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The Kell blood group is one of the major antigenic systems in human red blood cells. To determine the location of the Kell gene on human chromosomes, panels containing genomic DNA of human-hamster somatic cell hybrids were hybridized with radiolabeled cDNA probe specific for the Kell locus. Only the samples containing DNA from chromosome 7 gave positive hybridization signals. In situ hybridization analysis, using genomic clones isolated with the cDNA, localized the KEL gene to 7q33. Northern blot analysis of poly(A)+ RNA from human brain, kidney, lung, fetal and adult liver, and bone marrow showed that Kell transcripts were only present in fetal liver and bone marrow. This indicates that the Kell protein, which carries the Kell antigens, may only be expressed in erythroid tissues.

Red cells (RBCs) contain an abundance of protein methylase II, which catalyzes the transfer of methyl groups from S-adenosylmethionine to carboxyl groups of aspartyl and glutamyl residues in proteins. Enzyme-catalyzed transfer of methyl groups, labeled with 14C or 3H, from S-adenosylmethionine to membrane proteins of McLeod, Ko, and control RBCs was assayed by determining the acceptance of labeled methyl groups under standardized conditions. Membranes of control cells and Ko cells showed about 50 percent greater uptake than did those of McLeod cells. However, when ovalbumin was used as a methyl-accepting substrate, the levels of protein carboxymethyltransferase activity in all three types of cells were found not to differ significantly. In addition, no significant qualitative differences were apparent when methyl-labeled polypeptides from control and McLeod cells were separated by slab gel electrophoresis. The mechanisms responsible for changes in membrane protein methylation of McLeod cells remain unclear. However, these observations provide further evidence of the pleiotropic biochemical lesion associated with the acanthocytic morphology that characterizes McLeod RBCs.

The Kell blood group is a major antigenic system in human erythrocytes. Kell antigens reside on a 93-kDa membrane glycoprotein that is surface-exposed and associated with the underlying cytoskeleton. We isolated tryptic peptides and, based on the amino acid sequence of one of the peptides and by using the PCR, prepared a specific oligonucleotide to screen a lambda gt10 human bone-marrow cDNA library. Four clones were isolated, one containing cDNA with an open reading frame for an 83-kDa protein. All known Kell amino acid sequences were present in the deduced sequence; moreover, rabbit antibody to a 30-amino acid peptide, prepared from this sequence, reacted on an immunoblot with authentic Kell protein. The Kell cDNA sequence predicts a 732-amino acid protein. Hydropathy analysis indicates a single membrane-spanning region, suggesting that Kell protein is oriented with 47 of its N-terminal amino acids in the cell cytoplasm, and a 665-amino acid segment, which contains six possible N-glycosylation sites, is located extracellularly. Computer-based search showed that Kell has structural and sequence homology to a family of zinc metalloglycoproteins with neutral endopeptidase activity.

McLeod red cells (RBCs) lack Kx antigens and have weak expression of the Kell antigens. Individuals who carry the McLeod phenotype have acanthocytic RBCs and a compensated hemolytic state. To elucidate the role of the protein on which the Kx antigens reside in maintaining membrane deformability, the rheologic properties of McLeod RBCs were determined by ektacytometry. RBCs were obtained from normal individuals and from four patients with McLeod syndrome. Osmotic gradient deformability profiles of McLeod RBCs showed decreased whole cell deformability. Resealed ghosts from McLeod RBCs also showed decreased deformability, partly because of the decreased cell surface area and partly because of an intrinsic membrane stiffness in this syndrome. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytomer, and deformability was recorded continuously as the deformable ghosts fragmented into rigid spherical vesicles. Membranes from McLeod RBCs showed a noticeable increase in mechanical stability. Acquired causes of acanthocytosis, such as liver disease, did not cause the rheologic abnormalities observed in McLeod cells. Other abnormalities noted in McLeod RBCs were decreased RBC potassium content and an increased number of dense RBCs, as determined by centrifugation on a discontinuous density gradient. The data indicate that McLeod RBCs are rigid and have decreased surface area and that their membranes are intrinsically rigid with increased mechanical stability. These abnormalities may account for the reduced RBC survival observed in McLeod syndrome. The protein that carries the Kx surface antigen seems to be required for the maintenance of the normal physical function of RBC skeletal proteins.

Red cells of the McLeod phenotype in the Kell blood group system have an acanthocytic morphology. The membrane protein composition analyzed on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis, the ATP level and the activities of a large number of intracellular enzymes appear to be normal. Membranes prepared from McLeod red cells incubated with gamma AT[32P] and MgCl2 incorporated twice as much radioactivity into spectrin and also showed a slight elevation of phosphorylation in band 3 protein when compared to membranes from normal cells. Intact normal red cells incubated with carrier-free [32P] incorporated radioactivity into several proteins, with most incorporation in spectrin and band 3 protein. In comparison, McLeod cells incorporated three times more radioactivity into spectrin and band 3 protein but increased phosphorylation also occurred in other, but not all, membrane proteins. Intact McLeod red cells also showed increased phosphorylation of membrane phospholipids, but they incorporated [32P] into intracellular nucleotide phosphates in a normal manner.

An example of auto-anti-Kpb in a Kp(a+b-) patient is described. The antibody present in the patient's serum and in eluates from her red cells was IgG. It did not bind complement, and did not cause in vivo hemolysis. 9 months after recognition of the autoimmune state the direct antiglobulin test had become negative and anti-Kpb was no longer detectable. It is postulated that autoimmunity involving the Kell blood group may be precipitated by antigens or enzymes of microbial origin.

This report describes a healthy blood donor whose red cells have weakened expression of Kell blood group antigens. Kell antigen activity could not be detected by flow cytometric analysis and was demonstrable only by sensitive serologic techniques. As with normal-strength Kell antigens, reactivity could be abolished by treatment with 2-aminoethylisothiouronium bromide (AET). The donor's red cells have Kx antigen activity. Other commonly tested blood group antigens (MNSs, Rh, P1, Lewis, Duffy, and Kidd systems) appear normal. Clinical and serologic examination showed that this case is different from previously described examples of modified Kell expression. The propositus's phenotype has remained unchanged for 19 months, which suggests that it is not a transient condition. However, family studies provide no evidence that it is inherited. A 93-kD protein, which reacted weakly by Western blot with rabbit antibody to Kell protein, was isolated from the propositus's red cells by immunoprecipitation. This finding was not reproduced in subsequent studies, which suggests that the quantity of Kell protein recovered was at the threshold level detectable by the technique used. The red cell phenotype is categorized as Kmod, of which this is the first example reported in a healthy individual.

Anti-A and anti-K have been found in the serum of a 20-day-old child who had not been transfused but who was acutely ill with E. coli enterocolitis. Both antibodies are IgM proteins. The mother's serum does not contain either antibody and the anti-A and anti-K in the infant's serum are not of maternal origin. Both parents and the child are of the Kell phenotype K-k+. Stool cultures made from the child yielded E. coli O 125:B15, an uncommon B-variant pathogenic coliform. Cell-free preparations made from broth cultures of this organism have strong specific inhibitory activity against IgM anti-A and anti-K, and both antigens have been identified on the bacterial cells. At age 3 months the child had made a clinical recovery, stool cultures showed no pathogenic coliforms, and anti-A and anti-K were no longer detectable in her serum. These data indicate that absorption of metabolites with A-like and K-like activity produced by a pathogenic coliform in the intestinal tract were responsible for the appearance of apparent naturally occurring anti-A and anti-K in the child's serum.

Leukocytes of nine unrelated boys with X-linked chronic granulomatous disease lack Kx antigen. In three of these cases, the red cells also lack Kx and have the McLeod phenotype and abnormal morphology. X-linked chronic granulomatous disease CGD can thus be separated into two types. Type I cases have an antigenic deficiency that is restricted to the phagocytic leukocytes while in type II, the deficiency involves both leukocytes and red cells. Red cells of type II CGD patients have enhanced i antigen activity suggesting that they are under hemopoietic stress. Normal Kx synthesis is directed by an X-linked gene named X1k. Three variants, X2k, X3k, and X4k order the different permutations of leukocyte and red cell Kx antigen production that have been recognized.

Monospecific Kell blood group antibodies, of either human alloimmune or mouse monoclonal origin, react with a single surface-exposed protein of 93,000 daltons. Chymotryptic peptide maps of the 93,000-dalton protein isolated by antibodies of two different specificities (anti-K7 or anti-K14) indicate that Kell epitopes reside on the same protein. Kell protein is similar in size to band 3 protein but differs markedly in its tryptic and chymotryptic peptide maps, indicating that they are different proteins. In addition, sheep antibody to human band 3 does not react with Kell protein. Rabbit antibody to Kell protein reacts, by Western immunoblotting, with membrane proteins from Kell antigen positive red blood cells but not from those of a Ko (Kell null) cell. In intact red cells only a small portion of the Kell protein is available to lactoperoxidase-catalyzed iodination. Under nonreducing conditions Kell antigen is isolated not only as a 93,000-dalton protein but also as larger protein complexes ranging in size from above 200,000 to 115,000 daltons. Treatment of red cells with iodoacetamide, prior to isolation of Kell protein, reduces the amount of the very large complexes, but Kell protein occurs both as 115,000- and 93,000-dalton proteins.

Absence of KX antigen and of normal expression of the Kell system antigens is associated with bizarre red blood cell morphology when observed by either light or scanning electron microscopy. Numerous acanthocytes and dacryocytes have been observed in the peripheral blood smear of an apparently healthy individual with McLeod-phenotype blood, in a male patient with type II chronic granulomatous disease who had a shortened 51Cr red blood cell survival time, and in a minor population of the red blood cells of his carrier mother.

Human red cells incubated with a solution containing 6% 2-aminoethylisothiouronium bromide (AET) lose activity of antigens that are part of, or related to, the Kell blood group system. However, Kx antigen is not inactivated. Studies on a wide range of other blood-group antigens show no other evidence of changes and AET appears to react specifically with red-cell membrane structures that have Kell activity. The AET procedure produces an artificial K0 red cell that can be used in blood group serology, and allows easy recognition of antibodies that are associated with the Kell system. AET has been used by other workers to produce a red cell that has many serological and biochemical characteristics of a PNH cell. Our studies on red cells from PNH patients have not shown any changes in Kell blood-group antigens.

McLeod phenotype red cells of the Kell blood group system have acanthocytic morphology and reduced in vivo survival. The phenotype has an X-linked mode of inheritance and is found in some males who have no abnormality of leukocyte function and in some who have X-linked chronic granulomatous disease (CGD). We now describe an association between the McLeod phenotype and an abnormal elevation of serum creatine phosphokinase (CPK). The increase is of the MM isoenzyme type, derived from skeletal muscle or cardiac muscle, and muscle biopsy shows evidence of muscle cell changes. All of 11 males who have McLeod syndrome but do not have CGD have high levels of serum CPK. Males with McLeod syndrome and CGD may have normal or high levels of the enzyme. Individuals with other variant phenotypes in the Kell system have normal levels of serum CPK. Studies on a large kindred, which includes 5 people of McLeod phenotype, show high CPK levels only in the members of McLeod type. We conclude that the high level of CPK in the serum of these people is a reflection of a muscle cell anomaly and that in these individuals it is a pleiotropic effect of the X-linked gene that produces the McLeod red cell phenotype.

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.

The Kell blood group has 18 associated red cell antigens. One, named KX, is the product of an X-linked gene and appears to be a precursor in the Kell biosynthetic pathway. Lack of KX on red cells, caused by inheritance of a variant allele at the X-linked locus, results in gross changes in Kell antigenicity, an effect called the McLeod phenotype. Such cells also show striking morphologic changes. Normal phagocytic leukocytes lack Kell antigens but have strong KX. The leukocytes of boys with X-linked chronic granulomatous disease lack KX antigen and have defective bactericidal function. The fundamental defect in chronic granulomatous disease appears to be failure to inherit the X-linked gene that determines KX synthesis. The enzymatic and functional disorders of the leukocytes, and the structural changes in the red cells, are consequences that follow.

KX, an antigen related to the Kell blood group system, is present in trace amounts on normal red cells and is strongly active on the neutrophils of all of 50 persons thus far tested. Normal circulating monocytes are now shown to also bear KX determinants. Absence of neutrophil KX has been associated with all of three previously tested patients with chronic granulomatous disease. In this study two male siblings with CGD also have been shown to have KX negative leukocytes, and white blood cells from their heterozygous mother were found to have a reduced competency to absorb anti-KX. Five CGD boys are known to lack KX; the probability of this occurring by chance is greater 10(-6).

Red cells of the McLeod blood group phenotype have weak Kell antigens, lack Kx antigen and have acanthocytic morphology. We have immunoprecipitated Kell antigens from McLeod red cells and show that they are markers on the same 93 kDa membrane protein that carries Kell antigens on normal red cells. However, as determined by Western immunoblotting, McLeod red cells have a marked deficiency of this protein. We have also studied the near-neighbour relationship of McLeod and common Kell red-cell membrane proteins by cross-linking intrinsic sulphydryl groups by oxidation, catalysed with orthophenanthroline and copper, or by cross-linking amino groups with dimethyl-3,3'-dithiobispropionimidate. Results were analysed by diagonal mapping in two-dimensional gels. No abnormalities of membrane protein inter-relationship were detected in McLeod red cells. We have isolated Kx antigen from K0 red cells by immunoprecipitation with human alloimmune anti-Kx serum, isolation of immune complexes from detergent-solubilized cell membranes with protein A-Sepharose and analysis of the eluted immune complex by SDS-PAGE under reducing conditions. Kx antigen is a marker on a red-cell membrane protein of approximately 37 kDa. Ko (Knull) red cells have about twice the amount of Kx antigen as do red cells of common Kell type. McLeod red cells have no detectable Kx antigen by serological tests or by immunoprecipitation.

Fifteen antigenic determinants are known to be related to the Kell blood group. Some boys with X-linked chronic granulomatous disease have the very rare McLeod or Ko phenotype on their red cells. Serological studies of the McLeod type suggest that the weak Kell antigens that are present differ qualitatively and quantitatively from those on red cells of common Kell type. A new antigen, Kx, has been characterized and shown to be present on red cells and neutrophil leucocytes. Lack of red-cell Kx is associated with the McLeod phenotype, lack of leucocyte Kx is associated with chronic granulomatous disease.

The McLeod phenotype is inherited as an X-linked characteristic. The red cells have weak antigenicity in the Kell blood group and lack Kx, a precursor-like substance that appears to be necessary for proper biosynthesis of Kell antigens. Kx antigen is also required for establishment of normal cell morphology. Absence of Kx antigen causes a membrane abnormality, in which the most prominent feature is acanthocytosis, and a compensated haemolytic state. The X-linked gene that determines normal Kx production is called X1k. Inheritance of a variant allele at the Xk locus is responsible for lack of Kx synthesis and the McLeod phenotype. The Xk locus is inactivated by the Lyon effect, and female carriers of the variant gene exhibit blood group mosaicism in the Kell system and have a dual red cell population of acanthocytes and discocytes.

Some boys with X-linked chronic granulomatous disease (CGD) have red cells of the rare McLeod phenotype in the Kell blood group system. Only one example of this phenotype has previously been described in a non-CGD subject. We have studied a 10-year-old boy and a maternal brother who do not have CGD and whose red cells are of the McLeod type . The boy presented as a haematological problem with red-cell abnormalities. These were acanthocytosis, anisocytosis and 'tailing' in the osmotic fragility curve, changes now known to occur with the McLeod phenotype. Subsequent studies revealed his rare blood group. A family study has established that an uncle also has acanthocytic red cells and the McLeod phenotype. In addition the boy's sister, mother and maternal grandmother all show red-cell mosaicism with double populations of McLeod acanthocytes and normal red cells of common Kell type. The gene that determines inheritance of the McLeod phenotype is X-linked and the mosaicism present in female carriers is believed to result from X chromosome inactivation by the Lyon effect. The study provides further evidence that the McLeod phenotype arises by inheritance of a variant X-linked modifying gene and not through inheritance of a variant gene at the Kell autosomal locus. It also represents the first occasion that a person of rare blood group has been recognized because of an associated anomaly in red cell morphology.

The rare McLeod blood group phenotype is characterized by weak Kell antigens, lack of the common Kx antigen, and acanthocytic morphology. Previous studies that did not detect membrane or cytoskeletal protein abnormalities suggested a lipid disturbance. In normal red cells, dimyristoyl phosphatidylserine (DMPS) is transported across the membrane by an enzymatic process and accumulates in the inner leaflet of the membrane bilayer causing discocyte to stomatocyte shape changes. Scanning electron microscopy of McLeod red cells shows a mixture comprised of 15% discocytes, 51% with irregular surfaces, and 34% acanthocytes. On incubation with various concentrations of DMPS at 37 degrees C for periods up to two hours, McLeod red cells transported DMPS across the membrane and caused irregularly shaped and acanthocytic McLeod red cells to attain normal discocyte shape and later to become stomatocytes. Chlorpromazine, which at 0 degrees C preferentially partitions into the inner monolayer of the membrane, had a similar effect on the shape of McLeod red cells. This suggests that in McLeod cells acanthocytosis is due to a lack of lipid in the inner leaflet of the membrane bilayer but that the imbalance is not caused by defective transport of phosphatidylserine across the membrane.

A case of auto-immune hemolytic anemia caused by auto anti-K13 is described. Direct antiglobulin tests using monospecific reagents showed that IgG and the C3 component of complement were present on the patient's red cells. Eluted auto-antibody did not react with Ko or with K:--13 cells. Kell blood group antigenicity of the patient's red cells did not appear to be reduced.

An IgG autoantibody with Kx specificity was found in the blood of a 61-year-old white man. The antibody did not cause hemolysis of his own or transfused Kx-positive red cells. The patient is of common Kell blood type and does not exhibit any clinical or hematologic features of McLeod syndrome.

A 3-year-old black male child with X-linked chronic granulomatous disease and red cells of the rare McLeod phenotype is presented. The red cells showed acanthocytosis and did not react with anti-KL. Similarly the leukocytes were nonreactive with anti-Kx. The Xk and Xg linkage could not be investigated since all members of his family were Xg (a+).

An antibody in the serum of a gravida 4, para 3 woman reacted with red cells from two of her children, her husband, and his mother, but with none of more than 2100 reference red cell samples and blood samples from donors. The reactive antigen was inactivated by 2-aminoethylisothiouronium bromide or dithiothreitol-papain treatment. The antigen was immunoprecipitated from paternal red cells with maternal antibody and shown to migrate by sodium dodecylsulfate polyacrylamide gel electropheresis as a single protein of approximately 93,000 daltons. After transfer to nitrocellulose paper by Western blotting, the protein reacted with a rabbit antibody specific for Kell protein. The chemical inactivation and electrophoretic findings were characteristic of Kell group antigens. The reaction with the rabbit antibody establishes that the "new" low incidence antigen was an epitope on Kell group protein and must be coded for by the Kell gene. It has been designated K23.

Kell antigens on chimpanzee (Pan troglodytes) red cells were determined using specific human alloimmune and murine monoclonal antibodies. After avoidance of interspecies reactions, chimpanzee red cells were found to react with most Kell system antibodies. The chimpanzees had phenotypes similar to those of humans. The main difference was that all of 27 chimpanzee red cell samples tested were of the K:6, -7, phenotype, while in humans most are K:-6, 7. The most common chimpanzee Kell blood group phenotype was K:-1,2,-3,4,5,6,-7,11,12,13,14, 15,18,19,22. Murine monoclonal anti-K2 and -K14 immunoprecipitated a 97-kD protein from chimpanzee red cells and a 93-kD protein from human red cells. Enzymatic deglycosylation yielded proteins of about 79 kD for humans and 77 kD for chimpanzees. Both human and chimpanzee Kell proteins reacted equally well on Western blots with polyclonal rabbit antibody to human Kell protein, which indicated close homology.

McLeod syndrome is an X-linked condition in which individuals of McLeod blood group phenotype have weak Kell antigens, acanthocytic red cells, and a muscular disorder. We now report a family in which two brothers have McLeod syndrome. One is K:-1, while the other is the first known K:1 person with McLeod syndrome. The K1 gene in the latter is expressed weakly and was inherited from the father, in whom it is expressed normally. The brothers have the same clinical and laboratory manifestations of McLeod syndrome but have different Kell genes. Therefore, the Kell gene is unlikely to have any positive input into development of McLeod syndrome; its role is one of passive involvement in which its expression is modified.

Kell blood-group-active protein has been isolated by labeling red cell surface proteins with 125I, sensitizing intact cells with anti-K1, anti-K2, anti-K7, or anti-K22, solubilizing the cell membranes, isolating immune complexes, and separating their components by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Each antibody separated a protein of approximately 93,000 daltons. Periodic-acid Schiff (PAS) staining of Kell protein showed that it was glycosylated. When separated under non-reducing conditions, Kell protein had different SDS-PAGE characteristics with protein bands of approximately 85,000 daltons and 115,000 daltons. This suggests that in the red cell membrane Kell protein is complexed with other proteins. Quantitative experiments made with anti-K7, anti-K22, and a mixture of anti-K7 and anti-K22 indicate that both antigen specificities are present in the same molecule. These biochemical data support serological studies which indicate that K22 is part of the Kell system.

Approximately one in 250 people with autoimmunity involving their red cells have IgG autoantibodies with specificity in the Kell blood groups. Red cells of these individuals have an acquired temporary weakening of their Kell antigens. Some of the patients also have allo-anti-K in their serum. This report presents a case in which an IgG autoantibody may define a new high-incidence red cell antigen related to the Kell blood groups. The patient's Kell blood group antigens are depressed, and his serum contains allo-anti-K. It is postulated that reduced red cell Kell antigenicity is caused by enzymatic degradation, possibly of bacterial origin, and that the acquired loss of Kell antigens, the Kell-specific autoimmune state, and the serum all0-anti-K, are all related aspects of one phenomenon.

An 84-year-old woman with intestinal bleeding had marked reduction of red blood cell antigenicity in the Kell system, and a positive direct antiglobulin test caused by auto-anti-Kpb. KX antigen activity of her cell was increased, an observation which supports the belief that KX marks a precursor structure utilized in the normal Kell biosynthetic pathway. It is postulated that reduced Kell antigenicity was an acquired change that resulted from enzymatic degradation, possibly of bacterial origin.

A second example of anti-K19 has been identified in the serum of a black patient immunized by blood transfusions. The antibody was responsible for delayed hemolysis of transfused incompatible red blood cells. The patient's red blood cells are K:--19 but are otherwise of common Kell phenotype. The K19 antigen is closely associated with the Kell blood group although there is, as yet, no pedigree information to confirm that it is produced by the Kell gene. Tests on 10,757 group O blood donors have not revealed any further examples of the K:--19 phenotype.

An 18 year old man has red cells of the McLeod phenotype in the Kell system but does not have chronic granulomatous disease. His red cells show acanthocytic morphology and there is evidence of a compensated hemolytic state. Following an accident he was transfused with blood of common Kell type and made anti-K and anti-Km. Leukocytes from the patient are deficient in Kx antigen but the cells appear to have normal function. The mother is a carrier of the variant Xk allele and has a double red-cell populatio of McLeod cells and cells of common Kell type. The family may be informative with respect to linkage between the Xk and Xg loci. One of the mother's 5 brothers and one of her maternal uncles are possible Xk:Xg recombinants.