ABH antigens were found in human embryonic kidney cells by mixed agglutination technique in the late 1950s (1) and subsequently in the collecting tubules and calyces of adult human kidneys by immunofluorescence (2). Later, ABH antigens in human kidneys were identified in the vascular endothelium, the convoluted distal tubules and the collecting tubules while no expression was found in the glomerular mesangium and epithelium (reviewed in 3). Early immunohistochemical studies indicated that the expression of ABH antigens in human tissues differed in relation to ABO blood groups and secretor status (2, 3). Glycolipid-based blood group ABH and related carbohydrate antigens in the human kidney were shown to vary depending on the blood group A subtype (A1/A2) and Lewis status of the individuals (4) However, all these studies are limited to a low number of human individuals analyzed and, in many cases, an incomplete blood group typing of the tissue donors.
Renal transplantation across the ABO barriers was found, in the early days of transplantation, to result in hyperacute rejection in the majority of cases and it was therefore regarded as an absolute prerequisite to comply with the traditional ABO rules in allotransplantation. The first study in which the ABO barriers were crossed deliberately was started in the early 1970s when blood group A2 cadaveric donor (CD) kidneys were transplanted to O recipients (5, 6). In the 1980s, Alexandre performed the first series of ABO-incompatible live donor (LD) renal transplantation using A1 and B donors with an outcome similar to the ABO compatible cases (7). Thereafter, more than 400 cases of ABO incompatible LD renal transplantations have been reported worldwide (8).
The aim of this study was to demonstrate the expression of blood group A and B antigens in a large number of kidney biopsies obtained at transplantation and to correlate the antigen expression to the extended blood group ABO, Lewis and secretor typing of the donors. In addition the ABH antigen expressions in kidney biopsies available from the early A2 to O trial (5, 6) were correlated to graft outcome. Based on these results, a definition of “minor” and “major” blood group ABO-incompatible donors is proposed.
Kidney Specimens and Blood Samples
Wedge biopsies were taken from LD allografts at the end of the transplantation procedure. The biopsies were immediately fixed by immersion in 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 4–6 hr at room temperature. Biopsies were processed according to our routine methods for light microscopical (LM) and immunoperoxidase histochemical (IP) evaluation of allograft biopsies. Venous blood, for serological and genomic blood group typing, was collected from the kidney donors prior to transplantation. The study was approved by the Göteborg University Ethical Committee. Due to the low frequency of blood group B and AB individuals, biopsies from cadaveric donor cases where only standard ABO typing (no Lewis or secretor typing) was performed were also included. In addition, renal biopsies from an A2 to O CD renal transplantation trial performed 1974 to 1988 (5, 6) were investigated in those cases where paraffin blocks were available.
Blood Group ABO, Lewis, and Secretor Typing of Organ Donors
Monoclonal anti-A and anti-B reagents were used for serological typing of erythrocytes. Blood group A individuals were subtyped for A1 or A2 (non-A1) by the use of the Dolichos biflorus lectin (Gamma Biologicals Inc., Houston, TX). The Lewis status of the donor was established on erythrocytes using monoclonal anti-Lea and Leb reagents (Gamma Biologicals Inc., Houston, TX).
A1/A2/B, Lewis, and secretor status of living and cadaveric donors were determined using the SSP-PCR method as described (9).
In those cases where secretor status could not be established by genomic typing, the secretor status was deduced from the Lewis blood group type. Le(a-b+) individuals are defined as secretors and Le(a+b−) individuals as nonsecretors (10).
The formaldehyde-fixed renal biopsies were dehydrated in ethanol-xylene and embedded in paraffin (reagent grade, melting point 65°C) with a total processing time of 2.5 h in a programmable automatic tissue processor (TissueTek VIP, Miles Scientific). Paraffin blocks were stored at RT until further processing. Paraffin sections, 4 μm in thickness, were collected on polylysine-coated glass slides (DAKO, Denmark).
Immunostaining was performed on deparaffinized sections as described (11, 12) with modifications as follows. Antigen retrieval for histo-blood group antigens by protease digestion or microwave heating gave very similar results (unpublished data). Because the latter method is easier to standardize in the routine, it was selected for the present study. Briefly, the slides were immersed in 0.1 M Tris-EDTA buffer, pH 9.0, in a 150-ml glass jar, and heated at 600 W, 2×10 min, in a standard microwave oven. After blocking of endogenous peroxidase, the slides were incubated with primary antibodies, respectively anti-A (DAKO A0581) and anti-B (DAKO A0582) diluted 1:20 in PBS, pH 7.8, or the ChemMate antibody diluents (DAKO see below), for 30 min at RT. Optimum antibody dilution was predetermined by means of testing 10-fold serial dilutions on positive renal biopsy tissue from serologically typed A1, A2, and B individuals, choosing the level where the diaminobenzidine (DAB) reaction was distinct at maximum intensity with minimum nonspecific background. Two different detection systems were applied. The first was a two-step peroxidase-labeled streptavidin-biotin method (LSAB) as described previously (11), using the LSAB-kit (DAKO, K675) as a second step, in a programmable immunostainer (Cadenza, Shandon, UK). The second technique, applied recently, was based on the DAKO ChemMate EnVision Detection Kit peroxidase/DAB (DAKO, K5007), a biotin-free two-step indirect method with secondary antibodies (goat-antirabbit and mouse immunoglobulins) and horseradish peroxidase (HRP) conjugated to a dextran back bone (13). For this procedure, we used a Techmate500 computer-assisted immunostainer (DAKO). The peroxidase label was visualized with 0.1 M diaminobenzidine-HCl as a chromogen. As part of our routine assessment of allograft pathology, all base-line biopsies are stained by IP as described (12) for detection of immunoglobulin (Ig) G, IgA, IgM, C3c, C1q, and Fibrin, and also in selected cases CD31 a marker for endothelial cells (DAKO M0823).
The EnVision technique has a superior sensitivity and less unspecific background compared to previous methods. At the same dilutions, the anti-B antibody gave a stronger signal with EnVision compared to LSAB, while the result for the anti-A antibody showed very little difference. Mayer’s hematoxylin (diluted 1:2) was used for a slight nuclear stain. Positive and negative controls were from different serologically typed ABO individuals, and by omitting or replacing the primary antibody, accordingly. Each immunostained specimen included a section of positive tissue control present on each glass slide. Control tissues were from serologically typed A1 and B individuals for the A0581 and A0582 antibodies, respectively. The immunostained specimens were evaluated and classified by two of the authors (J.M., C.T.S.) without knowing the results of the extended blood group typing of the donors.
The A0581 antibody recognizes all structural different blood group A antigens irrespectively of the core saccharide chain while the A0582 antibody recognizes all mono-fucosylated blood group B antigens (4).
Blood Group A2 to O CD Renal Transplantation Trials 1974–1988
In Göteborg, this trial encompassed 21 blood group O recipients grafted with kidneys from A2 cadaveric donors. Pretransplant removal of anti-A/B antibodies and splenectomy were not performed. Immunosuppressive drugs used were prednisone/azathioprine in the first period followed by cyclosporin/prednisolone/azathioprine. The long-term follow up (6, 8) has been reported.
At the transplant unit in the University Hospital, Basel, eight blood group O patients were grafted with A2 cadaveric kidneys using a similar protocol without antibody removal and splenectomy.
All baseline biopsies in the present LD series showed renal cortical tissue with age-related changes but were otherwise normal and well preserved. Particularly, the intima including endothelium was intact at all levels of the vascular tree available in the superficial renal cortex. Staining for CD31 antigen gave a positive result marking all areas of endothelium in all baseline biopsies so far studied, i.e. confirming the LM observation (Fig. 1D). Immunostaining of renal biopsies from blood group A donors with the anti-A antibody showed three distinct different patterns with a major, minor, and minimal staining distribution, and intensity. These are hereafter designated as type 3+, type 1+, and type (+) respectively and these reflect a combination of the A antigen distribution and semi-quantitative expression level (Figure 1, A–C). The intensity and distribution of A antigen in renal cortex, according to the type of expression, correlated to the extended blood group typing of donors as shown in Table 1. In the type 3+, positive staining was seen in the entire vascular bed where the endothelium was strongly stained, focal strong staining of the epithelium in distal tubules but negative in proximal tubules and in the glomerular epithelium. In the type 1+, the endothelial staining was considerably weaker, which was most obvious in the glomerular capillaries. The distal tubules were strongly stained focally, whereas no positive reaction was found in the proximal tubules. In the type (+), the vascular tree was negative except for a weak positive reaction in the endothelium of single peritubular capillaries and in the distal tubules a focal positive reaction was present. The relative staining intensity and the distribution of the A antigen was similar with the two different staining methods (LSAB and EnVision) applied, in contrast to the result of B antigen staining (see below). The control tissues from blood group O individuals gave completely negative staining with the anti-A antibody. The wedge biopsies studied were only two to three mm in depth; therefore, neither collecting ducts nor urothelium were present in the specimens.
Correlating the A antigen expression and the extended blood group typing (Table 1) reveals that A1 individuals (14 secretor cases, two nonsecretor cases, and one A1B) all belong to the type 3+. All blood group A2 cases studied showed either type 1+ (n=8) or type (+) (n=9) A antigen expression and the two A2B cases were of type (+).
Staining of renal biopsies from blood group B donors with the anti-B antibody A0582 using the LSAB technique showed a low antigen expression and a pattern comparable to type (+) for A antigens using the same technique and anti-A antibodies. However, with the EnVision technique, the B antigen staining intensity and distribution gave a pattern more like the A type 3+, with the difference that the glomerular capillaries showed a weaker B antigen expression compared to A antigens in A1 donor tissue. Therefore, the B antigen expression in B secretor individuals studied was designated as type 2+. Table 2 shows the results of anti-B antibody A0582 staining with the EnVision detection system and typical staining patterns are illustrated in Figure 1. In two cases where the Lewis and the secretor status were not known, a weaker B antigen expression, in comparison with the B secretor individuals, was seen (Table 2). To date, we have not investigated any tissue from blood group B nonsecretor individuals due to their low frequency in the population (2.0%). The single A1B individual tested had very low B antigen expression, similar to type (+). In the two A2B individuals, the results for B antigen expression were divergent with one case having a stronger B antigen expression compared to the other case. The results shows that the expression of B antigens varies between B individuals but if this is correlated to the secretor status can not be established due to the limited number of cases studied.
In addition, the anti-B antibody A0582, gave a weak but significant staining in distal tubular epithelium (Fig. 1G) and focally in the media of interlobular arteries (Fig. 1H) from all blood group (A, B, and O) individuals, whereas the endothelium was negative in A and O cases. This staining may be due to cross-reactivity with Galα1-4Gal terminating compounds of the globo-series glycolipids which are present in tissues from individuals of all ABO groups (14).
The blood group A antigen expression in graft biopsies available from the 1974-88 A2 to O CD trial performed in Göteborg are listed in Table 3 together with some selected clinical data. Paraffin blocks available were from wedge biopsies obtained postreperfusion at transplantation, needle biopsies taken for suspected rejection and from explanted kidneys. The A antigen patterns were either of type 1+ or type (+). As for to the cases presented in Table 1, the 1+ and (+) pattern were found in both secretor and nonsecretor individuals with a similar frequency distribution. No type 3+ expression was found in these A2 kidneys. Hyperacute rejection did not occur in the 21 grafts transplanted and 13 cases had graft function for variable periods of time. Of the five cases that expressed type 1+ A antigens in their grafts, four did not gain any function. One graft of type 1+ functioned well at four months when the recipient died of a cytomegalovirus infection (Table 3). Five out of six grafts expressing A antigen type (+) had long-term function. One type (+) graft did not function, (Table 3, case 17) due to a long cold ischemia time and the biopsy taken at transplantation showed cortical necrosis.
Of the eight patients transplanted in Basel, four recipients had long-term function. Two patients died after 6 and 10 years with functioning grafts (same donor) and the other two grafts functioned for six and three years where after the patients returned to dialysis. Four grafts (from two donors) experienced hyperacute rejection. The biopsy tissues available were collected at rejection or from explanted grafts and all showed various degree of damage. Anti-A staining revealed a mixture of type 1+ and (+) antigen pattern and some tissue sections were completely negative. No type 3+ staining was found. No clear correlation with clinical outcome was found.
In this study, it was found that A antigens in the human renal vascular bed can be classified in three different groups with a major, minor, and minimal antigen expression pattern. The group, with major antigen expression designated type 3+, showed an intense generalized staining of the endothelium and a focal positive reaction in the distal tubules, whereas the glomerular epithelium and proximal tubules were negative. The other two groups, type 1+ and type (+), respectively, had considerably less amounts of A antigens (Table 1). When the A antigen expression was related to the blood group subtypes of the tissue donors, all type 3+ renal tissue specimens matched blood group A1 individuals, whereas the A2 donor tissues were divided equally between the type 1+ and type (+) groups, respectively. The secretor gene did not appear to influence the A antigen expression in the renal vascular bed. This may be due to the fact that vascular endothelium has been postulated to have mainly blood group ABH antigens based on the type 2 core carbohydrate chain (5) in contrast to glandular tissue where the type 1 core chain is dominating (15). The secretor gene coded fucosyltransferase mainly utilize the type 1 core chain precursor structures. Expression of A antigen is secretor gene dependent in collecting duct epithelium and human bladder urothelium of A1 individuals (3, 16). In the kidney cortex wedge biopsies here studied, collecting ducts and urothelium is lacking and no conclusion regarding the secretor gene expression in these tissue compartments can be made. This fact may explain the discrepancy in secretor gene influence on A antigen expression between the present and previous study (4) in which total kidney tissue (including collecting ducts and renal pelvic urothelium) were studied by immunostaining of glycolipid antigens with a tendency of more extensive expression in secretor positive individuals. Furthermore, immunohistochemistry of tissue sections stain both glycolipid and glycoprotein bound carbohydrate antigens compared to our previous study (4) where the analysis was restricted to biochemical purified glycolipid antigens.
The expression of B antigens in renal tissue from blood group B secretor donors showed a distribution similar to the A antigen type 3+ group but overall the intensity of the staining was less. In contrast to staining for A antigens, the B antigen staining was influenced by the immunohistochemical technique used. Studies of the glycolipid structures of human kidneys have revealed that B kidneys contained less B glycolipid antigens compared to A glycolipid antigens in A kidney and that B glycolipids based on the type 4 core saccharide were absent while the A type 4 core structure was the major A antigen (14).
Population data on blood group ABO (17) and the secretor distribution (18) in the Swedish population shows that A1Se individuals is the major group (29.5%), whereas A2Bse individuals are very rare (0.2%). Due to the low frequency, some blood group phenotypes are not represented in this material, even though the biopsies were collected over several years.
From the early ABO-incompatible trial using A2 CD kidneys into O recipients (5, 6), renal biopsy tissue from these A2 individuals all showed either a type 1+ or type (+) A antigen expression (Table 3). In this series, recipient anti-A antibodies were not reduced prior to grafting and splenectomy was not performed. This may contribute to the fact that the antigen expression in these A2 donors correlated to graft outcome in this series, which is not seen nowadays when antibody removal, splenectomy, and modern immunosuppressive drugs are used (8, 19–22). Theoretically, the A antigen expression could have been modified by anti-A antibody binding in these cases. However, in our recent series of ABO-incompatible renal transplants, we have analyzed several biopsies collected at various times postgrafting (23). In these cases, the A/B antigen pattern has been similar to that of the 30-minute biopsy.
Blood group B kidneys have been successfully transplanted to O recipients without splenectomy (24), which was postulated to be due to a low expression of B antigens in the renal grafts. However, severe antibody-mediated rejections have also been reported (25). Our initial studies using the LSAB technique revealed low amounts of B antigens in the kidneys from B donors. However, when the biopsies were reinvestigated using the more sensitive EnVision technique, a stronger B antigen expression was found. The EnVision DAB technique is two to four times more sensitive (13) and gives a stronger signal due to the heavy HRP labeling, which probably gives a higher molecular ratio of DAB to primary antibodies in the antigen target area and, due to the absence of avidin-biotin binding, a lower unspecific background.
In AB individuals, blood group A and B glycosyltransferases compete with each other for the precursor blood group H antigen compounds resulting in about half amounts of A and B antigens compared to that of corresponding A and B individuals. This is partly supported by the few AB cases analyzed in this work. Whether A1/2B donor to an A recipient and A1B to a B recipient should be regarded as “minor” or “major” incompatibilities cannot be defined at present.
Due to the disparity in the results of ABO incompatible transplantation using B donors (24, 25) and the amount of B antigens here detected in renal tissue by the new more sensitive immunostaining technique, all blood group B donors when crossing the ABO barriers should be regarded a major incompatibility (similar as for A1 donors) when selecting immunosuppressive protocol.
At present, pretransplant antibody removal and standard immunosuppressive drug induction as the only additional pretreatment is postulated to be insufficient when using A1 and B donors. In addition, splenectomy (7, 26) and/or use of anti-CD20 antibody (19, 20, 23, 27) administration seems to be needed. For A2 donors, splenectomy does not seem to be necessary (21, 22) even if reports of acute antibody-mediated rejection of A2 renal grafts in O recipients have been reported (22, 28, 29). The use of anti-CD20 antibody in recipients of A2 grafts should be considered in relation to possible long-term side effects. These are so far relatively unknown. Antibody rebound and subsequent graft loss has been reported to occur in blood group ABO-incompatible renal transplantation despite splenectomy (26, 30). Whether the use of anti-CD20 antibodies instead of splenectomy will increase the long-term graft survival in ABO-incompatible renal transplantation remains to be shown.
M. Mihatsch of the Basel Institute of Pathology, Schweiz, is acknowledged for help with tissue sections from the Basel trial.
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