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BACKGROUND: JMH is a high-frequency red cell blood group antigen that resides on a 76- to 80-kDa glycosylphosphatidylinositol-linked protein also known as CDw108. Antibodies with JMH specificity are often autoimmune and are usually, if not always, clinically benign. Some individuals with JMH-variant antigen produce alloantibodies to JMH, but little evidence concerning their clinical significance is available. This article reports on two patients who express a JMH-variant antigen and produced alloanti-JMH. STUDY DESIGN AND METHODS: Murine monoclonal antibodies and human antibodies to JMH were used in hemagglutination, radioimmunoassay, and Western blot testing of red cells from two JMH- variant patients; antiserum from one of these patients was also used in biochemical studies. In addition, in vivo survival of JMH-positive red cells was studied in the same patient. RESULTS: Biochemically, both examples of red cells with the JMH-variant phenotype expressed a JMH protein with a molecular weight similar to that of the normal JMH protein. For both patients, family studies suggested an autosomal recessive pattern of inheritance. Survival study demonstrated reduced in vivo red cell survival in one patient. CONCLUSION: JMH-variant phenotypes express a protein of normal molecular weight and are inherited in an autosomal recessive pattern. Furthermore, individuals with this phenotype can produce clinically significant antibodies.

A screening program was implemented to identify hrB– donors. D– C+, D–C–, and D+C– samples from African-American donors were typed with multiple examples of anti-hrB and anti-hrB-like, and one example each of anti-V and anti-VS. Of 75 D–C+ donors, 4 (5%) typed as hrB–, and 14 others had weak or variable expression of hrB. Of these 18 individuals, 15 were V–VS+, and 3 were V+VS+. No hrB– sample was found in 90 C– donors, 26 of whom were V+VS+, and 1 was V–VS+. A review of our records of 44 hrB– patients and donors studied earlier revealed that at least 12, and possibly as many as 30, carried r′ or r′s . All hrB– donors found in our screening program had D–C+VS+ RBCs, indicating an overrepresentation of r′. Our record review also showed that the presence of r′ and r′s more often results in hrB– RBCs, and that the most effective way to screen for hrB– donors is to type African Americans who have D–C+ RBCs. Immunohematology 1995;11:74–77.

In the course of investigating a patient with autoimmune hemolytic anemia in which the causative autoantibody had anti-Wrb specificity, it was demonstrated that an En(a-) red blood cell sample typed as Wr(a-b-). The only known example of Wr(a+b-) blood typed as En(a+) so that anti-Wrb and anti-Ena do not have the same specificity.

Background: It is known that some “warm”-reactive autoantibodies are directed against epitopes on the red cell anion exchanger, protein band 3. Some such antibodies (but not all) recognize Wrb. It is also known that Dia and Dib represent an amino acid polymorphism of band 3. Study Design and Methods: Autoantibodies from 119 patients were tested against Di(b-) red cells. Seventy-four of these autoantibodies were subsequently absorbed with Di(b-) red cells. Results: All 119 autoantibodies initially reacted with the Di(b-) red cells, which showed that none contained only anti-Dib. Among the 74 adsorbed with Di(b-) red cells, two were found to contain an unadsorbed anti-Dib component. Conclusion: No example of autoanti-Dib as the only autoantibody present was found among the 119 samples tested. However, 2 (2.7%) of 74 autoantibodies subjected to adsorption with Di(b-) red cells were seen to contain an anti-Dib component. This low incidence of autoanti-Dib is in marked contrast to the high incidence of autoanti-Wrb, although both antibodies define epitopes associated with the red cell anion exchanger, band 3.

The red cells of a white male blood donor typed as Rh:–1, –2, –3,w4,w5,6,–17,w19,–31,–32,–34, and –46. Although the donor has no history of transfusion, his serum contains an alloantibody that is weakly reactive with most red blood cells (RBCs) tested. Only Rhnull and D–– RBCs are nonreactive. Reactivity is enhanced with ficin- or papain-treated RBCs and is unaffected by AET or DTT treatment of the RBCs. Previously described Rh:–46 RBCs have been of deletion types D––, D••, and Rhnull or Rh:32. In three multitransfused patients, the Rh46 antigen was temporarily suppressed and the phenotype eventually reverted to normal. This is the first report of RBCs of the Rh:–32,–46 phenotype that are not of a rare Rh deletion or Rhnull type. In addition, the Rh:w5,w19,–31,–34 phenotype is rarely found in whites. Immunohematology 1994; 10:130–133.

D. J . Anstee, G. W. G. Bird E. Brodheim, J.-P. Cartron W. Dahr, C. P. Engelfriet P. D. Issitt, J . Jgrgensen L. Kornstad, M . Lewis C. Levene, A . Lubenko D. Mallory, P. Morel R . Nordhagen, Y. Okubo M . Reid, Ph. Rouger Ch. Salmon, S. Seidl, I? Sistonen S. Wendel, G. Woodfield 7: Zelinski International Society of Blood Transfusion Socihte lnternationale de Transfusion Sanguine Section Editor: B. Habibi, Paris

The Glycophorins (GPs = sialoglycoproteins) in erythrocyte membranes from various Black individuals, some of which exhibit the M1, Can, Sj, Tm, Sext and/or Hu antigens, and several Caucasian donors, including pooled fetal red cells, were studied. Using agglutination inhibition assays with GP fractions, GP fragments and chemically modified GPs as well as trypsin treatment of intact red cells, the antigens defined by anti-M1, anti-M+M1, anti-Can and anti-Tm sera were found to be located on the N-terminal tryptic peptide (T2, residues 1-31) of the major GP (GP A = MN sialoglycoprotein). Evidence was obtained that the N-terminal amino-acid residue, NeuNAc and/or (a) different sugar residue(s) are involved in the antigens. Amino-acid sequence and composition analyses excluded an amino-acid exchange within the N-terminal region (residues 1-31) of GP A. Carbohydrate analyses revealed the attachment of GlcNAc residues (up to about five, dependent on the strength of the above-mentioned antigens) to O-glycosidically linked oligosaccharides within the N-terminal portion (residues 1-31) of GP A. As judged from the carbohydrate compositions of peptides, the alteration of the O-glycosidic oligosaccharides is associated with a slight increase of the Gal and Fuc contents and a slight decrease of the NeuNAc level. Analyses of small, secondary cyanogen bromide and V8 proteinase peptides from the N-terminal region of GP A from Blacks, Caucasians and Caucasian fetal cells suggest that the variable attachment of small quantities of GlcNAc (about 0.03 to about 0.2 residues per peptide molecule) accounts, at least in part, for the polymorphisms detected by anti-Can and the original anti-Tm (serum Sheerin). Remarkably, the GlcNAc-containing O-glycosidic oligosaccharides occur only in small quantities, or not all at, within the positions 32-61 of GP A and the glycosylated domains of GP B and GP C.(ABSTRACT TRUNCATED AT 400 WORDS)

An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, 51Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.

A patient with warm autoimmune hemolytic anemia (AIHA) has been found to possess an autoantibody with Wrb specificity. While this is the first known description of Wrb specificity in this disease, additional studies on the Wrb status of En(a-) cells indicate that autoantibodies previously thought to be anti-Ena are in reality also anti-Wrb. Autoantibodies with Wrb specificity may thus be a rather common finding in patients with AIHA who have been thought to have "panagglutinins" on their red blood cells. Since anti-Wra alloantibodies are found frequently in patients with AIHA, it seems possible that the Wright system holds some clue to the pathogenesis of this disease.

In a retrospective study on samples from 10,000 recently transfused patients, 35 samples were found to contain an antibody that reacted with ficin-treated red cells but was not demonstrable by low-ionic- strength saline solution and indirect antiglobulin test (LISS-IAT). In those 35 patients, the specificity of the antibody was such that each patient would have been transfused with antigen-negative blood had the antibody reacted in LISS-IAT. Tests on red cells from the units already transfused showed that 19 patients had among them received, by chance, 32 antigen-positive and 74 antigen-negative units. The remaining 16 patients had among them received 57 units that were, again by chance, all antigen negative. One patient given antigen-positive blood suffered a delayed transfusion reaction; in two others the antibodies became LISS-IAT active after transfusion. However, similar changes to the LISS- IAT-active state were seen with two antibodies of patients given only antigen-negative blood. Also found in the 10,000 patients were 28 clinically insignificant antibodies, 77 sera in which the antibody was too weak to identify, and 216 autoantibodies that reacted only with ficin-treated red cells. These data support a belief, generally held in the United States but not necessarily elsewhere, that the use of protease-treated red cells for routine pretransfusion tests creates far more work than the accrued benefits justify.

A 64-year-old white man, who had never received a transfusion, was found to have anti-M in his serum. The antibody agglutinated all M+ red cells in room-temperature tests. When the ionic strength of the test milieu was reduced by use of an additive solution and the tests were incubated at 37 degrees C, the antibody hemolyzed M + N- but not M+N+ red cells. All M+ red cells reacted in indirect antiglobulin tests using polyspecific antiglobulin reagents when such tests followed an initial incubation at room temperature. When red cells and the patient's serum were warmed to 37 degrees C before being mixed, no antibody activity was demonstrable. The antibody was adsorbed to exhaustion onto M+N- and M+N+ red cells (including the patient's own), and its activity was destroyed by dithiothreitol. There was no evidence of in vivo red cell destruction by the autoantibody. No previously reported example of anti-M has been shown to activate complement in conventional in vitro tests. This example was extraordinary in that it caused sufficient complement activation to present as an in vitro hemolysin.

In tests on 50 single-donor sera containing anti-CD or anti-CDE, it was found that 37 of them reacted with rG red Mood cells. However, adsorption-elution experiments showed that only 15 of the sera contained anti-G. Those sera that reacted with rG red blood cells d but which lacked anti-G, apparently contained anti-C that accounted for their reactions with the C-,D-,G+,CG+, rG red blood cells used. In all, only 30 per cent of the singledonor anti-CD and anti-CDE antibodies contained anti-G. In muked contrast, a11 of 11 commercially prepared anti-CD and anti-CDE typing reagents contained potent anti-G. In tests on 46 anti-CD and 9 anti-C sera, it was found that most of them contained anti-rhi (anti-Ce) component. However, only 2 of the 55 sera (1 anti-CD and 1 anti-C, both single-donor sera) failed to react with ry red blood cells, showing that 53 of the anti-C antibodies would detect C in the absence of rhi. These findings are discussed in terms of some statements that exist in the literature about anti-G, and in term of the relative immunogenicity of D, C, G, CG and rhi.

We recently described6 an individual whose red blood cells appear to carry a hybrid MNSs sialoglycoprotein (SGP). The MN-derived portion of that SGP carries at least two determinants defined by some examples of antibodies that have been called anti-Ena. However, the red blood cells lack a different determinant that is defined by other examples of anti-Ena. This individual has now formed an anti-Ena antibody that reacts with the portion of Ena that her red blood cells lack, but not with the Ena determinants that have been shown7 to be carried on MN SGP. It is not yet clear whether the portion of Ena that her red blood cells lack and that her antibody defines is MN SGP-borne. The findings in this case provide further support for our conclusions6,7,9 that the terms “Ena” and “anti-Ena,” as previously used, describe heterogeneous groups of antigens and antibodies.

Tests in which 11 examples of anti-U were used in titration studies against the red blood cells of 9 obligate Uu heterozygotes, from 4 unrelated families, and random Negro and Caucasian donors (many of whom were of the presumptive UU genotype) have failed to demonstrate any dosage of the U antigen.

We describe a case of 'warm' antibody-induced haemolytic anaemia (WAIHA) in which marked depression of red cell Rh antigen expression resulted in the patient presenting with severe anaemia but a negative direct antiglobulin test (DAT). The serum contained potent IgG Rh antibodies. Unlike two previously reported cases (Koscielak, 1980; Veer et al. 1981) in which the diagnosis of WAIHA was established before the DAT became negative, this patient presented with negative serological findings during his first episode of anaemia. As a result, the serum antibodies appeared to be allo- not autoimmune in nature and to be unrelated to the patient's anaemia. Confirmation of the autoimmune nature of the Rh antibodies was not possible until nearly 2 years after the first episode of anaemia.

We describe an antibody, made by an individual of the phenotype M+, N+, S+, s+, U+, that reacted only with ficin or papain-treated red blood cells, and that initially appeared to have anti-S specificity. However, further studies revealed that the antibody, which did not have anti-U specificity, recognized a determinant present on S+, s-, U+, and S-, s+, U+, but missing from S-, s-, U-, and S-, s-, U+, red blood cells. The specificity of this antibody is discussed in terms of current knowledge of the structure of the Ss sialoglycoprotein.

A "normal" individual with a positive, direct antiglobulin test is described. In common with many other "normal" persons in whom a similar finding has been made, there was no evidence of an increased rate of in vivo red blood cell destruction in this patient. The patient successfully completed a pregnancy during the time that detailed serologic studies on her autoantibodies were being performed. Although the maternal autoantibodies were demonstrable in both an eluate made from the red blood cells of her newborn infant, and in the cord serum, there was no reason to believe that the antibodies caused red blood cell destruction in the infant. The case was of further interest because of the specificities of some of the autoantibodies. Although the mother and child were both C-negative, eluates from their red blood cells contained what ostensibly appeared to be anti-C. Studies that showed that the antibody could be totally adsorbed with C-negative, as well as C-positive red blood cells, proved that this was another example of an autoantibody mimicking an alloantibody. Although this autoantibody appeared initially to have anti-C specificity it was eventually shown to be more closely related to anti-Hr or anti-Rh34, than to anti-C.

Many lectins display blood group activity, and extracts from Dolichos biflorus (anti-A1), Ulex europaeus (anti-H), and Vicia graminea (anti-N) seeds provide an alternative to human sera as a source of blood-typing reagents. However, the major application of lectins in blood group serology undoubtedly lies in the recognition and elucidation of red celll polyagglutination. In this respect, lectins from Arachis hypogaea (anti-T/Tk), Salvia sclarea (anti-Tn). Salvia horminum (anti-Tn + Cad). Dolichos biflorus (anti-Tn/Cad) Glycine max, and the N-acetyl-D-glucosminyl-binding lectin, BS II (anti-Tk) from Bandeiraea simplicifolia seeds, provide an invaluable source of reagents for use in investigative immunohemotology. Because of their specific carbohydrate-binding properties, lectins have also been used as probes in studies on the topography of the red cell surface. This latter appliction has provided much information on the structure of the MN, T, and Tn red cell surface receptors and has aided in defining the red cell membrane abnormalities associated with certain uncommon phenotypes within the MN blood group system.

SUMMARY Eluates from the red blood cells (and sera whenever free autoantibody was present) of 150 individuals with positive direct antiglobulin tests, have been studied for antibody specificity. Of 87 patients with AIHA, 64 had autoantibodies reacting with all red cell samples including Rhnull. Of these 64 anti-dl autoantibodies, two were, and 32 contained, auto-anti-Wrb. Of 33 patients being treated with alphamethyldopa, who had developed positive direct antiglobulin tests, 23 had anti-dl autoantibodies four of which contained auto-anti-Wrb. Of 30 haematologically normal donors with positive direct antiglobulin tests, 23 had anti-dl autoantibodies, two of which were, and six of which contained, auto-anti-Wrb. The full specificities of autoantibodies, other than anti-Wrb and anti-dl, in the 150 patients are described, as are the natures of the protein red cell coatings that caused the positive direct antiglobulin tests. The presence of free serum autoantibody as a correlate of the three clinical conditions is reported. Several observations on auto-anti-Wrb are documented. The antibody can cause gross red cell destruction in vivo, but can be benign on other occasions; it occurs with approximately the same frequency in AIHA patients and ‘normal’donors with positive direct antiglobulin tests, but in fewer patients with alpha-methyldopa induced positive direct antiglobulin tests; it does not activate complement in vivo;, and finally it may eventually provide a clue to the aetiology of AIHA.

The nature of the common erythrocyte antigens EnaFR and Wrb, that are both absent from En(a-) cells, and the rare Wra receptor, apparently encoded by an allele of Wrb, was investigated. Various modification, fractionation or cleavage products of erythrocyte membranes were used in hemagglutination inhibition assays. The EnaFR and Wrb antigens were shown to represent labile structures within the residues approx. 62-72 of the major (MN) sialoglycoprotein that require lipids, at least for complete expression of antigenic activity. During the course of these experiments, the arrangement of the MN glycoprotein's peptide chain with respect to the lipid bi-layer was also studied, using various proteinases. Furthermore, the MN glycoprotein was found to aggregate with the major membrane protein (band 3) in the presence of Triton X-100. The Wra antigen was shown to exhibit properties that differ considerably from those of the Wrb receptor. Analyses on the MN glycoprotein, isolated from the red cells of the only known Wra homozygote and two WraWrb individuals, did not reveal any amino-acid exchange within the residues 40-96 of the molecule. Therefore, the Wr locus that determines the presence or absence of the Wrb antigen on the MN glycoprotein might influence the post-translational modification of amino-acid residues, the structure of tightly bound lipids or the aggregation of the MN glycoprotein with a different protein such as band 3.

Antiglobulin sera from nine different manufacturers have been tested, over a two-year period, for their ability to detect the complement components beta 1A, alpha 2D, and beta IE. The results demonstrate considerable variation in the abilities of sera from different manufacturers to detect these components and indicate that not all sera on the market are suitable reagents for diagnostic use and compatibility tests. The results also show that there is considerable variation between different lots of serum from some of the manufacturers. In general, the anti-complement levels of these reagents have increased during the two-year period of study but not all companies produce suitable reagents for routine use.

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.

An individual (J-1) was shown to be heterozygous for the MiV and Mk genes. Her red cells typed as M+(weak), N-, S-, s+(strong), U+, Hil+, Wr(a-b-), En(a+weak). Polyacrylamide gel electrophoresis analysis of her red cell membranes revealed absence of PAS-staining bands corresponding to normal MN and Ss sialoglycoprotein (SGP), and presence of a hybrid MNSs SGP [(alpha-delta)MiV] similar but not identical to that reported for an MiV homozygote. However, J-1 cannot be homozygous for MiV since the red cells of two of her children are Hil- and s-, carry only a single dose of M antigen, and have a sialic acid content that is consistent with the presumption that they are Mk heterozygotes. J-1's hybrid MNSs SGP is considered to be gene-fusion product resulting from unequal crossover between a normal alpha M and delta gene, and her red cells lack that portion of the Ena antigen that is resistant to ficin. Her hybrid MNSs SGP differs, therefore, from that reported for the MiV homozygote, which probably arose from unequal crossover between alpha N and delta genes. Further, the red cells of the MiV homozygote carry the ficin-resistant Ena determinant.

On rare occasions persons are found whose red blood cells react as D-positive yet have anti-D present in their serum. The usual explanation of this finding is that the red blood cells lack a portion of the D antigen mosaic and the individual has formed anti-D directed against the missing portions of the mosaic. This paper presents details of a case that closely resembled this situation but in which it was eventually possible to prove that misleading serological results had been obtained because of the Matuhasi-Ogata phenomenon. The specific antigen-antibody complex responsible for the Matuhasi-Ogata phenomenon in this case was Bgb-anti-Bgh. Investigation of a large number of anti-D typing sera revealed the presence of anti-Bg (anti-HL-A) antibodies in nearly 50 per cent of them. The implications of these findings are discussed.