Patients with Asian-type DEL can safely be transfused with RhD-positive blood.

Red blood cells (RBCs) of Asian-type DEL phenotype express few RhD proteins and are typed as serologic RhD-negative (D-) phenotype in routine testing. RhD-positive (D+) RBC transfusion for patients with Asian-type DEL has been proposed but has not been generally adopted because of a lack of direct evidence regarding its safety and the underlying mechanism. We performed a single-arm multicenter clinical trial to document the outcome of D+ RBC transfusion in patients with Asian-type DEL; 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 4045 serologic D- pregnant women throughout China; alloanti-D was found only in individuals with true D- (2.63%, 79/3009), but not in those with Asian-type DEL (0/1032). 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 after transfusion or pregnancy. Therefore, we recommend considering D+ RBC transfusion and discontinuing anti-D prophylaxis in patients with Asian-type DEL, including pregnant women. This clinical trial is registered at www.clinicaltrials.gov as #NCT03727230.

The study of variant s antigen expression revealing a novel c.160C>T (p.Arg54Cys) variant on GYPB*s allele associated with partial s phenotype.

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.

[Establishment of a genotyping method for the junior blood group and identification of a rare blood type with partial DVI.3 and Jr(a-)].

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.

A novel FUT1*c.889C>T allele associated with the para-Bombay AB phenotype and computational evaluation of the mutation effect.

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.

Molecular and serological analysis of the D variant in the Chinese population and identification of seven novel RHD alleles.

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.

Arsenic induces bronchial epithelial carcinogenesis with mitochondrial dysfunction through AKAP95-mediated cell cycle alterations.

Arsenic is a widely existing pollutant in the environment, but the mechanism of occurrence and development of lung cancer by long-term arsenic exposure needs to be elucidated further. How the high and low doses of arsenic induce human bronchial epithelial cell transformation is yet to be elucidated. In the present study, human bronchial epithelial cells were exposed to varying high-dose sodium arsenite (NaAsO2) for the short-term or treated with low dose for long-term. The data showed that both short- and long-term treatment promoted G1/S transition of Beas-2B cells, inducing a significant increase in the expression of AKAP95, cyclin D1, cyclin D2, and cyclin E1. However, silencing AKAP95 by treating cells with siAKAP95 exerted a protective function that inhibited G1/S transition, suggesting a regulatory mechanism of AKAP95 on the cell cycle during cell malignant transformation induced by NaAsO2. In addition, mitochondrial dysfunctions occurred during NaAsO2 exposure. Beas-2B cells exposed to low-dose NaAsO2 for long-term were subcultured for 20 generations, and the exposure time was positively proportional to the growth and migration rate of the cells. The exposed cells were used in a tumor-bearing transplantation experiment (mice), and the results showed that the longer the exposure time, the faster the tumor volume growth rate of As-Beas-2B cells. Tumor tissues were excised for hematoxylin-eosin staining, which showed altered cell morphology and increased volume.

Incidence of anti-D alloimmunization in D-negative individuals receiving D-positive red blood cell transfusion: A systematic review and meta-analysis.

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.

Secondary alloanti-D immunization post transfusion of "Asia type" DEL red blood cells.

BACKGROUND: "Asia type" DEL red blood cells (RBCs) express a very weak D antigen and cannot be detected by routine RhD typing. Thus, it is routinely typed as D-negative (D-) blood group and transfused to D- recipients. Here we described a case of secondary alloanti-D immunization that was associated with transfusion of DEL RBCs to D- recipients and was initially considered as primary alloanti-D immunization. CASE PRESENTATION: A 44-year-old D- woman (G2P2) with adenomyosis and anemia underwent transabdominal hysterectomy. She received four units of D- RBCs before operation. Before transfusion, the alloantibody screening test was negative. Four days after the first transfusion, she needed another RBC transfusion. Unexpectedly, the routine pre-transfusion alloantibody screening test became positive and anti-D (titer, 128-fold) was identified, indicating an alloanti-D immunization. The anti-D developed four days after the first transfusion was unexplained, so alloantibody identification was performed on the sample collected before the first transfusion, and weak anti-D combined with anti-E, which was not detectable during the previous routine pre-transfusion alloantibody screening test with non-enzyme-treated screening cells, was identified using bromelain-treated panel cells. The remaining blood samples of first transfusion in bag tails from two donors were collected for RHD genotyping analysis. One donor was later identified as "Asia type" DEL having RHD* 1227 A/01 N.01 genotype. CONCLUSION: Caution should be applied when we conclude that transfusion of "Asia type" DEL RBCs to true D- recipients could induce primary alloanti-D immunization, especially if the short time interval between transfusion and detection of anti-D is observed.

Preliminary mechanism in fetal alloimmune thrombocytopenia associated with anti-HPA 15b antibodies.

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.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

BACKGROUND: The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia , which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia -positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed. STUDY DESIGN AND METHODS: DNAs were extracted from the whole blood samples of 111 Mia -positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2-4 were performed in the Mia -positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast. RESULTS: The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals. CONCLUSION: The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.

Serological screening and genetic analysis of RhCE variants in the Chinese Southern Han donors.

OBJECTIVES: To screen RhCE variants in the Chinese Southern Han donors for molecular genetic analysis. BACKGROUND: More than hundreds of RhCE variant alleles have been described to resulting in weak and/or partial expression of RhCE antigens, generation of low-prevalence antigens and/or absence of a high-prevalence antigen of Rh system, which mainly reported in the people of African origin. In this study, the serological screening and molecular genetic analysis of RhCE variants were performed in the Chinese Southern Han donors. METHODS: The blood samples of E(+) donors were preliminarily collected. Then, RhCE antigens of the E(+) samples were further typed by using two sets of monoclonal anti-C, anti-c, anti-e and another anti-E. When weak expression of RhCE antigens was found, direct sequencing for 10 exons of RHCE gene, RH genotyping analysis by using multiplex ligation-dependent probe amplification, flow cytometric analysis and even cDNA sequencing were performed. RESULTS: A total of 4487 E(+) samples were collected and four samples with weak expression of antigens were detected. RHCE*Ce375G and RHCE*Ce667T variant alleles were identified in two samples with weak expression of e antigen, respectively. But no variant alleles were found in another two samples with weak expression of C antigen. CONCLUSION: The variant RHCE*Ce375G validated by mRNA sequencing and the deduced RHCE*Ce667T alleles were firstly identified in the Chinese population. The DCE haplotype might account for the weak expression of C antigen in two donors.

Identification of a novel DI*02(2558T) allele associated with weakened expression of DI2 antigen.

BACKGROUND: The distribution of DI1/DI2 antigens of the Diego blood group system is polymorphic in Mongoloid populations and the corresponding alloantibodies are clinically significant. Here a novel DI variant was found by donor screening, and the effect of the novel and previously reported mutations on expression of DI1/DI2 antigens and Band 3 protein was explored. STUDY DESIGN AND METHODS: DNA samples of 1150 Chinese donors were collected. DI*01/DI*02 genotyping was determined by Sanger sequencing. For the carrier of novel allele, the expression of Band 3 and DI1/DI2 antigens on red blood cells (RBCs) was detected by Western blot and flow cytometry, respectively. in vitro expression studies were conducted by transfecting the mutant (including the novel and three reported DI*02(2534T), DI*02(2358_2359insCAC), and DI*02(2572T) alleles) or wild-type DI*02 constructs into HEK 293T cells, the expression of Band 3 and DI1/DI2 antigens was analyzed. RESULTS: A novel heterozygous mutation (c.2558C>T, p.Thr853Met), which is located near the DI1/DI2 polymorphism site (c.2561T>C), was identified in a donor with DI:-1,2 phenotype. Reduced expression of DI2 antigen was observed on the RBCs, while weakened expression of Band 3 and absence of DI2 antigen were detected in cells transfected with the mutant DI*02(2558T) construct. In addition, absent or decreased expression of Band 3 and DI2 antigen was also detected in cells transfected with three reported mutant constructs. CONCLUSION: The novel DI*02(2558T) allele and three previously described DI mutations can affect the expression of Band 3 protein and/or DI2 antigen and/or interfere with DI*01/DI*02 genotyping result.

Distribution of maternal red cell antibodies and the risk of severe alloimmune haemolytic disease of the foetus in a Chinese population: a cohort study on prenatal management.

BACKGROUND: Haemolytic disease of the foetus and newborn (HDFN) is the most common aetiology of haemolytic anaemia and hyperbilirubinaemia in foetuses and neonates. Studies on the distribution of antibodies that cause haemolytic disease of the foetus (HDF) in China are limited, and the effects of multiple antibodies on the severity of HDF need further evaluation. METHODS: An observational cohort study from January 2005 to December 2019 was conducted in two hospitals affiliated with Sun Yat-sen University. Maternal red cell alloimmunization was identified by the Guangzhou Blood Centre. In total, 268 pregnant woman-foetus pairs were divided into four groups according to the type of maternal alloantibodies: anti-D, anti-D combined with other antibodies, other single-antibody and other multiple antibodies. The obstetric history, antibody characteristics, incidence of severe HDF and foetal outcomes were collected and compared. Logistic regression analysis of the risk factors for HDF and survival analysis of the severe HDF-free interval were conducted. RESULTS: Anti-D was the most common cause of HDF, followed by anti-M. No anti-K- or isolated anti-c-associated HDF was found. The incidence of severe HDF was higher in the group with anti-D combined with other antibodies than in the group with anti-D alone (P = 0.025), but no significant difference was found in haemoglobin level and reticulocyte count in the anaemic foetuses between these two groups. Foetuses in the other single-antibody group had a lower reticulocyte count (P = 0.007), more IUTs (P = 0.007) and an earlier onset of severe HDF (P = 0.012). The maximum antibody titre was significantly lower in the other single-antibody group than in the anti-D group (P < 0.001). A high maternal antibody titre (P < 0.001), multiple affected pregnancies (P < 0.001) and other single-antibody (P = 0.042) were independent risk factors for HDF. A higher reticulocyte count (P = 0.041) was an independent risk factor for severe HDF in anaemia foetuses affected by Rh(D) alloimmunization. CONCLUSIONS: The distribution of HDF-associated antibodies in China is different from that in Western countries. Other single non-Rh(D) antibodies could increase the risk of HDF, and anti-D combined with other antibodies would not influence the severity of foetal anaemia compared with anti-D alone.

Hemolytic disease of the fetus and newborn due to alloanti-M: three Chinese case reports and a review of the literature.

BACKGROUND: Alloanti-M was once regarded as not clinically significant, with a few exceptions in extremely rare cases. However, an increasing number of cases of severe hemolytic disease of the fetus and newborn (HDFN), resulting in fetal hydrops and recurrent abortion caused by alloanti-M, have been reported mainly in the Asian population. STUDY DESIGN AND METHODS: Three pregnant Chinese women with a history of abnormal pregnancy with hydrops fetalis were encountered. During this pregnancy, a series of clinical examinations and an alloantibody identification against RBCs and platelets were conducted. Intrauterine transfusion and postnatal transfusion were then performed in the fetuses. In addition, the HDFN cases caused by alloanti-M reported in different ethnic groups as well as their clinical and serologic features are also summarized. RESULTS: Three pregnant women were identified with an M-N+ phenotype and IgM mixed with IgG alloanti-M in serum. Their fetuses were found by ultrasound examination and cord blood testing to have severe anemia. Additionally, an M+N+ phenotype and IgG alloanti-M were detected in the cord blood of the three fetuses with titers ranging from 1:1 to 1:128. Moreover, low reticulocyte counts and negative direct antiglobulin tests were also shown in two of the fetuses. After receiving intrauterine transfusions and postnatal transfusions several times, these three fetuses eventually survived and then healthfully developed in the follow-up tracking. CONCLUSION: Alloanti-M immunization can cause severe HDFN with hyporegenerative anemia, often seen in the Asian population, and suppression of erythropoiesis might account for it.

A variant RhAG protein encoded by the RHAG*572A allele causes serological weak D expression while maintaining normal RhCE phenotypes.

BACKGROUND: The molecular events resulting in a weak D phenotype include missense mutations, in-frame insertion, or deletion mutations of the RHD gene and hybrid RHD-CE-D hybrid alleles. Mutations in genes encoding the proteins that are required for proper membrane expression of Rh proteins, such as RhAG and ankyrin 1, can lead to absent or weakened expression of Rh antigens. STUDY DESIGN AND METHODS: Blood sample from a Chinese blood donor with a serological weak D phenotype was collected. RhAG antigen expression, RhD, and RhCE phenotypes were determined. Analysis of the RHD and RHCE genotypes by RH multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing of the RHD exons, and next-generation sequencing (NGS) of the RHAG and ANK1 exons were performed. Expression studies in vitro were conducted by lentivirally transducing the mutant RHAG*572A or wild-type RHAG, in combination with either RHD or RHCE constructs, into HEK 293 T cells. The expression of RhAG, RhD, and RhCE antigens was analyzed by flow cytometry. RESULTS: Serological weak D and normal C + c- E- e + phenotypes, normal CCDDee genotype determined by RH-MLPA, and normal sequence of the RHD gene by Sanger sequencing were demonstrated. A homozygous variant (c.572G > A, p.Arg191Gln) of the RHAG gene was revealed by NGS analysis. Normal RhAG, weak RhD, and normal RhCE antigens were detected in cells transduced with the mutant RHAG*572A, the mutant RHAG*572A and RHD, and the mutant RHAG*572A and RHCE constructs, respectively. CONCLUSION: The homozygous presence of RHAG*572A allele results in weak D expression. It does not affect RhCE expression.

[Correlation between special A/O genotype and the O phenotype].

OBJECTIVE: To explore the correlation between special A/O genotype and the O phenotype. METHODS: Group O samples with partially reduced or lack of isoagglutinins were collected to determinate the ABO genotype with a PCR-sequence specific primer (PCR-SSP) assay. Seven samples with A/O genotype were selected for further study. Serological tests including forward and reverse typing, H antigen determination and adsorption/elution were carried out with a tube method. Genomic DNA was genotyped by amplifying and sequencing of the coding regions of exons 1 to 7 of the ABO gene. RESULTS: Seven samples were serotyped as group O by the forward typing test. However, reduced anti-A activity was found in 5 samples by the reverse typing test, reduced anti-A and anti-B activities were found in 1 sample, and no anti-A isoagglutinin activity was found with 1 sample. H antigen was determined in all samples by routine serologic method. Neither anti-A nor anti-B was eluted from red cells derived from all samples. Three samples were genotyped as Ael02/O02, whilst the remainders were Ael02/O13, Ael02/O65, Am04/O75, Ael06/O02, respectively. CONCLUSION: Special A/O genotype may not express the A antigen, leading to the generation of group O red cells. Reduced or missed anti-A activity is the typical serological feature of this special group of O phenotype, for which ABO*Ael02 and ABO*O02 are the major alleles. Group O individuals with isoagglutinin detection problem should be grouped by serological tests and genomic DNA analysis.

Genotyping analysis of MNS blood group GP(B-A-B) hybrid glycophorins in the Chinese Southern Han population using a high-resolution melting assay.

BACKGROUND: MNS hybrid GP(B-A-B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B-A-B) hybrid glycophorins based on high-resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population. STUDY DESIGN AND METHODS: DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B-A-B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation-dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles. RESULTS: A total of five GYP(B-A-B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B-A-B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild-type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5' inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single-nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles. CONCLUSION: The HRM assay could distinguish the GYP(B-A-B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B-A-B) genotyping kits for the Chinese population.

A Broad Blockade of Signaling from the IL-20 Family of Cytokines Potently Attenuates Collagen-Induced Arthritis.

Two heterodimeric receptors consisting of either IL-20R1 or IL-22R1 in complex with a common ß receptor subunit IL-20R2 are shared by three of the IL-20 family of cytokines: IL-19, IL-20, and IL-24. These proinflammatory cytokines have been implicated in the pathogenesis of some autoimmune diseases, including rheumatoid arthritis (RA), psoriasis, and atopic dermatitis. Although mAbs against IL-19 and IL-20 have each been shown to modulate disease severity of collagen-induced arthritis in animal models, and anti-IL-20 therapeutic Ab has exhibited some efficacy in the treatment of RA in clinical trials, benefits for a complete blockade of these functionally redundant cytokines remain to be explored. In this report, we show that recombinant human soluble IL-20R2-Fc fusion protein binds to IL-19, IL-20, and IL-24 with similar high affinity and blocks their signaling in vitro. In DBA/1 mouse collagen-induced arthritis model, recombinant human IL-20R2-Fc exhibits comparable efficacy as TNF blocker etanercept in the treatment of established arthritis, whereas the combined use of both biologics manifests little synergistic therapeutic effects. In situ ligand-receptor functional binding analysis shows that a large amount of immune infiltrates expressing high levels of TNFR and IL-20 subfamily cytokines congregate within the inflamed disease tissues. Colocalization experiments reveal that signals from IL-20R2 and TNF transduction pathways seem to converge in macrophages and function in tandem in orchestrating the pathogenesis of RA. Elucidation of this interaction provides a better understanding of cytokine cross-talk in RA and a rationale for more effective biologic therapies that target IL-20R2 instead of individual cytokines from IL-20 family.