Lennart Rydberg

ABO‐incompatibility in solid organ transplantation

. The most important transplantation antigen system in solid organ transplantation is the ABO histo‐blood group system. Crossing the ABO barrier in solid organ transplantation is usually not done except for emergency liver transplantations. Early experiences of crossing the ABO barrier in renal transplantation were very disappointing. In the 1970s, clinical trials were started transplanting kidneys of subgroup A2 into blood group O recipients. The tissues of the A2 subgroup expresses reduced amount of A antigens compared to subgroup A1 and the recipients had no special pretreatment and standard immunosuppression. A number of early graft losses were experienced but the trial also resulted in several long time surviving grafts. A few centres have adapted the concept of A2 to non A kidney transplantations with successful results, when the recipient anti‐A titres are low or reduced prior to transplantation.

ABO‐incompatible live donor renal transplantation using blood group A/B carbohydrate antigen immunoadsorption and anti‐CD20 antibody treatment

Abstract: Background: Blood group ABO‐incompatible live donor (LD) renal transplantation may provide a significant source of organs. We report the results of our first 14 cases of ABO‐incompatible LD renal transplantation using specific anti‐A/B antibody (Ab) immunoadsorption (IA) and anti‐CD20 monoclonal Ab (mAb) treatment.

Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system

In analogy with histo-blood group A antigen, Forssman (Fs) antigen terminates with α3-N-acetylgalactosamine and can be used by pathogens as a host receptor in many mammals. However, primates including humans lack Fs synthase activity and have naturally occurring Fs antibodies in plasma. We investigated individuals with the enigmatic ABO subgroup A(pae) and found them to be homozygous for common O alleles. Their erythrocytes had no A antigens but instead expressed Fs glycolipids. The unexpected Fs antigen was confirmed in structural, serologic, and flow-cytometric studies. The Fs synthase gene, GBGT1, in A(pae) individuals encoded an arginine to glutamine change at residue 296. Gln296 is present in lower mammals, whereas Arg296 was found in 6 other primates, > 250 blood donors and A(pae) family relatives without the A(pae) phenotype. Transfection experiments and molecular modeling showed that Agr296Gln reactivates the human Fs synthase. Uropathogenic E coli containing prsG-adhesin-encoding plasmids agglutinated A(pae) but not group O cells, suggesting biologic implications. Predictive tests for intravascular hemolysis with crossmatch-incompatible sera indicated complement-mediated destruction of Fs-positive erythrocytes. Taken together, we provide the first conclusive description of Fs expression in normal human hematopoietic tissue and the basis of a new histo-blood group system in man, FORS.

Studies on the removal of anti‐pig xenoantibodies in the human by plasmapheresis/immunoadsorption

Abstract: Removal of human preformed natural anti‐pig antibodies from the blood is a prerequisite before xenografting between pig and man can be performed. This work explores the effect of plasmapheresis and immunoadsorption (protein‐A sepharose) on the reduction and recurrence of anti‐pig antibodies in 14 patients. The anti‐pig antibody changes were evaluated by lymphocy to toxic, hemagglutinating, and endothelial cell ELISA techniques. The changes induced showed a similar pattern with all three techniques used. In addition, plasma from plasmapheresis treatments were perfused through pig kidneys and the reduction of anti‐pig antibodies was estimated by the mentioned in vitro techniques. The anti‐pig antibody titers could be reduced to low levels, but not completely eliminated, by 3–4 plasmapheresis sessions. The titers gradually returned to pretreatment levels or higher in a period of 1–2 weeks. A few patients showed signs of a more rapid resynthesis reaching pretransplant levels in 3–4 days. Protein A immunoadsorption satisfactory removed IgG but not IgM antibodies. In vitro perfusion of pig kidneys at 37°C showed a rapid reduction of anti‐pig antibody titers of 3–4 titer steps. The combination of 3–4 plasma exchanges followed by in vitro pig kidney perfusion completely removed all anti‐pig antibodies. Reduction of the anti‐pig lymphocyte and erythrocyte antibody titers by soluble oligosaccharides carrying terminal Galoc‐epitopes was only partly successful. A 40–60% inhibition was achieved by 5–10 mg saccharide/ml serum and no clear inhibition difference between di‐ and trisaccharides was found. Inhibition of plasma obtained after 3–4 plasmapheresis treatments with soluble Galα1‐di‐ and trisaccharides resulted in very low anti‐pig titers. Therefore one feasible pretreatment procedure, before pig to human xenotransplantation could be plasmapheresis for major reduction of anti‐pig antibody titer followed by neutralisation of the remaining antibodies by addition of soluble oligosaccharides or immunoadsorption with Galα‐1‐columns.

Lack of antibody production against Hanganutziu-Deicher (H-D) antigens with N-glycolylneuraminic acid in patients with porcine exposure history.

Abstract: The significance of non‐αgalactosyl antigens remains unclear in pig‐to‐primate xenotransplantation. Hanganutziu‐Deicher (H‐D) antigens with terminal N‐glycolylneuraminic acid (NeuGc) are widely expressed on endothelial cells of mammalian species, with the exception of humans. As baboons and monkeys also express H‐D antigens, a pig‐to‐non‐human primate experimental model cannot resolve the question of whether H‐D antigens can elicit a potent humoral response in human recipients. The purpose of this study was to elucidate the clinical significance of H‐D antigens by examining the sera from patients who have been previously exposed to porcine tissue.

Typing for the Human Lewis Blood Group System by Quantitative Fluorescence–Activated Flow Cytometry: Large Differences in Antigen Presentation on Erythrocytes between A1, A2, B, O Phenotypes

Background: Lewis phenotyping by hemagglutination is an unreliable routine method for Lewis antigen designation. Now genomic typing of the Lewis gene is available. Additionally, flow cytometry has been used for typing. We wanted to compare the results of Lewis typing in healthy individuals using the three methods. Materials and Methods: Ninety–three randomly selected plasma donors were genotyped for inactivating Secretor (FUT2) G428A and Lewis (FUT3) T59G, T202C, C314T, G508A and T1067A point mutations. All Le(a+b–) individuals (nonsecretors) were homozygous for the FUT2 G428A mutation and all Le(a–b–) individuals had inactivating mutations on both FUT3 alleles. Fixed erythrocytes were analyzed by fluorescence–activated flow cytometry and the results were compared with hem– agglutination and genotypic data. Antigen availability was expressed as median fluorescence intensity and as percentage positive cells with fluorescence intensities ≥102. Results: Using an anti–Lea reagent a mean of 99% of erythrocytes from Le(a+b–) individuals and 1% of erythrocytes from Le(a–b–) or Le(a–b+) individuals were stained positive. Using an anti–Leb reagent, a mean of 71% of erythrocytes from A1, 95% from B and 99% from O and A2 Le(a–b+) individuals and less than 10% of erythrocytes from Le(a–b–) or Le(a+b–) individuals were stained positive. After papain treatment 100% of the erythrocytes from A1 and A1B Le(a–b+) individuals stained positive without increase in background staining. The flow cytometric technique revealed large differences in staining intensities, within each ABO Le(a–b+) subgroup which was not directly correlated to plasma donation frequencies nor to Secretor or Lewis genotypes. Conclusion: Flow cytometry may prove valuable as a Lewis blood group typing technique but also as a research tool when investigating Lewis phenotypes of human erythrocytes.

Blood group A1 and A2 revisited: an immunochemical analysis

Background and Objectiveu2002 The basis of blood group A1 and A2 phenotypes has been debated for many decades, and still the chemical basis is unresolved. The literature generally identifies the glycolipid chemical differences between blood group A1 and A2 phenotypes as being poor or no expression of A type 3 and A type 4 structures on A2 red cells, although this assertion is not unanimous.

Evidence for an expression of blood group A antigen on platelet glycoproteins IV and V.

Summary. Blood group ABO antigens are known to be carried by several platelet glycoproteins (GP), e. g. GPIb, GPIIa, GPIIb, GPIIIa and PECAM. Beside these proteins, we recently observed that blood group A antigen was also expressed on some other uncharacterized platelet proteins (70–90 kDa) having electrophoretic mobilities closely resembling those of GPIV and GPV. These findings prompted us further to characterize these latter ABO‐expressing platelet proteins. By antigen capture ELISA, wherein the monoclonal antibodies (mAbs) CLB‐IVC7 and CLB‐SW16 were used to hold the corresponding antigens GPIV and GPV, human anti‐A specifically bound to these proteins derived from A1‐platelets; neither GPIV nor GPV derived from A2‐, B‐ or O‐platelets bound anti‐A. In a Western blot assay using immunoprecipitated GPIV and GPV as antigens, mAb anti‐A immunostained GPIV and GPV precipitated from A1, but not from A2 and O platelets. These results conclusively demonstrate that blood group A antigen is expressed on platelet GPIV and GPV.

Novel glycolipid variations revealed by monoclonal antibody immunochemical analysis of weak ABO subgroups of A

Background and Objectivesu2002 The chemical basis of the subgroups of A is largely unknown. We used thin‐layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups.

Complement activation in prestorage leucocyte-filtered plasma.

Summary.u2002 Complement activation and generation of pro‐inflammatory cytokines occur during storage of blood components. Prestorage leucocyte filtration of platelet concentrates and red cells diminishes the accumulation of leucocyte‐derived cytokines during storage, however, transfusion reactions are not eliminated. We investigated inflammatory mediator release during storage of plasma and whole blood and the effect of prestorage leucocyte filtration of plasma. Twenty‐four blood units were collected from healthy blood donors and stored for 35u2003days. Eight units were stored as whole blood, eight units as plasma and eight units as prestorage filtered plasma. Samples were collected weekly for analyses of potassium, leucocytes, free plasma haemoglobin, complement activation (C3a and SC5b‐9) and pro‐inflammatory cytokines [interleukin (IL)‐6, IL‐8 and tumor necrosis factor (TNF)‐α]. Elevated levels of C3a and SC5b‐9 were registered in filtered plasma, from the beginning of storage. C3a levels increased during storage. There was a higher rate of change during storage in C3a (Pu2003<u20030·01) and SC5b‐9 (Pu2003<u20030·05) in plasma compared with filtered plasma. Interleukin (IL)‐8 is released in whole blood. The cytokine levels generated in plasma and filtered plasma were low. Complement activation is present in whole blood, plasma and filtered plasma during storage. Prestorage filtration of plasma activates the complement cascade but does not influence cytokine generation.