JAHK: a low frequency antigen associated with the rG complex of the Rh blood group system

We have investigated a 'new' low frequency antigen JAHK, which is a marker for the rare Rh gene complex rG. The rG haplotype does not produce any D, c or E antigens, but does produce a strong G antigen. The rG haplotype [d(C)(e)G] is associated with weak C and weak e antigens. Three unrelated rG/dce individuals and one rG/rG propositus were JAHK+. In addition, three propositi whose red cells had a typical expression of C and/or e antigen, which could not be shown to be rG because of a normal D antigen produced by the haplotype in trans, were also JAHK+. Families of three of the propositi demonstrate the inheritance of JAHK as a Mendelian dominant character. It is likely that the JAHK antigen results from conformational changes in an RhCcEe protein that has the amino acid characteristic of c antigen at position 16 and the amino acid residues characteristic of C antigen at positions 60, 68, and 103. JAHK has been assigned the number RH53.     

Rh sytem. Genetics and function.

Little is known about the exact genetic control of the Rh system. The Rh antigen is a large protein molecule carrying many antigenic determinants. The structural genes controlling its production comprise a gene complex that has probably evolved by unequal crossover and mutation at the duplicated points. No convincing evidence for crossing-over within the gene complex has been found since the discovery of Rh. Studies of Rhnull families provide evidence for unlinked control genes that influence the ability of the structural Rh genes to function. It was hoped that the Rhnull bloods would provide information to establish the function of the Rh antigen, because the antigen appears to be necessary to maintain the integrity of the red cell membrane. However, the abnormalities of the Rhnull cells (increased Na+K+ pumps) have not been tied directly to their lack of Rh antigens.     

Murine monoclonal antibodies associated with Rh17, Rh29, and Rh46 antigens

After immunization with native human red cells and red cells infected with Plasmodium falciparum, mouse splenocytes were fused with a myeloma cell line to obtain hybridomas. Among the antibodies specific for blood group antigens, four antibodies (iB3C4, F12, MR432, and iB4) directed against epitopes related to the Rh antigen were selected for characterization. It is suggested that two monoclonal antibodies (iB3C4 and F12) recognized an epitope associated with the Rh29 antigen. The specificity of the monoclonal antibody iB4 seemed related to the CcEe series of antigens. MR432 did not react with red cells of people who are homozygous for the RN gene, and seemed to identify an epitope associated with the Rh46 antigen (recently numbered).     

中国汉族Rh血型基因多态性观察

为了观察中国汉族非血缘关系随机个体和家系Rh血型的基因多态性,采用聚合酶链反应一序列特异性引物(PCR—SSF)扩增技术,扩增Rh血型C／E基因,D基因外显子,内含子2和10,插入片段以及RhBox,检测160例Rh阳性个体,71例Rh阴性个体及7个先证为Rh阴性的家系的血样品。研究结果显示,带有C抗原的RhD阴性个体D基因结构具有多态性,D基因外显子有完整的、部分缺失和完全缺失的3种形式;先证为RhD阴性的家系中,大部分成员均出现插入片段和RhBox,且在遗传上符合孟德尔遗传定律,插入片段或RhBox并未影响D基因的表达;全部RhD阴性和阳性个体中没有发现“正常的”RhD外显子4。结论：中国人带C抗原的RhD阴性的基因结构具有多态性,具有完全缺失、部分缺失和不缺失3种情况,并可能存在特殊的D(nf)Ce单体型;本组样本不能确定插入片段和RhBox与D基因表达有关,插入片段和RhBox的生物学功能尚需进一步研究;中国汉族的Rh血型基因序列不同于白种人,应建立本民族的Rh基因库。     

RhD基因检测方法在Rh阴性血型基因鉴定中的应用

目的：探讨RhD基因检测方法在中国汉族人Rh阴血型鉴定中的应用价值。方法：对于68例各种表型的Rh阴性中国汉族样本,用扩增RhD基因外显子3,4,5,7,10的5种方法,检测RhD基因的存在与否。结果：68例样本,总阴性率为52．95,其中29例含有C抗原的Rh阴样性本,5种方法鉴定皆为阴性的仅有1例（3．4％）,而表型为C阴性的38例样本,34例（89．5％）为阴性。结论：以检测RhD基因存在与否为基础的Rh阴性血型基因鉴定方法,不适用于C抗原阳性的中国汉族Rh阴性血型基因鉴定,仅有扩增RhD外子3,4,10的方法,适用于含C抗原的Rh阴性血型的基因鉴定。     

FPTT is a low-incidence Rh antigen associated with a "new" partial Rh D phenotype, DFR

BACKGROUND: Several Rh D phenotypes with partial D antigens are recognized. Some partial D antigens are associated with low-incidence Rh antigens. New partial D antigens are revealed by an atypical pattern of reactions with anti-D. STUDY DESIGN AND METHODS: The reactions of D variant cells with panels of monoclonal anti-D and with antibodies to low-incidence antigens were compared to those of known D categories to identify a new Rh D phenotype. The inheritance of partial D antigens was studied by Rh phenotyping of the families of the probands. Standard serologic methods were used and family data were analyzed. RESULTS: A new Rh D phenotype, to be called DFR, was identified in 17 probands, two of whom had made anti-D. The partial D antigen carries epD3, epD4, and epD9 and lacks epD8. The presence of other D epitopes is ambiguous; different answers were obtained for the same sample with different monoclonal anti-D of the same apparent epitope specificity. The immunoglobulin class of the anti-D was important: IgG were more successful than IgM monoclonal anti-D in detecting the partial D of DFR. Family studies showed that DFR traveled with Ce more frequently than with cE. The low-incidence antigen FPTT (International Society of Blood Transfusion number 700048) was found on all DFR samples. Family studies demonstrated that FPTT is, as suspected, part of the complex Rh system. CONCLUSION: The partial D of the Rh D phenotype, DFR, is recognized by its pattern of reactions with monoclonal anti-D and its association with the low-incidence antigen FPTT, FPTT has now been numbered Rh50.     

Antigen site densities and ultrastructural distribution patterns of red cell Rh antigens

The ultrastructural distribution pattern and antigen site density of the major Rh antigens (C, c, E, e, and D) on red blood cell ghosts sensitized with WITH IgG Rh antibodies were determined using electron microscopy and ferritin conjugated rabbit anti-human IgG. The ferritin particle distribution pattern of all the Rh antigens studied was random and ranged from relatively isolated monodispersed clusters containing less than eight ferritin particles to large localized aggregates or masses of 20 or more ferritin particles. Evidence is presented to indicate that most of the antigen clustering was due to the conjugate during staining of the cell bound IgG on the stroma preparations. The conjugate-induced antigen clustering was influenced by both the titer of the conjugate and the staining time. Whether a similar degree of antigen mobility exists in the native membrane remains to be determined. Estimates of the number of cell bound IgG molecules (number of antigenic determinants) based on ferritin particle scoring of IgG Rh antibody sensitized red blood cells under nonsaturating conditions were in the range of 20,000 to 32,000 for each of the Rh antigens. These findings are consistent with the view that the Rh antigen complex is associated with a single membrane component.     

天津市滨海新区Rh+无偿献血者Rh血型表型的分布调查

目的 探讨天津市滨海新区Rh+献血者Rh血型表型的分布情况,建立Rh+血型表型数据库,确保临床输血安全,减少输血不良反应的发生.方法 采用简单随机抽样法选择2013年1月至2015年12月天津市第五中心医院输血科留存的2 672份Rh+库存悬浮红细胞为研究对象.研究对象纳入标准:①所有悬液红细胞均来自天津市滨海新区中心血站;②血液经Rh系统中抗-D检测,确定为Rh+血型;③血液入库时,严格核对运输条件、物理外观、血袋封闭及包装、标签等,均应符合血液入库标准.采用微柱凝胶法进行Rh血型系统的抗-D、抗-C、抗-c、抗-E、抗-e检测,并根据抗原检测结果计算Rh血型各表型频率.结果 本组2 672例Rh+无偿献血者的Rh抗原中,按照抗原阳性率由高至低排序,依次为抗-D(100.0％)、抗-e(90.8％)、抗-C(86.7％)、抗-c(58.2％)及抗-E(48.8％)抗原.本组2 672例Rh+无偿献血者中,共检测出9种Rh血型表型,按照抗原频率由高至低排序,依次为CCDee (40.5％)、CcDEe(35.5％)、CcDee(9.4％)、ccDEE(9.2％)、ccDEe(4.1％)血型表型,其他4种表型仅占1.3％(35/2 672).结论 天津市滨海新区Rh+无偿献血人群的Rh血型表型以CCDee为主.建立Rh+血型表型数据库,可为临床及时提供表型相合的Rh+血液,防止由于Rh血型系统不合引起的输血反应,确保临床输血安全.     

Major histocompatibility complex markers and red cell antibodies to the Rh (D) antigen. Absence of association

Between 20 and 35 percent of Rh(D) antigen-negative individuals do not develop antibodies to D even after multiple transfusions of Rh-positive red cells. To evaluate the possibility that antibody production after exposure to the D antigen was related to a major histocompatibility complex immune response gene, analysis of the HLA genotypes of 38 Rh-sensitized women and their families was performed. No significant deviations were found in the frequency of any individual HLA class I, II, or III allele or of any extended haplotype (fixed allelic combinations of HLA-B, HLA-DR, and the complement components BF, C2, C4A, and C4B). Type 1 errors due to the extreme allelic polymorphism of the HLA system, as well as the ethnic variation in patient groups, may have contributed to HLA allele-antibody responder relationships reported in earlier studies.     

Influence of Rh phenotype on the antigen density of C, c, and D: flow cytometric study using a frozen standard red cell

BACKGROUND: Flow cytometry is increasingly being used for the comparison of antigen density. Indirect immune fluorescence is more sensitive than direct immune fluorescence and thus allows the study of red cells (RBCs) with a weak D antigen. STUDY DESIGN AND METHODS: In indirect immune fluorescence, when the fluorescence is standardized by the use of aliquoted frozen standard RBCs, the coefficient of variation in fluorescence intensity was less than 5 percent, which allows accurate determination of minor variations of Rh antigen density. RESULTS: For D antigen, the well-known suppressive effect of C, and the low antigen density of the weak D phenotype, was demonstrated. Use of epitope-specific monoclonal antibodies yielded similar results and allowed the identification of a D category IV heterozygote; the relative antigen density measured with a monoclonal antibody that reacted with D(IV) was twice that measured with a monoclonal antibody that did not react with D(IV). RBCs from C and c homozygotes had significantly more antigen than those from heterozygotes. There was significant variation in antigen density, depending on Rh phenotype: for example, D+ RBCs had less C antigen than D- RBCs, and Rh:1,2,-3,4,5 (CcDee) RBCs had more c antigen than Rh:1,2,3,4,5 (CcDEe) RBCs. There was no difference in D, C, and c antigen density in neonatal and adult RBCs. CONCLUSION: Flow cytometry is an excellent tool for the demonstration of minor differences in antigen density.     

A "new" low-incidence red cell antigen, LOCR, associated with altered expression of Rh antigens

BACKGROUND: Mild cases of hemolytic disease of the newborn were being studied when it was recognized that the propositi's red cells carried a novel antigen. STUDY DESIGN AND METHODS: Serologic and genetic studies of a new low-incidence antigen, LOCR (International Society of Blood Transfusion series number 700.53), were performed. RESULTS: The antigen is associated with altered expression of the Rh antigen c in two unrelated families and in a third proposita and with an altered expression of e in a fourth proposita. The lods for the gene controlling LOCR relative to Rh are 2.107 at theta = 0.00; that is, they fall short of the requirement for inclusion of the antigen in the Rh blood group system. LOCR is excluded from the ABO, MNS, Lutheran, Kell, Duffy, Kidd, Xg, Chido/Rodgers, Kx, and Gerbich blood group systems. Genetic evidence suggests it is not part of the Yt, Colton, or LW systems. CONCLUSION: Serologic evidence that LOCR is associated with altered expression of c or e strongly suggests that LOCR is part of the Rh blood group system. However, the genetic evidence falls short of the level required for system assignment.     

Anti-Rh39—a "new" specificity Rh system antibody

Two examples of an autoantibody that defines a hitherto unrecognized Rh system antigen are described. Both were produced by C-negative individuals and ostensibly resembled anti-C in specificity. However, adsorption studies showed that the antigen that the autoantibodies define is present on all red blood cells with a "normal" Rh phenotype and on D–/D– and Dc–/Dc–samples. The antigen detected is not present on Rhnull red blood cells. Serologic studies have shown that the new antibody, that has been named anti-Rh39, has a different specificity from those that define the antigens C, Ce(rhi), G, Hro, Hr, CG, LW, Rh:29, Rh:34 and U. A possible relationship between auto-anti- Rh39 and allo-anti-C, in terms of the immune response, is discussed.     

Rh抗原缺失Dc-型抗体研究

目的研究Rh抗原缺失患者血清中抗体性质和效价。方法结合其临床表现和家系调查,利用血型血清学方法对RhDc-孕妇体内的抗体性质及效价进行分析。结果患者血型为B、Dc-型,其亲代血清学表型为父AB、Dccee型,母O、DCCee型,患者血清中存在IgM及IgG抗-Hr0,效价分别为128、512;IgM及IgG抗-e,效价分别为16、8。结论国内外有报道在RhD-和Rh-患者中查到IgG性质抗-Hr0,在RHDc-患者体内查出IgM和IgG性质的抗-Hr0和抗-e在国内外尚属首次报道;该患者体内RhEe缺失可能的原因为RHCE基因的遗传和变异;IgM型抗-Hr0会干扰血型检测,导致ABO正反定型不符,在临床工作中应引起注意。     

Stable solid-phase Rh antigen

Numerous investigators have attempted to isolate the Rh antigens in a stable, immunologically reactive form since the discovery of the Rh system over 56 years ago. We report here a successful and reproducible approach to solubilizing and adsorbing the human Rh antigen(s) to a solid-phase matrix in an antigenically active form. Similar results were obtained with rabbit A/D/F red blood cell antigens. The antigen preparation was made by dissolution of the red blood cell membrane lipid followed by fragmentation of the residual cytoskeleton in an EDTA solution at low ionic strength. The antigenic activity of the soluble preparations was labile in standard buffers but was stable in zwitterionic buffers for extended periods of time. Further studies showed that the antigenic activity of these preparations was enhanced, as was their affinity for plastic surfaces, in the presence of acidic zwitterionic buffers. Adherence to plastic surfaces at low pH maintained antigenic reactivity and specificity for antibody was retained. The data show that this approach yields a stable form of antigenically active human Rh D antigen that could be used in a red blood cell-free assay for quantitative analysis of Rh D antibody and for Rh D antibody immunoadsorption and purification.     

Immunochemical Studies of the Rh System. IV. Hemagglutination Assay of Antigenic Expression Regulated by Interaction Between Paired Rh Genes

Quantitative hemagglutination assays with anti-Rh1 (Rh₀ or D), anti-Rh2 (rh′ or C), anti-Rh3 (rh″ or E) and anti-Rh4 (hr′ or c) were performed on the erythrocytes of standard donors of known genotype and of members of one family showing segregation and recombination of four complex Rh genes: R^{1,2,–3,–4}, R^W1,–2,3,-4, R^?1,–2,-3,4, and R^{?1,2,–3,–4}. Within the pedigree, duplicate and triplicate examples of each genotype reacted identically. Hemagglutination scores provided concordant data but showed, additionally, that (1) the strength of expression of both Rh1 and Rhw1 (??Rh₀ or D^u) directly influenced the amount of agglutination obtainable with guinea pig anti-rhesus serum (anti-LW), and (2) in this family R^{?1,–2,–3,5,19} determines more Rh19 reactivity than does R^{?1,2,–3,5,19} and much more than R^{1,2,–3,5,w19}. The gene R^{?1,2,–3,–4} was found to depress not only the expression of Rh1 and Rh3 determined by the paired gene, as described previously by others, but Rhw1, Rh2 and Rh4 as well. The gene, R^{1,2,–3,–4}, was also found to depress the Rh3 and Rh4 antigenic expression of a paired gene but not to the same degree. These findings suggest that interaction between paired Rh genes may account for most, if not all, of the quantitative genotype differences previously attributed to single “gene dosage” and to pertinent portions of the so-called “cis-trans” effect. In contrast to Rh1 assays, those for Rh2, Rh3 and Rh4 were associated with parallel log-probit regression lines, a finding suggestive of more homogeneity in regard to specific binding affinities of these antiserums and antigenic determinants.     

Specificity and isotype of Rh specific antibodies produced by human B-cell lines established from alloimmunized Rh negative women.

Despite the successful outcome of anti-D prophylaxis program, alloimmunization still occurs. The aim of this study was to examine the specificity and isotype of anti-Rh antibodies in plasma samples of Rh negative alloimmunized individuals and to study the same parameters in lymphoblastoid cell lines (LCLs) generated from the same donors. Specificity of anti-Rh antibodies was determined in plasma of nine alloimmunized subjects by direct hemagglutination using a panel of known RBC genotypes and isotype of specific antibodies were identified by an antigen specific ELISA. Similar methods were employed to determine specificity and isotype of antibodies produced by Rh specific LCLs established from four donors. LCLs were generated by Epstein-Barr virus transformation of peripheral blood mononuclear cells isolated from each donor followed by their culture over a feeder of human fetal fibroblasts. Upon emergence of lymphoblastoid cells, culture supernatants were assayed for presence of Rh specific antibody by hemagglutination assay. Anti-D was the predominant antibody in both plasma samples and among the 128 established LCLs; however, antibodies to other Rh specificities namely C and E were also produced. The isotype of anti-Rh antibody in all plasma samples was found to be IgG, predominantly IgG1, combined in 7 samples with IgM. Similarly 76%, 9.2% and 14.8% of LCLs were determined to produce antibody of IgG, IgM and of both isotypes, respectively. The data supported that the D antigen is the immunodominant component of the Rh system as indicated by the in vitro and in vivo profiles of Rh specificities in our alloimmunized subjects.     

The Risk of Immunization to IgG Following Rh Immune Globulin Therapy

Three hundred thirteen women were typed for Gm and Inv factors and screened for the presence of antibodies to gamma globulin both before receiving Rh immune globulin and at six weeks, three, six, and nine months after injection. Thirty-five of the 313 women received multiple doses of Rh immune globulin following one or more pregnancies. In this group of patients, the incidence of anti-gamma globulins was no greater than in the women given a single 0.7 to 2.25 ml dose. Anti-gamma globulins were found in only 17 women (5.4%). Eleven had antibody prior to treatment and in six it first appeared in post injection specimens. Five of the women had allotype specific Gm agglutinators. In three, these were found in the pretreatment specimens. No evidence of Rh sensitization was demonstrable in the eleven Rh immune globulin recipients who had circulating anti-gamma globulins at the time of treatment. Our data indicates that the risk of sensitization to Gm and Inv factors is a function of the concentration of the antigen in the Rh immune globulin and the pheno-type of the recipients.     

Mapping assignment of the Rh and Duffy blood group genes to chromosome 1.

Blood group genes are inherited in a straightforward manner and their products are readily detectable. Because of this they are utilized over a wide area of scientific endeavor and provide excellent markers for use in gene mapping of the human chromosomes. Both the Rh and Duffy blood group genes have now been localized to the number 1 chromosome. Studies of a patient whose red cells exhibit mosaicism for the Rh blood group indicate that loss of the end of the short arm of chromosome number 1 is associated with loss of an Rh gene complex. The Rh gene can, therefore, be assigned with some precision to this region of the chromosome.     

ERIK, a low-frequency red cell antigen of the MNS blood group system associated with Sta

Summary. A new low-frequency red cell antigen, ERIK (MNS37), is associated with the St^a antigen of the MNS system. Four ERIK + propositi have been identified: all are St(a+). Thirteen other St(a+) samples, including one of the M^z type, and over 200 St(a-) samples were ERIK-. In two of the propositi ERIK is associated with an abnormal trypsin-resistant M antigen; in the others it is associated with N, which is shown to be expressed weakly in one family. Immunoblotting with an antibody to an epitope common to glycophorin A (GPA) and glycophorin B (GPB) gave identical results with St(a+) EiRIK + and St(a+) ERIK-cells, revealing GPA, GPB, an abnormal structure GPSt^a aggregates of these components. In the propositi with trypsin-resistant M, GPSt^acarries the unusual M antigen, whereas in the M - N + ERIK+ individual analysed by immunoblotting GPSt^a carries N. Immunoblotting with anti-St^a anti-ERIK showed that St^a is located on GPSt^a but that ERIK is located on GPA, presumably the GPA molecule encoded by the GYPA gene contiguous to the gene encoding GPSt^a.     

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.