Studies on Blood from the Original RhnullProposita and Relatives

A relative of the original Rh proposita, of group R1R2, shows weak expression of his Rh antigens, and is thought to be an Rhnull heterozygote. His wife and 3 of their 4 children show normal Rh antigen expression, but one daughter showed weak Rh antigen expression, as determined by quantitative haemagglutination. The observations support the proposition that the father is heterozygous for an unlinked modifier of Rh antigen expression. Stomatocytosis, observed in the Rhnull proposita and other Rhnull individuals, was also observed, but to a lesser degree, in the blood of an other individual thought to be an Rhnull heterozygote. This observation also supports the earlier conclusion that the Rhnull phenotype of the proposita is due to homozygosity for inactive alleles at a locus which controls the biosynthesis of precursor for Rh and LW antigens. Osmotic fragility tests showed that the Rhnull cells were more fragile than cells with normal Rh antigen expression, and cells from Rhnull heterozygotes had intermediate fragility. This is consistent with the proposition that Rh antigens are normal structural components of the red cell membrane, and the Rhnull heterozygotes show a deficiency of the Rh antigenic structures.     

Increased potassium transport and ouabain binding in human Rhnull red blood cells.

Potassium (K+) influx and 3H-ouabain binding were studied in human red cells completely lacking the rhesus (Rh) antigens (Rhnull cells) and compared with normal Rh(D) red cells. The Rhnull cells, originally described by Seidl, Spielmann, and Martin (Vox Sang. 23:182, 1972) were normal in size, cation, and water content, indicating no significant increase in cell volume as occurs in young human red cells. However, the ouabain-insensitive K+ permeability, as well as the ouabain sensitive active K+ transport, were increased 1.6 1.8-and 1.4-1.5-fold, respectively, above the values found in Rh(D) control cells. The Na+K+ ATPase activity of membranes from Rhnull cells was also higher than from Rh(D) cells. Binding studies with 3H-ouabain revealed that at 100% K+ pump inhibition Rhnull cells bound 670 and Rh(D) cells 450-500 ouabain molecules per cell. Since the rate of ouabain binding was identical in Rhnull and Rh(D) control cells, we concluded that the Rhnull cell had about 35%-45% more cation pumps than the Rh(D) cell. These additional pumps in Rhnull cells appeared to be indistinguishable from those in control cells. Anti-D or the serum from the Rhnull individual did not alter cation permeability in Rh(D) red cells. The data suggested that the Rhnull cell, known for its hematologic malfunction, was not a young or prematurely released red cell, but had a pleiotropic membrane defect which also affected the passive and active cation transport system on the molecular level. Our finding precludes a structural identity of the rhesus antigen with the molecules composing the Na+K+ pump system.     

Murine monoclonal antibodies against a unique determinant of erythrocytes, related to Rh and U antigens: expression on normal and malignant erythrocyte precursors and Rhnull red cells

Three murine monoclonal antibodies (Mabs) MB-2D10, LA-18.18 and LA-23.40 were prepared. They reacted with red cells of all common and most rare blood-group phenotypes, with the exception of those of the RhnullU negative and RhmodU negative phenotypes. So far, only a single example of an alloantibody (Duclos or anti-Rh38) of a similar specificity has been found. Serological studies indicated that the Mabs were probably not directed against an antigenic determinant of Rh polypeptides, the LWab glycoprotein or glycophorin B, all structures absent from or aberrantly expressed on Rhnull red cells. The antigen was found to be erythrocyte-specific, and was also present on pro-erythroblasts, erythroblasts and malignant erythroblastoid cells but not on erythroid progenitors in the bone marrow. The Mabs were found to block each other in an immune rosette method and are thus probably directed against the same epitope or against neighbouring epitopes on the same structure. In immunochemical studies, MB-2D10 precipitated the 30-32 kDa Rh polypeptides from red cell membranes and a protein or proteins which formed diffuse and overlapping bands in SDS-polyacrylamide gel electrophoresis, with Mrs of 40-200 kDa (probably the Rh-related glycoproteins). Under certain experimental conditions glycophorin B appeared to be coprecipitated. The 2D10 structure, detected by the Mabs, seems to be part of a complex of proteins and/or glycoproteins, which includes Rh polypeptides, the LWab glycoprotein and glycoproteins recognized by various Mabs with Rh-related specificities. In the red cell membrane, the complex may be associated with glycophorin B.     

A new blood group system,RHAG: three antigens resulting from amino acid substitutions in the Rh‐associated glycoprotein

Background and Objectives Rh‐associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol^a) have serological characteristics suggestive of expression on RhAG. Materials and Methods RHAG was sequenced from the DNA of one Duclos‐negative, one DSLK‐negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. Results The original Duclos‐negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti‐Duclos. An individual with DSLK‐negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh_mod phenotype had Ol(a+) red cells and was homozygous for 680C>T. Conclusion The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol^a is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).     

Spanish Rhnull family caused by a silent Rh gene: hematological, serological, and biochemical studies.

Another example of rare red cells that failed to react with all anti-Rh and anti-LW antibodies was discovered in a Spanish woman suffering from a severe hemolytic anemia typical of the Rhnull syndrome. Family study and Rh blood typings demonstrated clearly that the proposita was homozygous for a silent Rh gene complex (Rhnull of the amorph type) that she inherited from her parents who are first cousins. Western blot analysis carried out with glycosylation-independent antibodies directed against the Rh polypeptide and the LW glycoprotein, respectively, confirmed that these protein components were absent from the red cells of the proposita. In addition, the patient was typed U-positive, again in agreement with the presence on her red cells of 45-75 kDa glycoproteins detected with the murine monoclonal antibody 2D10.     

Immunochemical characterization of rhesus proteins with antibodies raised against synthetic peptides

Rabbit polyclonal antibodies were raised against synthetic peptides corresponding to hydrophilic regions of the human Rhesus (Rh) IX cDNA- encoded polypeptide predicted to be extracellularly or intracellularly exposed in the topologic model of the Rh blood group protein. Four antibodies encompassing residues 33-45 (MPC1), 224-233 (MPC4), 390-404 (MPC6), and 408-416 (MPC8) were characterized and compared with a polyclonal anti-Rh protein obtained by immunization with purified Rh proteins. All antibodies had specificity for authentic Rh polypeptides and reacted on Western blot with Rh proteins immunoprecipitated with human monoclonal anti-RhD, -c, and -E. MPC1, but not the other antibodies, agglutinated all human erythrocytes except Rhnull and Rhmod cells, which either lack totally or are severely deficient in Rh proteins, respectively. Immunoblotting analysis with membrane proteins from common and rare variants showed that MPC1 and MPC8 reacted in Western blot with 32-Kd Rh polypeptides from all common red blood cells except those from Rhnull and Rhmod, indicating that peptide regions 33- 45 and 408-416 may be common to several if not all Rh proteins, whatever the Rh blood group specificity. MPC4 reacted only with membrane preparations from cells carrying the E antigen, whereas MPC6 recognized preferentially the Rh proteins from E and Ee preparations, suggesting that the protein encoded by the RhIXb cDNA carries the E and/or e antigen(s). Immunoadsorption experiments using inside-out or right-side-out sealed vesicules from DccEE red blood cells as competing antigen showed that the MPC6 and MPC8 antibodies bound only to the cytoplasmic side of the erythrocyte membrane, thus providing evidence for the intracellular orientation of the C-terminal 27 residues of the Rh polypeptides. Attempts to transiently or stably express the Rh polypeptides. Attempts to transiently or stably express the Rh cDNA in eukaryotic cells were largely unsuccessful, suggesting that Rh antigen expression at the cell surface requires correct transport and/or folding of the Rh proteins, possibly as a complex with one-membrane proteins of the Rh cluster that are lacking in Rhnull cells.     

Homozygous D

A homozygote for the Rh complex .D. associated with the rare Evans antigen was identified and her family tested: .D./.D. can be clearly distinguished serologically from -D-/-D-. The immune antibody in the serum of the propositus reacts with all cells tested except homozygous -D-, CwD-, cD- and Rhnull cells.     

Differences between Rh(D) Negative Subjects in Response to Rh(D) Antigen

S ummary Further evidence is produced of the existence of two kinds of Rh negative subject: the first forms anti-Rh after a single injection although sometimes in such a low concentration that a clearance study with a small dose of Rh positive red cells is required to detect it; antibody usually becomes detectable serologically after a second, spaced injection; the second kind of Rh negative subject fails to form anti-Rh even after several injections and has a repeatedly normal survival of Rh positive red cells and may be incapable of becoming immunized to the Rh antigen. In the present series of 34 Rh negative subjects, approximately 50% became immunized following injections of Rh positive red cells; the number is not significantly different from the incidence of approximately 60% reported in other published series. Some further evidence is provided that the rate of clearance of Rh positive red cells is more sensitive than any serological test in indicating the presence of a low concentration of anti-Rh and that different examples of anti-Rh in presumably similar concentrations in the plasma produce very different rates of red cell clearance. Of seven subjects given at least two injections of D positive Bu^a-positive red cells, four formed anti-D, but only one formed anti-Bu^a.     

Comparison of the Reactions of the Rh-Related Murine Monoclonal Antibodies BS58 and R6A

The murine monoclonal antibodies BS58 and R6A are known to recognize epitopes related to the human Rh system: neither antibody reacts with Rhnull cells and the BS58 antigen is not expressed by -D- or .D. cells. It is shown here that the numbers of BS58 and R6A antigen sites vary with Rh phenotype. Both epitopes are well represented on cells of the CDe/CDe, CDe/cDE and CDe/cde phenotypes; BS58 sites are markedly reduced on cde/cde and cDE/cde and are only just detectable on cDE/cDE cells when compared with R6A sites. The number of R6A sites per red cell ranged between 20,000 and 150,000. The evidence indicates that the BS58 epitope is not on the polypeptides carrying D or R6A, nor is it uniquely on one of the polypeptides carrying either C, c, E or e. It is suggested that the BS58 epitope is either common to all the CcED polypeptides or that it is present on a polypeptide which has not yet been identified biochemically.     

The Detection and Quantification of Rh(D) Antigen Sites on Human Leukocytes

Radiolabeled eluates of human anti-D were used to measure the capacityof leukocytes to bind the D antibody in cell suspensions prepared from 16normal and 3 leukemic bloods from Rh(D) donors. The contamination of theleukocyte suspensions by D positive red cells was measured and the contribution of D antigen sites by these cells was estimated. After correction wasmade for the D antibody bound by the contaminant red cells, no specific binding of D antibody by Rh(D) leukocytes could be detected. Three pairs ofRh(D) and rh(d) leukocyte dilution curves of I¹³¹-labeled anti-D uptakewere compared with the uptake by D positive and D negative red cell dilutions. No significant differences among the D negative erythrocyte, the rh(d)leukocyte and the Rh(D) leukocyte curves were obtained. The results werecollated with previous serologic evidence concerning the presence of ABOand Rh antigens on human leukocytes.

Submitted on April 16, 1963 Accepted on June 7, 1963     

The Rh complex exports ammonium from human red blood cells

The Rh blood group system represents a major immunodominant protein complex on red blood cells (RBC). Recently, the Rh homologues RhAG and RhCG were shown to promote ammonium ion transport in yeast. In this study, we showed that also in RBC the human Rh complex functions as an exporter of ammonium ions. We measured ammonium import during the incubation of RBC in a solution containing a radiolabelled analogue of NH4Cl (14C-methyl-NH3Cl). Rhnull cells of the regulator type (expressing no Rh complex proteins) accumulated significantly higher levels (P = 0.05) of radiolabelled methyl-ammonium ions than normal RBC, at room temperature. Rhnull cells of the amorph type (expressing limited amounts of Rh complex proteins) accumulated an intermediate amount of methyl-ammonium ions. To show that decreased ammonium export contributes to its accumulation, the release of intracellular methyl-ammonium from the cells was measured over time. In 30 s, normal RBC released 87% of the intracellular methyl-ammonium ions, whereas Rhnull cells of the regulator type released only 46%. We conclude that the Rh complex is involved in the export of ammonium from RBC.     

Blocked D phenomenon, a rare condition with Rh D haemolytic disease of newborn - a case report

Accurate Rh testing can be difficult if the red cells are heavily coated with IgG anti D antibodies - a phenomenon called blocked D. Repeatedly, Rh D negative blood group report was obtained in a newborn male baby with severe haemolytic disease and features of kernicterus born to a 2nd gravida B Rh D negative mother. On investigation, the baby was grouped as B Rh D negative by direct grouping, but after elution, D antigen was detected and phenotyped as CcDe. Antibody was identified as anti D. All D antigens of the baby were fully saturated with anti D leaving any antigen to bind with antisera. Direct Coombs test was strongly positive even after three exchange transfusions. The baby also had free antibody apart from the red cell bound and the red cell eluate, gave a titre of 512. The mother was grouped as B Rh D negative and phenotyped as ce. She had IgM and IgG class of anti D with titres 32 and 1024 respectively. She also had IgM anti C (only in neat) and IgG anti-A with a titre of 512.     

Phosphatidylserine transport in Rhnull erythrocytes

Phosphatidylserine transport in normal and Rhnull red blood cells was determined by measuring characteristic morphologic changes induced by synthetic phospholipids. Treating normal A+ cells with commercial anti-A antisera, anti-Rho(D) antisera, or with saturating concentrations of purified Rho(D) antibodies had no effect on phosphatidylserine transport. Normal B- cells treated with purified anti-B antibodies transported phosphatidylserine at rates equal to those of cells not treated with antibody. Rhnull cells, deficient in the protein bearing the Rho(D) antigen, incorporated dimyristoylphosphatidylcholine and dimyristoylphosphatidylserine at rates and to extents similar to normal cells. Furthermore, incorporated phosphatidylserine, but not phosphatidylcholine, was rapidly transported across the membrane bilayer. Energy depletion or treatment with sulfhydryl reagents inhibited phosphatidylserine transport equally in normal and Rhnull cells. These results indicate that, although Rhnull cells have numerous membrane defects, they are capable of adenosine triphosphate-dependent transport of exogenously added dimyristoylphosphatidylserine. Normal phosphatidylserine transport in the presence of anti-Rho(D) antibodies or in cells deficient in the Rho(D) polypeptide indicates that this protein is not the aminophospholipid transporter.     

Le phénotype érythrocytaire Rh : 32,–46 : importance transfusionnelle et obstétricale

The red blood cells and sera from 21 R^N/R^N individuals were studied. The study confirmed the Rh type D+C+E−c−e+, C^W−, characterized by an increased expression of the D antigen, a markedly decreased expression of the C and e antigens, the presence of a low incidence antigen (Rh32) and the absence of a high incidence antigen (Rh46) associated with an epitope recognized by a murine monoclonal antibody (MR 432). Four individuals exposed to Rh46 cells by pregnancy and/or transfusion had an anti-Rh46 antibody. This antibody gave positive reactions with all red blood cells of common and rare Rh phenotype except Rh null, D--, D.., DC^W− and R^N/R^N cells. This antibody is considered to be of clinical significance in case of transfusion or pregnancy.     

Caprylate-Dependent Auto-Anti-e

A caprylate-dependent autoantibody with specificity for the e antigen of red cells is described. The antibody was completely inactivated by 2-mercaptoethanol and was probably of the IgM immunoglobulin class. The antibody had no apparent specificity for albumin. Reactivity of the described antibody may depend upon conformational change of red cell Rh antigen by caprylate.     

Three Sera Defining a New Granulocyte - Monocyte -T-Lymphocyte Antigen

Three sera containing antibodies recognizing a previously undescribed antigen on granulocytes were found during testing of sera from multiparous donors. All of the antibody producers were in good health. None had a history of transfusion. Using the granulocyte agglutination assay the sera recognize a single antigen which is not associated with the neutrophil antigens NA1, NA2, NB1, NC1, ND1, NE1, 5a, 5b, 9a, nor common red blood cell or HLA antigens. The three sera did not react with autologous cells or with the cells of the other antibody producers. Granulocytes from one antibody producer did not absorb antibody activity from any of the three sera. The antigen was found in large quantities on granulocytes and monocytes, in smaller quantities on T lymphocytes, and not on B lymphocytes, red cells, and platelets. The sera reacted with 340 of 343 random donors (99.1%) and were negative with the same donor cells. Family studies showed autosomal dominant inheritance of the antigen. Five of 12 sibs in three families lacked this antigen (not statistically different from the expected ratio). The calculated gene frequency for the gene controlling the production of this antigen is 0.906. There appeared to be no association to the HLA, NA or Rh loci or to the X or Y chromosomes. None of the infants of these three women showed clinical signs of alloimmune neonatal neutropenia.     

Autoantibodies of U Blood Group Specificity in Autoimmune Haemolytic Anaemia

Autoantibody specificity in acquired haemolytic anaemia of the warm antibody type has been thought to be directed against Rh antigens or the precursors from which they arise. In many cases Rh_null red cells fail to react with these antibodies, providing apparent support for the assumption that they have Rh specificity. Rh_null red cells are also non-reactive with anti-U sera, and in addition have abnormal S and s antigens. We have studied 50 cases of warm antibody autoimmune haemolytic anaemia looking particularly for possible U, S or s blood group specificity of the causative antibodies. In the majority of cases the antibodies were found to be directed against antigens of the Rh complex, but in three cases multi-specific autoantibodies were shown to have complex specificity involving the Rh and U blood groups.     

Rapid Clearance of Rh Positive Blood During Experimental Rh Immunisation

Summary. When Rh negative male volunteers were given successive small injections of Rh positive blood, it was repeatedly found that rapid clearance of the cells took place in the absence of detectable antibody. It is thought likely that primary immunisation to the D (Rh_o) antigen had occurred in these cases and that a similar state occurs as a result of Rh positive pregnancies in Rh negative mothers. These findings have a relevance to the assessment of trials in which gammaglobulin anti-D is given to prevent Rh immunisation.     

Rh血型抗原分析在建立实用型稀有血型库中的应用探讨

目的：从临床输血应用的角度,指导对稀有Rh血型的保存与实用型稀有血型库的建立。方法：对2000年～2006年的Rh阴性配血样本进行抗体分析,对1999年4月～2006年9月检出RhD阴性无偿献血者进行D抗原检测和D^Del血型的筛选,及对其他Rh血型抗原检测。结果：在364例Rh阴性配血样本中,有抗-D抗体36例,抗-E20例,抗-C14例,抗-C、E9例,抗-c4例,抗-e10例,抗-D、E2例,抗-D、c4例;在1215名RhD阴性的A、B、O和AB血型献血者中,Rh血型抗原表现型以dccee（51．60％）和dCcee（31．52％）居多,其次为dccEe和dCCee表现型,未见dCCEE和dCcEE表现型。1999年4月～2006年8月共查出D^u50人;在2005年5月～2006年1月的225名初筛RhD阴性献血者中共检出D“型13人,所占比例为5．8％。结论：把0型的dccEE和dC—Cee表型红细胞,在工作中发现后及时冻存并在临床对应使用,在建立实用型稀有血型库、解决临床对特殊血型的需求具有实际的应用意义。