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Introduction: Rh and Kell blood group systems are amongst the most important blood group systems; being highly immunogenic after ABO system. The aim of this study was to evaluate the frequencies of Rh antigens, haplotypes and K antigen among blood donors belonging to various ethnicities in Samtah, Jazan, Saudi Arabia., Methods: This study was conducted during January 2019 and August 2020 at Samtah General Hospital, Samtah. Records of all blood donors recruited during this period were included for data acquisition. A total of 4977 blood donors' records were reviewed and data were analysed. A total of 3863 donors' results were considered in the final analysis., Results: In comparison to Saudi blood donors, C antigen was less frequent in Sudanese donors (69.7% and 34.0%), the c antigen was less frequent in Indian (79.2% and 59.3%) and Philippine (79.2% and 40.0%) donors and more frequent in Sudanese (79.2% and 97.9%) donors, the E antigen was less frequent in Yemini (27.0% and 19.5%) and the e antigen was more frequent in Yemini (96.7% and 99.2%) donors. The DcE haplotype was less frequent (3.1% and 0.7%) and the ce haplotype was more frequent (4.3% and 7.6%) in Yemini donors. The K antigen was less frequent in Pakistani (11.9% and 4.1%; p = .041) and Indian (11.9% and 1.9%; p = .023) donors., Conclusion: Rh and K antigens showed marked variations in their frequencies among blood donors of different ethnicities. Utilization of blood from various ethnicities warrant extended phenotyping of Rh and K antigens to avoid the risk of alloimmunization in multiply transfused patients., (© 2022 John Wiley & Sons Ltd.)

The development of hemolytic erythrocyte alloantibodies and autoantibodies complicates transfusion therapy in thalassemia patients. These antibodies ultimately increase the need for blood and intensify transfusion complications. There is a scanty data on the frequency of RBC alloimmunization and autoimmunization in Egyptian β thalassemia patients as pretransfusion antibody screening is not routinely performed. We studied the frequency of alloimmunization and autoimmunization among 200 multiply transfused β thalassemia patients and investigated the factors that possibly affect antibody formation. Of the 200 patients in our study, 94 were males and 106 females, with the age range of 2-37 years. Alloantibodies were detected in 36 (18%) of the patients, while autoantibodies were detected in 33 (16.5%). The dominant alloantibodies were directed against Kell (33%) and Rh (24.4%) groups. Alloimmunization had a significant relationship with treatment duration and the frequency of transfusion (P = 0.007, 0.001, respectively). The presence of autoantibodies was significantly related to age (P = 0.001), total number of transfused units (P = 0.000) and splenectomy (P = 0.000). The high prevalence of alloimmunization in the study population disclosed the need for providing phenotypically matched cells for selective antigens especially for Kell and Rh subgroups to reduce risk of alloimmunization and increase the efficiency of blood transfusion.

Background: Several Kell-system antibodies are known to cause direct agglutination. Also, some specificities, such as anti-Ku, have been reported to react only via the indirect antiglobulin test (IAT)., Methods: Herein, we describe the case of a 61-year-old alloimmunized white woman who presented to an outside hospital with a gastrointestinal (GI) bleed and a "possible anti-Ku" was reported with 3+ reactivity at PEG-IAT and at Ficin-IAT; in addition to an unidentified cold antibody. Subsequently, when the patient presented to a second outside hospital, an anti-Ku that caused 3+ to 4+ reactions at saline-immediate spin (IS) was identified. The reactivity was evaluated with 0.01-M dithiothreitol (DTT) treatment of the plasma., Results: It was determined that the strong agglutination with saline-IS was caused by immunoglobulin (Ig)M anti-Ku., Conclusion: To our knowledge, this is the first reported case of an IgM anti-Ku., (© American Society for Clinical Pathology 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background & Objectives: : Sickle cell disease (SCD) patients require red cell transfusion during different clinical complications of the disease. Such patients are at a high risk for developing alloantibody against red cell antigens. From India, there are limited data available on alloantibody formation in multiply transfused SCD patients. The present study was thus undertaken to fill up this lacunae by looking at the development of red cell alloantibodies in SCD and β-thalassaemia patients on regular transfusion., Methods: : All sickle cell disease patients undergoing red cell transfusion between 2008 and 2016, were included. During this period, a large number of β-thalassaemia major patients also underwent regular red cell transfusion. These thalassaemia patients were also included to compare the tendency of antibody formation between SCD and β-thalassaemia major patients. All patients before regular transfusion were regularly assessed for the development of red cell antibody. Red cell antigen, antibody screen crossmatch and antibody identification were done using the standard technique., Results: : A total of 138 patients with SCD aged between 4 and 53 yr (mean 17.6 yr) consisting of 83 males and 55 females (male:female, 1.5:1) along with 333 transfusion-dependent β-thalassaemia patients were studied. Over the last eight years, 15 patients with SCD and four patients with thalassaemia developed alloantibody (P <0.001). Antibody specificity of their alloantibodies was against Rhc, RhE, Kell, Fy a and Fy b only. Sickle cell disease patients with and without alloantibody required on the average 11.8 and 8.6 units of red cell concentrate, respectively (P <0.05)., Interpretation & Conclusions: : About 11 per cent of the transfused sickle cells patients developed alloantibodies. The antibody specificity was restricted to Rh, Kell and Duffy blood group systems. Extended antigen matching involving Rh, Kell and Duffy antigens may prevent alloantibody in such patients., Competing Interests: None

Parvovirus B19 has tropism for red line blood cells, causing immune hydrops during pregnancy. A positive anti-Kell Coombs reaction usually happens during pregnancy when there is production of antibodies that target Kell antigens, but cross reactions to other antigens may occur. A 24-year-old Gypsy primigravida, 0 Rhesus positive, presented with persistent isolated hyperthermia for 2 weeks and a positive indirect Coombs test result with anti-Kell antibodies at routine tests. She had a 19-week live fetus. The blood tests revealed bicytopenia with iron deficiency anemia, leucopoenia with neutropenia, and elevated C-reactive protein. She was medicated with imipenem, and had a slow clinical recovery. Blood, urine and sputum samples were taken to perform cultures and to exclude other systemic infections. Escherichia coli was isolated in the urine, which most probably caused a transient cross anti-Kell reaction. Haemophilus influenza in the sputum and seroconversion to parvovirus B19 was confirmed, causing unusual deficits in the white cells, culminating in febrile neutropenia. Despite the patient's lack of compliance to the medical care, both maternal and fetal/neonatal outcomes were good. This a rare case report of 2 rare phenomena, a cross anti-Kell reaction to E. coli and parvovirus B19 infection with tropism for white cells causing febrile neutropenia, both events occurring simultaneously during pregnancy., Competing Interests: The authors have no conflicts of interest to report., (Thieme Revinter Publicações Ltda Rio de Janeiro, Brazil.)

Introduction: Kell antigen is highly immunogenic and is the common cause of antibody production in mismatched blood transfusions, haemolytic transfusion reaction (HTR) and maternal alloimmunization, which causes severe anaemia in neonates. The aim of this study is to determine the prevalence and ethnic variation of the Kell phenotype among pregnant women in Sokoto, Nigeria., Methods: Kell antigen status of 150 pregnant women aged 18-45 years and mean age 27.19 ±4.69 years attending antenatal clinic in UDUTH Sokoto Nigeria was determined using the conventional tube method and anti-Kell reagents (Lorne Laboratories, UK)., Results: Among the 150 subjects studied, 3 (2.0%) of subjects were positive and 147 (98.0) were negative for K antigen. Of the 150 pregnant subjects; 32 (21.3%) were primigravidae while 118 (78.7%) were multigravidae. Kell phenotype was more prevalent among primigravidae (3.1%) compared to multigravidae (1.7%) women. The distribution of Kell phenotype among the pregnant subjects was compared based on ethnicity. The prevalence of Kell antigen was significantly higher among the Hausa ethnic group (3.2%) compared to other ethnic groups which indicated zero prevalence (p = 0.001). Kell negative phenotype was ≥ 96.8% among all the ethnic groups., Conclusion: Our observed prevalence of Kell phenotype is consistent with previous studies among Blacks and Asians but significantly lower than values observed in previous studies among Caucasians. We recommend that all pregnant women should be screened for the presence clinically significant red cell antigens including Kell antigen on their first antenatal visit. Kell negative red cell should be routinely provided for all pregnant women and women with child bearing potential to reduce the risk of Kell-associated HDFN. There is need to introduce routine screening of pregnant women for clinically significant red cell antibodies to facilitate the effective management of HDFN as well as prevent HTR. There is also need for sustained health education of pregnant women in the area to encourage early booking for antenatal care.

We report a case of Kell sensitized pregnancy with good neonatal outcome. Anti-K antibodies were detected in maternal serum in early pregnancy as a part of routine antibody screening test. The middle cerebral artery doppler monitoring and serial titers were carried out to screen for foetal anaemia. Despite of rising antibody titers, serial middle cerebral artery doppler was normal and did not showed foetal anaemia. The pregnancy was carried out till term and patient delivered at 37 weeks of pregnancy with no evidence of foetal anaemia. This case underlines the need of general screening on rare antibodies in all pregnant women and that non-invasive monitoring of foetal anaemia can be done with anti-k titers and middle cerebral artery Doppler.

Fetal and neonatal haemolytic diseases result from maternal allo-immunisation to fetal antigens. Maternal antibodies cross the placenta causing red cell haemolysis, resulting in fetal anaemia and, in severe cases, hydrops and perinatal death. Intravascular intrauterine blood transfusion (IUT) has markedly reduced perinatal mortality and is now a standard procedure. IUT is considered to be a safe procedure with fetal loss rate reported to be less than 5% and no reported increase in the rate of neurodevelopment impairment. In this report, we are presenting a case of bilateral cystic encephalomalacia following fetal anaemia secondary to anti-Kell iso-immunisation treated with multiple IUTs. Such a significant adverse outcome following IUT for anti-Kell iso-immunisation has not been reported in the literature. This case highlights the need for appropriate parental counselling and routine postnatal head ultrasound in all babies delivered following multiple IUTs., (2015 BMJ Publishing Group Ltd.)

Background & Objectives: The development of alloantibodies can significantly complicate transfusion therapy and results in difficulties in cross-matching of blood. Most literature on alloimmunization is limited to multitransfused individuals, with very few studies on the general hospital patients. This study was aimed at assessing the frequency and type of unexpected red cell antibodies in the general patient population at a multispecialty tertiary care centre in New Delhi, India., Methods: The results of 49,077 antibody screening tests carried out on patients, from January 2009 to December 2012 were analyzed. The clinical and transfusion records were reviewed. The data were compiled and statistically analysed., Results: A total of 49,077 (29,917; 60.96% males and 19,160; 39.04% females) patient samples were screened for the presence of unexpected antibodies. Antibody screening was positive in 403 patients (0.82%). In the serum samples of 164 patients only autoantibodies were identified, 27 revealed autoantibodies with one or more underlying alloantibodies, while 212 patients had only alloantibody/ies in their serum. The overall alloimmunization rate was 0.49 per cent. Antibodies against the Rh system were the most frequent (64.1%), the most common alloantibody identified being anti E (37.2%), followed by anti D (19.2%)., Interpretation & Conclusions: Since clinically significant antibodies are frequently detected in our patient population, antibody screening and if required, identification is the need of the hour. Since antibodies against the common Rh and Kell blood group antigens are the most frequent, provision of Rh and Kell matched red cells may be of protective value.

Background: There is an ongoing controversy regarding provision of usually matched blood (i.e. matched for ABO-D antigens) or phenotypically matched blood (also matched for Rh and Kell antigens) for multiply transfused thalassaemics, especially in developing countries. A pilot study conducted at our centre revealed an alloimmunisation rate of 3.79% with Rh and Kell alloantibodies accounting for 90% of all antibodies. The present cross-sectional study was conducted to assess the impact of a policy of partial better matching (for Rh cDE and Kell antigens) of blood on alloimmunisation in thalassaemics., Material and Methods: In this cross-sectional study three groups of patients were considered. Group 1 comprised 211 thalassaemics who received usually matched (UM) blood until April 2009. Their rates of alloimmunisation have already been published in a prior study. Group 2 consisted of 46 thalassaemics who were enrolled after April 2009 and have received partially better matched (PBM) blood (matched for ABO, Rh cDE and Kell antigens) since the initiation of transfusion therapy. Group 3 (UM→PBM) comprised the patients from group 1 who, from April 2009, were given partial better matched blood. Antibody screening (using a 3-cell panel) and antibody identification (11-cell panel) were carried out to detect the presence of alloantibodies., Results: None of the thalassaemic patients in group 2 (PBM) developed alloantibodies. Eight thalassaemics in group 3 (UM→PBM) developed new alloantibodies (after April 2009)., Discussion: According to the results of the present study, providing at least partially better matched blood appears to improve the efficacy of transfusion for chronically transfused thalassaemics. Large-scale, comprehensive, multicentre studies need to be carried out to formulate realistic, evidence-based, economically feasible transfusion policies for thalassaemic children based on the red blood cell antigen profile of the population.

Exposure to nonself red blood cell (RBC) antigens, either from transfusion or pregnancy, may result in alloimmunization and incompatible RBC clearance. First described as a pregnancy complication 80 years ago, hemolytic disease of the fetus and newborn (HDFN) is caused by alloimmunization to paternally derived RBC antigens. Despite the morbidity/mortality of HDFN, women at risk for RBC alloimmunization have few therapeutic options. Given that alloantibodies to antigens in the KEL family are among the most clinically significant, we developed a murine model with RBC-specific expression of the human KEL antigen to evaluate the impact of maternal/fetal KEL incompatibility. After exposure to fetal KEL RBCs during successive pregnancies with KEL-positive males, 21 of 21 wild-type female mice developed anti-KEL alloantibodies; intrauterine fetal anemia and/or demise occurred in a subset of KEL-positive pups born to wild type, but not agammaglobulinemic mothers. Similar to previous observations in humans, pregnancy-associated alloantibodies were detrimental in a transfusion setting, and transfusion-associated alloantibodies were detrimental in a pregnancy setting. This is the first pregnancy-associated HDFN model described to date, which will serve as a platform to develop targeted therapies to prevent and/or mitigate the dangers of RBC alloantibodies to fetuses and newborns.

In this issue of Blood, Stowell et al describe a novel mouse model of hemolytic disease of the fetus and newborn (HDFN) that recapitulates many of the key features of human disease.1 Recently, this same group of researchers described a transgenic mouse that expresses the human KEL2 (Chellano) red cell surface protein from the Kell system on red cells, and subsequently demonstrated that Kell differences on transfused blood induce antibody responses and hemolytic transfusion reactions similar to those seen in patients. In this latest report, Stowell et al demonstrate that similar to some patients, Kell differences between mother and father can lead to maternal antibody generation and hemolytic disease in utero. In so doing, they provide experimental confirmation of a long sought after animal model of HDFN.

The mental decline of King Henry VIII from being a jovial, charismatic and athletic young man into an increasingly paranoid, brutal tyrant in later life, ever more concerned at his lack of one or more male heirs, has attracted many medical diagnostic theories. Previous hypotheses have included diabetes, syphilis and hypothyroidism, among others. However, these inadequately explain Henry's failure to produce a male heir, despite multiple pairings. The latest postulated diagnoses for Henry are the coexistence of both Kell blood group antigenicity (possibly inherited from Jacquetta Woodville, Henry's maternal great grandmother) causing related impaired fertility, and McLeod syndrome, causing psychotic changes. As the mutated McLeod protein of the syndrome significantly reduces the expression, effectively inactivating the Kell antigen, we critically review this theory, examining in detail the pathophysiology of these conditions and assessing the genealogy of Henry VIII and its effect in subsequent generations.

The K blood group remains an important target in hemolytic disease of the newborn (HDN), with no immune prophylaxis available. The aim was to characterize the Th response to K as a key step in designing specific immunotherapy and understanding the immunogenicity of the Ag. PBMCs from K-negative women who had anti-K Abs after incompatible pregnancy, and PBMCs from unimmunized controls, were screened for proliferative responses to peptide panels spanning the K or k single amino acid polymorphism. A dominant K peptide with the polymorphism at the C terminus elicited proliferation in 90% of alloimmunized women, and it was confirmed that responding cells expressed helper CD3(+)CD4(+) and "memory" CD45RO(+) phenotypes, and were MHC class II restricted. A relatively high prevalence of background peptide responses independent of alloimmunization may contribute to K immunogenicity. First, cross-reactive environmental Ag(s) pre-prime Kell-reactive Th cells, and, second, the K substitution disrupts an N-glycosylation motif, allowing the exposed amino acid chain to stimulate a Th repertoire that is unconstrained by self-tolerance in K-negative individuals. The dominant K peptide was effective in inducing linked suppression in HLA-transgenic mice and can now be taken forward for immunotherapy to prevent HDN because of anti-K responses.

Erythrocytic group antigens represent a genetically stably determined trait. Investigation of antigens of the said system in different regions is of the greatest importance in terms of both the creation of demographic data of the region as well as practical medicine, especially for transplantology and transfusiology. The peripheral or venous blood of 232 local natives (healthy donors) of Ozurgeti district of Guria region has been taken as the test subject. The test subject was taken by random methods in different vilifies (Bakhvi, Mshvidobauri, Ozurgeti, Likhauri, Gurianta, Bokhvauri, Dvadzu, Pampaleti) To identify the ABO, Rh-Hr, Kell, MN system antigens, an express-method using monoclonal antibodies has been applied. In studying the ABO system, it was fixed that the highest distribution frequency was characteristic of the 0(I) group (52.3±3.2%), then follows the group A(II) (38.5±3.2%). The distribution frequency of the B(III) group is (8.2±1.8%) and that of AB(IV)--(0.8±0.5). The population's 85.2±2.32% is the carrier of the Rh+ phenotypic group, while 14.7±2.3% belongs to the Rh-phenotypic group. In studying the concentration of alleles, the low concentration of p(K) allele was detected that equaled 0.2; the concentration of q(K) allele made 0.8, that of p(M)--0.65, and that of q (N) - 035.

Aim: 1) We have presented our experiment conducted to detect anti-K antibodies from the Kell-system in pregnant women and their connection with potential destruction of foetal red cells, which may result in haemolytic disease of the foetus and the newborn (HDFN). 2) We have also indicated serological and molecular methods important for a proper diagnosis., Material and Methods: 27 women with anti-K. Serological diagnosis of K antigen in fathers and children. KEL1 gene examination in foetuses from DNA isolated from foetal cells contained in amniotic fluid, using discrimination of alleles--real-time PCR method., Results: Anti-K were detected in most women after blood transfusion, the fathers were usually K negative. Foetomaternal incompatibility was found in 6 out of 27 women. Haemolytic disease was observed in 5 cases: 3-severe, 1-fatal, 1-mild. Foetal genotyping allowed us to avoid cordocentesis in two pregnant women, it appeared that both foetuses have not received KEL1 gene from heterozygous (Kk) fathers--in the previous pregnancies the children had died because of HDFN., Conclusions: 1) In every pregnant woman with anti-K, the K antigen should be examined in the father. 2) In every K+ father the phenotype should be evaluated and if he is heterozygote (Kk), the fetal KEL1 gene must be examined, to avoid unnecessary cordocentesis. 3) If KEL1 gene is not detected in the foetus, HDFN will not occur. 4/Foetal KEL1 genotyping may be performed in all mothers with anti-K and heterozygous father in our Department, after providing the material from the amniocethesis.

We describe a case of incidental detection of anti Kell antibody in a child with transfusion dependent thalassaemia. Kell antibody detection may be missed by routine indirect antiglobulin test (IAT) crossmatch procedure because of low prevalence of Kell antigen in the general population. A false negative result can be avoided by using sensitive cross matching techniques and screening cells representing antigens in homozygous state, against all clinically significant antibodies. A transfusion alert card describing the nature of antibody and future transfusion policy should be given to such allo-immunized patients.

Lectins are unique proteins of varying biological importance. They are characterized by specific binding to carbohydrate residues, whether monosaccharides, disaccharides or polysaccharides. The sugar heads on the surface of the erythrocyte specify the different blood groups. Lectins, as an antigenic determinant of blood group, have come to be an important tool in the identification of different blood groups. A handful of lectins may be considered excellent reagents for anti-A, anti-B, anti-N etc, but the anti-A and anti-M are not yet regarded as commercially suitable antisera. Lectin from Vicia cracca has been proved to be a good anti-A, lectin from Dolichus biflorus can be used as anti-A1, and lectin from Griffonia simplicifolia as anti-B. Lectin from Vicia graminea is said to be a good typing reagent as Anti-N. On the other hand, the lectins involved in polyagglutination are absolutely essential as the reagent of choice and these cannot as yet be replaced by antibodies of any kind. Erythrocytes with exposed cryptantigens are significantly more sensitive to agglutination by certain lectins than by polyclonal antibodies. Peanut agglutinin (PNA), Polybrene, and Glycine max lectins are frequently used for the identification of different cryptantigens. The application of lectins as an anti-B reagent has proven to be as useful as human polyclonal or mouse monoclonal antibodies. Besides their specificity, lectins are excellent reagents because of their lower cost and indigenous production. The importance of various lectins used as markers for blood grouping is discussed.

We assessed the clinical outcome of pregnancies with non-Rh-D antibody in Northern Ireland using retrospective case note review. During the study period (April 1999- March 2000) 186 women with clinically significant antibodies were identified from the records of the antenatal laboratory of the Northern Ireland Blood Transfusion Service. Eighty-five women were included in the study using the criteria mentioned above. None of the fetuses required intrauterine transfusion during this period. One baby required exchange transfusion, three were given top-up transfusions and 17 had phototherapy. Nine babies with a positive direct antiglobulin test (DAT) received no treatment. The incidence of anti-Kell could be reduced by transfusing Kell negative red cells to premenopausal women. It is important that all pregnant women are tested at least twice in their pregnancy to detect the antibodies formed late in the pregnancy. It is useful to formulate a standard protocol for antenatal interventions. Non Rh-D antibodies can cause significant anaemia for up to six weeks in the neonatal period, hence early detection of maternal antibodies is important so that the neonates are followed up for an appropriate length of time.

The Kell blood group system is polymorphic, and 23 antigens have been defined to date. The Kell antigens are located on a single red cell transmembrane glycoprotein, encoded by the 19 exons of the KEL gene. The different Kell phenotypes result from point mutations leading to amino acid changes in the Kell glycoprotein. An unusual phenotype, which is defined as the complete lack of all of the Kell antigens, has been identified and designated as the Kell-null or Ko phenotype. The coding region of the KEL gene of the Ko individual showed a normal KEL2/KEL4/KEL7 gene sequence; nevertheless, a G to C mutation at the splice donor site (5' splice site) of intron 3 was found to be present as a homozygote in the individual. The mutation destroys the conserved GT sequence of the splice donor site. Reverse transcription-polymerase chain reaction analysis showed the absence of the complete KEL mRNA. Instead, a major transcript with the exon 3 region skipped was found. The exon 3 of the KEL gene encodes the transmembrane domain of the Kell glycoprotein, and a transcript without exon 3 is predicted to have a premature stop codon that abolishes the translation of C-terminal segment. The segment contains all of the known positions responsible for characterizing different Kell antigens, and this explains the lack of all Kell antigens in Ko red cells.

The detection and identification of blood group antibodies in patients is crucial for successful allogeneic blood transfusions. Current methods are highly subjective and rely on red blood cells (RBCs), which simultaneously express many blood group antigens, have a short shelf-life, and carry potential biohazard risks. To overcome these problems, we have used the approach of expressing individual blood group antigen-bearing proteins in a heterologous system. We report here the high-level surface expression of type I (Knops), type II (Kell), and type III/multi-pass (Duffy) membrane proteins that carry blood group antigens in mouse erythroleukaemic (MEL) cells using a vector containing the beta-globin locus control region. Importantly, the antigens expressed were detected specifically by a panel of patients' sera containing alloantibodies at sensitivities that are comparable to antigen-positive RBCs. Furthermore, in contrast to other mammalian expression systems, antigen expression was stable following freezing and thawing of the cell lines. Thus, this system has the potential both to replace the current use of RBCs by providing a one step method to detect and identify blood group antibodies and to allow the automation of antibody identification for the clinical laboratory., (Copyright 2000 Wiley-Liss, Inc.)

Blood group polymorphisms have been used as tools to study the architecture of the red blood cell (RBC) membrane. Some blood group variants have reduced antigen expression at the cell surface. Understanding the underlying mechanism for this reduced expression can potentially provide structural information and help to elucidate protein trafficking pathways of membrane proteins. The Kp(a+) phenotype is a variant in the Kell blood group system that is associated with a single amino acid substitution (R281W) in the Kell glycoprotein and serologically associated with a weakened expression of other Kell system antigens by an unknown mechanism. We found by immunoblotting of RBCs that the weakening of Kell antigens in this variant is due to a reduced amount of total Kell glycoprotein at the cell surface rather than to the inaccessibility of the antigens to Kell antibodies. Using a heterologous expression system, we demonstrate that the Kpa mutation causes retention of most of the Kell glycoprotein in a pre-Golgi compartment due to differential processing, thereby suggesting aberrant transport of the Kell protein to the cell surface. Furthermore, we demonstrated that single nucleotide substitutions into the coding region of the common KEL allele, as predicted by the molecular genotyping studies, was sufficient to encode three clinically significant low incidence antigens. We found that two low incidence antigens can be expressed on a single Kell protein, thus showing that the historical failure to detect such a variant is not due to structural constraints in the Kell protein. These studies demonstrate the power of studying the molecular mechanisms of blood group variants for elucidating the intracellular transport pathways of membrane proteins and the requirements for cell surface expression.

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 study was conducted to report on the use of molecular biology methods and pregnancy outcome in women sensitized to either Rhesus D (RhD) or Kell 1 (K1) antigens. Paternal RhD genotype was determined by DNA amplification of an RhD-specific sequence from single sperm cells. Paternal Kell phenotype was determined by serologic assays using peripheral blood samples, and the fetal RhD or Kell-type status were established by the polymerase chain reaction (PCR) with amniotic cells. Thirteen women (14 pregnancies, one with twins) sensitized to RhD and four sensitized to K1 antigens, comprised the study group. All had paternal heterozygosity to either D or K1 antigens. Nine fetuses were RhD positive and five were RhD negative. An additional woman underwent early spontaneous abortion. The nine RhD-positive fetuses underwent a total of 41 invasive procedures. One fetus with hydrops fetalis died in utero after intrauterine blood transfusion. All the remaining RhD-positive fetuses were delivered after 33 weeks gestation, and all those who were RhD negative were delivered at term. Four women were sensitized to the K1 antigen; in three, the fetus was found to be K1 negative, and in one, K1 positive, necessitating intrauterine blood transfusion. In all cases, the results of RhD or K1 genotype analyses from amniotic fluid were compatible with fetal/neonatal red blood cell RhD or Kell phenotypes. In conclusion, the use of molecular biology techniques represents a major advance in the clinical management of RhD and Kell alloimmunization.

A 37-kDa protein was immunopurified from human erythrocytes as a complex with a monoclonal antibody directed against the Kell blood group protein of 93 kDa. A rabbit antibody raised against the purified complex reacted on a Western blot with the 93-kDa and 37-kDa proteins and was able to immunoprecipitate the 37-kDa component from K0 erythrocytes which express large amount of the Kx antigen, but not from erythrocytes of patients suffering from McLeod syndrome, a X-linked disorder in which the Kx antigen is lacking. Additional studies have shown that the 37-kDa protein is not glycosylated, and permitted the sequence of the 22 first N-terminal amino acids to be established. This sequence was identical to the predicted protein product of the XK gene cloned recently, which is deleted or mutated in McLeod patients [Ho, M., Chelly, J., Carter, N., Danek, A., Crocker, P. & Monaco, A. P. (1994) Cell 77, 869-880]. Our findings provide strong evidence that the 37-kDa red cell membrane protein is identical to the Kx protein produced by the XK structural gene and demonstrate that Kx and Kell proteins are two subunits expressed as a complex hold by disulfide bond(s) at the red cell surface.

The application of monoclonal antibodies to the quantification of blood group antigen sites on erythrocytes was examined. A second antibody technique using labelled anti-mouse IgG could not be used as it was not possible to predict the binding ratio between this and the monoclonal antibody. A series of monoclonal antibodies (R10, R18, BRIC 13, BRIC 14) to the erythrocyte sialoglycoprotein alpha (syn: glycophorin A) showed the number of antigen sites to be from 0.3 X 10(6) to 1.2 X 10(6) per erythrocyte and supported the conclusion that the Wrb antigen is located on this protein. An antibody with a specificity related to the Rh blood group system (R6A) showed 4.6 - 10.4 X 10(4) binding sites to be present on cells of phenotype cCDEe. On cells of phenotype -D- 1.24 X 10(4) binding sites were present but protease treatment increased the number of available sites to 1.3 X 10(5). An antibody to a Kell-related antigen (BRIC 18) recognized 2.5 - 5.9 X 10(3) sites per erythrocyte on cells of phenotype Kk. However, a similar number also appeared to be present on cells of the McLeod and Ko phenotypes although the affinity for the antigen on these cells was very much reduced. The potential of using monoclonal antibodies for this purpose and the value of this in the study of blood group systems has been demonstrated.

An automated technique for measuring haemagglutination kinetics is described. Equipment used in this test is very simple and already present in most blood transfusion centres. This method may be used with advantage to differentiate homozygous and heterozygous subjects in Rhesus, MNSs, and Kell systems, and to study antigen variations, especially in genetic investigations.

The IgG subclass distribution of anti Rh antibodies (anti-D, 'anti-Du', anti-c, anti-E), anti-Kell and anti-Duffy (anti-Fya) antibodies was measured by two haemagglutination techniques on microtitre plates. The first technique involved rabbit subclass specific antisera which were used to agglutinate red cells previously reacted with the patients' antibodies at high concentration. The second, which was more sensitive, had an additional step by introducing sheep anti-rabbit antibodies (sandwich technique). By the sensitive sandwich technique we revealed, for anti-D antibodies: IgG1 8/19, IgG3 1/19, IgG1/IgG3 8/19, IgG1/IgG2/IgG3/IgG 41/19, IgG1/IgG4 1/19; for the Du reactive anti-D antibodies: IgG1 1/8, IgG1/IgG3 1/8, IgG1/IgG3/IgG4 6/8; for the anti-E antibodies: IgG1/IgG2/IgG4 2/3, IgG1/IgG2/IgG3/IgG4 1/3; for the anti-c antibodies: IgG1 2/5, IgG3 1/5, IgG1/IgG3 1/5; for the anti-Kell antibodies: IgG1 9/20, IgG1/IgG3 1/20, IgG1/IgG4 8/20, IgG1/IgG3/IgG4 2/20; and for anti-Duffy antibodies: IgG1 1/8, IgG1/IgG4 7/8. These results are partly at variance with previously published results.

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.

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.

A human monoclonal anti-Kell (K1) antibody secreted by an Epstein-Barr virus (EBV)-transformed B-cell line was used for binding studies and immunopurification of the K1 blood group antigen. The 125I-labeled antibody bound to 4 to 5 x 10(3) and 2.5 to 3 x 10(3) antigenic sites on K1K1 and K1K2 erythrocytes, respectively, with an affinity constant of 5 x 10(8) mol/L-1. Immunoprecipitation analysis showed that the K1 antigen is carried by a 93 Kd glycoprotein containing several cysteine residues, and approximately six N-glycosidically linked sugar chains but no detectable O-linked sugar. A minor labeled component of 32 Kd was also immunoprecipitated from K1K1 RBCs but the 93- and 32-Kd components were absent from K2K2 and Kell null erythrocytes. Under nonreducing conditions, three bands were detected at 200 (weak), 120, and 93 Kd. We suggest that the 120-Kd component represents a heterodimer of the 93- and 32-Kd proteins covalently linked by disulfide bridge(s). The 93-Kd glycoprotein is a transmembrane component which interacts with the membrane skeleton but is distinct from band 3 as shown by one-dimensional peptide mapping. The site density of K1 antigen blood group on Gerbich-negative RBCs (Ge:-2,-3) was threefold lower than on K1K1 erythrocytes, but the qualitative properties of the 93-Kd component were not modified.

Acanthocytosis represents an unusually pathological variant of red cell morphology which is encountered in a diverse group of inherited and acquired disease states. While the morphological features are similar in all instances, the biochemical lesions frequently differ. Most demonstrable abnormalities involve lipids although those acanthocytes associated with the McLeod phenotype are probably due to an alteration in a membrane protein. Acanthocytes, regardless of their etiology, usually have a decreased survival in the circulation owing to splenic sequestration and destruction.

Hemolytic anemia occurred in a 70-year-old female after a five-day course of intravenous cefamandole. The patient's serum contained an IgG antibody which was reactive with red blood cells which had been coated in vitro with cefamandole but not with uncoated cells. An in vitro assay of allogeneic mononuclear phagocytosis of cefamandole-coated red cells sensitized with the patient's anti-cefamandole indicated that the anti-cefamandole could induce significant phagocytosis. The anti-cefamandole was easily inhibited in vitro by cefamandole as well as by a variety of related cephalosporins indicating broad cross-reactivity, with the antigenic site primarily the 7-amino-cephalosporanic acid nucleus. Penicillins could inhibit the anti-cefamandole but only when using concentrations 3-10 X those of cephalosporins. Eleven examples of anti-penicillin tested failed to react with cefamandole-coated red cells. Screening of 344 random sera from hospitalized patients found only five (1.5%) reactive with cefamandole-coated red cells; three of these sera were also reactive with penicillin-coated red cells. The patient's hemolysis subsided following cessation of the drug. This is the first report of anti-cefamandole-induced hemolytic anemia.