Identification of human erythrocyte blood group antigens on decay-accelerating factor (DAF) and an erythrocyte phenotype negative for DAF.

Decay accelerating factor (DAF) is a glycoprotein present on the surfaces of many types ofcells in contact with plasma, including erythrocytes, leukocytes, and platelets (reviewed in reference 1). A small amount of DAF is also present in serum. Numerous investigators have demonstrated that DAF inhibits the action of C3 convertases on cell surfaces, and its absence has been shown to be at least partially responsible for the abnormal sensitivity to lysis by complement exhibited by erythrocytes of patients with the acquired stem cell disorder paroxysmal nocturnal hemoglobinuria (PNH) (2). Hereditary absence of DAF has not been previously described. Tc(a) and Cr(a) are high-frequency human erythrocyte antigens . These antigens are part of a family of blood group antigens, designated Cromer related, which are all absent from the null phenotype cell IFC(-) , or Inab (3). Recently, Spring and colleagues (4) have identified two monoclonal antibodies which bound to high frequency red cell antigens absent from the Inab phenotype. They also demonstrated that these antibodies, as well as several human antisera to Cromer-related antigens, bound to a 70-kD glycoprotein when used to stain immunoblots of human erythrocyte membrane proteins . Because the wide tissue distribution of mAb reactivity, along with some of the biochemical characterization and immunoblotting data, was similar to that of DAF, we investigated whether the Cromer-related antigens Cr(a) and Tc(a) resided on the DAF molecule.

The C3b/C4b receptor is recognized by the Knops, McCoy, Swain-langley, and York blood group antisera.

Erythrocytes (E) lacking high incidence blood group antigens were screened by an antiglobulin test with a monoclonal antibody to human complement receptor type 1 (CR1; C3b/C4b receptor; CD35). Some examples of E lacking Knops, McCoy, Swain-Langley, and York antigens, a serologically related group, were not agglutinated. Moreover, E of the null phenotype for these same antigens were nonreactive. To further explore this relationship, E expressing these antigens were surface labeled, solubilized, and incubated with the corresponding blood group-specific antisera. CR1 was immunoprecipitated, indicating that the epitopes recognized by each of these antisera are expressed on CR1. E of two individuals, putative null phenotypes for the Knops, McCoy, and Swain-Langley blood group antigens, expressed a very low number of CR1 (less than 30/E; approximately 10% of the normal mean). This observation accounts for their lack of reactivity in the antiglobulin test and their prior designation as null phenotypes. Also, the previously reported low as well as variable expression of CR1 on E explains prior difficulties in the serologic analyses of these blood group antigens.

Mutations in aquaporin-1 in phenotypically normal humans without functional CHIP water channels.

The gene aquaporin-1 encodes channel-forming integral protein (CHIP), a member of a large family of water transporters found throughout nature. Three rare individuals were identified who do not express CHIP-associated Colton blood group antigens and whose red cells exhibit low osmotic water permeabilities. Genomic DNA analyses demonstrated that two individuals were homozygous for different nonsense mutations (exon deletion or frameshift), and the third had a missense mutation encoding a nonfunctioning CHIP molecule. Surprisingly, none of the three suffers any apparent clinical consequence, which raises questions about the physiological importance of CHIP and implies that other mechanisms may compensate for its absence.

Blood group associations with parasites, bacteria, and viruses.

Although recent investigations into the human blood groups have proceeded mainly at the molecular level, the RBC remains an exquisite model to study the expression of various genes and their related proteins. Although DNA may be informative, it may not always give meaningful information regarding protein expression on cell surfaces, which is where binding occurs. Because of their easy accessibility, RBCs will continue to be used as a major tool in the investigation of the causative agents for disease, whether they be viral, bacterial, or parasitic in nature.

Anti-Holley detected in a primary immune response.

Anti-Holley (Hy) has been reported as an IgG antibody occurring in previously transfused or multiparous black patients. In this case anti- Hy was identified in a 16-year-old black, primigravida female admitted at 32 weeks gestation because of premature rupture of the membranes. On admission, her blood type was determined to be A2B, D-positive and an antibody screen was negative. A second antibody screen, performed 4 days later, was positive in all three cells. Anti-Hy was subsequently identified. The antibody was reactive at room temperature, 37 degrees C, and in the antiglobulin phase. IgG and IgM components of anti-Hy were demonstrated in the maternal serum, documenting a primary immune response. This resulted in serologic findings not previously described for anti-Hy. A direct antiglobulin test on the newborn red cells was negative and there was no clinical evidence of hemolytic disease of the newborn (HDN). A monocyte monolayer assay performed with maternal serum yielded negative results. Recent scientific information has resulted in the placement of Hy in the Dombrock blood group system. Alloantibodies to Dombrock system antigens have not been associated with severe HDN.

Blood grouping using a galvanic immunoelectrode sensor.

Traditional blood grouping techniques have been performed using either direct or indirect hemagglutination or adherence methods. Most procedures are time-consuming to perform, labor intensive and, for the most part, have subjective interpretation. An immunoelectrode system using a pair of electrodes, with either monoclonal antibody or red cell membrane attached to one of the electrode surfaces, has been developed. The fluid (whole blood) to be analyzed is used as an electrical bridge between the electrodes. The analysis of the fluid sample for predetermined immunological reactions can be evaluated by controlling and measuring either the current or the voltage across the two electrodes of the pair. Tests using a printed electrical circuit card of pairs of electrodes (one of the pair coated with a reactant), or a series of electrodes (each coated with a different reactant) with one common reference electrode indicate that the test procedure is rapid (less than 60 seconds) and specific. Tests results read in one hundredth of a second intervals and read at the millivolt or microvolt levels can be electronically scanned, processed through the logic table and immediately interpreted.

High-frequency antigens of human erythrocyte membrane sialoglycoproteins, IV. Molecular properties of the U antigen.

The nature of the common erythrocyte antigen U, that is absent from S-s-U-cells, which lack glycophorin B (Ss sialoglycoprotein), was investigated using six different antisera. The molecular features of a U-like antigen (Duclos), detected by a hitherto unique serum, were also studied. The U and Duclos antigens are complex in that they exhibit relationships with the MNSs and Rh blood group systems. Various fractionation, cleavage, or modification products of normal erythrocyte membranes were used in hemagglutination inhibition assays. Both, the U and Duclos antigens were found to represent labile structures that require lipids, at least for optimum expression of antigen activity. The antigens could be solubilized using conditions of Triton X-100 extraction that release glycophorin B, but solubilize the Rh antigens only to a small extent. Anti-U and anti-Duclos were also inhibited, albeit weakly, by glycophorin B-containing fractions obtained by chromatographic separation of Triton X-100 extracts. The residues approx. 33-39 of glycophorin B represent essential parts of the U antigen, as judged from proteolytic digestion and chemical modification. Conversely, the expression of Duclos activity seems to require a region of glycophorin B (C-terminal of the positions approx. 34-36) that could not be cleaved by various proteinases. Data obtained with anti-Duclos have to be interpreted with caution, since there is evidence that this serum might contain a mixture of antibodies.

MNSs and Gerbich blood group systems.

Human red blood cell membranes contain four sialic acid-rich glycoproteins, denoted as glycophorins (GPs), that carry the antigens of the MNSs and Gerbich (Ge) blood group systems. The MNSs locus corresponds to two related and adjacent genes that encode the polypeptide sequences of two of these molecules: GP A and GP B. The structural differences between the major polymorphic antigens M and N or S and s are determined by amino acid heterogeneities within the glycosylated NH2-terminal domain of GP A or GP B, respectively. Because the NH2-terminal 26 residues of GP B are identical with those of GP A possessing blood group N specificity, the former molecule carries an additional N antigen, denoted as 'N'. Apart from the major antigens, GP A and GP B carry several high- or low-frequency receptors that are encoded by the MNSs locus. Additional alleles are apparently silent or produce GP A-GP B hybrid molecules. The Ge locus is similar to the MNSs locus in that it appears to correspond to the adjacent genes encoding the polypeptide chains of GP C and GP D. However, the Ge system does not include antigens that are polymorphic in Caucasoids. Because all GPs are heavily glycosylated, oligosaccharides, in addition to protein, are involved in antigens of the MNSs and Ge systems. The carbohydrate units on all GPs account for additional antigens that are not part of the MNSs or Ge systems.

Doa (Dombrock) blood group antigen in the Japanese: tests on further population and family samples.

A low frequency of the Doa antigen in Japanese was confirmed by a further testing of 502 donors and 79 families. In the family studies, Doa was shown to be independent of the Diego or Jr systems.

Membrane glycophorins of Dantu blood group erythrocytes.

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.

Isolation of the JMH antigen on a novel phosphatidylinositol-linked human membrane protein.

JMH is a high-frequency human erythrocyte blood group antigen. Previous work has shown that JMH is absent from complement-sensitive erythrocytes of patients with paroxysmal nocturnal hemoglobinuria (PNH); such cells have a broad defect in expression of phosphatidylinositol (PI)-linked proteins. Using both human JMH antisera and a JMH-like murine monoclonal antibody, we have identified a 76-Kd membrane protein present in JMH-positive but not JMH-negative erythrocytes. A similar 76-Kd JMH protein was also identified on a human lymphoid T-cell line, HSB-2. Using PI-specific phospholipase C, a small amount of JMH antigen could be cleaved from intact erythrocytes and immunoprecipitated from the supernate of treated erythrocytes, thus confirming that the protein bearing the JMH antigen is anchored by a PI-linkage to the erythrocyte membrane. This protein was further shown not to be identical to decay accelerating factor (70 Kd), a previously identified PI-anchored protein of somewhat similar molecular weight.

Structural analysis of the major human erythrocyte membrane sialoglycoprotein from Miltenberger class VII cells.

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the red blood cells of an individual, homozygous for the Mi-VII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic peptide comprising the residues 40-61 of glycophorin A was deduced from manual sequence analyses. The Mi-VII-specific glycophorin A was shown to exhibit an arginine----threonine and a tyrosine----serine exchange at the positions 49 and 52 respectively. The threonine-49 residue was found to be glycosylated. Inhibition assays demonstrated that one of the Mi-VII-specific antigen determinants (Anek) is located within the residues 40-61 of glycophorin A and comprises sialic acid residue(s) attached to O-glycosidically linked oligosaccharide(s). Our data contribute to an understanding of the Miltenberger system and provide an explanation at the molecular level for the previous finding that the erythrocytes from the Mi-VII homozygote lack a high-frequency antigen (EnaKT), located within the residues 46-56 of normal glycophorin A.

The rapid identification of Chido and Rodgers antibodies using C4d-coated red blood cells.

A simple method for the recognition of anti-Ch and anti-Rg, using red blood cells coated in vitro with C4d, is described. Of 58 anti-Ch and 20 anti-Rg sera studied, all caused direct agglutination of C4d-coated red blood cells in tests incubated at room temperature. Moreover, selected sera were shown to react strongly (3+ to 4+) with C4d-coated red blood cells when tested by the indirect antiglobulin test without prior incubation at 37 C. Sera containing other high-titer, low avidity (HTLA) antibodies were either nonreactive with C4d-coated red blood cells, or reacted to the same degree with "non-C4d-coated" trypsin-treated red blood cells. Similar findings were obtained in tests with non-HTLA antibodies of assorted specificities, including a variety of alloantibodies to high-incidence red blood cell antigens. These findings indicate that the use of C4d-coated red blood cells in investigative immunohematology may aid in the rapid identification of Ch and Rg antibodies.

Doa (Dombrock) blood group antigen in the Japanese.

Blood samples from 100 unrelated Japanese were tested for Doa antigen. It was found that the frequency of the antigen in the Japanese (18%) is lower by far than in Caucasians, Negroes or American Indians.

Shortened long-term survival of incompatible red cells in a patient with anti-McCoy-like antibody. Immunoglobulin characteristics of this antibody.

The long-term survival of incompatible 51Cr-labeled red cells in a patient with a high titer, low avidity antibody of anti-McCoy-like specificity was decreased (half-life = 14 days), although the 24-hour survival was normal (91.9%). This antibody was composed of IgA and IgG, the latter consisting of subclasses 1, 3, and 4.

Chloroquine dissociation of antigen-antibody complexes. A new technique for typing red blood cells with a positive direct antiglobulin test.

We have investigated a method using the quinoline derivative chloroquine diphosphate (200 mg/ml, pH 5.0) to dissociate antibody without denaturing red blood cell antigens. All samples treated with chloroquine diphosphate demonstrated some dissociation of the coating immunoglobulin, and in most cases, the ability to dissociate the coating immunoglobulin was related to the strength of the direct antiglobulin test (DAT). Complete dissociation of antibody was observed in 22 of 40 strongly in vitro sensitized samples and 47 of 56 in vivo sensitized specimens, with no apparent loss of ABH, Rh, MNSs, P1, Lewis, Kell, Duffy, or Kidd antigens. We have found the chloroquine dissociation technique to be of value in the examination of red blood cells with a positive DAT, either for the qualitative or quantitative expression of antigens.