TSEN: a novel MNS-related blood group antigen.

We report an antibody (anti-TSEN) that recognizes an antigen (TSEN) at the unique amino acid sequence that results from the junction of GPA58 to GPB27 if the GPB carries the S antigen. Red cells from several unrelated donors that possess this specific GP(A-B) hybrid molecule were agglutinated by anti-TSEN. Since a synthetic peptide with the amino acid sequence at this junction (Pro-Glu-Glu-Glu-Thr-Gly-Glu-Met-Gly-Gln-Leu-Val-His-Arg) specifically inhibited anti-TSEN, it must detect an antigen within this novel amino acid sequence. The TSEN antigen has been provisionally assigned the MNS blood group system number 002.033 on behalf of the ISBT Working Party on Terminology for Red Cell Surface Antigens.

Synthetic peptides homologous to human glycophorins of the Miltenberger complex of variants of MNSs blood group system specify the epitopes for Hil, SJL, Hop, and Mur antisera.

The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, "Hil", "SJL", "Mur", and "Hop" to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha-delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta-alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur.

Two cases of "hrB-like" autoantibodies appearing as alloantibodies.

Two cases are described in which autoantibodies mimicked alloantibodies. The direct antiglobulin test (DAT) on the red cells (RBCs) from both patients was negative when routine manual hexadimethrine bromide (Polybrene) and enzyme-linked antiglobulin techniques were used. The RBCs also did not react on direct bromelin and direct Polybrene tests. However, an "hrB-like" antibody was eluted from the RBCs of both patients. The sera from these patients reacted with all e+ hrB+ RBCs but not with e+ hrB-, e-, or their own RBCs. The antibody in the serum of one patient was not adsorbed by R2R2 RBCs. Serologic tests initially suggested (by direct testing and adsorption studies) that the serum antibodies were alloantibodies rather than autoantibodies. RBCs taken from one patient, 8 months after her sample was first referred to our laboratory, reacted with a serum sample from her first admission. An RBC sample taken from the other patient, initially typed e+ and hrB- but 1 month later typed e+ and hrB+ by using the same anti-hrB sera, was used to test the earlier samples.

Autoanti-Ge associated with severe autoimmune hemolytic anemia.

Severe autoimmune hemolytic anemia due to anti-Ge is described. The patient's red cells had a positive direct antiglobulin test, and they typed as Ge+ using saline reactive reagents. Anti-Ge was eluted from her RBCs, and her serum had an IgG and IgM anti-Ge2,3.

Amino acid sequence of an alpha-delta-glycophorin hybrid. A structure reciprocal to Sta delta-alpha-glycophorin hybrid.

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of alpha- and delta-glycophorins (glycophorins A and B) in a alpha-delta arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-alpha-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-alpha-glycophorin and whose carboxyl terminus corresponded to S-delta-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1-8) was identical to the amino-terminal octapeptide of M-alpha-glycophorin. The proceeding peptide (residues 9-61) contained a segment identical to residues 9-58 of alpha glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-delta-glycophorin (residues 27-29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30-52 and 53-68 of delta-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1-58 were identical to residues 1-58 of M-alpha-glycophorin, and residues 59-100 were entirely identical to residues 27-68 of S-delta-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69-72 of delta-glycophorin. The amino acid sequence arrangement of the variant alpha-delta-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a delta-alpha hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.

Structural analysis of glycophorin A from Miltenberger class VIII erythrocytes.

The major human erythrocyte membrane sialoglycoprotein (glycophorin A or MN glycoprotein) was purified from the erythrocytes of two individuals heterozygous for the Mi-VIII gene in the Miltenberger subsystem of the MNSs blood-group system. The complete structure of a tryptic glycopetide from glycophorin A comprising the residues 40-61 was deduced from automated and manual sequence analyses. The Mi-VIII-specific glycophorin A was found to exhibit an arginine----threonine exchange at position 49. The threonine residue was found to be glycosylated. Hemagglutination and hemagglutination inhibition assays demonstrated that one of the Mi-VIII-characteristic antigenic determinants (Anek) is located within the residues 40-61 of glycophorin A. Furthermore, erythrocytes from the two Mi-VIII heterozygotes reacted only weakly with anti-EnaKTsera, suggesting that the Mi-VIII-specific glycophorin A does not express the EnaKT antigen that is located within the positions 46-56 of normal glycophorin A. Our data suggest that the Mi-VIII-specific glycophorin A represents the evolutionary link between normal glycophorin A and the Mi-VIII-specific molecule which exhibits arginine----threonine and tyrosine----serine exchanges at the positions 49 and 52, respectively. Our data also provide an explanation for the close serological similarity between Mi-VII and Mi-VIII erythrocytes.

Immunochemical specificity of autoanti-Gerbich from two patients with autoimmune haemolytic anaemia and concomitant alteration in the red cell membrane sialoglycoprotein beta.

Blood samples from two patients with autoimmune haemolytic anaemia, whose autoantibodies failed to agglutinate certain examples of red cells that lack Gerbich blood group antigens, were studied using immunochemical analyses. One of these autoantibodies differed from all other anti-Ge in that it showed a unique beta sialoglycoprotein (SGP) specificity. It reacted with normal beta but not with the abnormal beta-related SGPs associated with Gerbich-negative red cells of the Gerbich and Yus types. Red cells from this patient had an alteration of beta SGP, while the alpha, gamma and delta SGPs appeared to be normal. The autoantibody from the other patient did not show this unique characteristic. Its immunochemical specificity was similar to alloanti-Ge3 in that it reacted with both beta and gamma SGPs from normal red cell membranes and with the abnormal beta-related SGPs found in red cell membranes from individuals with Gerbich-negative red cells of the Yus type. Red cells from this patient could not be analysed because she had recently received a massive transfusion of red cells.

Acquired loss of red cell Kell antigens.

A 19-year-old patient with a long history of idiopathic thrombocytopenic purpura developed a potent antibody against a high-incidence antigen in the Kell blood group system. The direct antiglobulin test on his red cells was negative. His cells exhibited profound depression of Kell blood group antigens, but antigens of other blood groups were normal. Transfusion of incompatible blood was well tolerated and differential agglutination tests, using selected Rh antisera, showed in vivo survival of the transfused red cells for more than 8 weeks. However, the transfused red cells also showed acquired loss of Kell antigens. Five months after the initial findings, Kell-related antibody disappeared and Kell antigens reappeared on his red cells. The patient's serum stored from the initial investigation now reacted with his freshly collected red cells. These data suggest that an environmental agent in the patient's plasma was responsible for the temporary loss of Kell antigens from red cells in his circulation.

Evaluation and comparison of four reticulocyte enrichment procedures.

Four reticulocyte enrichment techniques were compared: differential centrifugation, and density gradient centrifugation with phthalate esters, Percoll-Renografin (PR), and silicone oil. The reticulocyte yields were 2.6, 7.4, 13.5, and 13.1 percent, respectively. The differential centrifugation procedure did not separate a statistically significant higher number of reticulocytes than were counted in an unseparated sample. The PR density gradient procedure was the best for our needs because it yielded the highest number of reticulocytes and was the easiest and quickest of the four procedures to perform; however, the method requires a high-speed centrifuge.

The distribution of ABO and Rh(D) and selected high- and low-frequency antigens in the people in the Kingdom of Tonga.

The incidence of red cell A, B, and D antigens in 7903 people from the Kingdom of Tonga is reported. ABO and D typings were performed by the slide method while establishing a registry of potential blood donors. The results of selected high-frequency antigens and one low-frequency antigen typing of 1009 donors also are reported.

A new test useful in identifying red cells with a (delta-alpha) hybrid sialoglycoprotein.

St(a+) and Dantu+ red cells (RBCs), treated with 0.1 percent ficin solutions, reacted strongly with Vicia graminea lectin (anti-NVg). No other RBC sample tested gave these results. This test (ficin-pretreated RBCs tested with anti-NVg [FT-NVg]) was used to screen the RBCs of 300 Oriental and 100 black donors. Three percent of the Oriental donor samples tested were FT-NVg+, whereas no FT-NVg + RBCs were found in the black donor samples tested. The FT-NVg test is easy to perform and gives selective reactions with antigens associated with (delta-alpha) hybrid sialoglycoproteins, such as Sta and Dantu. This test can be used for mass screening of RBCs in a search for St(a+) or Dantu+ RBCs. Another application of the FT-NVg test would be to screen RBCs suspected of having a low-incidence antigen before using scarce anti-Sta and -Dantu reagents.

Isolation and serological characterization of a monoclonal antibody recognizing the N blood group antigen.

A mouse hybridoma has been isolated which secretes a hemagglutinating monoclonal antibody (MoAb) recognizing the N and 'N' blood group antigens. This conclusion is based upon the following observations. First, all red cells expressing either N or one of the alleles of Ss (ie, 'N' were strongly agglutinated by the MoAb diluted fourfold. The only cells not reactive were of the M+N-S-s-(U-) and M+N-S-s-(U+) phenotype and cells (J.R. and A.G.) expressing Lepore-type hybrids of the MN and Ss sialoglycoproteins, which do not express N or 'N'. Secondly, red cells of the N+S-s-(U-) phenotype were rendered unreactive to MoAb following treatment of the cells with trypsin, which is known to destroy N antigen activity. Conversely, cells expressing S and/or s maintained their reactivity with MoAb following trypsin treatment, which does not cleave 'N' from the Ss sialoglycoprotein. When spent culture medium containing MoAb was diluted and tested against a panel of red cells, the antibody titer fell into two distinct categories depending upon the MNSs phenotype of the target. Red cells expressing either homozygous or heterozygous N-sialoglycoprotein (N-SGP) were agglutinated by 128-fold diluted MoAb. In contrast, a 16-fold dilution of MoAb was the endpoint for agglutination of cells lacking N-SGP, but expressing S and/or s.

Serologic and IgG subclass characterization of Cartwright (Yt) and Gerbich (Ge) antibodies.

Examples of anti-Yta and anti-Ge were tested for reactivity with red cells treated with proteolytic enzymes and with antisera of known IgG specificity. Eight of 14 examples of anti-Yta did not react as well with cells treated with either papain or ficin; treatment with trypsin did not reduce reactivity. Four examples of the anti-Yta were IgG4; the others tested did not react with IgG subclass antisera. Of 13 examples of anti-Ge, seven failed to react with red cells treated with trypsin, ficin, or papain. Nine examples were IgG1, one was IgG1 plus IgG3, and three did not react with IgG subclass antisera.

An unusual inhibition of an anti-P1.

Tests for unexpected antibodies using commercially prepared red blood cells demonstrated no agglutination even though crossmatching and testing with saline washed commercially prepared reagent red blood cells identified an anti-P1. This antibody was inhibited when tested with red blood cells suspended in a modified Alsever's solution and several commercially prepared reagent red blood cell diluents. The inhibitory substance was identified as inosine. Hypoxanthine, adenine, and thymine also inhibited the anti-P1. Partial inhibition was produced by uracil, 5-methylcytosine, and xanthine. No other example of anti-P1 has been found to exhibit this unusual inhibiting characteristic.

Studies on the blood of an MiV homozygote.

An individual, whose parents are third cousins, has been shown to be homozygous for the rare Mi.V. condition. The proposita's red blood cells type as M-, N+(weak), S-, s+(strong), U+, Mi(a-), Vw-, Hil+; Wr(a-b-). The cells react, albeit less strongly than most other samples, with anti-Ena. However, from studies on the red blood cells of the proposita and on those of another person of the En(a+), Wr(a-b-) phenotype, it is apparent that the term "anti-Ena" actually describes a number of antibodies of differing specificities. Inhibition studies with sialoglycoprotein (SGP) isolates, and tests on protease-modified red blood cells illustrate some of the differences in specificity. Biochemical analyses of the SGPs of the red blood cells of the MiV homozygote and those of her parents confirm that the Mi.V condition is associated with the absence of normal MN SGP (alpha) and normal Ss SGP (delta), the appearance of a hybrid SGP molecule comprised of a portion of the MN SGP at its NH2 terminal end, and a portion of the Ss SGP at its C terminal end.