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Background: Mutation in the KLF1 gene is the cause of the In(Lu) (Inhibitor of Lutheran) Lu(a-b-) phenotype and more than 60 alleles have been associated with this phenotype. Here we describe findings from investigation of seven cases: six presenting with a Lu(a-b-) phenotype including the historical index case and one referred from a patient with chronic anemia., Study Design and Methods: Serologic testing was by standard methods. DNA testing included amplification and sequencing of KLF1 and LU coding regions. A StuI polymerase chain reaction-restriction fragment length polymorphism was designed to target c.304T>C in KLF1., Results: Five different KLF1 alleles were identified. Three are new: KLF1*90A (p.Trp30Ter), KLF*911A (p.Thr304Lys), and KLF1*304C,318G (p. Ser102Pro, Tyr106Ter) present in two unrelated individuals. Two, including the index case, had c.954dupG (p.Arg319Glufs*34), that is, KLF1*BGM06. The child with unexplained anemia had c.973G>A (p.Glu325Lys), associated with congenital dyserythropoietic anemia. The common c.304T>C was found in two of the seven samples investigated and in 60 of 100 blood donors., Conclusion: Mutations in KLF1 are pleiotropic and although most are benign, others are associated with hematologic abnormalities. We report three new KLF1 alleles associated with benign In(Lu) and document both the molecular basis of the original In(Lu) phenotype using a frozen sample stored for more than 50 years and the cause of unexplained anemia in a child. We also confirm previous observations that c.304C (p.102Pro) is not, by itself, associated with an In(Lu) phenotype in donors self-identified as U.S. minorities., (© 2017 AABB.)

The Lutheran glycoprotein (Lu), also known as basal cell adhesion molecule (B-CAM), is an Ig superfamily (IgSF) transmembrane receptor for laminin α5. Although Lu is not present in normal hepatocytes, its expression is significantly increased in hepatocellular carcinoma (HCC). In this study, we isolated thirteen phage antibodies to Lu from a phage library of peripheral blood from HCC patients, suggesting that these patients produced autoantibodies against endogenous Lu. To characterize the phage antibodies, we determined the Lu domains they recognize. The extracellular domain of Lu contains five IgSF domains, D1-D2-D3-D4-D5. The epitope of one phage antibody (A7) was localized to the D5 domain. The other phage antibodies recognized the D2 domain, which is also recognized by a function blocking mouse monoclonal antibody. One of the antibodies to D2 (C7) inhibited the binding of Lu to ligand, and it also prevented tumor cell migration on laminin-511 (LM-511). However, the C7 scFv purified from the periplasm fraction of bacteria did not exhibit the inhibitory effects, indicating that the scFv form could not sterically inhibit the binding of Lu to LM-511. We also identified the amino acid residues that form the epitope recognized by the C7 phage antibody. Mutagenesis studies showed that Arg247 is necessary for forming the epitope. The C7 phage antibody and its epitope may be useful for developing drugs to prevent HCC progression and/or metastasis., Competing Interests: The authors have declared that no competing interests exist.

Life-long red blood cell (RBC) transfusions remain the main treatment for severe thalassemia. We hereby report a case of anti S and anti Lu(a) in a β-thalassemia major patient detected incidentally on antibody screening. The patient was a known case of β-thalassemia major and was on regular blood transfusion every 3 weeks from the institute from the age of 6 months. Subsequently, on one occasion, patient's crossmatch was compatible despite positive antibody screen using microcolumn gel technique. Autocontrol and direct antiglobulin test were negative on microcolumn gel. Anti S and anti Lu(a) antibodies were identified. Blood unit found compatible was negative for S and Lu(a) antigens. Antibody titers were 1:1 for both anti S and anti Lu(a) in AHG phase using tube technique and antibodies were of IgG type. Blood unit was transfused uneventfully to the patient. Donors were traced back (last three donations) and called for repeat blood sample testing for S and Lu(a) antigen. Two out of three donors were found to be S antigen positive and one out of these two was Lu(a) antigen positive. Anti S and anti Lu(a) antibodies were again identified on patient's subsequent visit for transfusion. The present case re-emphasize the importance of antibody screening at each visit in earlier detection of antibodies in multi transfused patients. Encouraging patients to receive transfusion from one center and dedicating donors could reduce alloimmunization rate but larger studies are required., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin's action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.

Background: The Lutheran blood group glycoprotein (Lu), an Ig superfamily (IgSF) transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a major component of basement membranes in various tissues. Previous reports have shown that Lu/B-CAM binding to laminin α5 contributes to sickle cell vaso-occlusion. However, as there are no useful tools such as function-blocking antibodies or drugs, it is unclear how epithelial and sickled red blood cells adhere to laminin α5 via Lu/B-CAM., Methodology/principal Findings: In this study, we discovered a function-blocking antibody that inhibits Lu binding to laminin α5 using a unique binding assay on tissue sections. To characterize the function-blocking antibody, we identified the site on Lu/B-CAM recognized by this antibody. The extracellular domain of Lu/B-CAM contains five IgSF domains, D1-D2-D3-D4-D5. The antibody epitope was localized to D2, but not to the D3 domain containing the major part of the laminin α5 binding site. Furthermore, mutagenesis studies showed that Arg(175), the LU4 blood group antigenic site, was crucial for forming the epitope and the antibody bound sufficiently close to sterically hinder the interaction with α5. Cell adhesion assay using the antibody also showed that Lu/B-CAM serves as a secondary receptor for the adhesion of carcinoma cells to laminin α5., Conclusion/significance: This function-blocking antibody against Lu/B-CAM should be useful for not only investigating cell adhesion to laminin α5 but also for developing drugs to inhibit sickle cell vaso-occlusion.

Objective: To assess the presence of irregular xenoantibodies against human red blood cells (RBCs) in 6 primate species used in xenotransplantation and other experimental procedures., Materials and Methods: Serum samples from 109 baboons of 4 different species (olive, chacma, sacred, and Guinea), 38 rhesus macaques, and 30 squirrel monkeys were tested for irregular xenoantibodies using an agglutination test using human RBCs of known phenotype for Rh, Kell, Kidd, Lewis, Lutheran, P1, and Duffy antigens, commercially available as RBC I, II, and III., Results: We found hemagglutination for RBC I in 49%, 22%, 100%, 57%, 32%, and 33% of olive, chacma, sacred, and Guinea baboons, rhesus macaques, and squirrel monkey, respectively. The frequency for RBC II was 49%, 50%, 100%, 57%, 37%, and 33%, respectively, and for RBC III was 56%, 37%, 100%, 79%, 34%, and 33%, respectively. There were differences in frequency depending on the sex of the rhesus macaques; all 3 RBCs tested were higher in the females: 44% vs 0%, P = .008; 48% vs 1%, P = .02, and 44% vs 9.1%, P = .04 for RBC I, II, and III, respectively. There were differences due to age in only olive baboons, and a higher frequency in younger animals compared with juvenile, subadult, and adult animals for all 3 human RBCs., Conclusions: Assessment of irregular antibodies in the presence of primate serum should be taken into account during any experimental xenotransplantation protocol.

Background: Until now, it was not possible to identify antibodies to red blood cells (RBCs) except with pretyped RBCs. Here, a novel method with particles coated with recombinant Lu(b) protein for detection of anti-Lu(b) is described., Study Design and Methods: Prokaryotic recombinant Lu(b) proteins were generated and coupled onto superparamagnetic particles coated with streptavidin. The coated particles were tested in the presence of different serum and plasma samples (13 anti-Lu(b), 6 anti-Lu(a), 20 other antibodies, and 35 serum samples from blood donors) with the particle gel immunoassay (ID-PaGIA)., Results: Lu(b)-coated particles reacted with all 13 samples containing anti-Lu(b), but not with any samples lacking anti-Lu(b). In addition, the anti-Lu(b) titers were higher with Lu(b)-coated particles than with Lu(a-b+) RBCs in almost all cases., Conclusion: Recombinant blood group proteins may be able to dispense with the need for RBCs for identification of certain RBC alloantibodies.

Background: At present, identification of antibodies against high-frequency antigens is limited to reference laboratories having panels of rare red blood cell (RBC) specimens in stock. Antibodies against Lu(b) are among the most frequent clinically relevant antibody specificities directed against high-frequency antigens., Study Design and Methods: Soluble recombinant Lu(b) fusion proteins consisting of the first three N-terminal immunoglobulin superfamily domains and a V5-His tag were generated. Eukaryotic recombinant Lu(b) proteins were isolated from cell culture supernatant of stably transfected HEK293 cells with anti-V5 Sepharose. Prokaryotic Lu(b) fusion proteins were expressed in Escherichia coli, purified by Ni-NTA, and refolded by chromatographic procedures. Ten anti-Lu(b) serum samples, 6 anti-Lu(a) serum samples, 30 serum samples directed against other blood group antigens, 10 serum samples from patients with RBC autoantibodies, and 100 serum samples from randomly selected donors were used for antibody screening., Results: Eukaryotic and prokaryotic recombinant Lu(b) proteins proved to be equally suited for identification of anti-Lu(b). Recombinant Lu(b) protein-based enzyme-linked immunosorbent assay correctly identified samples containing anti-Lu(b) sera, and the titers were at least two times higher than those measured by the gel agglutination-based indirect antiglobulin test. In hemagglutination inhibition assays, recombinant Lu(b) protein neutralized all anti-Lu(b), but none of the other alloantibodies decreased in reactivity., Conclusion: Antibody detection systems based on soluble eukaryotic or prokaryotic recombinant blood group proteins have the potential to replace current systems with rare RBCs for identification of alloantibodies against high- or low-frequency antigens. This innovation could bring routine laboratories one step closer to specialized antibody diagnostics.

Background: Monoclonal antibodies (MoAbs) are gradually replacing human polyclonal sera as typing reagents. Many blood group specificities, however, are not amenable to classic hybridoma technology. The phage display technology, aimed at isolating peptides or antibody fragments, offers an alternative strategy. Recombinant antibodies derived from this technology would greatly facilitate phenotyping and decrease analysis cost., Study Design and Methods: A human single-chain Fv (scFv) phage-displayed library was panned on red blood cells (RBCs) in an attempt to isolate clones recognizing human RBC specificities. Three rounds of biopanning were performed. Enrichment was monitored by phage titration, and selected phage populations were analyzed further., Results: Three major clones were identified by clone diversity analysis. One of them showed a specificity for Lua. This scFv was reconstructed into a human IgG1 by recombinant DNA methods. The reactivity of the reconstructed human IgG1 toward Lua is indistinguishable from its parent scFv. Moreover, the specificity of the antibody was confirmed by serologic assays, flow cytometry, and biochemical analysis with RBCs of different Lu phenotypes and a recombinant cell line expressing Lu glycoproteins., Conclusion: With phage display and standard recombinant DNA methods, isolation of a scFv of Lua specificity was successful, from which a complete human IgG1 MoAb of equivalent reactivity was reconstructed. To our knowledge, this is the first MoAb specific for Lua.

Background and Objectives: The Lutheran blood group system comprises 18 antigens numbered LU1 to LU20, with two numbers obsolete. Thirteen antigens are of high frequency., Materials and Methods: Serological tests were performed by conventional methods. The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was carried out with monoclonal antibodies to Lutheran glycoprotein. All exons of the LU gene were amplified by the polymerase chain reaction (PCR) and directly sequenced from genomic DNA., Results: A patient was found to have an antibody to a high-frequency red cell antigen during her second pregnancy. The antibody was shown to be Lutheran-related and was distinguished from all reported Lutheran antibodies. MAIEA suggested the antibody was defining a novel epitope in domain 1 of the Lu-glycoprotein. Sequencing of the LU gene revealed a new homozygous single-point mutation, C282G, in exon 3, encoding an Asp94Glu change in the first domain of the Lu-glycoprotein., Conclusions: The antibody detected a new high-frequency Lutheran antigen, numbered LU21, that appears to result from an Asp94Glu substitution in the first, N-terminal domain of the Lu-glycoprotein.

Background: There is only little information on the transfusion support of patients with antibodies to high-frequency RBC antigens., Study Design and Methods: In cooperation with reference laboratories and transfusion services in Austria, Germany, and Switzerland, the transfusion support provided to hospitalized patients identified as having such antibodies was reviewed during a 20-month period., Results: A total of 52 patients with antibodies to high-frequency antigens were treated in hospitals. Twenty-two of them received 104 units of antigen-negative RBCs. In 23 cases, a deviation from the standard transfusion policy (e.g., transfusion of antigen-incompatible units) occurred. The use of frozen or fresh units varied amongst the different countries but did not affect the rate of deviation from protocol. About 20 percent of all units were supplied internationally. Four antibody specificities, anti-Kpb, anti-Vel, anti-Lub, and anti-Yta, were identified in two-thirds of the patients., Conclusion: This survey indicated that transfusion support was unsatisfactory in about one-third of the hospitalized patients with antibodies to high-frequency antigens. Maintaining a rapidly accessible stock of just four types of rare blood units would ensure adequate transfusion support for most of these patients.

Background: Lu6 is a high-incidence antigen of the Lutheran blood group. Examples of anti-Lu6 are rare and are of uncertain clinical significance., Case Report: Three patients were encountered in whom anti-Lu6 was detected on pretransfusion screening. The patients were all Iranian Jews and were not known to be related. In vitro studies to ascertain the potential clinical significance of the antibody using the monocyte monolayer assay (MMA) were negative in two patients. The third patient received a two-unit transfusion of incompatible Lu6 RBCs with no signs of hemolysis. However, after the transfusion, the MMA and a chemiluminescence test were positive, whereas a chromium survival study was normal. Thus, the antibody may have changed in its clinical significance., Conclusion: Although anti-Lu6 does not appear to be a clinically significant RBC antibody in all circumstances, transfusion of Lu6 RBCs in patients with anti-Lu6 should be performed cautiously.

Background: The In(Lu) gene reportedly suppresses several blood group antigens that are not part of the Lutheran system, including the high-incidence antigens of the Knops blood group system. Because complement receptor 1 (CR1), which is known to carry the Knops system antigens, has a red cell (RBC) expression polymorphism, the role of In(Lu) in the expression of the Knops system antigens was reinvestigated., Study Design and Methods: Blood samples from nine donors having the Lu(a-b-) phenotype were obtained and immediately phenotyped for Lu(b), Kn(a), McC(a), Sl(a), and Yk(a). The samples were also tested for Lu(a), P1, and AnWj. Immunoblots were performed to study both the CR1 and Lutheran glycoproteins from these donors. RBC expression of CR1 was quantified with an enzyme-linked immunosorbent assay, and the genetic inheritance of the high-expression (H) or low-expression (L) allele for CR1 was determined by Southern blot., Results: Lu(b) was demonstrable only by absorption and elution techniques on all nine samples; however, the high-incidence Knops system antigens were readily detectable by hemagglutination. Two Lu(a-b-) donors (sibs) demonstrated weak Lutheran glycoprotein bands of 78 and 85 kDa on immunoblots, while the other seven Lu(a-b-) samples had no detectable glycoprotein. All donors had CR1*1, and one donor also had CR1*2 on immunoblot. Only one donor was homozygous for the L allele, and all had RBC copy numbers of CR1 within the normal range., Conclusions: Nine donors with the Lu(a-b-) phenotype showed suppression of the Lutheran system antigens but normal expression of CR1 glycoprotein and the Knops system blood group antigens. This suggests that the genes that suppress Lutheran system antigens do not suppress CR1 or its related blood group antigens.

Phenotypic analysis of hematopoietic stem and progenitor cells has been an invaluable tool in defining the biology of stem cell populations. We use here flow cytometry to examine the expression of human erythroid-specific surface markers during the maturation of early committed erythroid cells derived from cord blood in vitro. The temporal order of the expression of erythroid specific markers was as follows: Kell glycoprotein (gp), Rh gp, Landsteiner Wiener (LW) gp, glycophorin A (GPA), Band 3, Lutheran (Lu) gp, and Duffy (Fy) gp. The time at which some of these markers appeared suggests possible roles for some of these erythroid-specific polypeptides during the differentiation of these committed progenitors. The early appearance of Kell gp raises the possibility that it may have an important role in the early stages of hematopoiesis or cell lineage determination. Kell gp may also be a useful marker for the diagnosis of erythroleukemia. The late expression of Lu gp suggests it may be involved in the migration of erythroid precursors from the marrow. Fy gp is also expressed late consistent with a role as a scavenger receptor for cytokines in the bone marrow and circulation. Rh c antigen appeared before Rh D antigen, and it is suggested that this may reflect a reorganization of the developing erythroid cell membrane involving the Rh polypeptides and other components, including GPA and Band 3.

The density of CR1, the C3b/C4b receptor (CD35), on erythrocytes (E) (CR1/E) is genetically determined. However, the broad distribution of CR1/E within a given genotype suggests that other genetic elements might contribute to the regulation of CR1/E. In some pathological conditions, including systemic lupus erythematosus (SLE), AIDS and hemolytic anemia, CR1 deficiency parallels the severity of the disease. When compared to healthy individuals, an accelerated decrease in CR1/E in these patients has been demonstrated, but other mechanisms interfering with CR1 density regulation during erythropoiesis might also contribute. In exceptional circumstances, CR1/E can be dramatically decreased in healthy individuals by the effect of a regulatory gene, In(Lu), that switches off various surface molecules on E, the structure genes of which are located on four different chromosomes, suggesting a transcription regulatory role for In(Lu) gene products. The hypothesis that products of this gene could physiologically regulate the surface density of all these molecules has been tested by determining Lub density on E (Lub/E) using quantitative flow cytometry. Lub antigenic sites were then compared to CR1/E among healthy individuals of the different CR1 density phenotypes, SLE patients with and without CR1 deficiency, and an exceptional SLE patient totally lacking CR1/E and reticulocytes. No quantitative relationship was found between CR1 and Lub expression in either normal or pathological conditions. These data establish that In(Lu) products are not involved in normal or pathological CR1 density regulation.

The Lutheran antigens are recently characterized glycoproteins in which the extracellular region contains five immunoglobulin like domains, suggesting some recognition function. A recent abstract suggests that the Lutheran glycoproteins (Lu gps) act as erythrocyte receptors for soluble laminin (Udani, M., Jefferson, S., Daymont, C., Zen, Q., and Telen, M. J. (1996) Blood 88, Suppl. 1, 6 (abstr.)). In the present report, we provided the definitive proof of the laminin receptor function of the Lu gps by demonstrating that stably transfected cells (murine L929 and human K562 cell lines) expressing the Lu gps bound laminin in solution and acquired adhesive properties to laminin-coated plastic dishes but not to fibronectin, vitronectin, transferrin, fibrinogen, or fibrin. Furthermore, expression of either the long-tail (85 kDa) or the short-tail (78 kDa) Lu gps, which differ by the presence or the absence of the last 40 amino acids of the cytoplasmic domain, respectively, conferred to transfected cells the same laminin binding capacity. We also confirmed by flow cytometry analysis that the level of laminin binding to red cells is correlated with the level of Lu antigen expression. Indeed, Lunull cells did not bind to laminin, whereas sickle cells from most patients homozygous for hemoglobin S overexpressed Lu antigens and exhibited an increased binding to laminin, as compared with normal red cells. Laminin binding to normal and sickle red cells as well as to Lu transfected cells was totally inhibited by a soluble Lu-Fc chimeric fragment containing the extracellular domain of the Lu gps. During in vitro erythropoiesis performed by two-phase liquid cultures of human peripheral blood, the appearance of Lu antigens in late erythroid differentiation was concomitant with the laminin binding capacity of the cultured erythroblasts. Altogether, our results demonstrated that long-tail and short-tail Lu gps are adhesion molecules that bind equally well laminin and strongly suggested that these glycoproteins are the unique receptors for laminin in normal and sickle mature red cells as well as in erythroid progenitors.

The monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) technique is an immunoassay devised primarily for locating blood group antigens on specific red-cell membrane proteins. The assay involves the incubation of intact red cells with two antibodies, one human alloantibody, the other a nonhuman antibody, usually a rodent monoclonal antibody, but polyclonal antibodies of rabbit origin have been utilized. For a positive result, both antibodies must bind to the same membrane protein. The red cells are lysed, the membrane solubilized and the trimolecular complex of two antibodies and membrane protein is captured in a well coated with goat antirodent (or rabbit) immunoglobulin. The immobilized complex is then detected by the use of peroxidase-conjugated goat antihuman (or rodent) immunoglobulin. Negative results, due to mutual blocking between the human and animal antibodies when their epitopes are close together on the same molecule, have permitted a degree of localization of epitopes on some proteins. This has been most effective in the mapping of Cromer blood group system antigens on the complement control protein domains of decay-accelerating factor (DAF, CD55), but has also proved informative in the clustering of antigens on the Lutheran and Kell glycoproteins. MAIEA is an effective tool for the identification of antibodies to Knops-system antigens on complement receptor 1 (CR1, CD35) in immunohaematology reference laboratories. These antibodies are clinically unimportant, but must be identified before they can be ignored for transfusion purposes.

Twenty Mabs of human or murine origin were studied. Serological characterization was achieved by the gel technique with common, rare, and enzyme-modified RBCs. Three clones bound to cell surface proteins on immunoblots: 1 anti-Lub and 2 Mabs raised to a purified Kell glycoprotein. Four anti-K Mabs precipitated the expected 93kD protein and a dimeric form under non-reducing conditions.

The past year has seen further advances in our understanding of the molecular biology of the most abundant erythrocyte proteins associated with blood group antigens (band 3, the glycophorins, and the Rh antigen-related proteins). There have also been several important developments in the structural and functional identification of some of the less abundant antigens. These developments include the association of the Colton antigens with the erythrocyte water channel, aquaporin; the cloning of the Duffy antigen and its identification as a chemokine receptor; the cloning of the Kx antigen, which is associated with McLeod syndrome; and the cloning of the LW, CD47, and Xga antigens.

Anti-LU8, an antibody defining a high-frequency antigen in the Lutheran system, was identified in the serum of a gravida 4 para 0 pregnant women who had never been transfused. The antibody consisted of both an IgM and an IgG1 component and was nonreactive in the antibody-dependent cell-mediated cytotoxicity (ADCC) assay. The red cells of the mother and 3 siblings of the patient typed as LU:8,14 and were incompatible with the patient's serum. The baby was born at term with a negative direct antiglobulin test and typed as LU:8,14. There was no clinical evidence of haemolytic disease of the newborn.

The Lutheran (Lu) blood group antigens are a family of human erythrocyte antigens which reside on two closely-related erythrocyte integral membrane proteins. Sixteen Lutheran or so-called para-Lutheran antigens have thus far been described, and human antisera to many of them have been shown to immunoblot two proteins, of 78 and 85 kDa. Lu cDNA encodes an integral membrane protein of 597 amino acids that is a member of the Ig superfamily. Lu proteins comprise five Ig superfamily domains, along with a single transmembrane domain and a cytoplasmic domain of about 60 amino acids. The two proteins seen in biochemical studies of red cell membranes appear to be derived from 2 mRNA species that differ only in their 3' ends, suggesting that they arise from alternate splicing of a single preRNA. Three genetic backgrounds for the Lu(a-b-) [Lu null] phenotype have been described. A recessive Lu null phenotype is rarely observed as a result of homozygosity for two amorphic LU alleles. However, the most common Lu(a-b-) phenotype appears to be caused by an independently segregating, dominant gene, designated In (Lu), which inhibits expression of all Lutheran antigens to nearly undetectable levels. This gene also affects the expression of other cell surface proteins and blood group antigens that are genetically unlinked to the Lutheran locus, including CD44 and MER2. CD44, a member of the cartilage link family of proteins, bears the In and AnWj blood group antigens. A widely distributed protein CD44 is expressed at normal levels on all tissues except erythrocytes in the presence of the In (Lu) gene. A second Lutheran regulatory gene, XS2, is responsible for the third Lu(a-b-) phenotype, which exhibits an X-linked inheritance pattern. The XS2 gene down-regulates but does not abolish expression of LU genes and does not affect expression of CD44.

A new assay, the monoclonal antibody-immobilization of erythrocyte antigens (MAIEA) is described for the assignment of red cell antigens recognised by human allo-antisera, to particular membrane components of the red cell membrane. This technique detects tri-molecular complexes formed by the reaction of a human antibody and a mouse antibody with a particular red cell protein. A positive reaction, in an ELISA-type detection procedure, occurs if the epitopes recognised by the human and mouse antibodies are present on the same membrane component but different regions. The MAIEA assay has been developed to investigate blood group antigens and in this report its application to the study of the Lutheran, Yt and Kell blood group systems is described. The technique has been used to show that the Lu(a) and Lu3 antigens are carried on the Lutheran glycoprotein, Yta and Ytb on the enzyme acetylcholinesterase and K, k, Kpa and Kpb on the Kell glycoprotein.

The Lutheran blood group system consists of several genes of which Lua and Lub are the best characterized. Over 98% of the Western population is Lu (b+), and consequently antibody formation against Lub is rare. Cases reported in the literature suggest that the expression of Lutheran antigens on red cells of the newborn is weak, and Lutheran antibodies are not known to have caused severe haemolytic disease of the newborn. We describe a case of anti-Lub immunization during pregnancy. Despite increasing anti-Lub antibody titre and antibody-dependent, cell-mediated cytotoxic activity of the anti-Lub, two parameters associated with haemolytic disease of the newborn, the baby did not suffer from erythroblastosis.

A 43-year-old Arab woman was found to be negative for the high incidence AnWj antigen and her serum contained anti-AnWj. Two of her seven siblings were also AnWj-negative, which provides evidence for the first time that the AnWj-negative phenotype may be an inherited character. Blood groups of the family, in which the parents of the proposita are consanguineous, show that AnWj is not part of the ABO, Rh, MNSs, Kell, Duffy, Kidd, Xg and, notably, Lutheran blood group systems and neither is it X or Y linked.

Immunoblots of red cell membranes stained with eluates of alloanti-Aua and alloanti-Aub show that these antibodies recognize 2 membrane components from Au(a+) and Au(b+) cells, respectively. These structures, of apparent molecular weight (Mr) 79,000 and 85,000, are identical in appearance and mobility on a 10% SDS polyacrylamide gel to the Lutheran glycoproteins identified by alloanti-Lub. Like the Lutheran glycoproteins, they showed a reduction in apparent Mr of about 1,500 after sialidase treatment. Red cell membrane components immunoprecipitated by a Lutheran-related monoclonal antibody, were analysed with Lutheran and Auberger antibodies by immunoblotting. The Lutheran glycoproteins were revealed by anti-Lub in precipitates from Au(a+b-) Lu(a-b+) and Au(a-b+) Lu(a-b+) cells, by anti-Aua in precipitates from Au(a+b-) Lu(a-b+) cells and by anti-Aub in precipitates from Au(a-b+) Lu(a-b+) cells. The same components were also recognized by anti-Lua in precipitates from Lu(a+b-) cells. Thus Aua and Aub antigens appear to be carried on the same red cell membrane structures as those carrying the Lutheran determinants. These results are particularly significant in the light of the very close phenotypic association between the Auberger and Lutheran blood groups which have been shown, by one family, to be controlled by genes at separate loci.

An IgG autoantibody, named anti-Wj, reacts with all random and most selected red cell samples. It does not agglutinate cells of Lu(a-b-) type caused by the In(Lu) dominant inhibitor gene, but cells of recessive Lu(a-b-) type are reactive. These data establish that synthesis of the Wj antigen is suppressed by the In(Lu) gene, but it is not a part of the Lutheran blood group system. The Wj antigen is not well developed on fetal red cells. This example of auto anti-Wj is a non-complement-binding IgGl protein that did not appear to cause accelerated in vivo destruction of the patient's red cells.

Heterologous anti-lymphocyte globulin (ALG) is known to contain panagglutination activity and may cause a positive direct antiglobulin test in a renal allograft recipient. Three patients are reported in whom the passively acquired antibody showed apparent specificity in the Lutheran blood group system. The sera of these patients and eluates from their red cells displayed reactivity consistent with a Lutheran-related antibody. While the neat ALG acted as a panagglutinin, the Lutheran system specificity appeared only following dilution of the ALG. This specificity did not appear to represent a separate, distinguishable antibody in the ALG product. Before considering the utilization of units of the rare Lu(a-b-) phenotype for transfusing a renal allograft patient who has a Lutheran-related antibody, investigation of ALG as a source of that antibody should be conducted.

Pregnant women with antibodies to high incidence blood group antigens should be diagnosed as early as possible and red blood cells should be stored frozen for future possible needs. Either autologous, compatible siblings or unrelated individuals' red blood cells should be frozen. The application of such a program is described in three pregnant women with exceedingly rare antibodies to high incidence blood group antigens (anti-Lutheranb, and PP1Pk, and anti c with blood type B CCddee Du negative).

Immunoblotting was performed, after sodium dodecyl sulphate polyacrylamide gel electrophoresis of solubilised red cell membranes under non-reducing conditions, with human Lutheran antibodies, anti-Lua, -Lub, -Lu3, -Lu6 and -Lu8, and with human para-Lutheran antibodies, -Lu4, -Lu12 and -Lu17. On antigen-positive red cells all antibodies revealed two components of apparent molecular weight (Mr) 83,000 and 76,000. Anti-Lua and -Lub gave no bands with Lu(a-b+) or Lu(a+b-) cells, respectively. With the exception of anti-Lu17, which reacted normally, and anti-Lu3, which reacted weakly, none of the antibodies showed bands with membranes from Lu(a-b-) cells of the In(Lu) type. Treatment of the red cells with sialidase led to a small reduction in the apparent Mr of the Lutheran glycoproteins which, therefore, appear to contain sialic acid bearing O-linked oligosaccharides.

We have previously shown that a murine monoclonal antibody (A3D8) identifies a human erythrocyte protein antigen whose expression is regulated by the Lutheran inhibitor [In(Lu)] gene. In the present study, we demonstrated by immunoprecipitation and Western blot techniques that the antigen defined by A3D8 was on an 80-kD erythrocyte membrane protein. A second 170-kD protein was coprecipitated with the 80-kD protein but failed to show antigen activity by Western blot analysis. The 170-kD protein, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in two dimensions, was composed of 50- and 30-kD disulfide-linked subunits. In(Lu) Lu[a-b-) erythrocytes differed from Lu(a+b+) or Lu(a-b+) erythrocytes in that In(Lu) deoxycholate erythrocyte membrane extracts contained trace amounts of immunoprecipitable 80-kD protein compared with detergent-solubilized erythrocyte membrane extracts prepared from Lu(a+b+) or Lu(a-b+) subjects.

The authors analyse numerous publications dealing with hemolytic disease of the newborn due to erythrocyte antigens other than A, B and Rhesus D. They emphasize the frequency of these diseases and the clinical presentation according to the antibody specificity. Finally, they suggest a practical management to treat these hemolytic diseases of the newborn.

A patient with Hodgkin's disease became temporarily Wj-negative with alloanti-Wj in his serum. Four human autoantibodies, and 1 of 2 murine monoclonal antibodies, with serological characteristics of anti-Wj were nonreactive with his red cells, confirming that they have anti-Wj specificity. Six siblings of the patient are all Wj-positive. The patient was also temporarily Anton-negative, and cross-testing between Wj and Anton red cells and antisera showed mutual compatibility, indicating that the antigens are the same. The patient and 3 of his 6 siblings are also of the rare Lu: - 13 phenotype, providing the first evidence that this is an inherited characteristic.