Background: Mutations of ABO gene may cause the dysfunction of ABO glycosyltransferase (GT) that can result in weak ABO phenotypes. Here, we identified two novel weak ABO subgroup alleles and explored the mechanism that caused Ax phenotype. Materials and methods: The ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the mutations was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. Results: The results of serological showed the subject RJ23 and RJ52 both were Ax phenotypes. The novel A alleles, Avar-1 and Avar-2 were identified according to the gene analysis. Both Avar-1 and Avar-2 harbored recombinant heterozygous alleles, specifically A2.05 and O.01.02. These alleles showcased substitutions at positions c.106G > T, c.189C > T, c.220C > T, and c.1009A > G in their respective exons. It is worth noting that the crossing-over regions of these two alleles differed from each other. In vitro expression study showed that GTA mutant impaired H to A antigen conversion, and the mutant did not affect the production of GTA though the Western bolt. In silico analysis showed that GTA mutant may change the local conformation and the stability of GT. Conclusion: The Avar-1 and Avar-2 alleles were identified, which could cause the Ax phenotype through changing the local conformation and reducing stability of the GTA.

RHD variants are classified into three types: weak D, partial D, and DEL. Pregnant women with the D variant phenotype require RhD serological typing and/or RHD genotyping.1 Partial D, which shows qualitative changes in RhD protein expression, usually caused by RHD/RHCE hybrid alleles, in-frame deletions or missense mutations in the external loops of the RhD protein or at the entrance of the RhD channel (RhD protein vestibule).2 Partial D individuals may produce alloanti-D after exposure to D+ red blood cells (RBCs) during transfusion or pregnancy. Here we report a novel RHD allele caused by a c.687_689delAAG variant in a 40-year-old Chinese pregnant woman (G2P2) with a partial D phenotype. Initial RhD blood group screening was determined using the ABO/RhD blood group test card (Bio-Rad, Switzerland). If the result was negative, verification tests were performed using saline or indirect antiglobulin test (IAT) with IgM/IgG blend anti-D (Dominion Biological Ltd, Canada) and IgG anti-D (Shanghai Hemo-Pharmaceutical &Biological Co., Ltd, China). To determine the presence or absence of D epitopes, RBCs were tested using a D-screen identification kit (DIAGAST, France). The RhCE phenotype was verified with monoclonal IgM anti-C (clone: MS-24), anti-c (clone: MS-33), anti-E (clones: MS-80+258) and anti-e (clones: MS-62+69) using the Rh Blood Grouping Card (Jiangsu Libo Medicine Biotechnology Co., Ltd, China). Unexpected antibodies were screened by panel cells (SHPBC). To exclude a false positive result from an IAT, a direct antiglobulin test (DAT) was performed. DAT and unexpected antibody screening were performed with gel card technology in an automated system (Bio-Rad IH-system, Switzerland). After informed consent, genomic DNA was isolated from peripheral blood using the TIANamp blood DNA kit (Tiangen Biotech Co., Ltd, China). RHD zygosity was assessed by sequence-specific primer polymerase chain reaction (SSP-PCR), as previously reported.3 DNA sequencing of RHD exons 1 to 10 and flanking intron regions was performed by the Sanger dideoxy method using a cycle-sequencing kit (Tianjin Super Biotechnology Development Co., Ltd, China). To determine the associated RHCE allele, RHCE genotyping was determined by real-time PCR using allele-specific primers and a commercially available RHCE genotyping kit (Jiangsu ZojiWat Biomedical Co., Ltd, China). This study was approved by the Ethics Committee of the Women and Children's Hospital, School of Medicine, Xiamen University. The RBCs of the proband showed no agglutination with IgM monoclonal anti-D [LHM59/20(LDM3), 175-2] by microcolumn gel card, while the serotyping with IAT using IgM/IgG monoclonal anti-D (D175-2, D415 1E4) and IgG monoclonal anti-D (MS-26) showed a 1+S and 3+S result, respectively. Anti-D monoclonal antibodies in the D-screen identification kit were non-reactive except for P3X21223B10, P3X249 and P3X290 which were reactive (1+ to 3+). This pattern of reactivity seen on the antibody panel (Table 1) was inconclusive according to the manufacturer's interpretation table, but was similar to that reported by Flegel et al. for RHD*DVL1.2 The proband exhibited a partial D phenotype. The RhCE phenotype was C-c+E+e+. The DAT result is negative. The proband had not received transfusion therapy or Rh immunoglobulin prophylaxis. Interestingly, although the proband had two RhD-positive children, the screening test for unexpected antibodies in the proband's plasma was still negative, and no alloanti-D antibody was detected. The result of the RHD zygosity test indicated that the proband was hemizygous (RHD+/RHD−). The RHCE genotype was RHCE*cE/ce. RHD DNA sequencing results revealed a novel c.687_689delAAG variant in exon 5 of the RHD gene (Figure S1), causing a p.Arg229del change in the RhD protein. The c.687_689delAAG variant was not found in the dbSNP, RhesusBase, or ISBT databases. The sequence of this novel RHD allele has been submitted to GenBank under accession number ON745527. The amino acid change at residue 229 was predicted to be located in the vicinity of the RhD protein vestibule and adjacent to the extracellular loop 4. The p.Arg229del could result in the loss of D epitopes. In reviewing previous reports, it was found that RHD*DVL1(c.684_686delGAG) also results in a partial D phenotype due to the p.Arg229del change.2 The in-frame triplet deletion (c.687_689delAAG) can be explained in terms of replication slippage,4, 5 like the previously reported RHD*DVL and RHCE*ceBP. In conclusion, we have identified a novel RHD allele with c.687_689delAAG, p.Arg229del variant, which causes partial D phenotype in a Chinese pregnant woman. This study was supported by grant from the Foundation of Xiamen Science and Technology Bureau (3502Z20209206), Fujian Provincial Health Technology Project (2019-ZQN-32), and Key Clinical Specialty of Fujian Province (Department of Clinical Laboratory at Women and Children's Hospital, School of Medicine, Xiamen University). The authors have disclosed no conflicts of interest. Figure S1. Sequence analysis of the partial exon 5 region of the RHD gene. The variant sequence and the corresponding wild-type sequence are shown in the bottom and top rows, respectively. The arrow indicates a novel c.687_689delAAG variant and the corresponding wild-type nucleotides are underlined. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Figure S1. Direct sequence analysis of partial Exon 7 region in the ABO gene. Direct sequencing revealed a heterozygous sequence (T and C) at nucleotide position 586. Figure S2. Cloning sequence analysis of partial Exon 7 region in ABO gene. The T nucleotide at position 586 is replaced by C. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Background: Here we report a case of para-Bombay phenotype due to a novel mutation FUT1 c.361G>A p.(Ala121Thr) and a nonfunctional allele FUT1*01N.13(c.881_882delTT) which showed a discrepancy in the routine ABO blood group typing. Materials and Methods: The ABO phenotype and the Lewis blood group were typed with serological methods. The ABH antigens in saliva were determined by a hemagglutination inhibition test. The CDS region of ABO, FUT1and FUT2 were amplified with polymerase chain reaction and then directly sequenced. The novel mutation was confirmed by cloning and sequencing. Three-dimensional (3-D) structural analysis of the mutant and wild-type Fut1 were performed by the Chimera software. Results: A, B and H antigens were not detected on the surface of red blood cells (RBCs) by the serological technique, and the B and H blood group substances were detected in the saliva, while the Lewis phenotype was Le(a–b+). Sequencing and cloning analysis showed the presence of a novel FUT1 mutation c.361G>A and a nonfunctional allele FUT1*01N.13(c.881_882delTT). The ABO genotype was ABO*B.01/ABO*O.01.01. The in silico analysis showed that the mutation p.(Ala121Thr) of FUT1did not change the 3-D structure of the whole enzyme but caused a certain amplitude of turnover in the loop region where Ala121 was located. Conclusions: A novel FUT1 allele (FUT1*c.361G>A) was identified in a Chinese individual with para-Bombay B phenotype. The FUT1c.361G>A mutation may significantly downregulate the expression of H antigens on RBCs by damaging the enzyme conformation.

Fig. S1. The results of serology and sequencing. (A) The results of ABO typing by gel card test. (B) The ABO gene Exon 7 partial DNA sequence and cloning results. The upper part demonstrates the heterozygous sequence (C and G) were detected at the c.618. The lower part reveals the sequences of the isolated variant A allele after cloning. The nt. in box denotes the mutation site Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

目的 探讨急性等容稀释性自体输血对围生期妇女免疫功能的影响.方法 选取2016年1月-2018年1月在厦门市妇幼保健院行剖宫产术分娩的孕妇88例为研究对象,采用随机数字表法分为异体输血组和自体输血组,每组各44例.异体输血组在产后或产程即将结束时输入400～1 000ml异体血,自体输血组在产后或产程即将结束时输入400 ～800 ml等容稀释性自体血.比较产前、产后第1天和第5天两组研究对象血常规指标[血红蛋白(Hb)、血小板(PLT)、红细胞(RBC)、红细胞压积(HCT)]、免疫球蛋白(IgA、IgM、IgG)及T细胞亚群(CD3+、CD4+、CD8+、CD4+/CD8+)水平的差异.结果 产前两组研究对象Hb、PLT、RBC、HTC水平比较差异无统计学意义(P＞0.05),产后第1天两组研究对象血常规指标水平低于产前,且异体输血组Hb、RBC、HTC水平均显著低于自体输血组,差异有统计学意义(P＜0.05),产后第5天异体输血组研究对象Hb、RBC、HTC水平仍低于产前(P＜0.05),而自体输血组基本恢复正常,PLT水平均无明显变化.产前两组研究对象IgA、IgM、IgG水平比较差异无统计学意义(P＞0.05),产后第1天两组研究对象免疫球蛋白指标水平均低于产前,且异体输血组IgA、IgM、IgG水平均显著低于自体输血组,差异有统计学意义(P＜0.05),产后第5天异体输血组研究对象免疫球蛋白指标水平仍低于产前(P＜0.05),而自体输血组基本恢复正常.产前两组研究对象CD3+、CD4+、CD4+/CD8+数量比较差异无统计学意义(P＞0.05),产后第1天两组研究对象上述指标均低于产前,且异体输血组CD3+、CD4+、CD4+/CD8+水平均显著低于自体输血组,差异有统计学意义(P＜0.05),产后第5天异体输血组研究对象T细胞亚群数量仍低于产前(P＜0.05),而自体输血组基本恢复正常.结论 急性等容稀释性自体输血对围生期妇女免疫功能无明显抑制作用,是一种安全有效的输血方式.

目的 比较长春博讯(BioXun)、达亚美(Bioad)和戴安娜(Diana)3种不同厂家微柱凝胶抗人球蛋白检测卡检测低效价抗体的敏感度,并与经典试管法进行比较.方法 用3种不同微柱凝胶检测卡和经典试管法同时检测我院门诊159例Rh阴性孕妇的不规则抗体.结果 BioXun、Bioad、和Diana卡测得的不规则抗体真阳性率分别为3.2％、12.6％、31.4％,试管法为11.3％.3种微柱凝胶抗人球蛋白卡检测敏感度组间差异有统计学意义(P<0.05).Diana卡、BioXun卡与试管法检测的敏感度差异有统计学意义(P<0.05);Bioad卡检测与试管法检测的敏感度差异无统计学意义(P>0.05).结论 各种微柱凝胶抗人球蛋白检测卡敏感度有差异,可以购买多种厂家的微柱凝胶卡进行检测,以备必要的时候可以互相验证以免漏检或者出现假阳性.

目的:了解厦门地区RhD阴性孕产妇的血清学表型和抗体产生原因、种类及抗体效价强度对胎儿的影响.方法:通过对2014-01-2014-12进行产检的30 496例孕妇进行RhD阴性的初筛、RhD阴性确诊、Rh阴性表型鉴定、抗体鉴定、抗体效价监测及胎儿治疗情况进行分析.结果:30 496例孕妇初筛检出RhD阴性400例(1.31％),其中不规则D4例,占RhD初筛阴性的1％;确诊RhD阴性的396例中,A型有134例(33.83％),B型有76例(19.20％),O型有160例(40.41％),AB型有26例(6.56％);5种Rh表型所占比例依次为ccdee(57.58％),Ccdee(29.82％),CCdee(8.58％),ccdEe (3.02％),CcdEe(1.00％);检出不规则抗体13例,其中Rh系统抗体为10例(2.53％).结论:通过对厦门地区Rh阴性孕产妇的检测,初步掌握了本地区的RhD阴性孕产妇的血型频率,血清学表型及不规则抗体产生情况,有利于做出合理的孕期监测及诊疗方案,对保证RhD阴性孕产妇的输血安全起到积极的推动作用.

目的 了解厦门孕产妇人群的Rh血型C、c、E、e抗原分布情况,并探讨RhD与RhCE之间的相互关系.方法 使用抗D、抗C、抗c、抗E,抗e血清进行Rh血型分型,对RhD初筛阴性者用间接抗人球蛋白试验确定其阴性表型.结果 在厦门孕产妇(育龄女性)人群中随机检测RhD阳性1000例,Rh表型分布规律为DCCee (49.50％)＞DCcEe (31.50％)＞DCcee (9.50％)＞DccEE (6.50％)＞DccEe (2.00％)＞ DCcEE (0.60％)＞Dccee (0.40％),以DCCee为主;检出RhD阴性396例,表型分布dccee (57.58％)＞dCcee (29.79％)＞dCCee(8.59％)＞dccEe (3.03％)＞dCcEe (1.01％),以dccee为主.而且C、c、E、e抗原在Rh阳性和阴性人群中的分布差异存在统计学意义.结论 了解本地区的育龄女性Rh表型的多态性,制定合理的输血策略,减少输血反应具有重要意义.

目的 调查2014年本院临床用血现状,分析临床输血适应证符合率及用血分布情况,进一步提高本院临床输血的技术水平.方法 查阅715份输血病历,按照相关标准对输血适应证符合率进行统计分析.结果 共输用各类成分血2 243.35 U,适应证符合率为95.9％ (686/715),其中手术科室红细胞、血浆、冷沉淀和血小板适应证符合率分别为91.7％(210/229)、57.9％(11/19)、90.9％(10/11)、100％ (6/6),非手术科室红细胞、血浆、冷沉淀和血小板适应证符合率分别为94.3％(345/366)、100％ (45/45)、100％(30/30)、100％ (9/9);新生儿科用血人数占总输血人数的53.4％ (382/715).结论 本院非手术科室对成分血的输注指征掌握的较好,手术科室欠佳,其中手术科室输用血浆的适应证符合率最低,有待进一步加强血浆的使用管理;新生儿人均用血量不多,但是本院主要的输血人群.

目的:探讨孕妇不规则抗体检测在非ABO新生儿溶血病(HDN)中的意义.方法:用试管法、微柱凝胶法、抗人球蛋白法、盐水法对2 772例孕妇进行血型不规则抗体筛选和鉴定,阳性者进一步检测其抗体特异性,抗体效价测定和免疫球蛋白类型检定.结果:2 772例孕妇中检测25例不规则抗体,其中抗D8例,抗E3例,抗D+C 2例,抗A+抗Ec 1例,抗M4例,抗Mur合并抗C1例,抗Lea 1例,抗LebH 1例,抗HI 1例,其他未检出抗体3例.10例新生儿被来自母体的不规则抗体所致敏,出现不同程度HDN.结论:孕妇不规则抗体的筛选、鉴定、性质类型和抗体效价等检测都有助于非ABO-HDN的早期诊断,评估HDN的严重程度,从而有效的预防并及时治疗核黄疸,水肿等严重并发症有较大意义.