Glomerular C3c deposition and intravascular macrophage accumulation in early humoral renal allograft rejection signifies a poor short-term outcome.

C4d deposition in the walls of peritubular capillaries is considered the key phenomenon in the histopathological diagnosis of humoral, i.e. antibody-mediated, allograft rejection. We have earlier proposed that deposition of C3c in glomerular capillaries and simultaneous intravascular accumulation of macrophages in allografts with immediate or early humoral rejection indicates a potentially serious condition with very poor prognosis. The clinical outcome of 45 cadaveric grafts with this phenomenon among 1960 renal allografts transplanted at our centre during 1984-1999, and the recipients of the contralateral kidneys, was retrospectively evaluated. Graft failure occurred in 44/45 grafts within 3 weeks, with graft loss in 33/45 (77%) within 4 months and 37/45 (82%) within 1 year. From the contralateral kidneys, 5/33 (15%) were lost within 1 year. In a recent series of early biopsies, we recognised that of 13 cases showing C4d positivity in peritubular capillaries but lacking C3c in glomeruli, 10/13 (77%) were still functioning after 4 months. The mean number of CD68(+) macrophages per glomerular profile, i.e. the glomerular macrophage index, shows a significant difference between C4d(+)C3c(-) and C4d(+)C3c(+) cases (p<0.001). Our results indicate the existence of a clinically important subgroup of early humoral rejection with particular morphological features.

Blood group A and B antigen expression in human kidneys correlated to A1/A2/B, Lewis, and secretor status.

BACKGROUND: In the revived interest in crossing ABO barriers in organ transplantation renal A/B antigen expression has been correlated with donor ABO, Lewis, and secretor subtype to predict antigen expression. METHODS: A/B antigen expression was explored by immunohistochemistry in LD renal biopsies. Donor A1/A2/B, Lewis, and secretor status were determined by serology and polymerase chain reaction. RESULTS: In the renal vascular bed, three distinct A antigen expression patterns with a major, minor, and minimal staining distribution, and intensity (designated as types 3+, 1+ and (+) respectively) were identified. Type 3+ had a strong A antigen expression in the endothelium of arteries, glomerular/peritubular capillaries and veins. The type 1+ showed an overall weaker antigen expression, whereas type (+) had faint staining of peritubular capillaries only. In all cases, distal tubular epithelium was focally stained, whereas proximal tubules were negative. Type 3+ were all from blood group A1 subtype individuals while A2 cases expressed either a 1+ or (+) pattern. The secretor gene did not appear to influence renal A antigen expression. All B kidneys examined showed a B antigen pattern slightly weaker but otherwise similar to A type 3+. CONCLUSION: Renal vascular A antigen expression correlates to donor A1/A2 subtypes, whereas B individuals show one singular antigen pattern. From antigen perspective, A1 and B donors are a "major" and A2 individuals a "minor" antigen challenge in ABO-incompatible renal transplantation.

Adult ABO-incompatible liver transplantation, using A and B donors.

BACKGROUND: The longer waiting time for a liver graft in patients with blood group O makes it necessary to expand the donor pool for these patients. This applies in both urgent situations and for elective patients. We report on our experience with ABO-incompatible liver transplantation using A2 and B non-secretor donors here. PATIENTS AND METHODS: Between 1996 and 2005, 12 adult blood group O recipients (seven male/five female) received ABO-incompatible cadaveric liver grafts (10 A2 donors, two B non-secretor donors). The indications were either rapid deterioration of liver function or hepatocellular cancer, in blood group O recipients, where an ABO-identical/compatible graft was not available. Mean recipient age was 54+/-8 (mean+/-SD) yr. All pre-operative CDC crossmatches were negative. The initial immunosuppression was induction therapy with antithymocyte globulin (n = 3), interleukin 2 receptor antagonists (n = 3) or anti-CD20 antibody (rituximab) (n = 1), followed by a tacrolimus-based protocol. Three patients underwent plasmapheresis post-transplantation. Baseline biopsies were taken before or immediately after reperfusion of the graft and after grafting when clinically indicated. No pre-operative plasmapheresis, immunoadsorption or splenectomies were performed. RESULTS: Patient and graft survival was 10/12 (83%) and 8/12 (67%), respectively, with a 6.5-month median follow-up (range 10 days to 109 months). Two patients (B non-secretor grafts) died of multiorgan failure probably because of a poor condition before transplantation. Three patients were retransplanted. Causes of graft loss were bacterial arteritis (n = 1), death with a functioning graft (n = 1) and portal vein thrombosis (n = 2). In one of the patients with portal vein thrombosis, an anti-A titer increase occurred concomitantly, and ABO incompatibility as the cause of the thrombosis cannot be excluded. Seven acute rejections occurred in five patients and all were reversed by steroids or increased tacrolimus dosage. The pre-transplant anti-A titers tested against A1 red blood cells were 1 to 128 (NaCl technique) and 4 to 1024 (indirect antiglobulin technique, IAT); the maximum postoperative titers were 16 to 2048 (NaCl) and 256 to 32,000 (IAT). CONCLUSION: The favorable outcome of A2 to O grafting, with a patient survival of 10/10 and a graft survival of 8/10, makes it possible to also consider this blood group combination in non-urgent situations. The use of non-secretor donor grafts is interesting but has to be further documented. There was no hyperacute rejection or increased rate of rejection. Anti-A/B titer changes seem not to play a significant role in the monitoring of ABO-incompatible liver transplantation.

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

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. PATIENTS AND TREATMENT PROTOCOL: Recipients were blood group O (n = 12), A (n = 1) and B (n = 1). Donors were A1 (n = 2), A2 (n = 3), A2B (n = 1) and B (n = 8), and all were secretor positive. Anti-human leukocyte antigen (HLA) Ab panel reactivity was negative in all recipients except one. All recipients were pre-treated with 3 to 6 IA sessions, using A or B carbohydrate antigen columns, until their anti-A1/B RBC panel indirect antiglobulin test (IAT) titers were < or =8. CDC crossmatch was negative in all cases. Recipients received preoperative mycophenolic acid, and steroids/tacrolimus were started at transplantation. No splenectomy was performed. Eight recipients received one dose of anti-CD20 mAb (rituximab, 375 mg/m2) pre-operatively and 11 recipients had postoperative protocol IA. RESULTS: In the initial protocol, anti-CD20 mAbs were used only for recipients receiving A1 grafts. One B graft (HLA-identical donor, 84% panel reactivity) was lost in a severe anti-B Ab-mediated acute rejection. Subsequently, the protocol included anti-CD20 for recipients of both A1 and B grafts and postoperative protocol IA to all recipients. The subsequent 10 grafts had excellent function, giving a total graft survival of 13/14 (observation range 2 to 41 months). At 1 yr, mean serum creatinine was 113 micromol/l (n = 8) and mean glomerular filtration rate was 55 ml/min/1.73 m2 (range 24 to 77). In the remaining five cases, with less than 1 yr follow up, mean serum creatinine was 145 micromol/l at 2 to 9 months follow up. Pre-IA anti-A/B titers were in the range of 2 to 32 (NaCl technique) and 16 to 512 (IAT). More than 90 IA sessions were performed in 14 recipients without any significant side effects. Recipient anti-A/B titers returned after transplantation to pre-IA levels or slightly lower. Postoperative renal biopsies were performed in 10 patients. In the 13 patients with long-term function, one patient experienced cellular rejection (Banff IIB) at 3 months without anti-B titer rise. This rejection was concomitant with low tacrolimus plasma levels and was easily reversed by steroids. In 8 of 10 cases, C4d staining was positive in peritubular capillaries. CONCLUSION: Blood group ABO-incompatible LD renal transplantation using A and B carbohydrate-specific IA and anti-CD20 mAbs has excellent graft survival and function.

Immunoperoxidase versus immunofluorescence in the assessment of human renal biopsies.

BACKGROUND: For half a century, immunofluorescence (IF) on frozen sections has been the gold standard for immunohistochemical evaluation of renal biopsy specimens. In routine diagnostic immunohistopathologic evaluation, traditional IF has been replaced to a large extent by immunoperoxidase (IP) methods applied to paraffin sections of formaldehyde-fixed tissue. This is caused in part by the practical disadvantages inherent in the IF method, eg, separate tissue specimen and handling, UV microscopy, fading and impermanence of the label-making archiving, and difficult later investigation. Our aim for the present study is to evaluate IP as an alternative to IF in the diagnostic assessment of renal biopsy specimens. METHODS: Proteolytic antigen retrieval, antibodies effective on deparaffinized sections, a sensitive detection system (Dako EnVision HRP; Dako, Copenhagen, Denmark), and a standardized and rigorously controlled procedure were applied to a series of renal biopsy specimens (n = 81) previously classified by means of light microscopy (LM) and IF. Staining for immunoglobulin G (IgG), IgA, IgM, C1q, and C3c were recorded as positive or negative for IF and IP in paired proportions, presuming that IF was the test standard. RESULTS: Concordant observations were 71% for all (282 of 398 observations), 82% for IgG (65 of 79 observations), and 89% for IgA (72 of 81 observations). The majority of discordant observations (74 of 116 observations) were positive by means of IP, with mesangial deposits of IgM and C1q that were not found by IF. Statistically, there was no significant difference in outcomes between IF and IP for IgG, IgA, and C3c ( P > 0.2). In addition, IP staining allowed simultaneous evaluation of tissue by LM and therefore correlation between tissue structure and immune deposits not readily attained by IF. CONCLUSION: In the present study, it is documented that for the detection of IgG, IgA, and C3c, IP applied to protease-digested deparaffinized sections of formaldehyde-fixed renal tissue is, with few exceptions, equal to IF on frozen sections. The EnVision HRP method used here is several times more effective in terms of primary antibody dilution than earlier existing IP methods, and because the avidin-biotin system is not involved, very little nonspecific background staining will occur. Discordant observations (116 of 398 observations; 29%) were in the majority (91 of 116 observations) due to positive IP findings of IgM and C1q, which deserve additional investigation.