Diagnostic value of C4d in renal biopsies

PURPOSE OF REVIEW: Capillary C4d is now an established marker of antibody-mediated rejection in graft biopsies. The technique is widely used to further define the clinical relevance of humoral alloreactivity in various patient subgroups. These include highly sensitized patients, recipients with late graft failure and also some with 'stable' graft function. RECENT FINDINGS: The C4d technique compares favourably with other techniques that are explored, for example detection of C3d. Capillary C4d can be associated with any graft pathology, including transplant glomerulopathy. C4d is related to circulating alloantibodies but not autoantibodies, and is probably not derived from local sources. Presensitization and de-novo sensitization are important scenarios of humoral alloreactivity that require refined analysis and treatment. SUMMARY: Detection of C4d in graft biopsies has emerged as an important tool that could substantiate the clinical significance of antibody-mediated rejections. The comprehensive analysis of humoral alloreactivity in the posttransplantation period is still ongoing and will hopefully result in improved patient care and better long-term graft survival.     

Significance of donor-specific antibodies in acute rejection

Significant advances in recent years in the diagnosis of antibody-mediated graft rejection have led to the re-evaluation of humoral alloreactivity in organ transplantation. By introducing the "C4d-test" into the work-up of transplant biopsies, donor-specific antibodies were claimed to be directly involved in about 30% of acute rejection episodes. The diagnostic criteria for antibody-mediated rejections of renal grafts are now incorporated in the "Banff classification" as refined at a recent consensus conference. Capillary C4d is not always concordant with circulating anti-HLA-antibodies, even if these are assayed with improved techniques. Antibody absorption within the graft and antigens other than HLA, therefore, have to be considered. Effective therapy of humoral rejection is now available. Serial assessment of humoral alloreactivity also in the posttransplantation period is now mandatory to identify at-risk patients.     

C4d staining of perioperative renal transplant biopsies

BACKGROUND: Deposition of C4d in peritubular capillaries (PTCs) has been shown to be a sensitive marker for antibody-mediated (humoral) rejection in renal transplant biopsies. Some studies also suggest that C4d in PTCs is specific for humoral rejection or, at least, for the presence of donor-specific antibodies. However, in other studies, PTC C4d deposits were noted in more than 40% of renal transplant biopsies performed for graft dysfunction and capillary C4d deposition in heart transplants may result from ischemic injury. METHODS: To test the specificity of C4d staining as a marker for acute humoral rejection ACR in renal allografts, indirect immunofluorescence using a monoclonal anti-C4d antibody and a fluorescein-isothiocyanate-conjugated secondary antibody was performed on cryostat sections of 90 renal transplant biopsies, including 35 pairs of preimplantation and 1-hr postreperfusion biopsies of the same graft, postreperfusion biopsies of 12 additional grafts, and 8 positive controls (biopsies with known C4d-positive AHR). Eighteen grafts were cadaveric, 17 grafts were liviing-related, and 12 grafts were living-unrelated (excluding controls). Included in these grafts were 13 grafts that developed AHR 3 to 34 days posttransplantation. RESULTS: Only 2 of 82 perioperative biopsies showed C4d staining in PTCs. Both perioperative biopsies were postreperfusion biopsies of grafts diagnosed with AHR 5 and 34 days posttransplantation, respectively, and, in each case, the recipient had been treated with plasmapheresis before transplantation because of a positive crossmatch (cytotoxic and flow cytometric) and continued to have a weakly positive flow crossmatch at the time of transplantation. In one biopsy, C4d staining was focal, and in the other biopsy, it was diffuse; in both biopsies, C4d staining was relatively mild (1+ on a 0-4+ scale). No C4d staining was noted on preimplantation biopsies of each graft. All biopsies that contained glomeruli showed linear capillary loop or blotchy mesangial staining, or both, which was similar in prereperfusion and postreperfusion biopsies. All positive controls showed diffuse C4d staining in PTCs.CONCLUSIONS: C4d staining in PTCs may be seen as early as 1 hr posttransplantation in some recipients with low levels of antidonor antibodies. However, this was not observed as a feature of ischemic or ischemia-reperfusion injury in perioperative renal transplant biopsies, including those of cadaveric grafts with cold ischemia times of as long as 41 hr.     

Complement C4d in Graft Capillaries – the Missing Link in the Recognition of Humoral Alloreactivity

Staining of C4d in graft capillaries has emerged as a useful method to detect antibody-mediated rejections in situ. Demonstration of capillary C4d has provided substantial clinical results and allows several conclusions: Antidonor antibodies (preformed or produced de novo) activate complement directly in the graft. Capillary C4d is present in about 30% of biopsies with acute and chronic rejections and separates rejections with a humoral component from 'pure' cell-mediated rejections. Recognition of humoral alloreactivity is important, since effective treatment is now available. Since capillary C4d can appear and disappear at any time post transplantation, every transplant biopsy should be tested. Capillary C4d is now incorporated in the 'Banff classification'. The incidence of C4d-positive cases will probably decline because of the 'routine' application of potent immunosuppressants, including mycophenolate mofetil, that can inhibit antibody production. Presensitization, however, will remain a potential threat to allografts.     

Posttransplant HLA Alloreactivity in Stable Kidney Transplant Recipients—Incidences and Impact on Long‐Term Allograft Outcomes

Humoral alloreactivity is well established to predict adverse allograft outcomes. However, in some recipients, alloantibodies may also occur in the absence of graft dysfunction. We evaluated if and how often complement‐ and noncomplement‐fixing alloantibodies are detectable in stable recipients and whether, in this context, they affect long‐term outcomes. Sera obtained from 164 kidney transplant recipients at 2, 6 and 12 months were evaluated by FlowPRA screening and single‐antigen testing for detection of IgG‐ or C4d‐fixing HLA panel reactivity and donor‐specific antibodies (DSA). Applying stringent criteria, we selected 34 patients with an uneventful 1‐year course (no graft dysfunction or rejection) and excellent graft function at 12 months [estimated glomerular filtration rate (eGFR) ≥60 mL/min and proteinuria ≤0.5 g/24 h]. Nine (27%) and 5 (15%) of these recipients tested positive by [IgG] and [C4d]FlowPRA screening, respectively. In five cases, DSA were identified. Frequencies of positive test results and DSA binding intensities were not significantly lower than those documented for patients who did not fulfill the above criteria. In recipients with an excellent 1‐year course, FlowPRA reactivity was not associated with lower eGFR or increased protein excretion during 68‐month median follow‐up. Our results suggest cautious interpretation of antibody monitoring in patients with normal‐functioning grafts.     

Renal allograft C4d deposition in Chinese: Hong Kong perspective

Background: Acute rejection constitutes a significant proportion of renal allograft loss. Peritubular capillary deposition of C4d has been recognized as the footprint of humoral alloimmunity and proven to be a sensitive and specific marker for humoral rejection in the appropriate clinical context. Its presence in indication biopsies is the most important independent risk factor for graft failure. Data are, however, scarce among Chinese subjects. Methods: We retrospectively reviewed all renal graft biopsies performed from 1 April 2002 to 31 March 2006 for unexplained acute renal dysfunction or delayed graft function. Renal outcomes were assessed at the time of renal biopsy and at 1 month, 3 months, 6 months and 1 year afterwards. Survival was assessed by Kaplan–Meier analysis. Multivariate analysis was used to determine if C4d positivity is an independent risk factor for poor renal outcome. Results: Fifty‐two biopsies were included, of which 16 were positive for peritubular capillary C4d. Peritubular capillary C4d was associated with lower glomerular filtration rate and higher serum creatinine at 6 and 12 months after renal biopsies. The C4d‐positive group fares worse in terms of death‐censored graft failure, doubling of serum creatinine and reaching 50% of glomerular filtration rate at the end of the study. Peritubular capillary C4d deposition was the only significant risk factor that predicts graft failure in multivariate analysis. Conclusion: Our findings confirmed the independent prognostic value of peritubular capillary C4d staining on renal allograft survival in Chinese.     

Diagnostic value of C4d in renal allograft biopsies in different clinical settings: absence of C4d in grafts from non-heart-beating donors

Humoral mechanisms of rejection after kidney transplantation (TX) can be identified through the detection of diffuse complement C4d deposits in peritubular capillaries (PTC) in graft biopsies or donor-specific antibodies (DSA) in serum samples. It has been hypothesized that ischemic injury in the graft may facilitate humoral responses. Kidney grafts from non-heart-beating donors (NHBD) present more often severe ischemia lesions than grafts from heart-beating or living donors. METHODS: We reviewed kidney TX biopsies performed from May 2002 to November 2004 with special interest paid to recipients from NHBD. We checked corresponding frozen tissue for the detection of C4d in PTC using immunofluorescence with a monoclonal antibody against C4d. We also collected post-TX contemporaneous DSA data, either flow crossmatches or cytotoxic PRA. RESULTS: During this period, we performed 22 kidney TXs from NHBD of a total of 326 kidney TX (either single or combined with other grafts). Nine patients of this group underwent 12 biopsies for delayed graft function over 15 days or deteriorating scans. All biopsies showed acute tubular necrosis, but one also presented IA Banff acute rejection and another one had neutrophils in PTC. Frozen tissue from these 12 biopsies did not have diffuse C4d deposits in PTC. Serum samples of seven of nine patients were available: four had negative DSA flow crossmatches and three had 0% PRA within the same period. We diagnosed acute humoral rejection (AHR) in 13 patients-with acute renal dysfunction, C4d in biopsies and DSA after kidney TX-of 38 with high clinical suspicion for AHR. We detected C4d in seven biopsies of 30 patients performed more than 6 months after TX. CONCLUSIONS: Severe ischemic injury does not necessarily determine the activation of humoral mechanisms of rejection mediated through DSA. Therefore, C4d is extremely interesting for the identification of humoral rejection in any clinical setting after kidney TX.     

Clinical impact of complement deposition findings on biopsies in acute rejection episodes of pediatric renal transplant patients

IntroductionRejection is the most important problem for renal graft function and survival. Complement system plays a key role in immune responses from host to graft. It was demonstrated that complement system activation is related with renal fibrosis. We evaluate clinical impact of complement deposition findings on biopsies in acute rejection episodes of pediatric renal transplant patients.MethodDemographics of the patients, graft functions, acute rejection episodes and graft loss were recorded from data files of 165 pediatric renal transplant patients. Findings of 98 renal biopsies were retrospectively evaluated.ResultsThirty three patients with kidney transplant had 44 acute rejection episodes (32 pure cellular acute rejection episodes / 1 pure humoral acute rejection episode / 11 combined acute cellular and acute humoral rejection episodes) proven by biopsy. C1q staining was positive in 7 biopsies, C3 staining in 15 biopsies and, C4d staining in 15 biopsies. 26 patients had graft fibrosis. All patients with a rejection history had a significant decrease in GFR value during follow-up. Patients who did not have fibrotic changes in first biopsy had same level of deterioration of GFR when compared with patients who had fibrotic changes in first biopsy.ConclusionWe could not demonstrate a significant relation between complement deposition and renal fibrosis, and between complement deposition and GFR values. Our data demonstrated that graft outcomes and graft loss after acute rejection episodes cannot be predicted only with complement deposition on graft or only with graft fibrosis.     

Capillary deposition of complement C4d and C3d in pediatric renal allograft biopsies

BACKGROUND: Peritubular capillary deposition of C4d (C4d(PTC)) is a marker of antibody-mediated alloresponse and is associated with poor graft survival in adults. C3d(PTC) has received less attention; its significance is unclear. To date no information has been gained in children. METHODS: The prevalence of C4d(PTC) and C3d(PTC) in pediatric renal allograft biopsies (n=77, 31 cadaveric kidneys) was analyzed retrospectively. Associations with histology, donor-specific antibodies (DSAs), and outcome were investigated. RESULTS: The overall prevalence of C4d(PTC) and C3d(PTC) was 52% and 48%, respectively. C3d(PTC) was associated with C4d(PTC) (P<0.0001). Thirty-six percent of acute rejections were cellular, 28% were humoral, and 36% were combined cellular and humoral. C3d(PTC) was found in 57% of acute rejection biopsies. C4d(PTC), but not C3d(PTC), was associated with accumulation of polymorphonuclear cells in peritubular capillaries (P=0.02). Fifty-one percent of late biopsies (>6 months posttransplantation) had features of chronic allograft nephropathy: 50% were C4d(PTC_ positive, and 50% were C3d(PTC) positive. C4d(PTC) positive chronic allograft nephropathy biopsies had more transplant glomerulopathy (P=0.020) and mesangial matrix increase (P=0.026). C3d(PTC) tended to be associated with transplant glomerulopathy (P=0.06), but not with mesangial matrix increase. C4d(PTC) was correlated with DSA (P=0.011). Excluding early nonrejection graft losses, more grafts were lost in the C4d(PTC) positive group (P=0.019). C3d(PTC) was not associated with DSA or graft outcome. CONCLUSIONS: Our results support C4d(PTC) being a hallmark of humoral rejection in pediatric renal transplantation; its presence was associated with DSA and poorer immunologic graft outcome. In contrast, C3d(PTC), although highly associated with C4d(PTC), did not correlate with DSA or outcome.     

C4d deposition and cellular infiltrates as markers of acute rejection in rat models of orthotopic lung transplantation

BACKGROUND: C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. METHODS: We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6-) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. RESULTS: The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6- recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. CONCLUSIONS: The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.     

Adult living-donor liver transplantation with ABO-incompatible grafts

BACKGROUND: Liver transplantation using ABO-incompatible grafts is rarely performed because the reported outcome is poorer than with compatible grafts. We report our positive experience with adult-to-adult living-donor liver transplant (LDLT) using ABO-incompatible grafts. METHODS: The immunosuppressive protocol consisted of plasmapheresis/intravenous immunoglobulin infusion before LDLT followed by thymoglobulin induction and splenectomy, maintenance with tacrolimus/cyclosporine (FK/CSA), mycophenolate mofetil, and a rapid steroid taper. Plasmapheresis was planned for up to 3 months after LDLT aiming at maintaining the anti-ABO titers level below 1:16. Liver biopsies were routinely stained for humoral rejection with complement 4d (C4d) and for biliary damage with cytokeratin 7. RESULTS: Between January 2003 and September 2004, five patients, mean age 59 years, received an ABO-incompatible LDLT. Patient and graft survival was 80% at mean follow-up of 43 months (range, 34-54) for the four surviving patients. One patient died 4 months after LDLT. Humoral rejection occurred in one patient whereas acute cellular rejection was diagnosed in four patients. CONCLUSIONS: ABO-incompatible LDLT can be performed with patient and graft survival similar to compatible LDLT. Minimization of immunosuppression is possible, and chronic biliary damage is not the norm. Better tools than complement 4d staining must be researched to diagnose the features of immunologic damage to the graft. If these results will be confirmed in a greater number of patients, ABO-incompatible LDLT may be proposed when ABO-compatible donors are not available or when the ABO-incompatible donor is the better candidate.     

Chronic Antibody Mediated Rejection of Renal Allografts: Pathological, Serological and Immunologic Features in Nonhuman Primates

The pathogenesis of late renal allograft loss is heterogeneous and difficult to diagnose. We have analyzed renal allografts in nonhuman primates to determine the relationship between alloantibodies and the graft pathology of late graft loss. Seventeen Cynomolgus monkeys were chosen from among those on several protocols for renal allotransplantation with mixed chimerism induction so that animals with and without alloantibodies were included. All animals received transient CD154 blockade and short-term cyclosporine treatment until day 28. Serial blood samples were tested for alloantibodies. Protocol biopsies and autopsy kidneys were scored for pathology and C4d deposition. Group 1, defined by complete lack of C4d deposition (24 tissue samples; 8 recipients), had no detectable alloantibodies (33 serum samples; 1-7 samples per recipient) and no evidence of chronic rejection. Three survived greater than 2 years with normal function and histology. Group 2, defined as having C4d deposition in peritubular capillaries, all made alloantibodies (100%), and most grafts later showed chronic allograft glomerulopathy (89%), and/or arteriopathy (89%). All grafts in Group 2 failed (3-27 months). Pathologic lesions of typical of chronic rejection in humans develop in monkeys, correlate with antecedent alloantibodies/C4d deposition and predict chronic rejection rather than durable accommodation.     

C3D deposition in peritubular capillaries indicates a variant of acute renal allograft rejection characterized by a worse clinical outcome

BACKGROUND: C4d deposition in peritubular capillaries (PTCs) is a sign of humoral renal allograft rejection and an independent predictor of graft survival. Few investigators have focused on the meaning of capillary C3 deposition in rejecting grafts. Because C3 production can result from both classic and alternative pathway activation of the complement cascade, it is not clear whether C3 deposition indicates a distinct entity of acute rejection (AR) or merely represents a separate form of C4d-positive AR. METHODS: We examined the deposition of C3d in the PTCs of recipients with AR in the first year posttransplantation (n=30). Clinical outcome variables and histology were compared with C3d-negative control patients (n=82). RESULTS: C3d-positive patients demonstrated more frequent preexisting T-cell antibodies (57%) and more re-transplants (37%), and they received more blood transfusions (mean 10.3 units). C3d-positive patients experienced more frequent multiple AR episodes (57%) and delayed graft function (36.7%). All nine C3d-positive recipients screened for posttransplantation donor-specific human leukocyte antigen antibodies demonstrated positive results. Graft failure occurred in 23% of C3d-positive recipients (7.3% in the control group) (P=0.03). C3d-positive biopsies showed significantly less tubulitis (P=0.03), whereas congestive PTCs with intraluminal accumulation of polymorphonuclear leukocytes were conspicuous. Thrombi, fibrinoid necrosis, and acute tubular necrosis were not more pronounced. In 19% of rejection biopsies, C3d deposition in PTCs was present without C4d deposition. In the remaining biopsies, C3d and C4d deposition was found simultaneously. CONCLUSIONS: The deposition of complement factor C3d in PTCs indicates a variant type of AR characterized by a worse clinical outcome.     

The Significance of Donor‐Specific HLA Antibodies in Rejection and Ductopenia Development in ABO Compatible Liver Transplantation

The role of humoral alloreactivity in ABO‐compatible liver transplantation remains unclear. To understand the significance of donor‐specific HLA alloantibodies (DSA) in liver rejection, we applied the currently used strategy for detection of antibody‐mediated rejection of other solid allografts. For this purpose we reviewed the data on 43 recipients of ABO identical/compatible donor livers who had indication liver biopsy stained for complement element C4d and contemporaneous circulating DSA determination. Seventeen (40%) patients had significant circulating DSA in association with diffuse portal C4d deposition (DSA+/diffuse C4d+). These DSA+/diffuse C4d+ subjects had higher frequency of acute cellular rejection (ACR) 15/17 versus 13/26 (88% vs. 50%), p = 0.02, and steroid resistant rejection 7/17 versus 5/26 (41% vs. 19%), p = 0.03. Based on detection of the combination DSA+/diffuse C4d+, 53.6% of cases of ACR had evidence of concurrent humoral alloreactivity. Six of the 10 patients with ductopenic rejection had circulating DSA and diffuse portal C4d, three of whom (2 early and 1 late posttransplantation) developed unrelenting cholestasis, necessitating specific antibody‐depleting therapy to salvage the allografts. Thus, in ABO‐compatible liver transplantation humoral alloreactivity mediated by antibodies against donor HLA molecules appears to be frequently intertwined with cellular mechanisms of rejection, and to play a role in ductopenia development.     

Detection of humoral rejection in human cardiac allografts by assessing the capillary deposition of complement fragment C4d in endomyocardial biopsies.

BACKGROUND: There are no well-established diagnostic criteria to detect humoral rejection in organ transplantation. The value of commonly used markers in immunohistochemistry, such as C1q, C3c, IgG, IgM and fibrinogen, is questioned by some groups. Complement fragment C4d is a more stable marker of complement activation as it is covalently bound to graft capillaries. C4d has been shown to identify clinically relevant, but otherwise undetectable humoral anti-graft reactions in human kidney transplants. METHODS: Immunohistochemical techniques were used to evaluate 155 endomyocardial biopsies from 56 heart transplant recipients less than 3 months post transplantation for the presence of capillary C4d staining. In a subset of patients, C4d staining was compared with C1q, C3c, IgM and fibrin staining and was correlated with clinical outcome. RESULTS: Within 3 months 9 of 56 patients died. Five of these nonsurvivors had prominent C4d staining (p < .05), whereas C1q, C3c and IgM showed no correlation with clinical outcome. Presence of fibrin correlated with clinical outcome and C4d staining (p < .05). CONCLUSIONS: The capillary deposition of complement split product C4d in human endomyocardial biopsies was significantly associated with graft loss. Determination of fibrin deposition may yield additional information to establish a diagnosis of humoral rejection. The immunohistochemical assessment of capillary deposition of C4d and fibrin appears to be an appropriate tool for the identification of patients, who may require additional or alternative immunosuppressive therapy targeted against the humoral immune system.     

Capillary deposition of complement split product C4d in renal allografts is associated with basement membrane injury in peritubular and glomerular capillaries: a contribution of humoral immunity to chronic allograft rejection

Endothelial deposition of the complement split product C4d is an established marker of antibody-mediated acute renal allograft rejection. A contribution of alloantibody-dependent immune reactions to chronic rejection is under discussion. In this study, the association of immunohistochemically detected endothelial C4d deposition in peritubular capillaries (PTC) with morphologic features of chronic renal allograft injury was investigated in a large study cohort. C4d deposits in PTC were detected in 73 (34%) of 213 late allograft biopsies performed in 213 patients more than 12 mo after transplantation (median, 4.9 yr) because of chronic allograft dysfunction. Endothelial C4d deposition was found to be associated with chronic transplant glomerulopathy (CG) (P < 0.0001), with basement membrane multilayering in PTC (P = 0.01), and with an accumulation of mononuclear inflammatory cells in PTC (P < 0,001). Furthermore, C4d deposits in PTC (in biopsies with normal glomerular morphology) were associated with development of CG in follow-up biopsies. Other morphologic features of chronic allograft nephropathy (with exception of tubular atrophy) were not associated with C4d deposits in PTC. Analyses of previous and follow-up biopsies revealed that C4d deposits may occur de novo and may also disappear at any time after transplantation. In conclusion, the data suggest that complement activation in renal microvasculature, indicating humoral alloreactivity, contributes to chronic rejection characterized by chronic transplant glomerulopathy and basement membrane multilayering in PTC.     

C4d complement split product in diagnosis of immunological activity of chronic allograft nephropathy

INTRODUCTION: Activation of the humoral branch of the immunological response is currently believed to play an important role in pathogenesis of chronic allograft nephropathy. The impact of humoral alloreactivity, indicated by the presence of C4d deposits in peritubular capillaries of a renal allograft, on the development of chronic allograft nephropathy is a significant problem in transplantation. The aim of the study was to assess and correlate C4d expression in patients with chronic allograft nephropathy, with clinical and morphological variables, as well as to assess the impact of a change in immunosuppression regimen on posttransplant course and renal allograft morphology. PATIENTS AND METHODS: Twenty-six patients with chronic allograft nephropathy underwent biopsies to correlate C4d expression with clinical parameters and morphological findings. In all patients azathioprine was replaced with mycophenolate mofetil with additional CsA dose reduction in 12 patients. After 1 year, 14 protocol biopsies were performed. RESULTS: The frequency of C4d peritubular capillary deposition among patients with chronic allograft nephropathy was 30%. C4d expression appeared later after transplantation, was correlated with chronic allograft glomerulopathy and proteinuria but not other clinical or histological variables. C4d deposits displayed no independent impact on serum creatinine level. Proteinuria was significantly more reduced in the C4d(+) group. Progression of chronic morphological changes was significantly accelerated in the C4d(+) group. CONCLUSION: C4d peritubular capillary expression did not differentiate patients after immunosuppression enhancement, but it predisposed to progression of chronic morphological findings during 1-year observation.     

C4d deposition in allografts: current concepts and interpretation

Alloantibody responses are not prevented by the latest immunosuppressive regimens and contribute to increased early and late renal allograft graft loss. Numerous papers have set forth the clinical, pathological, and immunopathological features of acute humoral rejection, in particular the strong correlation between the presence of C4d deposition and circulating antidonor HLA class I and class II antibodies. Humoral rejection also occurs in a chronic setting, associated with chronic allograft glomerulopathy and arteriopathy. C4d deposition can also be found in stable grafts without concurrent graft pathology, a finding that may indicate accommodation. The central diagnostic criterion for humoral rejection is the demonstration of C4d in peritubular capillaries. The criteria for humoral rejection are not as widely accepted for other organs, such as the heart, lung, and pancreas, although humoral rejection, including C4d deposition, has been described. This review focuses on the practical aspects of this test, particularly as applied routinely in renal allografts since 1998 in our laboratory.     

Use of C4d as a Diagnostic Adjunct in Lung Allograft Biopsies

PURPOSE: Humoral allograft rejection is a defined mechanism for cardiac and renal graft dysfunction; C4d deposition, a stable component of complement activation, inversely correlates with graft survival. With the recent recognition of humoral rejection in lung grafts, we examined C4d's role as a prognostic adjunct in lung allografts. MATERIAL AND METHODS: Twenty-three lung recipients underwent biopsies for deterioration in clinical status or routine surveillance. Clinically unwell patients possessed acute rejection or bronchiolitis obliterans syndrome (BOS). Biopsies attributable to infection were excluded from the study. In addition to routine light microscopy, an attempt was made to correlate the clinical status and morphologic findings with the pattern of C4d deposition and also to compare these clinical and morphologic parameters with the other assessed immunoreactants. Panel reactive antibody testing was also carried out at various points in their post transplantation course whereby in 6 of the cases the samples were procured at exactly the same time as the tissue samples. RESULTS: The patients were segregated into two groups: those patients with recurrent acute rejection and those with BOS. In those patients with symptomatic acute rejection, all biopsies showed light microscopic and immunofluorescent evidence of humoral allograft rejection. The level of C4d was positively correlated with the degree of parenchymal injury, the hallmark being one of septal capillary necrosis. In addition, high and intermediate levels of C4d correlated with a clinical diagnosis of acute rejection. C4d was the strongest predictor of parenchymal injury and of the clinical status (p <.0001) compared to other the immunoreactants C1q, C5b-9 and immunoglobulin. There was no specific correlation between C4d deposition and the presence of acute cellular rejection. In those patients fulfilling clinical criteria of BOS, deposits of C4d as well as other immunoreactants were found in the bronchial wall as opposed to the rarity of this finding in bon-BOS patients. However the only statistically significant predictor of BOS was bronchial wall deposition of C1q. In no case were panel reactive antibodies at significant levels discovered post transplantation. CONCLUSIONS: In the context of acute rejection, C4d deposition correlates with clinical evidence of rejection and the degree of humoral rejection assessed pathologically; there is no association with the presence of histocompatibility related antibodies. It is a more specific predictor of allograft status compared to other immunoreactants. C4d deposition within the bronchial wall is a feature of BOS and hence may be used as a marker of chronic graft dysfunction. The antigenic target resulting in C4d deposition may not be histocompatibility related.     

Clinical significance of C4d deposition in stable renal allografts in the early post‐transplantation period

Abstract: The clinical significance of C4d positivity in patients with stable graft function is undetermined. This study evaluated the clinical outcome of protocol biopsy‐proven C4d‐positive renal transplants with stable graft function in the early post‐transplantation period. Protocol biopsies (n = 79) were performed on stable allografts on the 14th post‐transplant day, and indication biopsies (n = 74) were performed on dysfunctioning allografts within one yr after transplantation. Clinical and histological findings, graft function and graft survival rates were compared between C4d‐positive and C4d‐negative grafts in each group. The incidence of C4d positivity was 5.1% in protocol biopsies and 9.5% in indication biopsies. In protocol biopsies, C4d‐positive allografts showed minimal tubulointerstitial inflammation, and the graft function and graft survival rate did not differ from C4d‐negative allografts. All C4d‐positive allografts maintained stable graft function without anti‐rejection therapy, and follow‐up biopsies of two patients showed no C4d deposition or evidence of rejection. On the other hand, C4d‐positive allografts in indication biopsies showed severe tissue injury, and the graft survival rate was significantly lower than C4d‐negative allografts. In conclusion, C4d‐positive allografts with stable graft function in the early post‐transplantation period take an indolent course.