfusion-transmitted SARS-CoV is at least theoretically possible. Although the risk is low, with its rapid spread of the disease, appearance of alarmingly high infectivity and high fatality rate, public health authorities need to consider strategies for blood donor recruitment and virus inactivation during an epidemic to further ensure blood safety.

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.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.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).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.

The RhD mRNA was analyzed in both Rh-positive and D(el) phenotypes with RHD 1227A allele through sequencing. As a result, five and six transcripts were detected in Rh-positive and D(el), respectively. Four of them have identical sequences between Rh-positive and D(el). 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 D(el) individuals. Unexpectedly, two additional transcripts were found in D(el) 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 D(el) 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.

As an emerging metropolis with population expansion from 2 million to 10 million from 1993 to 2012, the clinical demand for blood in Shenzhen has increased 20 times. To deal with this big challenge, Shenzhen utilized voluntary nonremunerated blood donation (VNRBD) in 1993 for the first time in China. After two decades of efforts, Shenzhen has achieved self-sufficiency in its blood supply and guaranteed its blood security by nonpaid blood donation.We summarized the strategies to achieve self-sufficiency and security in the blood supply in Shenzhen during two decades, including the legal construction of VNRBDs and the continuously improving strategies to recruit and retain nonpaid donors. The collection data of whole blood (WB) and apheresis platelet (PLT) donations were retrieved, and donor demographic and donation characteristics were analyzed.From 1993 to 1998, paid and nonpaid blood donations coexisted in Shenzhen. From the year 1999, all WB for clinical use came from VNRBDs. From 1999 to 2012, the donors who chose to donate 400 mL each time and repeat and regular donors increased sharply to meet the fast growth of clinical demand. From the year 2005, the clinical demand for PLTs was entirely satisfied by nonpaid donations.After two decades of practice, we believe that the legal regime of VNRBD is fundamental guarantee for long-term self-sufficiency and security in the blood supply. In addition, strengthening the publicity to improve the public's awareness and improving donation services and measures to recruit more nonpaid donors and retain repeat and regular donors are very important.

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 (P0.05), but the differences were no statistically significant after treatment with glucocorticoid (P0.05). 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 (P0.05), but the there were no statistically significant differences after treatment (P0.05). There were positive correlation between the Breg cells and IL-10 expression in CITP patients (P0.05), 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 (P0.05). Seven 7 DEL samples were tested to be negative (P0.05), 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.

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.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.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.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.

Previously, both primary and secondary anti-D alloimmunizations induced by “Asian type” DEL (RHD1227A allele) were observed in two incidents. We investigated how often these alloimmunization events occur. The transfusions of any D-negative patients were investigated in the First Affiliated Hospital of Xi’an Jiaotong University Medical College, China, during the entire 2009. The antigens of D, C, c, E, and e were routinely serotyped. The “Asian type” DEL variant was genotyped and the RHD heterozygote was determined through two published methods. The changes in anti-D levels were monitored by the indirect antiglobulin test (IAT) and flow cytometry. Thirty D-negative transfused patients were included in the study. We focused on 11 recipients who were transfused with packed red blood cells (RBCs) from DEL donors at least one time. Of those 11 recipients, seven were anti-D negative before transfusion and four were anti-D positive (one patient with an autoantibody). One of the seven pre-transfusion anti-D negative patients produced a primary-response anti-D after being transfused with 400 ml of DEL blood twice. All four pre-transfusion antibody positive patients were not observed hemoglobin (Hb) levels increased, as expected after transfusions. Two patients had an increase in anti-D from 1:8 to 1:64 by IAT, which was also shown by flow cytometry. None of the patients experienced an acute hemolytic episode. Our data indicated that the primary anti-D induced by DEL transfusion or the secondary anti-D elevated by DEL in a truly D-negative patient might not be unusual. We suggest that a truly D-negative childbearing-aged woman should avoid DEL transfusion to protect her from primary anti-D allosensitization. In addition, anti-D positive recipients should also avoid DEL red cell transfusion due to the delayed hemolytic transfusion reaction (DHTR).

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.

To investigate the RHD zygosity of Rh(D)-positive Chinese Hans in order to study the mother-fetus Rh isoimmunization prophylaxis.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.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.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.

Background In order to further improve blood safety, mini-pool (MP) nucleic acid testing (NAT) was implemented to screen samples negative for hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (anti-HCV), anti-human immunodeficiency virus (anti-HIV), syphilis (anti-Treponemal antibody) and with normal ALT. Study design and methods From August 2006 to February 2008, 41,301 donations were screened using commercial HIV/HCV RNA and HBV DNA Real-Time PCR NAT assays in pools of 8. Reactive pools were re-tested as individual samples using the appropriate screening test and confirmed using an alternate commercial NAT assay. Donors reactive on both NAT assays were considered ‘confirmed’ positive for the virus concerned and recalled for additional follow-up testing and counseling. Results Of the 41,301 samples screened, no HIV or HCV RNA-positive/seronegative donations were detected but two HBV DNA positive/HBsAg negative blood donors (Donors 1 and 2) were identified. Their respective hepatitis immunological markers were: Donor 1 – anti-HBc positive/anti-HBe positive/HBeAg negative/ALT normal and HBV DNA viral load of 112 IU/ml; Donor 2 – anti-HBc positive/anti-HBe negative/HBeAg negative/ALT normal and HBV DNA viral load 2750 IU/ml. Conclusions MP NAT identified two HBsAg negative donors with presumed occult infection but no HIV or HCV seronegative/NAT positive (yield) donors. The HBV yield rate of 1 in 20,650 (95%CI – 1 in 5663 to 1 in 75,303) is comparatively high, exceeds the predicted rate based on previous modeling for the population and demonstrates the incremental blood safety value of NAT in countries where HBV is highly epidemic. The low viral load of the two yield samples underscores the importance of optimizing the sensitivity of the HBV NAT assay selected for screening.

Whole length of RHD introns 7 and 9 of one normal Rh D-positive individual and 2 DEL samples, carrying RHD1227A allele, were sequenced and aligned. Thirty-three and 27 nucleotide variants were totally observed in intron 7 and intron 9, respectively (EMBL/GenBank/DDBJ EU372940 approximately 2). Among them, 8 variants in intron 7 and 7 in intron 9 were observed commonly in all 3 samples, whereas 2 variants in intron 7 and one in intron 9 were only found in 2 DEL samples, but not in the normal D-positive. The variants observed in intron 7 in DEL cannot explain enough for that DEL mRNA has one segment of 170 base pairs sequence from intron 7. But the nucleotides AG-GT at both sides of the segment may be related to this molecular even as AG-GT may cut intron 7 with its normal splicing site (GT-AG) into two "new introns" although the mechanisms are complicated in fact. We also have not found any suspicious splicing-affecting variants in intron 9 of DEL allele. However, this may make out further that the reason of whole exon 9 spliced out in DEL mRNAs may be no more than the 1227A>G mutation in DEL allele.

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.

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).

To identify HLA novel allele in Chinese Han individual.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.There were 4nt changes from B*5610 in exon 3, at nt379 where CG (codon 127 CTGGTG, 127 LeuVal); nt412 where AG (codon 138 AACGAC, 138 AsnAsp), nt419 where TC and nt420 where AC (codon 140 TTATCC, 140 LeuSer). The sequence was submitted to Genbank and the accession number was EF016753.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 observe the effect platelet antigen modification by mPEG-SPA with different molecular masses.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.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.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.

To study the genetic feature of weak D type 15 allele (RHD845A) in a Chinese family.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.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.The proband is the weak D type 15 allele homozygous. The weak D type 15 gene is an ancestral allele, but not a mutation.

BACKGROUND: The D antigen of the polymorphic Rh blood group system is of particular clinical importance regarding transfusion- and pregnancy-induced alloimmunization. Different RhD variants with specific clinical implications have been characterized. The least expressed D variants collectively called DEL are serologically detectable only by adsorption-elution techniques, with so far only poorly defined antigenic properties. STUDY DESIGN AND METHODS: A comprehensive immunohematologic analysis of five of the six currently known DEL genotypes was performed. DEL phenotypes associated with the RHD(M295I), RHD(IVS3+1g>a), RHD(K409K), RHD(X418L), or RHD(IVS5-38del4) allele were characterized with extended serology and flow cytometry. RESULTS: Epitope mapping with adsorption-elution revealed a prominent D epitope loss in the RHD(IVS3+1g>a)-associated DEL phenotype, whereas in the other four DEL types no signs of qualitative D antigen alteration were detected. The observation of alloanti-D in two RHD(IVS3+1g>a) cases confirmed the partial nature of this DEL phenotype. The RHD(M295I) phenotype exhibited the highest D antigen expression among all investigated DEL types, as determined by a semiquantitative adsorption-elution approach and flow cytometry. CONCLUSION: In conclusion, evidence is provided that different DEL genotypes code either for partial or complete D antigen expression and that this finding is clinically relevant. he Rh blood group system is determined by the highly homologous RHD and RHCE genes encoding the RhD and RhCE polypeptides, respectively. The D antigen (RH1) carried by the RhD polypeptide is of particular clinical importance with respect to transfusion- and pregnancy-induced alloim

Previously a few Rh-negative individuals in Caucasian and Chinese were found existing exons 1 and 10 of RHD through genomic DNA testing. The molecular mechanisms, however, remain disputed. In this study, 2 individuals carrying RHD positive, D antigen negative allele (with exon 1 and 10 of RHD) and their mRNA were investigated by using reverse transcriptase PCR (RT-PCR) and sequencing through one pair of specific primers for 5'- and 3'-non-coding region of RHD, taking a Rh-positive (Ccee) sample as control. As a result, the control sample had a normal RHD mRNA, whereas other transcripts were detected in both RHD-positive, D antigen negative individuals, which have same length and exons as the normal RHD or RHCE mRNA. Those transcripts had the same sequences with RHD in exon 1 and exon 10, while the sequences in exons 2-9 were in concordance with RHCE(e) mRNA. It indicated that a hybrid RHD allele, RHD-CE(2-9)-D, was existed in the 2 individuals, in which the exons 2-9 of RHD were substituted by homologous RHCE(e). Thus, this allele could not code normal RhD protein and therefore resulted in Rh-negative phenotype.

There are 3' non-coding region, downstream Rhesus box, SMP1 gene et cetera. after RHD stop code. This study was intended to determine the sequence of 3' non-coding region. One pairs of primer was designed and then a polymerase chain reaction (PCR) was established for specific amplification of whole length of 3' non-coding region of RHD in 10 Rh-positive and 10 D(el) samples. The PCR products were purified and directly sequenced. The results showed that all Rh-positive and D(el) samples were identical, which revealed that there were 103 bp between 3'-end of RHD coding region and 5'-end of downstream Rhesus box. The D(el) samples showed the same result with the normal Rh-positive sample. It suggests that lower expression of D antigen in D(el) red cells does not associate with 3' non-coding region of D(el) gene.

The study was purposed to analyze DNA and allele structure of the partial D phenotypes D(Va) and D(VI) of the Rhesus blood group in Chinese. Through polymerase chain reaction (PCR) and direct genomic DNA sequencing, the RHD gene was detected in three weak D individuals identified serologically. The results showed that among the three weak D individuals, one was identified as partial D phenotype D(Va) (Hus) type and genotyped DccEe; another two were testified as D(VI) III type and genotyped DCcee. Moreover, the breakpoints of the replaced region by RHCE in D(Va) (Hus) were 5' end of the exon 5 and 3' end of the intron 5, and there were 7 novel polymorphisms in intron 5: 23-25(GCA)2, 98GA, 168-169insG, 205-206insT, 494-495insA, 1256-1257insC, 1347GT. In conclusion the whole exon 5 and intron 5 are replaced by RHCE in D(Va) (Hus) detected in Chinese.

To establish a direct method for the determination of RHD zygosity.Two pairs of primers were designed specific for hybrid Rhesus box and exon 1 of RHD, respectively. Combined with a pair of internal control primers, a new dual-tube PCR method was established. One hundred and fifty-two DNA samples, of which the sequence of RHD and RH genotypes were known, and samples from 359 donors or patients, of which the Rh phenotypes were determined, were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference.The RHD zygosities of 152 samples, including 92 Rh-negative, 28 D(el), 3 partial D, 5 weak D and 24 Rh-positive, were determined in concordance with the known genotypes. It showed that the PCR method could detect RHD(+)/RHD(+), RHD(+)/RHD(-) and RHD(-)/RHD(-) zygosities in one test. The PCR results of 359 known phenotype samples including 12 husbands, 18 family members, 48 Rh-negative and 281 Rh-positive, were identical with RFLP except for one Rh-positive sample, which was tested as RHD(+)/RHD(-) heterozygote by PCR, whereas RHD(-)/RHD(-) homozygote by RFLP. Apparently, RFLP revealed a false negative result detecting downstream Rhesus box.The dual-tube PCR is a less-labored and more rapid method for the determination of RHD zygosity comparing RFLP. It can be used routinely in the laboratories in clinics and blood banks.

To explore a molecular method for the determination of RHD zygosity.Two pairs of primers were designed specific for downstream rhesus box and hybrid rhesus box according to the sequences in GenBank. Together with a pair of internal control primer, a dual-tube polymerase chain reaction (PCR) method was established for determination of the RHD zygosity. Thirty RhD-positive, D-negative and weak D phenotype samples were evaluated by taking a recent restriction fragment length polymorphism (RFLP) method as reference.The results of the dual-tube PCR and RFLP are identical in 29 samples, which are also in concordance with the Rh phenotype, respectively. In one weak D sample, however, both methods tested as RHD(-)/RHD(-), which showed that there was one RHD(-) haplotype in this individual, but a false negative result in testing downstream rhesus box.The dual-tube PCR is a less-labored method for RHD zygosity detection. It can be used clinically in prenatal test to determine RHD zygosity of pregnant women's partner for prediction of fetus RhD phenotype.

Previously the weak D and D category allele were investigated in Caucasian and Japanese families. The current study is aimed at an RHD positive, D antigen negative allele in a Chinese family.A pair of primers specific for RHD 270A allele were designed, and a sequence specific primer-PCR (SSP-PCR) method was then established to detect RHD 270A allele in 6 members of a family. Furthermore, RFLP method was used to determine the RHD zygosity in all family members.The RHD 270A allele was detected in the proband, her father and uncle but not grandmother. Therefore this allele may be from grandfather and is inherited through 3 generations. The RHD zygosity test showed that the father and uncle possess one normal RHD gene as RHD 270A carriers, the mother is RHD(+)/RHD(-)heterozygote and the individual is RHD 270A/RHD(-)which causes an RHD positive, D antigen negative trait.The RHD 270A allele is an ancestral allele, but not a spontaneous.

To the Editor: The RhD status of transfusion recipients and donors is routinely matched for red-cell transfusion. This worldwide practice is due to the potent immunogenicity of RhD. In East Asians, the frequency of RhD-negative status is only about 0.3%, which sharply limits the supply of RhD-negative blood. However, approximately 30% of RhD-negative persons carry an RhD variant, termed “Asia type” DEL.1 Beginning in 2008, my colleagues and I organized a collaborative group of 10 laboratories, located in 10 cities in northern, central, and southern China. This group retrospectively evaluated 104 RhD-negative pregnant women with anti-D alloantibodies from 2005 through . . .

We report the identification of a novel human leukocyte antigen (HLA)-B*56 allele, B*5618 and an extension of B*2736 that were found during routine high-resolution sequence-based typing in Chinese Han individual. The B*5618 allele has 4nt changes from B*5610 in exon 3, The B*2736 allele has 10nt changes from B*270401 in exons 3-4.

We report the identification of the novel allele HLA-A*3018 that was found during routine high resolution sequence-based typing of a Chinese bone marrow donor. The A*3018 allele has three nucleotides that differ from A*300101 at codon 17 (AGT-->CGC) and codon 18 (GGA-->GGG) in exon 2.