Objective To develop a solid-phase red cell adherence(SPRC A)reagent kit for unexpected alloantibody i-dentification.Methods A solid-phase carrier for bounding red blood cell(RBC)was prepared by coating microstrip with succinimide,then panel cell suspensions were dispensed to microwells to produce RBC monolayers,after that,1×PBS(pH7.40)was used to lysis the RBC monolayer to form stroma monolayer.Prior to the drying procedure,drying medium suspension was added to microwell to protect stroma monolayer.The solid-phase microplate was successfully produced after the stroma had been dried in refrigerator at 4℃.IgG anti-E and anti-Fya with low titer were tested every two months with the reagent kit to observe its stability.Seventy-two plasma samples containing unexpected antibodies and 152 normal plasma samples were determined with SPRCA reagent kit and column agglutination technique(CAT).Performances including sensi-tivity and specificity of the two reagents were compared by paired chi-square test.Results RBC stroma monolayer could be attached well on microwell bottom coated with succinimide.Drying medium suspension could provide effective protection for stroma monolayer.Both IgG anti-E and anti-Fya with low titer were determined well by the reagent kit during the storage peri-od.Of the 72 positive samples,70 were identified correctly by SPRCA kit and 71 by CAT[Sensitivity:97.22％(70/72)vs 98.61％(71/72)].Of the 152 negative plasma samples,149 were correctly detected by SPRCA kit and 152 by CAT[Spe-cificity:98.02％(149/152)vs 100％(152/152)].Paired chi-square test showed no significant differences between the two reagents(P＞0.05).Conclusion A SPRCA reagent kit for IgG unexpected antibody identification had been developed suc-cessfully.

Objective To investigate the distribution,phenotype and genotype of D-elute type(Del)in blood donors with RhD negative blood in Guangzhou,so as to understand the molecular biological background of DEL blood group in this area.Methods During the period from November 1,2021 to June 30,2022,the RhD-negative blood initially screened by saline method was confirmed by indirect anti-human globulin test(IAT)serology,and RhCE phenotype was determined by RhCE typing card.A total of 1 146 RhD-negative samples,including all RhD-negative samples with RhCE C+(n=459)and a randomly selected subset of RhCE C-(n=175),were subjected to adsorption-elution(Del)screening(a total of 634 sam-ples).DNA from Del-positive samples was extracted for real-time fluorescent PCR detection of the RHD gene c.1227 locus using high-resolution melting curve analysis(HRM).For samples without mutations detected at the RHD∗1227 locus by HRM,restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP)was performed to amplify the prod-uct which was subsequently digested with Pst I enzyme and analyzed by electrophoresis to determine RHD gene haplotypes.Sanger sequencing was performed for exon sequencing(exon 1-10)of the RHD gene,and gene mutations were analyzed u-sing SeqMan software.Suspected Del-positive samples were subjected to RHD whole gene analysis using third-generation sin-gle-molecule sequencing technology.Results Among the 634 confirmed RhD-negative samples,229(36.1%)displayed Del phenotype,accounting for20%(229/1 146)of the total confirmed RhD-negative samples.The RhCE phenotypes of the 229 DEL cases were as follows:Ccee in 181 cases,CCee in 40 cases,CcEe in 7 cases,and ccEe in 1 case.HRM combined with RHD haplotype analysis showed that there were 170 cases with RHD gene as RHD∗1227A/01N.01,32 cases with RHD gene as RHD∗1227A/1227G,26 cases with RHD gene as RHD∗1227A/1227A,and 6 cases with RHD gene as RHD∗1227G/1227G(sequencing results included 1 case of weak D type 12,4 cases of D-,and 1 case of RHD∗01EL.02).Con-clusion The individual genotype of DEL blood donors in Guangzhou area is mainly characterized by RHD∗1227A/01N.01,and their RhCE phenotypes are all C+.HRM can be used as a molecular biology method for routine screening of Asian-type DEL blood type genes.

TransfusionEarly View ONE-PAGE REPORT OF NEW ALLELES OR ANTIGENS Identification of a novel A4GALT*299A allele associated with the rare p phenotype in one Chinese family Chunyan Mo, Chunyan Mo orcid.org/0009-0003-4943-9524 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorShuangshuang Jia, Shuangshuang Jia orcid.org/0000-0002-0092-8576 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorGuangping Luo, Guangping Luo Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorYanli Ji, Corresponding Author Yanli Ji [email protected] Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China Correspondence Yanli Ji, 31 Luyuan Road, Yuexiu District, Guangzhou, 510095, China. Email: [email protected]Search for more papers by this author Chunyan Mo, Chunyan Mo orcid.org/0009-0003-4943-9524 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorShuangshuang Jia, Shuangshuang Jia orcid.org/0000-0002-0092-8576 Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorGuangping Luo, Guangping Luo Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, ChinaSearch for more papers by this authorYanli Ji, Corresponding Author Yanli Ji [email protected] Institute of Clinical Blood Transfusion, Guangzhou Blood Center, Guangzhou, China Correspondence Yanli Ji, 31 Luyuan Road, Yuexiu District, Guangzhou, 510095, China. Email: [email protected]Search for more papers by this author First published: 19 March 2024 https://doi.org/10.1111/trf.17795Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. CONFLICT OF INTEREST STATEMENT The authors have disclosed no conflicts of interest. REFERENCES 1Di Ciaccio P, Cutts B, Alahakoon TI, Dennington PM, Soo LA, Curnow J. Clinical consequences of the extremely rare anti-PP1Pk isoantibodies in pregnancy: a case series and review of the literature. Vox Sang. 2020; 116: 591–600. 10.1111/vox.13042 PubMedGoogle Scholar 2Westman JS, Stenfelt L, Vidovic K, Moller M, Hellberg A, Kjellstrom S, et al. Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT. Blood. 2018; 131: 1611–1616. 10.1182/blood-2017-08-803080 CASPubMedWeb of Science®Google Scholar 3Hellberg A, S-MA, Reid ME, Olsson ML. Expression of a novel missense mutation found in the A4GALT gene of Amish individuals with the pphenotype. Transfusion 2007; 48: 479–487. 10.1111/j.1537-2995.2007.01552.x PubMedGoogle Scholar 4Daniels G. Human blood groups. 3rd ed. Hoboken, NJ: Wiley-Blackwell; 2013. 10.1002/9781118493595 Google Scholar 5Wei L, Ji YL, Luo H, Mo CY, Zhang RQ, Zhao Y, et al. Serological and genetic study of a pedigree featuring a rare p phenotype [article in Chinese]. Zhonghua Yi Xue Yi Chuan Xue Za Zhi. 2012; 29: 701–704. CASPubMedGoogle Scholar Early ViewOnline Version of Record before inclusion in an issue ReferencesRelatedInformation

OBJECTIVE:To develop a genotyping method for the Junior blood type and report on a rare blood type with Jr(a-).METHODS:Healthy O-type RhD+ volunteer donors of the Shenzhen Blood Center from January to May 2021 (n = 1 568) and a pedigree with difficult cross-matching (n = 3) were selected as the study subjects. Serological methods were used for proband's blood type identification, unexpected antibody identification, and antibody titer determination. Polymerase chain reaction-sequence specific primer (PCR-SSP) method was used for typing the proband's RhD gene. ABCG2 gene coding region sequencing and a PCR-SSP genotyping method were established for determining the genotypes of the proband and his family members and screening of Jra antigen-negative rare blood type among the 1 568 blood donors.RESULTS:The proband's ABO and RhD blood types were respectively determined as B and partial D (RHDDVI.3/RHD01N.01), Junior blood type Jra antigen was negative, and plasma had contained anti-D and anti-Jra. Sequencing of the ABCG2 gene revealed that the proband's genotype was ABGG201N.01/ABGG201N.01 [homozygous c.376C>T (p.Gln126X) variants], which is the most common Jr(a-) blood type allele in the Asian population. Screening of the voluntary blood donors has detected no Jr(a-) rare blood type. Statistical analysis of the heterozygotes suggested that the allelic frequency for ABCG2*01N.01 (c.376T) was 0.45%, and the frequency of Jr(a-) rare blood type with this molecular background was about 0.2‰.CONCLUSION:A very rare case of partial DVI.3 type and Jr(a-) rare blood type has been identified. And a method for identifying the Junior blood type through sequencing the coding regions of the ABCG2 gene and PCR-SSP has been established.

Objective To identify the specificity of alloantibody against high-frequency antigens in one case suffering with severe hemolytic diseases of the fetus and newborn(HDFN)and to screen for matching blood for transfusion.Methods The HDFN test and the antibody serological identification tests in the mother were performed.Several common high fre-quency antigens of maternal red blood cells(RBCs)were determined.IgG subtype coated on the RBCs of the newborn was determined.The phagocytic efficiency of the antibody was tested using the monocyte phagocytosis of sensitized erythrocyte by flow cytometry in vitro.Sanger sequencing of DI gene was performed in the mother,father and mother's brother.The diluted maternal plasma was used for large scale screening of matching blood using IAT in Coomb's gel card.Results Di(b-)phenotype was identified in the mother of the newborn and anti-Dib(titer:512)related HDN was detected in the newborn.IgGl and IgG2 subtypes of anti-Dib were detected and the rate of monocyte phagocytosis was 88.83％(74.7/84.09).The compatible blood was not detected in the maternal relatives.Subsequently,the newborn received the matching RBCs of two Di(b-)donors identified from 5 520 blood donors and discharged from the hospital.We screened out 17 Di(b-)donors out of 51 334 blood donors,indicating that the distribution frequency of Di(b-)among blood donors in Guangzhou was about 0.033％(17/51 334).Conclusion By serology and molecular biology methods,the newborn was identified with HDFN caused by anti-Dib,and an effective large-scale screening method for Di(b-)rare blood types was established to find matching blood,which supported the establishment of rare Di(b-)blood database.

BACKGROUND:Little s antigen is mainly defined by a single nucleotide polymorphism at c.143C (p.Thr48) on the GYPB gene. Several variants on GYPB can alter the expression of s antigen. The aim of this study was to investigate the molecular basis of variant s antigen expression in the Chinese population.STUDY DESIGN AND METHODS:A total of 4983 whole blood samples were collected to screen the individuals with discrepant s typing results using two different monoclonal anti-s. Then, the sequence of GYPB exon 4 was analyzed by Sanger sequencing. Flow cytometry analysis was performed to quantify s antigen expression on red blood cells (RBCs). In vitro expression study was performed to verify the effect of the GYPB variants identified on the expression of s antigen.RESULTS:Four donors were identified to have discrepant s typing results. Sanger sequencing showed that three donors carried the c.173C > G variant (p.Pro58Arg) specific for sD antigen, the other one carried a novel GYPB (c.160C > T, p.Arg54Cys) variant. Flow cytometry identified a partial and weak expression of s antigen on the RBCs of the four donors. Furthermore, in vitro expression study confirmed the effect of the two variants on the s antigen expression.CONCLUSION:The results demonstrated that in addition to p.Thr48, the two extra amino acids p.Arg54 and p.Pro58 are also important for full expression of s antigen. Since the individuals with partial s antigen are at risk for the development of alloanti-s, it is important to select at least two different monoclonal anti-s for correct s typing.

BACKGROUND:Mutation in the FUT1 gene can impact the structure and function of α-(1,2)-fucosyltransferase 1 (α2FucT1). To explain the para-Bombay phenotype of a novel FUT1 allele, three-dimensional (3D) modeling and mutation effect analysis of α2FucT1 were performed by bioinformatic tools.MATERIALS AND METHODS:Blood and saliva samples were collected from a patient who was suspected to be a para-Bombay phenotype. H, A, and B antigens were determined with routine serologic methods for those samples. FUT1 and FUT2 coding regions were determined by Sanger sequencing. The novel heterozygous mutation was confirmed by cloning and sequencing. 3D model of mutant α2FucT1 was built by Phyre 2 and the mutation effect was evaluated by Chimera, PROVEAN, and Polyphen-2.RESULTS:Weak H, A, and B antigens were detected on RBCs of the proband and normal quantities of H, A, and B antigens were observed in his saliva. Cloning sequencing showed that the proband carried a novel FUT1 allele (c.889C>T, p.Leu297Phe) and a null FUT1*01N.06 allele. 3D model showed that the p.Leu297Phe variant in α2FucT1 reduced the number of hydrogen bonds and the mutation effect was predicted to be deleterious and possibly damaging, which suggested that the conformation and activity of the enzyme might be significantly damaged.CONCLUSION:A novel missense mutation led to an amino acid variant p.Leu297Phe in α2FucT1, which was a potential cause of the inactivation of the enzyme. Computational evaluation was a convenient and useful approach for the mutation effect analysis of the enzyme.

Objective To study the effect of RHAG variants identified in Chinese population on mRNA splicing by minigene splicing assay(MSA) in vitro. Methods The pSplicePOLR2G minigene expression plasmids were constructed for 10 RHAG mutations with relatively high distribution frequency in Chinese population near splicing sites or synonymous mutations by analyzing the RHAG gene data in the KMxD database. Then, the wild-type and mutant plasmids were transfected into HEK 293T cells, and RNA was extracted 48 hours after transfection. After reverse transcription, specific primers were used for PCR amplification, and then agarose gel electrophoresis and capillary electrophoresis were performed to determine whether the mutations will affect the normal splicing of exons. Results MSA in vitro showed that 2 mutations (c.158-5delT, c. 807+ 3A>C) near the splicing site reduced the amount of normal transcripts slightly. The remaining 8 synonymous mutations(c.312G>A, c. 341+ 3G>A, c. 609C>T, c. 681G>A, c. 861G>A, c. 957T>A, c. 984T>C and c. 1139-7G>A) had no impact on the splicing of RHAG mRNA. Conclusion This study showed that RHAG gene was conservative in terms of splicing, and the mutations near splicing sites and synonymous mutations were less likely to cause abnormal splicing of RHAG gene.

Objective To solve the difficulty of RhD blood group typing in a patient with double population(DP) of red blood cells for RhD antigen by serological and genotyping analysis. Methods Separation of the two populations of red blood cells of the patient was performed using capillary centrifugation method. ABO, RhD and RhCE typing, direct anti-human globulin test (DAT), irregular antibody screening, antibody identification and blood crossmatching of the patient were conducted using the standard serological methods. The hybrid Rhesus zygosity analysis of the RHD gene was performed by PCR-RFLP method. RHD and RHCE genotype of the patients were identified by PCR-SSP method. Results The patient was B type but with DP of red blood cells for RhD, Rhc and RhE antigens. DAT of the patient was positive and the alloanti-D was detected in serum. The RHD zygosity was D-/D- homozygote. PCR-SSP testing showed the RHD gene deletion (RHD * 01N. 01/01N.01 genotype) and Ccee of RHCE genotype in the patient, which was consistent with RHD zygosity analysis. Conclusion This is a special case with D-negative phenotype which was wrongly detected as D-positive type after D-positive red blood cells transfusion in emergency. When the DP of red cells for D antigen encountered like this case, the RhD typing can be accurately determined by using RHD genotyping analysis to provide strong evidence to the clinical blood transfusion.

Red blood cells (RBCs) of the Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) in routine testing. RhD-positive (D+) RBC transfusion for Asian-type DEL patients has been proposed but has not been generally adopted due to a lack of direct evidence regarding its safety and underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in Asian-type DEL patients; none of the recipients (0/42; 95% confidence interval, 0%-8.40%) developed alloanti-D after a median follow-up of 226 days. We conducted a large retrospective study to detect alloanti-D immunization in 4,045 serologic D- pregnant women throughout China; alloanti-D was found only in true D- individuals (2.63%, 79/3,009), but not in those with Asian-type DEL (0/1,032). We further retrospectively examined 127 serologic D- pregnant women who had developed alloanti-D and found none with Asian-type DEL (0/127). Finally, we analyzed RHD transcripts from Asian-type DEL erythroblasts and examined antigen epitopes expressed by various RHD transcripts in vitro, finding a low abundance of full-length RHD transcripts (0.18% of the total) expressing RhD antigens carrying the entire repertoire of epitopes, which could explain the immune tolerance against D+ RBCs. Our results provide multiple lines of evidence that individuals with Asian-type DEL cannot produce alloanti-D when exposed to D+ RBCs following transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in Asian-type DEL patients, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as NCT03727230.

Objective To establish a high-throughput detection method for ABCG2*376T allele of Jr(a-), and apply it to the study of the frequency of this allele in the Chinese population. Methods The specific primers were designed and synthesized, the sample carrying homozygous ABCG2*376T alleles, obtained in the previous study, was used as the homozygous positive control, and the sample carrying heterozygous allele as the heterozygous positive control. The wild-type sample was used as a negative control, and a high-resolution melting curve(HRM) method for detecting this allele was established. The established method was used to screen DNA samples from blood donors in Guangzhou, and the samples carrying ABCG2*376T alleles were sequenced to confirm the accuracy of the HRM method. Results A HRM method, which can detect ABCG2*376T allele and accurately type homozygotes and heterozygotes at the same time, had been established successfully. Fifteen individuals with heterozygous alleles were screened out of 1 560 blood donors in Guangzhou, while none homozygous allele was detected. Conclusion The HRM method can be used to accurately screen and type ABCG2*376T allele. The frequency of this allele in Chinese population is about 0.48%(15/3120).

目的 制备能用于Mur抗原血清学检测的抗-Mur试剂,并将其应用于献血者标本的筛查中.方法 采用偶联了载体蛋白——钥孔血蓝蛋白(KLH)的Mur抗原特异性多肽免疫BALB/c小鼠,通过淋巴细胞杂交瘤技术获得分泌抗体的细胞株,用Mur抗原阳性细胞筛选阳性克隆,利用谱细胞鉴定单克隆抗体的特异性,利用制备的单克隆抗体对1 062例献血者标本的Mur抗原进行检测.结果 经筛选及鉴定后共获得4个能稳定分泌抗-Mur的单克隆,均为IgM型Kappa轻链,效价分别为64、128、256和256.制备的抗-Mur单克隆抗体与日本惠赠的人源单克隆抗-Mur分别检测了 1 062例献血者标本,其检测结果符合率为100％.结论 成功建立了能稳定分泌抗-Mur单克隆抗体的杂交瘤细胞株,可用于临床上Mur抗原的血清学快速检测.

Objective To identify the antibody specificity in a pregnant women who had no history of blood transfusion but presented the antibodies against high-frequency antigens. Methods ABO, RhD blood group antigens were identified by saline. Antibody screening and identification were performed by saline and indirect Coomb’s technique. Further antibody identification tests were conducted using papain, trypsin and chymotrypsin-treated cells. Antibody titer in serum was tested. PCR amplification and sequencing analysis of 16 exons of ABCG2 gene were conducted. Results The blood type of the patient were B, RhD positive. The serum reacted with antibody screening/identified cells by indirect antiglobin test(both 2+ ) but not by saline. The agglutination was enhanced after papain treatment (4+ ), but remained unchanged after trypsin and chymotrypsin treatment (2+ ). The IgG titer was 1∶2. The sequencing analysis of ABCG2 gene revealed a homozygous nonsense mutation(c.376C>T, p. Gln126X) in exon 4 of the women. Conclusion In this case, the development of anti-Jra in Jr(a-) mother was stimulated by mother-child serology incompatibility during pregnancy.

Background and Objectives The transfusion of D-negative red blood cells (RBCs) to D-negative patients has been widely adopted to prevent anti-D alloimmunization, especially in women of childbearing age. Still, transfusion of D-positive RBCs to D-negative recipients is occasionally inevitable in practice, and the resulting incidence of anti-D in different D-negative groups of patients has not been well summarized. Materials and Methods We searched the relevant literature using PubMed, Cochrane Library, and Embase databases from inception date to 30 September 2021. We looked for studies of anti-D occurring in D-negative recipients who received D-positive RBC transfusions. The anti-D incidence was summarized with 95% confidence intervals (CIs). Data with similar characteristics were combined using a random-effects model. Results About 42 studies (2226 cases), which found anti-D, the exact volume of D-positive RBC transfused, and the follow-up time for anti-D detection, met the inclusion criteria. The pooled anti-D incidence was 64% (95% CI, range 55%-74%) in volunteers receiving small volumes of D-positive RBCs, 84% (95% CI, 74%-94%) in those receiving whole units, 26% (95% CI, 19%-32%) in mixed patients, 12% (95% CI, 8%-16%) in oncology patients, 27% (95% CI, 13%-40%) in trauma patients, 4% (95% CI, 0%-8%) in immune-compromised transplant patients, and 6% (95% CI, 1%-39%) in those with AIDS. Conclusion Compared with the high frequency of anti-D in healthy D-negative volunteers given D-positive RBCs, we found a lower rate of anti-D immunization in various D-negative patients and almost none in transplant and AIDS patients.

Background: Common alloantibodies leading to severe hemolytic disease of the fetus and newborn (HDFN) could vary among different ethnic groups. The MNS blood group hybrid glycophorin GP.Mur distributes with a high frequency in the regions of Southeast Asia. Alloantibodies against GP.Mur (anti-‘Mia’) often present as mixture of antibodies against several low frequency antigens. In this study, we first described a case of severe HDFN in Guangzhou, China, which was caused by alloantibodies of anti-E in combination with specificities to the GP.Mur including Mia, Mur and Hil. Methods: Blood samples from the newborn boy and parents have been subjected to antibody screening and identification analysis followed by GYP*Mur genotyping. The direct antiglobulin test (DAT) and the eluate technique were also performed for the newborn. Results: The mother was group B, CCDee, Mur−, the father was group B, ccDEE, Mur+, and the newborn was group B, CcDEe, Mur+. Genotyping results showed the mother was absent for GYP*Mur, while the father and the newborn carried heterozygous GYP*Mur allele. DAT test of the newborn was strongly positive with anti-IgG. Anti-E and anti-‘Mia’ were detected in the maternal serum and the newborn’s eluate, whereas anti-E alone was detected in the newborn’s serum. The anti-‘Mia’ specificity was further identified as combination of anti-Mia, anti-Mur and anti-Hil. Conclusions: Because alloantibodies to GP.Mur could cause severe HDFN, it is highly recommended to include GP.Mur red cells in antibody screening cells to avoid miss detection of the alloantibodies in the populations of Southeast Asia.

Objective Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a bleeding disease that can cause fetal hydrops, a rare but life-threatening condition in which abnormal amounts of fluid accumulate in one or two areas of the fetus's body. A case of FNAIT with fetal hydrops caused by anti-HPA-15b antibodies was involved in this study, as we investigated whether or not anti-HPA-15b antibodies can induce endothelial angiogenesis and apoptosis. Methods The monoclonal antibody immobilization of platelet antigens assay (MAIPA) was used to identify anti-HPA-15b antibodies. The three groups in Tube formation and apoptosis assays were the PBS group, the AB serum IgG group, and the anti-HPA-15b serum IgG group, all reacted with HPA-15bb HUVEC. Results The presence of anti-HPA-15b antibodies was found in this case by MAIPA assay. The OD values are 0.33 and 0.21, reacted with HPA-15bb and HPA-15ab platelets, respectively (cutoff OD value = 0.2). Quantitative analysis revealed that the length of capillary-like tube induced by anti-HPA-15b antibodies was significantly decreased over that of AB serum IgG (*p = 0.0005), but weaker than when incubated with thrombin (**p = 0.0009). The apoptosis results show a significantly increased number of apoptotic endothelial cells in the anti-HPA-15b antibody IgG group when compared with the PBS and AB serum IgG groups (*p < 0.0001, **p < 0.0001). In addition, there is no statistical difference between the PBS and AB serum groups. Conclusion Anti-HPA-15b antibodies can inhibit angiogenesis and induce apoptosis. This may associate with hydrops fetalis (HF), or fetal hydrops of FNAIT.

目的 探讨罕见的抗-Chido/Rodgers抗体的鉴定方法并分析其特性.方法 应用试管法鉴定患者ABO血型,Rh分型卡及IAT法鉴定患者Rh血型;对患者血清进行不规则抗体筛选及抗体鉴定;进一步将患者血清分别与用胰酶和菠萝酶处理的谱细胞进行反应,初步判定其抗体的特异性;最后用混合新鲜献血员血浆对患者血清进行中和抑制实验,鉴定抗体.结果 患者血型为O型,Ccdee;患者血清仅与2个谱细胞不反应、2个谱细胞弱阳性反应,与其余谱细胞反应凝集强度均为2+;与胰酶和菠萝酶处理后的谱细胞均不反应;患者血清与新鲜血浆混合孵育后,与谱细胞反应均为阴性,其血清中的抗体可被新鲜血浆中和,经判定为抗-Chido/Rodgers抗体.结论 经鉴定疑似属于Chido/Rodgers血型系统抗体.当患者血清中不规则抗体与大多数或者全部谱细胞反应,且反应强度不一致,但与不同的酶处理后的谱细胞反应均为阴性时,应考虑是否是抗-Chido/Rodgers抗体,可应用混合新鲜血浆对其进行中和抑制实验进一步鉴定.

Objective To establish an experimental method for detecting phagocytosis of sensitized red blood cells in vitro by flow cytometry. Methods Mononuclear cells were isolated from the peripheral blood of blood donors and cultured in a cell incubator for 1 hour, and then adherent monocytes were isolated and obtained. Dib-positive red blood cells (RBCs) were labeled with PKH26 and then sensitized with IgG anti-Dib. The sensitized RBCs were added to monocytes for in vitro phagocytosis assay. Monocytes were labeled with FITC anti-human CD14, then phagocytosis was measured by flow cytometry, and the phagocytic efficiency was calculated. The method was used to detect the phagocytic efficiency of monocytes on human IgG anti-D sensitized RBCs with different titers. Results The phagocytic efficiency of monocytes was averaged at 5% (1.2%~7.6%, SD 3.30) versus 81% (71.4%~92.7%, SD 8.65) in the negative versus positive control group, respectively. Phagocytic activity of monocytes mediated by anti-D was correlated with the antibody titer. The phagocytosis efficiency was within 10% when the antibody titer was lower than 32 and increased sharply when the titer was between 32 to 128, it entered a plateau and stabilized at 80% at the titer above 256. Conclusion A detection platform for detecting phagocytosis-sensitized RBCs in vitro by flow cytometry has been successfully established. It can be used to assess the clinical significance of red blood cell allotype or autologous IgG antibodies.

BACKGROUND:The molecular basis of the D variant phenotype in the Chinese differs greatly from that of the Caucasian. Adapting a specific D typing strategy to the spectrum of prevalent RHD variant alleles is necessary.STUDY DESIGN AND METHODS:Blood samples with ambiguous D phenotypes were collected in the Southern Chinese population. A special three-step typing strategy was applied. First, the common DVI type 3 was identified from epitope profiles of D antigen. Then, another common weak D type 15 (RHD*845A) was identified by epitope profiles of D antigen and Sanger sequencing of RHD exon 6. Finally, the remaining D variants were genotyped mainly by Sanger sequencing. For the novel RHD alleles in the coding region and exon-intron junction, in vitro transfection and minigene splicing assays were performed, respectively. The anti-D investigation was performed.RESULTS:DVI type 3 (65/253, 25.7%) and weak D type 15 (62/253, 24.5%) were common Chinese D variants, and RHD*960A, DFR, RHD*weak D type 25, 72, and 136 were frequent variant RHD alleles. Besides, twenty-two sporadic and seven novel RHD alleles (RHD*188A; RHD*688C; RHD*782 T; RHD*1181C; RHD*165 T, 993A; RHD*148 + 3G > T and RHD*1227 + 5G > C) were identified. The deleterious effect of the novel RHD alleles on D antigen or mRNA expression was confirmed. Anti-D was detected in two DVI type 3 pregnant women.DISCUSSION:The three-step typing strategy provides an effective approach for Chinese D variant typing. It can be anticipated that commercially available RHD genotyping kits have limitations for testing Chinese D variants, as some of the frequent variants are not interrogated.

Objective To identify three cases of pregnant women with the D variant phenotype using serological and molecular tests, and discuss the strategy of prenatal examination. Methods The peripheral blood samples from three pregnant women with the D variant phenotype were collected. RhD variant phenotype was determined using routine serological methods with two different kinds of monoclonal anti-D. The serological characteristic for the epitope of D antigen was further analyzed using the commercial panel anti-D reagents (D-Screen, Diagast). The hybrid RHD-CE-D allele was analyzed by the Multiplex Ligation-dependent Probe Amplification (MLPA) assay and polymerase chain reaction with sequence specific primers (PCR-SSP) method. Further Sanger sequencing of RHD gene exons was also performed. Results DFR phenotype was primarily determined by serological characteristic for the epitope of D antigen. RHD*DFR2/01N.01(n=2) and RHD*DFR1/1227A(n=1) genotypes were identified by the MLPA assay, PCR-SSP and Sanger sequencing. Conclusion Two pregnant women with RHD*DFR2/01N.01 genotype should be treated as D negative patients clinically, while the pregnant woman with RHD*DFR1/1227A genotype can be treated as Asia type DEL to avoid unnecessary antibody screening and anti-D prophylaxis.