Determinants of ABH expression on human blood platelets

Platelets express ABH antigens, which can adversely effect platelet transfusion recovery and survival in ABH-incompatible recipients. To date, there has been no large, comprehensive study comparing specific donor factors with ABH expression on platelet membranes and glycoconjugates. We studied ABH expression in 166 group A apheresis platelet donors by flow cytometry, Western blotting, and thin layer chromatography relative to donor age, sex, A1/A2 subgroup, and Lewis phenotype. Overall, A antigen on platelet membranes, glycoproteins, and glycosphingolipids was linked to an A1 red blood cell (RBC) phenotype. Among A1 donors, platelet ABH varied significantly between donors (0%-87%). Intradonor variability, however, was minimal, suggesting that platelet ABH expression is a stable, donor-specific characteristic, with 5% of A1 donors typing as either ABH high- or low-expressers. Group A2 donors, in contrast, possessed a Bombay-like phenotype, lacking both A and H antigens. Unlike RBCs, ABH expression on platelets may be determined primarily by H-glycosyltransferase (FUT1) activity. Identification of A2 and A1 low expressers may increase the availability and selection of crossmatched and HLA-matched platelets. Platelets from group A2 may also be a superior product for patients undergoing A/O major mismatch allogeneic progenitor cell transplantation.     

ABH and Lewis antigen and antibody expression after bone marrow transplantation

We have tested 10 bone marrow transplant donors and recipients for ABH and Lewis antigens and ABH antibodies. Two group A2 recipients stably engrafted with group O marrow produced anti-A1. Small quantities of recipient-type ABH substance were found on the posttransplant erythrocytes in cases where the recipient was a secretor but not in a single nonsecretor recipient. Lewis antigens were always of recipient type. The incidence of graft-versus-host disease and graft rejection were similar in 41 patients who received ABO-mismatched and 61 patients who received ABO-matched grafts.     

Circulating immune complexes involving the ABO system after platelet transfusion

Summary. It has been proposed that when ABO unmatched platelets are transfused circulating immune complexes (CIC) may be formed between the patient's soluble ABH antigens and the transfused antibodies. Platelets might then be destroyed by bystander mechanisms or by the binding of CIC to the Fc receptor or to C3 binding membrane proteins on the platelet. An ELISA Clq assay was used to detect CIC in 40 patients with haematological diseases who had received multiple platelet transfusions. A significantly larger number of refractory patients were positive (41%) in the assay than non-refractory patients (13%) or normal blood donors (0%). The presence of circulating IgG anti-A was sought in six group A refractory patients who had been transfused with ABO unmatched platelets. To determine whether the IgG anti-A was monomeric or in high molecular weight complexes, the serum was fractionated by gel exclusion chromatography and fractions were tested for the presence of IgG anti-A. In all six patients the peak of IgG anti-A binding occurred in fractions of high molecular weight (200–900 kD). Five out of six patients also demonstrated anti-A activity in fractions corresponding to monomeric IgG (about 150–180 kD). Fractions containing high molecular weight anti-A were purified using a protein G column and the eluates were tested for the presence of group A antigen using dot immunoblotting. Group A antigen was associated with the purified IgG anti-A in 4/5 patients tested. Appropriate transfused and non-transfused controls had no anti-A in any fractions. Although not unexpected, these studies demonstrate for the first time that refractory patients receiving ABO unmatched platelets have CIC composed of ABO antigens and their corresponding antibodies present in their serum that circulate for at least several days. It also confirms the hypothesis that some CIC in haematological patients are induced by transfusion.     

Possible effect of secretor locus on plasma concentration of factor VIII and von Willebrand factor

A significant fraction (30%) of the genetically determined variance in plasma concentration of the von Willebrand factor antigen (vWf:Ag) has been shown to be related to ABH determinants. Individuals with blood group O, who have the highest amounts of blood group H substance, have the lowest concentration of vWf:Ag. The Lewis substances, Le(a) and Le(b), are biochemically closely related to the ABH substances as both can be produced from the same precursor substance. We studied the effect of the presence of the Lewis antigens on the plasma concentration of vWf:Ag and factor VIII antigen (VIII:Ag) in 323 individuals of different ABO groups from a series of twins and in 58 blood donors of blood group O. Among persons belonging to blood group O, those with the Le(a) antigen had a higher concentration of both vWf:Ag and VIII:Ag than individuals lacking Le(a). Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. Thus, the lowest concentration of vWf:Ag and VIII:Ag was found in group O secretors. The effect is most likely due to an effect of the secretor locus. This finding may be of importance for the detection of carriers of hemophilia A and for the diagnosis of type I von Willebrand disease.     

Acquired Zwa antigen on Zwa negative platelets demonstrated by Western blotting

Zwa-negative platelets that had been incubated in random donor AB plasma for several hours and subsequently cryopreserved, were found to be Zwa-positive when tested 3 years later by Western blotting. Following this serendipitous observation, we demonstrated that the Zwa antigen could be passively adsorbed onto the membrane of Zwa-negative platelets incubating for 4 h in the plasma from Zwa-positive donors. This observation supports the hypothesis that passive acquirement of Zwa antigens is a pathogenic mechanism for inducing post-tranfusion purpura, and suggests that Zwa-negative platelets should not be stored in Zwa-positive plasma before transfusion.     

Role of A and B blood group antigens in the expression of adhesive activity of von Willebrand factor

ABO (H) blood group antigens are covalently linked to the oligosaccharide side-chains of von Willebrand factor (VWF). In this study, we investigated the role of the A and B antigens in the expression of VWF adhesive activity. VWF of type A, B or O was purified from fresh frozen plasma. Presence of A or B antigen on the VWF was confirmed by enzyme-linked immunosorbent assay (ELISA) and by immunoblotting with monoclonal anti-A or anti-B. The A or B antigen was also detected in the 48/52-kDa fragment of the respective VWF after trypsin digestion. Removal of A antigen with alpha-N-acetylgalactosaminidase or B antigen with alpha-galactosidase did not affect its multimer size or antigenic level, but decreased the ristocetin cofactor (RCoF) activity of the respective VWF by 33-39% (P < 0.01-0.002). Removal of A or B antigen from VWF did not affect the binding of the VWF to immobilized type III collagen. A and B antigens were not detected in platelet VWF. These results indicate that AB structures play a role in platelet aggregating activity of VWF.     

Expression of ABH and X (Lex) antigens on platelets and lymphocytes

We used a panel of reagents, polyclonal and monoclonal antibodies, and lectins to define the expression of the ABH- and Lewis-related specificities on platelets and lymphocytes. We also determined the expression of the alpha 2- and alpha 3-L-fucosyltransferases necessary for their biosynthesis. The antigens that could be detected by immunofluorescence and Western blot analysis were based on type 2 monofucosylated structures. Antibodies directed toward types 1, 3, and 4 ABH-, X- and Lewis-related antigenic determinants were always negative because the small amounts of ABH and Lewis antigens adsorbed from the serum could not be detected by these techniques. The presence of the type 2 ABH antigens on intrinsic glycoproteins was controlled by the H gene. This correlates with the presence of alpha 2-L- fucosyltransferase and the absence of alpha 3-L-fucosyltransferase on platelets. In contrast, ABH antigens were not detected by immunofluorescence on normal peripheral lymphocytes. These cells thus have only the small amounts of antigens adsorbed from the serum, these being under control of the secretor and Lewis genes. This correlates with the absence of alpha 2-L-fucosyltransferase on lymphocytes. When lymphocytes were transformed in vitro by the Epstein-Barr virus (EBV), however, they strongly expressed the X and sialylated X antigens, which are specific markers of normal granulocytes and monocytes, respectively. Treatment of EBV-transformed lymphoblastoid cell lines with 12-O-tetradecanoylphorbol-13-O-acetate significantly decreased the expression of X and sialylated X antigens along with that of surface immunoglobulins, whereas it induced a significant expression of the H antigen under control of the H gene.     

Blood group A and B antigens are strongly expressed on platelets of some individuals

It is widely thought that expression of ABH antigens on platelets is insufficient to materially affect the survival of ABH-incompatible platelets in transfusion recipients, but anecdotal reports of poor survival of A and B mismatched platelets suggest that this is not always the case. The A and B antigen expression on platelets of 100 group A(1) and group B blood donors was measured, and 7% and 4%, respectively, had platelets whose A and B antigen levels consistently exceeded the mean plus 2 SD. On the basis of flow cytometric and statistical analysis, donors whose platelets contained higher than normal levels of A antigen were subdivided into 2 groups, designated Type I and Type II ("high expressers"). Serum A(1)- and B-glycosyltransferase levels of A and B high expressers were significantly higher than those of group A(1) and B individuals with normal expression. H antigen levels were low on the red cells of high expressers, indicating that the anomaly affects other cell lineages. Immunochemical studies demonstrated high levels of A antigen on various glycoproteins (GPs) from high-expresser platelets, especially GPIIb and PECAM (CD31). The A(1) Type II high-expresser phenotype was inherited as an autosomal dominant trait in one family. The sequences of exons 5, 6, and 7 of the A(1)-transferase gene of one Type II A(1) high expresser and exon 7 from 3 other genes were identical to the reported normal sequences. Further studies are needed to define the molecular basis for the high-expresser trait and to characterize its clinical implications. (Blood. 2000;96:1574-1581)     

ABO compatibility and platelet transfusions of alloimmunized thrombocytopenic patients.

With data on 91 alloimmunized thrombocytopenic patients and 389 donor-recipient pairs matched or selectively mismatched for HLA antigens, it was observed that ABO incompatibility significantly reduced the effectiveness of platelet transfusions. The mean 24-hr recovery of platelets from histocompatible donors and from donors selectively mismatched for cross-reactive HLA antigens was decreased by approximately 23% if the donor typed for blood group A and/or B not found in the recipient. Thus, the reduction in platelet recovery associated with ABO incompatibility is not of a magnitude that would contraindicate transfusion of ABO-mismatched platelets.     

Plasma blood group glycosyltransferase activities after bone marrow transplantation

Human blood groups (ABO) are known to be determined by the terminal glycosyl residues attached to common carbohydrate chains of the red cell surface. N-acetylgalactosaminyltransferase (A-enzyme) in blood group A persons and galactosyltransferase (B-enzyme) in blood group B persons are responsible for producing A and B substances on the red cell surface, with both enzymes absent in blood group O persons. The plasma transferase (A - and B-) activity was assayed after the complete replacement of the bone marrow of patients with acute leukemia or aplastic anemia by transplantation bone marrow from donors with ABO blood group differing from the recipient. The patient's blood type completely changed from the recipient's type to the donor's type. However, the A- and B-enzyme activities of the patients changed only slightly after bone marrow transplantation. The results indicate that most of the A- and B-enzymes in the circulatory plasma is not derived from the bone marrow, lymphoid, or macrophage tissue. Other tissues must be the primary source of the enzymes in plasma.     

Normal human serum contains natural antibodies reactive with autologous ABO blood group antigens.

It is widely accepted that the serum of healthy individuals contains natural antibodies only against those blood group A or B antigens that are not expressed on the individual's red blood cells. The mechanisms involved in tolerance to autologous blood group antigens remain unclear. In the present study, we show that IgM and IgG antibodies reactive with autologous blood group antigens are present in the immunoglobulin fraction of normal human serum. Natural IgG anti-A antibodies purified by affinity chromatography from IgG of individuals of blood group A exhibited an affinity for A trisaccharide antigen in the micromolar range and agglutinated A red cells at sixfold higher concentrations than those required for agglutination with affinity-purified anti-A IgG of individuals of blood group B. Whereas autoantibodies reactive with self A and B antigens are readily detected in purified IgG and IgM fractions, their expression is restricted in whole serum as a result of complementary interactions between variable regions of antibodies. These observations suggest that tolerance to autologous ABO blood group antigens is dependent on peripheral control of antibody autoreactivity.     

HLA antigens on platelet membranes. In vitro and in vivo studies

In order to determine whether HLA-A,B antigens of platelets are integral membrane constituents or rather represent adsorbed plasma proteins, their presence in plasma and their adsorbability onto platelet membranes was studied by in vitro and in vivo experiments. The amount of HLA antigens was quantitated by inhibition of lymphocytotoxicity (LCT) and by enzyme-linked immunosorbent assay (ELISA) using operationally monospecific polyclonal HLA antibodies or murine HLA-specific monoclonal antibodies, respectively. We found that in 11 out of 13 HLA-A2 and in 9 out of 10 HLA-B13 experiments, platelets from antigen-negative donors pretreated with plasma from the same number of antigen-positive donors inhibited LCT to the same extent as platelets from antigen-positive donors. Nevertheless, the in vitro adsorbed HLA antigens onto antigen-negative platelets were, unlike those on antigen-positive platelets or in plasma, not reactive with monoclonal antibodies as quantitated by ELISA. Similarly, infusion of HLA-A2-negative platelets from single donors into 3 HLA-A2-positive, thrombocytopenic patients with bone marrow failure led to a good platelet increment, but did not convert the HLA type of donor platelets, neither at 2 h nor at 18 h posttransfusion. On the basis of these results, we conclude that soluble HLA antigens can be taken up by human platelets from plasma in small amounts. However, the major portion of HLA antigens appears to be integral membrane constituents.     

An association of ABO non-identical platelet and cryoprecipitate transfusions with altered red cell transfusion needs in surgical patients

Background Transfusion of ABO non‐identical plasma, platelets and cryoprecipitate is routine practice even though adverse effects can occur. Methods and Materials Our hospital changed transfusion practice in 2005 and adopted a policy of providing ABO‐identical blood components to all patients when feasible. We retrospectively compared the transfusion requirements, length of stay and in‐hospital mortality in relation to ABO blood group in surgical patients who received platelet transfusions before and after this change to determine whether it resulted in any benefit. Results Prior to the change in practice, both group B and AB patients received more ABO non‐identical platelet transfusion (P = 0·0004), required significantly greater numbers of red cell transfusions (P = 0·04) and had 50% longer hospital stays (P = 0·039) than group O and A patients. Following the policy change, there was a trend for fewer red cell transfusions (P = 0·17) and length of stay in group B and AB patients than group O or A patients. Overall, the mortality rate per red cell transfusion decreased from 15·2 per 1000 to 11·0 per 1000 (P = 0·013). Conclusions These results, in the context of previous findings, suggest that providing ABO‐identical platelets and cryoprecipitate might be associated with reduction in transfusion requirements and improve outcomes in surgical patients.     

Lack of ABH-antigen expression on human cardiac valves

BACKGROUND AND AIM OF THE STUDY: Seeding of heart valve prostheses with human umbilical vein endothelial cells (HUVEC) and human saphenous vein endothelial cells (HSVEC) has been applied to create a viable valve surface and improve valve performance. HUVEC and HSVEC are well characterized and have been used as a model of endothelial antigenicity, but antigenicity of the valve endothelium is less well characterized. To clarify this issue, we studied the expression of blood group antigens by human valvular endothelium, HSVEC and HUVEC. METHODS: Human aortic and mitral valves and myocardial tissue were freshly harvested from explanted hearts of patients undergoing heart transplantation (blood group A, n = 4; group O, n = 4) or valve replacement (blood group B, n = 4). After fixation in Carnoy's or formalin solution, paraffin sections were stained with anti-A (blood group A), anti-B (blood group B), and anti-H (blood group O) antibodies. Human umbilical cords were freshly harvested postpartum, and human saphenous veins were obtained from patients undergoing coronary bypass grafting (each blood group, n = 2) and similarly fixed and stained to detect ABO antigens. The preservation of endothelium was confirmed by staining with anti-CD 31 monoclonal antibody. All sections were examined by light microscopy. RESULTS: CD 31 staining demonstrated vascular and valve endothelial preservation. Human umbilical cords, saphenous vein and myocardium showed strongly expressed A, B and H blood group antigens on vascular endothelium. However, no A, B and H antigens were detected on the valvular endothelium. CONCLUSIONS: Valve endothelial cells appear to be a class of specialized endothelial cells that does not express the ABO antigens. Due to the strong expression of A, B and H antigens by HUVEC and HSVEC blood group cross-matching should be considered for non-autologous endothelialization of valve prostheses.     

ABO blood group system and bone marrow transplantation.

The role of the ABO blood group system in determining the outcome of bone marrow transplantation was investigated in 53 patients with aplastic anemia and acute leukemia grafted from HLA-identical siblings. There was no correlation between ABO compatibility and marrow engraftment, graft rejection, or graft-versus-host disease. In 5 recipients with antibodies prior to transplantation to antigens of the ABH system present on the cells of their donors, plasma exchange and antibody absorption in vivo were effective in permitting engraftment of ABO-incompatible bone marrow. These findings indicate that the ABO system is not a clinically significant barrier to successful bone marrow transplantation in otherwise histocompatible individuals.     

Response to Immunization with A and B Human Glycoproteins for the Procurement of Blood Grouping Reagents

37 subjects have been immunized with A or B human blood group substances. All subjects showed at least a threefold increase in potency of their ABO antibodies. Use of the resulting plasma has considerably improved the quality of our ABO grouping reagents. The IgG component of these antibodies increased in all subjects and an IgA component was seen in the majority of post-immunization samples. All group O subjects developed cross-reacting anti-A,B antibodies.     

Influence of the combined ABO, FUT2, and FUT3 polymorphism on susceptibility to Norwalk virus attachment

The binding of Norwalk virus (NV) recombinant capsids was tested in a panel of saliva samples collected from 96 donors with different ABO, secretor, and Lewis phenotypes. As previously reported, binding occurred specifically to saliva from secretors, regardless of their Lewis phenotype status. Blood group B saliva was poorly recognized, whereas binding to blood group O saliva was higher and binding to blood group A saliva was highest. Transfection of either blood group A or B enzyme into H epitope-expressing cells showed that masking of H epitopes by the A and B antigens blocked the attachment of NV capsids. The high level of binding to blood group A secretor saliva could be explained by an optimal H type 1 ligand density, which was lower than that in blood group O saliva and much higher than that in blood group B saliva. Indeed, despite a higher ligand density, saliva from homozygotes with 2 functional FUT2 alleles was less strongly recognized than saliva from heterozygotes with 1 functional and 1 inactivated FUT2 allele. Partial fucosidase treatment of duodenal tissue sections and binding to a synthetic probe with varying densities of H type 1 trisaccharide indicated that optimal attachment occurred at medium ligand density.     

Study of antigenic varieties of group A red cells, platelets and lymphocytes using liquid phase electrophoresis.

The authors attempt to study the presence of subgroups of the phenotype A on lymphocytes and platelets with the help of liquid phase electrophoresis. The results show that anti-A of B serum reduces the electrophoretic mobility of red cells, platelets and lymphocytes of group A but leaves unchanged the mobility of group O or B cells. The same variations are observed in red cell lymphocytes and platelets. The authors suppose that the distribution of antigenic sites is identical in these elements.     

Dissociation of ABH antigen expression from von Willebrand factor synthesis in endothelial cell lines

ABO blood group determines plasma von Willebrand factor (VWF) levels and ABH antigens are present on VWF. To investigate whether ABO influences the rate of VWF synthesis, we performed stable transfection of A transferase in a phenotypically group-O endothelial cell line (EAhy926). A transferase expression did not affect the rate of VWF synthesis. Although high levels of A antigen were expressed on the cell surface, no A determinants were added to VWF synthesized within these cells. Further studies demonstrated H structures were not present on EAhy926-derived VWF, despite the fact that H antigen is constitutively expressed by these cells.     

Detection of Immune Anti-A and Anti-B Allo-Antibodies with Groupamatic® System

An inhibition technique using soluble AB group substances for screening of 'immune' anti-A and anti-B allo-antibodies has been set up on Groupamatic. This screening is performed on all blood units of group O, A and B to be transfused. The ratio of potentially dangerous donors in case of non-isogroup transfusion is 2.86% of the total number of screened donors. This detection is performed simultaneously with other immunohaematological and serological tests: ABO and Rh grouping, screening of irregular allo-antibodies, screening of syphilis.