C4d-positive acute humoral renal allograft rejection: effective treatment by immunoadsorption.

There is increasing evidence for an important pathogenetic role of alloantibodies in acute renal allograft rejection. Acute humoral rejection (AHR) has been reported to be associated with a poor transplant survival. Although treatment modalities for cellular rejection are fairly well established, the optimal treatment for AHR remains undefined. Ten of 352 kidney allograft recipients transplanted at the authors' institution between November 1998 and September 2000 were diagnosed as having AHR, supported by severe graft dysfunction, C4d deposits in peritubular capillaries (PTC), and accumulation of granulocytes in PTC. AHR was diagnosed 18.9 +/- 17.5 d posttransplantation. All patients were subjected to immunoadsorption (IA) with protein A (median number of treatment sessions, 9; range, 3 to 17). Seven recipients with additional signs of cellular rejection (according to the Banff classification) received also antithymocyte globulin. In nine of ten patients, AHR was associated with an increase in panel reactive antibody reactivity. A pathogenetic role of alloantibodies was further supported by a positive posttransplant cytotoxic crossmatch in all tested recipients (n = 4). In nine of ten recipients, renal function recovered after initiation of anti-humoral therapy. One patient lost his graft shortly after initiation of specific therapy. Another recipient with partial reversal of AHR returned to dialysis 8 mo after transplantation. Mean serum creatinine in functioning grafts was 2.2 +/- 1.2 mg/dl after the last IA session (n = 9) and 1.5 +/- 0.5 mg/dl after a follow-up of 14.2 +/- 7.1 mo (n = 8). In conclusion, this study suggests that AHR, characterized by severe graft dysfunction, C4d staining, and peritubular granulocytes, can be effectively treated by timely IA. In the majority of patients, IA treatment can restore excellent graft function over a prolonged time period.     

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.     

C4D deposition and positive posttransplant crossmatch are not necessarily markers of antibody-mediated rejection in renal allograft recipients

The aim of the study was to search for serologic, immunopathologic, and morphologic evidence of antibody-mediated rejection (AMR) among patients with acute renal allograft dysfunction. The study included 19 patients with episodes of acute rejection (ARE) within the first year after transplantation. All patients had negative crossmatch tests before transplantation. Patients underwent biopsy for histologic and C4d examinations. All patients were monitored for donor-specific HLA alloantibodies during the first posttransplant year. Complement-dependent cytotoxic crossmatches were performed with donor lymphocytes. In eight patients, the crossmatch test results changed to positive during ARE. In all biopsies except one with cortical infarction, we observed C4d staining (group 1). The biopsies of four patients showed histologic changes of AMR, and all of their grafts were lost. In one patient, cellular and vascular rejection (Banff II) were present; in two, Banff I; and in one, borderline lesions. These results were compared with 11 patients with ARE but negative posttransplant crossmatches and negative staining for C4d (group 2). The histologic findings in the biopsies of these patients were cellular interstitial and vascular rejection (Banff I and Banff II). With no features suggestive of AMR. During the first year after transplantation, the creatinine levels of group 1 patients, were significantly higher than group 2 patients. One-year graft survival was 50% in group 1 and 91% in group 2. CONCLUSIONS: C4d and a positive posttransplant crossmatch were not associated with histologic features of AMR in half of the ARE. Nevertheless, C4d deposition and positive posttransplant crossmatches correlated with allograft injury among renal transplant patients.     

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 and C3d Staining in Biopsies of ABO- and HLA-Incompatible Renal Allografts: Correlation with Histologic Findings

Biopsies of ABO-incompatible and positive crossmatch (HLA-incompatible) renal allografts were retrospectively examined to compare results of C4d and C3d staining, and the correlation between such staining and histologic findings suggestive of antibody-mediated rejection (AMR). A total of 75 biopsies (55 protocol, 17 for graft dysfunction, 3 for other indications) of 24 ABO-incompatible grafts and 244 biopsies (103 protocol, 129 for graft dysfunction, 12 for other indications) of 66 HLA-incompatible grafts were examined; all were stained for C4d and approximately 40% for C3d. In ABO-incompatible grafts, 80% of protocol biopsies and 59% performed for graft dysfunction showed C4d staining in peritubular capillaries (PTC); this staining was not correlated with neutrophil margination in PTC. In HLA-incompatible grafts, PTC C4d was present in 26% of protocol biopsies and 60% of biopsies for graft dysfunction; 92% of biopsies with >1+ (0-4+ scale), diffuse PTC C4d had > or =1+ margination and/or thrombotic microangiopathy (TMA), compared with 12% of C4d-negative biopsies. C3d was somewhat more predictive of margination than C4d in ABO-incompatible, but not HLA-incompatible, grafts. In summary, while PTC C4d deposition indicates probable AMR in biopsies of HLA-incompatible grafts, including protocol biopsies, there is no histologic evidence that C4d deposition is correlated with injury in most ABO-incompatible grafts.     

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.     

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.     

C4d-negative antibody-mediated rejection in renal allografts: evidence for its existence and effect on graft survival

The use of C4d staining as a tissue marker for humoral immunity has served an important role in allowing pathologists to more accurately diagnose acute antibody-mediated rejection (AMR) in renal and other allograft biopsies, and also to recognize the contribution of humoral immunity to lesions of chronic renal allograft rejection, including transplant glomerulopathy. However, while C4d remains a specific marker of a humoral response, recent evidence indicates that a considerable fraction of renal allograft biopsies showing antibody-mediated injury are C4d-negative, even by immunofluorescence. This review summarizes the current evidence supporting the existence of C4d-negative AMR, as well as evidence that this entity may, if untreated, lead to the development of scarring within the graft, transplant glomerulopathy and even graft loss.     

Impact of humoral alloreactivity early after transplantation on the long-term survival of renal allografts

BACKGROUND: The contribution of humoral alloreactivity to the rejection of renal allografts is not well defined because humoral antigraft reactions are not easily detectable in transplant biopsies, and serial measurements of circulating allo-antibodies in the post-transplantation period are not routinely performed. We have developed diagnostic techniques that improve the assessment of humoral alloreactivity in vivo and in vitro. METHODS: Humoral alloreactivity in transplant biopsies derived from 218 single kidney grafts was detected by assessing the deposition of complement fragment C4d in interstitial capillaries. Circulating alloantibodies were determined in corresponding serum samples by flow cytometry using lymphoblastoid cell lines of donor DR-type as target cells and by a conventional microcytotoxicity test. The impact of capillary C4d and other selected variables on renal graft survival was calculated by univariate and multivariate analysis. RESULTS: Capillary C4d, present in 46% of biopsies from first grafts and 72% of regrafts, is related to circulating alloantibodies. Grafts with capillary C4d have a markedly shorter survival than grafts without capillary C4d (50% graft survival, 4 vs. 8 years, P = 0.0001). Among several risk factors, capillary C4d is the strongest predictor of subsequent graft loss in a multivariate analysis (relative risk, 2.1, 95% CI, 1.4 to 3.1). Humoral alloreactivity detectable within six months after transplantation has a much stronger impact on graft survival than alloreactivity detected beyond this period. CONCLUSIONS: Humoral alloreactivity, manifested by the capillary deposition of complement C4d in about 50% of biopsied renal grafts, exerts a strong impact on graft survival when it operates within six months after transplantation.     

Late developing C4d-positive humoral renal allograft rejection associated with withdrawal of mycophenolate mofetil

In renal transplantation, C4d-positive acute humoral rejection (AHR) usually develops in the early stage posttransplantation. It is clear C4d can be detected late after the operation, when it is associated with chronic renal allograft rejection. We report a case of a renal allograft recipient who experienced C4d-positive acute renal allograft rejection associated with withdrawal of mycophenolate mofetil (MMF) at 10 months after transplantation. This 21-year-old single male patient received his first cadaveric renal allograft under immunosuppression with cyclosporine, MMF, and prednisolone. The serum creatinine recovered to the normal range within 4 days. A protocol biopsy performed at 1 month after transplantation revealed no signs of rejection. The graft function was stable until 10 months postoperation, when MMF was converted to mizoribin. Three days later a biopsy showed a C4d-positive rejection. Patient had no response to the MMF combined with tacrolimus and steroid bolus therapy, which generally improves 85% of AHR among Chinese. He finally returned to dialysis. Our report suggested that C4d positive AHR may occur late after transplantation. MMF is important to suppress the body's humoral response to allograft; when MMF was converted to a weaker immunosuppressant, the dose of the other immunosuppressants (cyclosporine for example) must be adjusted properly.     

Loss of peritubular capillaries in the development of chronic allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN) remains the most important cause of late renal graft loss. In this study, we examined the role of peritubular capillary (PTC) injury in the development of CAN. METHODS: We studied renal biopsies (n = 79) obtained from grafts with CAN. PTC injury was examined morphologically by immunohistochemistry for CD34. These findings were correlated with interstitial fibrosis and graft dysfunction. Humoral immunity involved in CAN was studied by C4d staining. RESULTS: The CAN cases in the present study included chronic rejection (CR) (n = 14, 17.8%) and C4d-positive chronic humoral rejection (CHR; n = 6, 42.9% in CR cases). Irrespective of CR, CHR, or other CAN, the development of CAN was characterized by injury to and loss of identifiable PTCs, accompanied with the development of interstitial fibrosis. In CR and CHR cases, the loss of PTCs was prominent and seemed to progress within a relatively short period after transplantation. A decrease in the number of PTCs significantly correlated with the development of interstitial fibrosis (r = -0.75, P < .001) and impairment of graft function (r = -0.69, P < .001). CONCLUSIONS: Irrespective of whether CR, CHR, or other factors contribute to CAN, the processes involved in its development appear similar and are characterized by progressive injury and loss of PTCs, with the development of renal scarring. Immunohistochemistry for CD34 in human renal biopsies is a useful method for the detection of microvascular injury.     

Association between C4d staining in renal transplant biopsies, production of donor-specific HLA antibodies, and graft outcome

BACKGROUND: We carried out a retrospective study of C4d staining in paraffin sections from renal transplant biopsies to determine the association between C4d staining, donor-specific antibodies (DSA), histological features, and graft outcome. METHODS: We studied 92 patients who had been biopsied for graft dysfunction. Biopsies were classified using Banff 97 criteria and features suggestive of antibody-mediated rejection were noted. Paraffin sections were stained with a polyclonal antibody using an immunoperoxidase technique. The presence of DSA in concurrent sera was determined by enzyme-linked immunosorbent assay and clinical data were reviewed. RESULTS: Of the 92 cases, 15% showed diffuse and 24% showed focal C4d positivity. The grafts failed in 36% of the diffuse (P<0.025), 23% of the focal, and 7% of the negative group at between one month and 15 years posttransplantation. Only patients in the group with diffuse C4d positivity had concurrent DSA (five cases, P<0.001). Of the five DSA-positive patients, three had type II acute rejection and two of these transplants subsequently failed. The remaining two had chronic allograft nephropathy with features of alloimmune injury. Only two of the nine DSA-negative/C4d-positive transplants had failed at the time of writing, in one case due to recurrent disease. CONCLUSION: We demonstrated a significant association between diffuse C4d staining, production of DSA, and graft failure. Although the concurrent detection of DSA and C4d positivity is uncommon in our patients, these results indicate that outcome in this group is poor and they may benefit from therapies directed at the humoral response.     

C4d deposition in early renal allograft protocol biopsies

BACKGROUND: Deposition of the complement protein C4d in renal allograft biopsies obtained during graft dysfunction and rejection has been proposed to be a sensitive marker of antibody-mediated acute rejection. To determine the diagnostic specificity of C4d deposition, it is important to study biopsies from allografts with no evidence of dysfunction. In this study, we examined C4d deposition in protocol biopsies obtained irrespective of clinical status. METHODS: Immunohistochemistry for C4d was performed on routine protocol biopsies preimplantation and on day 7 posttransplantation from 48 unselected renal allografts. Serum samples obtained up to 1 month after transplantation were assayed for donor-reactive antibodies (DRA). Results were correlated with histopathology and clinical outcome measures. RESULTS: Diffuse C4d deposition was detected in the peritubular capillaries of 6 of 48 (13%) biopsies. C4d deposition was present in 5 of 15 (33%) biopsies that showed acute rejection (Banff 97, category 4) but only in 1 of 33 (3%) biopsies with no rejection (P=0.003, 97% specificity). Posttransplant DRAs were detected in 21 of 48 (44%) patients. All five recipients with C4d deposition and rejection had posttransplant DRA; the recipient whose biopsy showed C4d positivity, but not rejection, did not have detectable DRA. C4d deposition was not treated with plasmapheresis or intravenous immunoglobulin and was not associated with poor posttransplant graft outcome at 1-year follow-up. CONCLUSIONS: Our results show that in early posttransplant protocol biopsies, C4d is a specific marker for the presence of humoral rejection, as indicated by its association with DRA and acute histologic rejection.     

C4d—The Witness of Humoral Rejection

Objective

Acute antibody-mediated (humoral) rejection is a major cause of morbidity, graft loss, and mortality among heart transplant patients. Herein we have presented our experience using C4d to characterize humoral rejection.

Materials and Methods

All nonformalin-fixed cardiac graft biopsies (protocol or emergency) received between May 2007 and May 2008 were examined by immunofluorescence for C4d.

Results

One hundred twelve endomyocardial biopsies from 25 transplanted patients included 20 males and 5 females of ages ranging from 3 to 71 years. The number of biopsies per subject varied from 1 to 11; the timespan between transplantation and the diagnostic biopsies ranged from days to 8 years. Thirteen biopsies showed acute humoral rejection (intramyocardial capillaries positive for C4d); 31, acute cellular rejection (grades 1R, 2R); 7, both humoral and cellular rejection; and 1, acute humoral rejection and allograft vasculopathy. Some of the positive biopsies belonged to the same person, and some to transplanted individuals with signs and symptoms suggestive of rejection, while others did not. The persistence of humoral rejection, despite the disappearance of a cellular component, correlated with slower clinicoechocardiographic improvement.

Conclusions

C4d positivity is a morphologic sign of humoral rejection. It may hasten the appearance and/or worsening of allograft vasculopathy independent of patient age or posttransplantation time.     

Acute humoral rejection in kidney transplantation: II. Morphology, immunopathology, and pathologic classification

The incidence of acute humoral rejection (AHR) in renal allograft biopsies has been difficult to determine because widely accepted diagnostic criteria have not been established. C4d deposition in peritubular capillaries (PTC) of renal allografts has been proposed as a useful marker for AHR. This study was designed to test the relative value of C4d staining, histology, and serology in the diagnosis of AHR. Of 232 consecutive kidney transplants performed at a single institution from July 1995 to July 1999, all patients (n = 67) who developed acute rejection within the first 3 mo and had a renal biopsy with available frozen tissue at acute rejection onset, as well as posttransplant sera within 30 d of the biopsy, were included in this study. Hematoxylin and eosin and periodic acid-Schiff stained sections were scored for glomerular, vascular, and tubulointerstitial pathology. C4d staining of cryostat sections was done by a sensitive three-layer immunofluorescence method. Donor-specific antibodies (DSA) were detected in posttransplant recipient sera using antihuman-globulin-enhanced T cell and B cell cytotoxicity assays and/or flow cytometry. Widespread C4d staining in PTC was present in 30% (20 of 67) of all acute rejection biopsies. The initial histologic diagnoses of the C4d(+) acute rejection cases were as follows: AHR only, 30%; acute cellular rejection (ACR) and AHR, 45%; ACR (CCTT types 1 or 2) alone, 15%; and acute tubular injury (ATI), 10%. The distinguishing morphologic features in C4d(+) versus C4d(-) acute rejection cases included the following: neutrophils in PTC, 65% versus 9%; neutrophilic glomerulitis, 55% versus 4%; neutrophilic tubulitis, 55% versus 9%; severe ATI, 75% versus 9%; and fibrinoid necrosis in glomeruli, 20% versus 0%, or arteries, 25% versus 0%; all P < 0.01. Mononuclear cell tubulitis was more common in the C4d(-) group (70% versus 100%; P < 0.01). No significant difference between C4d(+) and C4d(-) acute rejection was noted for endarteritis, 25% versus 32%; interstitial inflammation (mean % cortex), 27.2 +/- 27% versus 38 +/- 21%; interstitial hemorrhage, 25% versus 15%; or infarcts, 5% versus 2%. DSA were present in 90% (18 of 20) of the C4d(+) cases compared with 2% (1 of 47) in the C4d(-) acute rejection cases (P < 0.001). The pathology of the C4d(+) but DSA(-) cases was not distinguishable from the C4d(+), DSA(+) cases. The C4d(+) DSA(-) cases may be due to non-HLA antibodies or subthreshold levels of DSA. The sensitivity of C4d staining is 95% in the diagnosis of AHR compared with the donor-specific antibody test (90%). Overall, eight grafts were lost to acute rejection in the first year, of which 75% (6 of 8) had AHR. The 1-yr graft failure rate was 27% (4 of 15) for those AHR cases with only capillary neutrophils versus 40% (2 of 5) for those who also had fibrinoid necrosis of arteries. In comparison, the 1-yr graft failure rates were 3% and 7%, respectively, in ACR 1 (Banff/CCTT type 1) and ACR 2 (Banff/CCTT type 2) C4d(-) groups. A substantial fraction (30%) of biopsy-confirmed acute rejection episodes have a component of AHR as judged by C4d staining; most (90%), but not all, have detectable DSA. AHR may be overlooked in the presence of ACR or ATI by histology or negative serology, arguing for routine C4d staining of renal allograft biopsies. Because AHR has a distinct therapy and prognosis, we propose that it should be classified separately from ACR, with further sub-classification into AHR 1 (neutrophilic capillary involvement) and AHR 2 (arterial fibrinoid necrosis).     

The impact of c4d staining as a humoral injury marker

Purpose

Acute and chronic humoral injuries in renal tranplant recipients are the main reasons for graft rejection and failure. Histological and clinical characteristics of humoral rejection and symptoms are variable and not always helpful for differential diagnosis. Clinical monitoring of the allograft, an elevated serum panel-reactive antibody (PRA), and the presence of donor-specific antibody (DSA) during immune monitoring as well as C4d staining of biopsy material can establish the differential diagnosis. Even without a cellular component, humoral rejection reaction is serious because the target tissue is the graft endothelium. Because the kidney graft has a rich vascular structure this attack causes permanent injury to the kidney in the long term. Graft dysfunction in this setting is usually more severe, requiring dialysis therapy, compared with acute cellular reactions. Positive C4d staining of peritubular capillaries in biopsy material represent a hallmark of complement-dependent cytotoxicity, supporting the diagnosis of humoral rejection. We analyzed C4d staining as a hallmark of humoral rejection.

Methods

From 2009 to 2011, we analyzed the relationship between pathological findings of C4d immunohistochemistry staining and the clinical outcomes of 45 kidney transplant recipients who underwent a kidney biopsy because of graft dysfunction due to possible humoral rejection.

Results

Biopsy specimens of 15 patients stained C4d positive; the remaining 30 showed negative results. Intravenous steroids, PP + IVIG with or without antithymocyte globulin (ATG), was administered for treatment. Sixty six percent (n = 10) of patients were C4d positive with 16% (n = 5) of those showing C4d-negative biopsy results, losing their grafts, and returning to hemodialysis.

Conclusions

C4d staining refractory humoral rejection injury was related to poor graft outcomes.     

Positive C4d in Kidney Transplantation Biopsy: Clinical Impact

BackgroundDeposition of C4d in peritubular capillaries of renal graft is normally associated with the presence of antibody-mediated rejection. The clinical impact of its presence in patients with renal transplant in Colombia is uncertain, as well as the association in acute rejection and the response to the management and survival of the graft. The aim of this study was to determine the risk of having positive C4d in biopsies of patients with episodes of acute cellular rejection.MethodsWe retrospectively reviewed 226 biopsies of kidney transplantation, all of them with acute rejection and histopathological findings classified according to Banff criteria 2009 and performed between January 2005 and December 2012 for graft dysfunction. C4d staining was performed by immunohistochemistry.ResultsC4d staining was positive in 25 of 226 biopsies. Rejection time in patients with positive C4d was 15 months in average vs 8 months with negative C4d.ConclusionsWith the use of a multivariate analysis, we found that the unique risk for C4d in our population was the positive panel reactive antibodies and elapsed time between transplant and the rejection (odds ratio: 2.12, P = .034) and that the other variables analyzed are not related to the expression of C4d.     

Incidence and importance of c4d deposition in renal allograft dysfunction

Recent studies showed that peritubular capillary deposition of C4d is a marker of humoral immune responses directed against a renal allograft. The aim of this retrospective study was to investigate the incidence, clinical features, and prognostic implications of C4d deposition in renal allograft biopsy specimens. The biopsies had been performed due to acute graft dysfunction. This study of 104 renal allograft biopsies performed in 2004 classified histopathological findings according to Banff criteria. All paraffin-embedded biopsy samples were stained with an immunohistochemical method for C4d deposition. Demographic data, clinical findings, and biochemical findings were obtained from patients' charts. C4d staining was positive in 15/104 (14%) samples. The staining pattern was diffuse in 8 and focal in 7 patients. Nine patients were males. The overall mean age was 33 +/- 6 years. Ten received live-donor grafts. The biopsy occurred at a mean of 1007 +/- 1415 (range, 15-4712) days after the operation with a mean serum creatinine (SCr) level of 2.8 +/- 1.5 (1.25-6.0) mg/dL. Patients were divided into 2 groups according to the occurrence time: early (before 100 days) and late (after 100 days). Among the early group (n = 5), the mean SCr level was 2.8 +/- 1.5 mg/dL; a diffuse staining pattern was seen in 4 (80%) patients. Histological findings were acute rejection in 3, borderline changes in 1, or thrombotic microangiopathy in 1 patient. Two patients were treated with pulse steroids and 3 with ATG, intravenous immunoglobulin, and plasmapheresis. Three patients lost their grafts at the mean of 118 +/- 100 days after the biopsy. In the late group (n = 10), the mean SCr level was 2.8 +/- 1.7 mg/dL with a diffuse staining pattern in 4 (40%) patients. The histological findings included acute rejection in 6, chronic vascular rejection in 2, thrombotic microangiopathy in 1, and chronic allograft nephropathy in 1 patient. Six patients were treated with pulse steroids, and 3 with ATG and intravenous immunoglobulin. Five patients lost their grafts at a mean of 200 +/- 270 days. The overall incidence of C4d deposition was 14%; it was seen both in the early and late posttransplantation period. Although a diffuse staining pattern was more frequently seen in the early period, C4d deposition indicated a poor allograft prognosis in both periods. Introduction of C4d staining into the routine may guide more specific treatments directed toward the humoral alloresponse.     

Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries.

The distinction between acute humoral rejection (AHR) and acute cellular rejection (ACR) in renal allografts is therapeutically important, but pathologically difficult. Since AHR is probably mediated by antibodies to the donor endothelium that activate the classical complement pathway, it was hypothesized that peritubular capillary C4d deposition might distinguish this group. Renal biopsies (n = 16) from 10 patients with AHR who had acute graft dysfunction, neutrophils in peritubular capillaries, and a concurrent positive cross-match were stained for C4d by immunofluorescence. Control biopsies for comparison showed ACR (n = 14), cyclosporin A toxicity (n = 6), or no abnormality (n = 4). Peribiopsy sera were tested for anti-donor HLA antibody. C4d deposited prominently and diffusely in the peritubular capillaries in all AHR biopsies (16 of 16). IgM and/or C3 were also present in 19 and 44%, respectively. With two-color immunofluorescence, C4d was localized in basement membranes (type IV collagen+) and in the endothelium (Ulex europaeus agglutinin-I+). In ACR, no more than trace C4d was found in peritubular capillaries (P < 0.0001 versus AHR), and no patient had anti-donor HLA antibodies (0 of 8); 27% had neutrophils in peritubular capillaries. One of six biopsies with cyclosporin A toxicity had similar C4d deposits, and circulating anti-donor class I antibody was detected. Grafts with AHR were lost (40%) more often than those with ACR (0%; P < 0.02). C4d in peritubular capillary walls distinguishes AHR from ACR, is more specific and sensitive than traditional criteria, and is a potentially valuable adjunct in the diagnosis of graft dysfunction.