Blood Group Specificity Research Articles

Potential implications of ABO blood group for vascular rejection in pig to human kidney xenotransplantation.

A substantial hurdle for successful xenotransplantation is to negate the effect of xenoreactive natural antibodies [mainly Galalpha1-3Galbeta1-4GlcNAc (alpha-Gal) specific] that cause hyperacute xenograft rejection. Galalpha1-3Gal molecules (alpha-Gal) have close structural homology with human ABO blood groups and therefore an individual's blood group might influence the formation of alpha-Gal specific antibodies. Genetic heterogeneity controlling alpha-Gal specific antibody formation could have important implications for future pig to human xenotransplantation clinical trials. We have investigated the relationship between ABO blood group and immunoglobulin M (IgM) and immunoglobulin G (IgG) alpha-Gal specific antibody titres in sera obtained from renal dialysis patients and healthy blood donors. Serially diluted sera (n = 166) obtained from renal dialysis patients awaiting kidney transplantation (n = 116) and healthy blood donors (n = 50) were tested for IgM and IgG alpha-Gal antibodies using an enzyme-linked immunosorbent assay (ELISA) specific for alpha-Gal. The study cohort comprised 62, 48, 36 and 20 sera obtained from blood group O, A, B and AB individuals, respectively. Reciprocal alpha-Gal specific antibody titres were calculated from ELISA titration curves and stratified by individual blood group. No significant heterogeneity was found in IgM alpha-Gal specific antibody titres across ABO blood groups. In contrast, marked heterogeneity was observed in IgG alpha-Gal specific antibody titres when stratified by blood group. IgG alpha-Gal specific antibody titres were higher in sera obtained from blood group O renal dialysis patients [median titre 40, interquartile range (IQR) 14 to 72], compared with blood group A (median titre 18, IQR 7 to 54, P = 0.05), blood group B (median titre 6, IQR 0 to 15, P < 0.001) and blood group AB patients (median titre 3.5, IQR 0 to 16, P = 0.002). A similar correlation was found for IgG alpha-Gal specific antibody titres in sera obtained from healthy blood donors with median titres of 20 (IQR 12 to 34), 37 (10 to 91), 9 (0 to 20), and 5.5 (0 to 12) in blood groups O, A, B and AB individuals, respectively. There was a strong interrelationship between alpha-Gal specific antibody class and blood group, with both IgM and IgG alpha-Gal specific antibodies found in 84% of the blood group O sera, 73% of blood group A sera, 50% of blood group B sera and 40% of blood group AB sera (P < 0.001). In a subgroup of 39 renal dialysis patients, IgM and IgG alpha-Gal specific antibody titres were measured in two serum samples obtained at different time-points (median time interval 581 days, range 42 to 4414), and showed a high degree of stability (correlation coefficient 0.88 and 0.90 for IgM and IgG, respectively). IgG alpha-Gal specific antibody titres are significantly higher in the sera of blood group O and A renal dialysis patients and healthy individuals compared with blood groups B and AB. These data indicate that future clinical trials of pig to human xenotransplantation may be more problematic for non-blood group B patients who are likely to have high levels of IgG alpha-Gal specific antibodies that are associated with acute vascular rejection.

Professor Walter Morgan

Professor Walter Morgan

Isolation, purification, and physicochemical characterization of a d-galactose-binding lectin from seeds of Erythrina speciosa

A lectin was isolated from the saline extract of Erythrina speciosa seeds by affinity chromatography on lactose–Sepharose. The lectin content was about 265 mg/100 g dry flour. E. speciosa seed lectin (EspecL) agglutinated all human RBC types, showing no human blood group specificity; however a slight preference toward the O blood group was evident. The lectin also agglutinated rabbit, sheep, and mouse blood cells and showed no effect on horse erythrocytes. Lactose was the most potent inhibitor of EspecL hemagglutinating activity (minimal inhibitory concentration (MIC)=0.25 mM) followed by N-acetyllactosamine, MIC=0.5 mM, and then p-nitrophenyl α-galactopyranoside, MIC=2 mM. The lectin was a glycoprotein with a neutral carbohydrate content of 5.5% and had two p I values of 5.8 and 6.1 and E 1% 1 cm of 14.5. The native molecular mass of the lectin detected by hydrodynamic light scattering was 58 kDa and when examined by mass spectroscopy and SDS–PAGE it was found to be composed of two identical subunits of molecular mass of 27.6 kDa. The amino acid composition of the lectin revealed that it was rich in acidic and hydroxyl amino acids, contained a lesser amount of methionine, and totally lacked cysteine. The N-terminal of the lectin shared major similarities with other reported Erythrina lectins. The lectin was a metaloprotein that needed both Ca 2+ and Mn 2+ ions for its activity. Removal of these metals by EDTA rendered the lectin inactive whereas their addition restored the activity. EspecL was acidic pH sensitive and totally lost its activity when incubated with all pH values between pH 3 and pH 6. Above pH 6 and to pH 9.6 there was no effect on the lectin activity. At 65 °C for more than 90 min the lectin was fairly stable; however, when heated at 70 °C for 10 min it lost more than 80% of its original activity and was totally inactivated at 80 °C for less than 10 min. Fluorescence studies of EspecL indicated that tryptophan residues were present in a highly hydrophobic environment, and binding of lactose to EspecL neither quenched tryptophan fluorescence nor altered λ max position. Treating purified EspecL with NBS an affinity-modifying reagent specific for tryptophan totally inactivated the lectin with total modification of three tryptophan residues. Of these residues only the third modified residue seemed to play a crucial role in the lectin activity. Addition of lactose to the assay medium did not provide protection against NBS modification which indicated that tryptophan might not be directly involved in the binding of haptenic sugar d-galactose. Modification of tyrosine with N-acetylimidazole led to a 50% drop in EspecL activity with concomitant acetylation of six tyrosine residues. The secondary structure of EspecL as studied by circular dichroism was found to be a typical β-pleated-sheet structure which is comparable to the CD structure of Erythrina corallodendron lectin. Binding of lactose did not alter the EspecL secondary structure as revealed by CD examination.

Clinical features of Fabry's disease in Australian patients.

Anticipating the prospect of specific treatment, we studied a large group of Australians with Fabry's disease. We aimed to: (i) document the clinical features of Fabry's disease in Australian patients, (ii) test the hypothesis that clinical features vary with specific mutation and blood group and (iii) assess small-fibre peripheral nerve function. A questionnaire was forwarded to all Australian patients known to us. Patients were invited to attend for clinical, renal cardiac, ophthalmological and neurological assessment. Sixty-seven patients (29 men and 38 women) from 18 families participated. Diagnosis in index cases was delayed by > or = 10 years in nearly all families. Common clinical features are: (i) episodic acroparaesthesia (100% of hemizygotes; 53% of heterozygotes), (ii) anhydrosis (93%; 1%), (iii) characteristic rash (93%; 13%), (iv) renal disease (69%; 21%), (v) ischaemic heart disease (28%; 26%), (vi) palpitations (62%; 29%), (vii) mitral valve murmurs (37%; 23%) and (viii) premature cerebrovascular disease (31%; 5%). Ophthalmic findings of cornea verticillata (96%; 76%) and anterior cataract (48%; 14%) were common. Findings were variable within and between families. In women, anhydrosis reliably predicts the presence of significant Fabry's renal disease. Small nerve fibre testing using quantitative sensory testing was clearly abnormal in 95% of male patients, and in those female patients with paraesthesiae. Symptoms of anhydrosis, acroparaesthesiae, rash and renal disease suggest diagnosis of Fabry's. Women are commonly symptomatic, and the advent of therapy highlights the practical advantage of earlier diagnosis.

Interrelation between ABH blood group 0, Lewis(B) blood group antigen, Helicobacter pylori infection, and occurrence of peptic ulcer.

Helicobacter pylori is a human pathogen that causes chronic gastritis and peptic ulcers. Epidemiological studies demonstrated that individuals who are blood group 0 positive or represent non-secretors of their blood group antigens are more likely to develop peptic ulcers. The Lewis(b) blood group antigen has been reported to mediate the attachment of H. pylori to human gastric mucosa. The aim of this study was to examine the interrelation between Le(a-b+) phenotype, blood group 0, H. pylori infection, and peptic ulcer occurrence. The study population consisted of 330 consecutive patients (185 men, 145 women) referred to endoscopy of the upper gastrointestinal tract for various reasons. AB0(H) blood groups and Lewis(a,b) phenotype were carried out by standard haemagglutination assays. Antibodies (IgG) against H. pylori were determined by a quantitative enzyme-linked immunosorbent assay (ELISA). 49 of the 330 patients (14.8 %) showed duodenal or gastric ulcers with a H. pylori seroprevalence of 87.8 %. The IgG immune response to H. pylori was not dependent on ABH blood group phenotype. There was also no significant association between the secretor status and the presence of H. pylori infection. Secretors, 35/238 (14.7 %), were no more likely to have gastroduodenal ulcer compared with non-secretors, 9/65 (13.8 %). Our data show no association between secretor status or specific ABH blood group on the one hand, and H. pylori infection or occurrence of gastroduodenal ulcers on the other. Determination of ABH blood groups or secretor status is, therefore, not a useful tool to characterize the individual risk for gastroduodenal ulcer or to guide any diagnostic procedures.

ChemInform Abstract: Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H‐Type 2 Human Blood Group Specificity.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Age of blood in inventory at a large tertiary care hospital.

This major tertiary care centre transfers over 1000 red cell units monthly. Units are rotated as part of inventory management to ensure minimum wastage. In 1998, the expiration date of AS3 red cells was extended from 35 to 42 days, potentially affecting inventory. The average age of the red cell units in inventory on any given day was evaluated to determine whether the extended expiration date would affect blood availability and to determine the feasibility of using blood at different ages for various purposes. Over a 6-month duration, 20 days were selected for review: units were categorized according to ABO group and Rh type and then analysed for age within certain categories. The average age of the blood in inventory was 1 to 2 weeks. The probability of having units less than 1-week old was highest for Group O and zero for Group B Rh(+) and Group AB Rh(+). More than 60% of the O Rh(-) blood was older than 28 days. The age of units in inventory varies with respect to ABO group, Rh type and weekday. In practice, the stock rarely reaches 42 days of age. Future studies on the effects of age of blood on patient outcome must consider the logistics of supply and the availability of blood of each group. Transfusion of large numbers of units of the same age and of a specific blood group and type may not always be possible.

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

Toward Fully Synthetic Homogeneous Glycoproteins: A High Mannose Core Containing Glycopeptide Carrying Full H-Type 2 Human Blood Group Specificity

A DNA-based immunization protocol to produce monoclonal antibodies to blood group antigens.

A major challenge facing transfusion medicine is the establishment of immunological methods to produce specific and avid blood group typing reagents to the many polymorphic blood group antigens. This is especially true when sources of human antibody are limited. Based on the knowledge that inoculation with plasmid DNA can induce a humoral response in the host animal, we inoculated mice with plasmid DNA followed by a single boost injection with plasmid-transfected cells that have a high level of expression of the same target protein. Using this method, several hybridoma clones that produced strongly reactive antibodies specific for the Kell polymorphic antigens (anti-K, anti-k, anti-Kp(a)) were isolated. The monoclonal antibodies that were produced with this method have potential clinical utility for identifying a patient's blood type and for screening for antigen-negative donor blood.

Tissue distribution of histo-blood group antigens.

The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo-blood group antigens and related carbohydrate structures. The present paper summarizes the available data concerning the histological distribution of histo-blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo-blood group antigens carried by type 1, 2, and 3 chain carrier carbohydrate chains. Histo-blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histoblood group antigens, i.e. the ABO, Lewis, and saliva-secretor type of the individual, and the cell- and tissue type. Oligosaccharides with blood-group specificity are synthesized by the stepwise action of specific gene-encoded glycosyltransferases. In general, this stepwise synthesis of histo-blood group antigens correlates with cellular differentiation. The H and the Se genes both encode an al-2fucosyltransferase, which is responsible for the synthesis of blood group antigen H from precursor disaccharides. A new model for the participation of the Se/H-gene-encoded glycosyl transferases in synthesis of terminal histo-blood group antigens in human tissues is proposed; the type and degree of differentiation rather than the embryologic origin determines whether it is the H or the Se gene-encoded transferases that influence expression of terminal histo-blood group antigens in tissues.

Host determinants of Helicobacter pylori infection and its clinical outcome.

Greater than one-half of the world's population harbors Helicobacter pylori. The majority of infected individuals, however, remain asymptomatic, with only 10% to 20% developing diseases, including peptic ulcer disease, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. This article reviews host factors that may predispose an individual to both the acquisition of H. pylori infection and subsequent clinical outcome. Individuals with specific blood group antigens and human leukocyte antigen genotypes may be more susceptible to H. pylori infection. Additional factors, such as the age of acquisition, the host immune response, the site of infection, acid secretion, and interactions with nonhost factors (including bacterial virulence factors and environmental influences) may play a role in determining clinical outcome. Further investigation is required to clarify the mechanisms by which these interactions occur and, more critically, to determine their relative importance. This knowledge will enable the identification of individuals at risk of developing clinical disease with H. pylori infection.

The Molecular Basis for the B(A) Allele: An Amino Acid Alteration in the Human Histoblood Group B α-(1,3)-Galactosyltransferase Increases Its Intrinsic α-(1,3)-N-Acetylgalactosaminyltransferase Activity

The formation of the subgroup B(A) phenotype is thought to be due to an overlapping specificity of the human blood group A and B transferases. A new molecular basis for the B(A) allele, resulting from the C700 to G substitution which predicts the alteration of Pro234 to Ala, just ahead of the second of the four amino acid residues which differentiates the specificities of the A and B transferases, is reported here. Compared to normal group B sera, a relatively lower B-transferase activity was demonstrated in the B(A) serum, which correlated well with the observation of a smaller amount of B antigen on the B(A) red cells. Also a much higher A-transferase activity was demonstrated in the B(A) serum in contrast to the minute amount of A-transferase activity found in normal group B sera. The formation of the B(A) phenotype in this report is most likely due to the shifting of the specificity of the B transferase rather than an enhanced B-transferase activity which was previously presumed to be responsible for the formation of this phenotype. The Pro234 to Ala alteration is suggested to be responsible for the shifting of the specificity with a subsequent increase in A- but a decrease in B-transferase activity. This new B(A) allele shows that not only the four critical residues but also the neighboring areas may influence the specificity of the A and B transferases.

Synthesis of Rh Fv phage-antibodies using VH and VL germline genes.

Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.

A Newly Identified Horseshoe Crab Lectin with Specificity for Blood Group A Antigen Recognizes Specific O-Antigens of Bacterial Lipopolysaccharides

A 14-kDa lectin, named tachylectin-3, was newly identified from hemocytes of the Japanese horseshoe crab, Tachypleus tridentatus. This lectin exhibited hemagglutinating activity against human A-type erythrocytes, but not against the B- and O-types of erythrocytes and animal erythrocytes, including those of sheep, rabbit, horse, and bovine. The hemagglutinating activity of tachylectin-3 was equivalent to that of a previously identified lectin, named tachylectin-2, with affinity for N-acetyl-D-glucosamine or N-acetyl-D-galactosamine. However, the activity of tachylectin-3 was not inhibited by these two N-acetylhexosamines at 100 mM but was inhibited by a blood group A-pentasaccharide at a minimum inhibitory concentration of 0.16 mM. Furthermore, the hemagglutinating activity was strongly inhibited by bacterial S-type lipopolysaccharides (LPSs) from Gram-negative bacteria but not by R-type LPSs lacking O-antigens. One of the most effective S-type LPSs was from Escherichia coli O111:B4, with a minimum inhibitory concentration of 6 ng/ml. These data suggest that tachylectin-3 specifically recognizes Gram-negative bacteria through the unique structural units of O-antigens. Ultracentrifugation analysis revealed that tachylectin-3 is present in dimer in solution. A cDNA coding for tachylectin-3 was isolated from a hemocyte cDNA library. Tachylectin-3 consisted of two repeating sequences, each with a partial sequence similarity to rinderpest virus neuraminidase. Tachylectin-3 and three previously isolated types of tachylectins were all predominantly expressed in hemocytes and released from hemocytes in response to external stimuli. These lectins present at injured sites suggest that they probably serve synergistically to accomplish an effective host defense against invading microbes.

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

A New Solid-Phase Method for ABO Grouping, Rh Phenotyping, and Kell Determination

Background: In comparison with a well established agglutination method, a newly developed solid-phase method for ABO grouping, Rh phenotyping, and Kell determination was examined for its suitability in routine testing. This new method is characterized by the use of monoclonal IgM antibodies of blood group specificities which are immobilized onto the polystyrene microplate wells during the test procedure. Red blood cell suspensions are added to the prepared wells and are allowed to react immunologically with the solid phase. Positive reactions are indicated by the adherence of red blood cells over the entire surface of the wells, whereas negative reactions form discrete red blood cell buttons in the center of the wells. The uniformity of the reaction patterns permits an objective reading of the results, both visually and spectrophotometrically. Material and Methods:Blood samples of 631 healthy blood donors and 237 patient samples were determined by the solid-phase method, and the results were compared with those obtained by the conventional agglutination test. In addition, a panel of weak A and B subgroups and D variants was determined. Results:Determining the ABO blood groups as well as the Rh phenotypes and the Kell antigen, we found complete accordance between both methods. Detecting A and B subgroups and D variants, the solid-phase method was found to be more sensitive when compared with the agglutination method. Conclusions:Due to the ease of handling of the new solid-phase assay and the unequivocal test results, the method is suitable for manual routine testing in small- to medium-sized laboratories as well as for automation. The reaction patterns can easily be interpreted visually or by computer software-supported readers. Storing the microplates in a refrigerator, the results remain stable for several days.

Kx, a Quantitatively Minor Protein from Human Erythrocytes, Is Palmitoylatedin Vivo

Kx is a quantitatively minor blood group protein of human erythrocytes which is thought to be a membrane transporter. In the red cell membrane, Kx forms a complex stabilized by a disulfide bond with the Kell blood group membrane protein which might function as a metalloprotease. The palmitoylation status of these proteins was studied by incubating red cells with [3H] palmitic acid. Purification of the Kell-Kx complex, by immunochromatography on an immobilized human monoclonal antibody of Kell blood group specificity demonstrated that the Kx but not the Kell protein is palmitoylated. Six cysteines in Kx are predicted to be intracytoplasmic and might be targets for palmitoylation. Three of these cysteines are present in a portion of sequence which is predicted to form an amphipathic alpha helix. Palmitoylation of one or several of these cysteines might contribute to anchor the cytoplasmic portion of the Kx protein to the inner surface of red cell membrane.

Cross-Contamination of Red Blood Cell Unit Samples Associated With Scissors Cutting of Segments

Use of scissors for cutting segments from RBC units is the prevalent technique for obtaining blood samples to type, crossmatch, and screen for specific blood group antigens. Concerns associated with this technique include safety issues for technologists (eg, blood splatter, sharp injuries) and the potential for blood sample cross-contamination between sequential segment cuttings. We documented unequivocally that the use of improper scissors technique (ie, inadequate cleaning of scissors between sequential segment cuttings) can be associated with cross-contamination of RBC samples from segments. The study underscores the need for improved segment sampling techniques to eliminate the potential of cross-contamination. This problem could be obviated by implementation of single-use segment sampling devices or by enforcing rigorous standard operating procedures for cleaning and wiping scissors between sequential segment cuttings.