Detection Of C4d Research Articles

Analyses of the clinical and immunological characteristics of patients with lupus erythematosus.

Background:Lupus erythematosus (LE) is a broad-spectrum, heterogeneous disease. At one end of the spectrum is the cutaneous LE (CLE) without systemic involvement, and at the other end is the systemic LE (SLE) with multisystem involvement. Analyses of clinical and immunological indicators and pathological examinations are helpful for early diagnosis, differential diagnosis, and prognosis of LE.Aim and Objectives:We described the clinical and laboratory characteristics of patients with LE and assessed the diagnostic value of immunohistochemical detection of C3d, C4d, IgG, IgG4, and CD123 in skin lesions of LE.Materials and Methods:Clinical and laboratory data of 62 patients with LE were collected. The expression levels of C3d, C4d, IgG, IgG4, and CD123 in skin lesions of LE were detected by immunohistochemistry (IHC).Results:Clinical manifestations such as hematological involvement, C3, C4, ESR, hematuresis, proteinuria, anti-Sm, anti-ribosomal P-protein, anti-U1-RNP, anti-histone, and anti-nucleosome antibodies are helpful for classificatory diagnosis of LE. The positive rate of C3d and/or C4d along the basement membrane zone in LE skin lesions by IHC was 74.6%, which was higher than that by direct immunofluorescence (47.5%) (P = 0.002). The expression of CD123 protein and the number of CD123+ plasmacytoid dendritic cells (PDCs) in skin lesions of patients with LE were higher than those of dermatomyositis (DM), while the distributed form of CD123 + PDCs in the dermis was different between LE and DM.Conclusions:The diagnosis of CLE and SLE requires a combination of clinical manifestations, laboratory indicators, and pathological examination. Immunohistochemical detection of C3d, C4d, and CD123 in skin lesions is important for the classificatory diagnosis of LE.

Intrapulmonary Autoantibodies to HSP72 Are Associated with Improved Outcomes in IPF.

Rationale Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic interstitial lung disease, with high mortality. Currently, the aetiology and the pathology of IPF are poorly understood, with both innate and adaptive responses previously being implicated in the disease pathogenesis. Heat shock proteins (Hsp) and antibodies to Hsp in patients with IPF have been suggested as therapeutic targets and prognostic biomarkers, respectively. We aimed to study the relationship between the expression of Hsp72 and anti-Hsp72 antibodies in the BAL fluid and serum Aw disease progression in patients with IPF. Methods A novel indirect ELISA to measure anti-Hsp72 IgG was developed and together with commercially available ELISAs used to detect Hsp72 IgG, Hsp72 IgGAM, and Hsp72 antigen, in the serum and BALf of a cohort of IPF (n = 107) and other interstitial lung disease (ILD) patients (n = 66). Immunohistochemistry was used to detect Hsp72 in lung tissue. The cytokine expression from monocyte-derived macrophages was measured by ELISA. Results Anti-Hsp72 IgG was detectable in the serum and BALf of IPF (n = 107) and other ILDs (n = 66). Total immunoglobulin concentrations in the BALf showed an excessive adaptive response in IPF compared to other ILDs and healthy controls (p = 0.026). Immunohistochemistry detection of C4d and Hsp72 showed that these antibodies may be targeting high expressing Hsp72 type II alveolar epithelial cells. However, detection of anti-Hsp72 antibodies in the BALf revealed that increasing concentrations were associated with improved patient survival (adjusted HR 0.62, 95% CI 0.45-0.85; p = 0.003). In vitro experiments demonstrate that anti-Hsp72 complexes stimulate macrophages to secrete CXCL8 and CCL18. Conclusion Our results indicate that intrapulmonary anti-Hsp72 antibodies are associated with improved outcomes in IPF. These may represent natural autoantibodies, and anti-Hsp72 IgM and IgA may provide a beneficial role in disease pathogenesis, though the mechanism of action for this has yet to be determined.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

Antibody-mediated rejection: analyzing the risk, proposing solutions.

Polyreactive Antibodies Developing Amidst Humoral Rejection of Human Kidney Grafts Bind Apoptotic Cells and Activate Complement

Antibody mediated rejection (AMR) is associated with a variety of graft-reactive antibodies following kidney transplant. To characterize these antibodies, we immortalized 107 B cell clones from a patient with AMR. In a previous study, we showed that six clones were reacting to multiple self-antigens as well as to HLA and MICA for two of them, thus displaying a pattern of polyreactivity. We show here that all six polyreactive clones also reacted to apoptotic but not viable cells. More generally we observed a nearly perfect overlap between polyreactivity and reactivity to apoptotic cells. Functionally, polyreactive antibodies can activate complement, resulting in the deposition of C3d and C4d at the surface of target cells. Testing the serum of 88 kidney transplant recipients revealed a significantly higher IgG reactivity to apoptotic cells in AMR patients than in patients with stable graft function. Moreover, total IgG purified from AMR patients had increased complement activating properties compared to IgG from non-AMR patients. Overall, our studies show the development of polyreactive antibodies cross-reactive to apoptotic cells during AMR. Further studies are now warranted to determine their contribution to the detection of C4d in graft biopsies as well as their role in the pathophysiology of AMR.

Banff Initiative for Quality Assurance in Transplantation (BIFQUIT): Reproducibility of C4d Immunohistochemistry in Kidney Allografts

Detection of C4d is crucial for diagnosing antibody-mediated-rejection. We conducted a multicenter trial to assess the reproducibility for C4d immunohistochemistry on paraffin tissue. Unstained slides from a tissue microarray (TMA) comprising 44 kidney allograft specimens representing a full analytical spectrum for C4d were distributed to 73 institutions. Participants stained TMA slides using local protocols and evaluated their slides following the Banff C4d schema. Local staining details and evaluation scores were collected online. Stained slides were returned for centralized panel re-evaluation. Kappa statistics were used to determine reproducibility. Poor interinstitutional reproducibility was observed (kappa 0.17), which was equally due to limitations in interobserver (kappa 0.44) and interlaboratory reproducibility (kappa 0.46). Depending on the cut-off, reproducibility could be improved by omitting C4d grading and only considering ± calls. Heat-induced epitope recovery (pH 6-7, 20-30 min, citrate buffer) with polyclonal antibody incubation (<1:80, >40 min) appeared as best practice. The BIFQUIT trial results indicated that C4d staining on paraffin sections varies considerably between laboratories. Refinement of the current Banff C4d scoring schema and harmonization of tissue processing and staining protocols is necessary to achieve acceptable reproducibility.

Predictive Value of HLA Antibody Characteristics in Progression of Subclinical Antibody Mediated Rejection in Heart Transplant Recipients

Purpose Donor specific antibody (DSA) is a risk factor for antibody-mediated rejection (AMR). Concomitant detection of C4d & C3d in heart biopsies correlates with DSA and allograft dysfunction. Detection of C4d only in surveillance biopsies may not be associated with allograft dysfunction. However, conversion from C4d only to C4d + C3d is noted in ~20% of cases (C4d converters). We studied DSA patterns in clinical (C4d+C3d+ & C4d converters) & subclinical (C4d+) AMR. Methods and Materials Of 1589 transplants, 610 were screened for AMR (last 6 yr). We studied DSA patterns in C4d+ asymptomatic patients, C4d+C3d+ biopsies symptomatic patients & patients who started as C4d+ & converted to C4d+C3d+. We also compared cPRA & crossmatches. Results Significant differences in DSA incidence, mean cPRA, & DSA mean MFI were noted across all groups with C4d+ patients having the lowest values ( table 1 ). DSA mean MFI was significantly higher in patients with clinical AMR ( table 1 ). C4d converters had significantly higher mean cPRA & DSA, driven by higher DQ DSA but not class I DSA compared to patients who remained C4d+ ( Table 2 ). Having any DSA, DQ DSA or cPRA≥50% at time of biopsy was associated with C4d conversion with high -ve predictive value (NPV) but low +ve predictive value (PPV). Conclusions Our data suggest that HLA antibody characteristics may be relevant in determining which subclinical C4d+ may not progress to clinical AMR & warrants investigation in larger cohorts. Table 1 Any DSA DSA Cl I DSA Cl II DSA Cl I +II mean ± SE cPRA mean ± SE MFI C4d+: n=34 19, 56% 9, 26% 12, 35% 2, 6% 44.8 ± 5.1 7398.5 ± 1525.1 C4d+C3d+: n=30 28, 93% 20, 67% 26, 87% 18, 60% 87.4 ± 5.4 12633.0 ± 1222.8 C4d conv: n=11 11, 100% 4, 36% 10, 91% 3, 27% 81.1 ± 8.9 8378.2 ± 1950.9 p .0003 .005 .022 Table 2 Any DSA DSA Cl I DSA Cl II DR DSA DQ DSA cPRA≥50 C4d+: n=34 19, 56% 9, 26% 12, 35% 3, 9% 11, 32% 16, 47% C4d conv: n=11 11, 100% 4, 36% 10, 91% 6, 55% 9, 82% 10, 91% p .008 .70 .002 .003 .004 .014 PPV .38 .30 .46 .67 .45 .39 NPV 1 .78 .96 .86 .92 .95

C4d/CD34 double-immunofluorescence staining of renal allograft biopsies for assessing peritubular capillary C4d positivity

Immunofluorescence detection of the complement split product C4d along peritubular capillaries in renal allograft biopsies is the mainstay for the diagnosis of antibody-mediated rejection. The extent of peritubular capillary C4d positivity may have significant clinical ramifications; however, peritubular capillary density in the renal cortex is often difficult to assess with single-channel immunofluorescence. In this study, we report a C4d/CD34 double-immunofluorescence staining protocol for renal allograft frozen sections that allows rapid and sensitive detection of C4d positivity, as well as improved accuracy in estimating the C4d-positive fraction of peritubular capillaries. In addition, this method aids in determining whether C4d-positive structures correspond to peritubular capillaries or whether they represent common mimics of peritubular capillaries such as tubular basement membranes. C4d/CD34 double immunofluorescence provides rapid, convenient, and low-cost implementation for laboratories currently utilizing single-channel C4d immunofluorescence.

Optimal Cutoff Point for Immunoperoxidase Detection of C4d in the Renal Allograft: Results From a Multicenter Study

Although C4d deposition in peritubular capillaries has been identified as a strong risk factor for subsequent renal allograft loss, the optimal cutoff for the fraction of peritubular capillaries needed to establish a positive stain in formalin-fixed, paraffin-embedded material has not been defined systematically. The objective of this study was to establish the threshold for positive staining that best predicts renal outcome in renal biopsies in a multicenter study in which local and central pathologic conditions were compared. Unstained renal biopsy slides were obtained from 296 patients. The percentage of peritubular capillaries staining positively for C4d was detected by immunoperoxidase staining. The percentage C4d deposition ranged from 0% to 90% with 44% (129/296) having a positive percentage of C4d staining. The median for positive cases was 25%. Local C4d+ results were reported qualitatively, with 28% recorded as positive for C4d. Using a centrally determined cutoff of 10%, tests for agreement of local and central C4d staining were fair (κ 0.40, 95% confidence interval 0.29-0.51). Raising the centrally determined cutoff to 25% or 50% did not change the κ values (0.44 and 0.41, respectively). By Cox proportional hazards model, C4d positivity (centrally determined assessment) using a cutoff of 10% was the strongest predictor of time to graft loss (hazard ratio 2.66, 95% confidence interval 1.68-4.21). Centrally determined C4d positivity correlated with Banff scores indicative of acute inflammation but not with scores indicative of fibrosis/atrophy or transplant glomerulopathy. Our findings indicate that C4d positivity, defined as more than or equal to 10% by immunoperoxidase, is a strong predictor of graft loss.

ELISA-based detection of C4d after liver transplantation — A helpful tool for differential diagnosis between acute rejection and HCV-recurrence?

Hepatitis-C is the most common indication for liver transplantation. Recurrence of HCV is universal leading to graft failure in up to 40% of all patients. The differentiation between acute rejection and recurrent hepatitis-C is crucial as rejection treatments are likely to aggravate HCV-recurrence. Histological examination of liver biopsy remains the gold standard for diagnosis of acute rejection but has failed in the past to distinguish between acute rejection and recurrent hepatitis-C. In a retrospective study we have recently reported that C4d as a marker of the activated complement cascade is detectable in a hepatic specimen in acute rejection after liver transplantation and may serve as a valuable tool in differential diagnosis between ACR and HCV-recurrence. We performed a prospective analysis by ELISA measurement of C4d concentration in cryo-preserved liver biopsies of LTX patients who had either experienced acute rejection, hepatitis-C recurrence or displayed no pathological alterations (controls). Opposed to our immunohistologically based findings in paraffinized tissue we were unable to detect significant differences of C4d concentration in ELISA of cryo-preserved liver tissue. Consequently the role and potential value of C4d as a diagnostic marker may not be determined using ELISA-based tissue evaluation.

The Prevalence of C4d-Positive Renal Allografts in 134 Consecutive Biopsies in Thai Patients

Antibody-mediated rejection (AMR) has been recognized recently as an important cause of graft failure. Detection of C4d in renal allograft biopsies is a proven ancillary technique in the diagnosis of AMR. The prevalence of C4d staining in Western countries varies from 17% to 60% among indication biopsies. There are only a few C4d prevalence studies in an Asian population. The objective of this study was to identify prevalence of C4d among Thai renal transplant patients. Consecutive renal allograft biopsies from 99 patients from 1999 to 2007 were stained for C4d by an immunoperoxidase technique. The biopsy slides were evaluated for the diagnosis according to the Banff'07 classification and histological scores. The relevant clinical data were obtained from clinical records. The prevalence of C4d in renal allografts was reported as a percentage using a descriptive analysis. Chi-square and unpaired Student t tests were used to evaluate the association between clinicopathologic findings and C4d positivity. P values less than .05 were considered significant. The prevalence of positive C4d staining was 16.4%. Fourteen biopsies (10.4%) showed diffuse staining while 8 (5.9%) revealed focal staining. Transplant glomerulopathy, glomerulitis, and peritubular capillaritis were associated with C4d positivity. Most inflammatory cells in peritubular capillaritis were mononuclear cells. Banff score elements, including tubulitis, intimal arteritis, interstitial infiltrate, interstitial fibrosis, tubular atrophy, mesangial matrix increase, vascular fibrous thickening, and arteriolar hyaline thickening, were not associated with C4d positivity. Many factors contribute to the varied prevalence of C4d positivity, including immunologic risks for AMR, type of allograft biopsy, and technique of C4d staining. Our study showed no difference in C4d prevalence among Thai renal allograft patients compared to the Western population. The suggestion to use C4d staining on all allograft biopsies should applied to Thai patients as well.

Analysis of Different Staining Techniques for C4d Detection in Renal Allograft Biopsies

Capillary C4d deposition has been recognized as a marker of antibody-mediated rejection (AMR). Although the detection of capillary C4d by means of immunofluorescence (IF) in cryostat sections is well established, frozen tissue is not always available, thus limiting the diagnosis of AMR. The aim of the present study was to analyze different techniques for C4d staining and the prevalence of C4d in renal allograft biopsies. Detection of C4d was carried out using IF or immunohistochemistry (IHC) on frozen and paraffin sections of renal allograft biopsies available from the same patients. Biopsies obtained from 20 patients were classified into 3 groups: no rejection, acute rejection, and chronic allograft nephropathy (CAN). The capillary C4d deposition prevalence in frozen-IF, considered the gold standard technique for C4d detection, was 45% (9/20 cases). Compared with frozen-IF, the frozen-IHC technique presented an 85% concordance rate (17/20 cases; r = .70; P < .001; sensitivity = 77.8%; specificity = 90.9%). The paraffin-IF technique showed similar results, with an 80% concordance rate (16/20 cases; r = .64; P < .005; sensitivity = 55.6%; specificity = 100%), whereas C4d detection occurred in only 65% of paraffin-IHC cases (13/20; r = .30; not significant; sensitivity = 66.7%; specificity = 63.6%). No capillary C4d deposition was detected in cases without evidence of rejection. However, 4/7 cases (57%) of acute rejection were C4d positive. In the CAN group, 5/11 cases (45%) were C4d positive. In conclusion, these results demonstrated that frozen-IHC and paraffin-IF can be considered alternative techniques to frozen-IF for C4d detection. The paraffin-IHC technique displayed the lowest concordance rate for C4d detection.

Screening biomaterials for functional complement activation in serum

Complement plays an important role in the immune attack against invading microorganisms. However, blood-contacting medical device biomaterials lacking specific complement inhibitors, with free hydroxyl and/or amino groups, or with absorbed antibodies, may inappropriately activate complement. Inappropriate activation by either the antibody-mediated classical or the antibody-independent alternative pathway may have well-known acute or poorly understood chronic effects on the host or device. This article describes methods for screening biomaterials for functional whole complement activation using normal human serum, or specifically for alternative pathway activation using C4-deficient guinea pig serum, or for classical pathway activation using both sera in combination. Detailed protocols are available as standard methodologies from the American Society for Testing and Materials (ASTM F1984, ASTM F2065, and ASTM F2567). Results obtained with these functional tests are confirmed by detection of classical and alternative pathway-specific markers C4d and Bb, respectively. These methods demonstrate dose and time-course activation of complement to beaded agarose, cellulose acetate, and purified alginate as examples of biomaterials. Significant difference in a functional endpoint denoting specific complement pathways is an appropriate screening method for complement activation by medical device biomaterials which might result in adverse events when the device is implanted or contacts human blood.

Diagnostic efficacy of C4d immunostaining in the detection of the humoral component of renal allograft rejection and therapeutic implications

In the current scenario of renal transplantation, the role of immunological methods in the detection of C4d has emerged as a useful adjunct in the recognition of acute humoral rejection (AHR). Few reports of this nature are available from the Indian context although there are several from the Western literature. To study the humoral component of renal allograft rejection in patients presenting clinically with graft dysfunction by histopathological detection of polymorphs in the peritubular capillaries and the expression of C4d using immunological techniques, as well as the response of patients to appropriate antirejection therapy. This study from a tertiary care center reemphasizes the importance of recognition of AHR as a cause of renal allograft dysfunction. Percutaneous renal biopsies were obtained from 40 postrenal transplant patients and evaluated for C4d using immunofluorescence and immunohistochemical methods. SPSS software. Positive expression of C4d was seen in a total of 19/40 cases (44.4%) indicating immunological evidence of AHR. Diffusely positive cases were treated with IV immunoglobulin therapy, plasmapheresis and Rituximab following which graft function was restored. Patients with minimal to focal positive expression of C4d responded well to pulse steroids and change in immunosuppressive therapy. C4d staining is a useful adjunct to routine histopathological methods in evaluating the humoral component of acute renal allograft dysfunction and helps in planning appropriate antirejection therapy with the goal of achieving long-term graft survival.

Humoral and cellular rejection after combined liver-kidney transplantation in low immunologic risk recipients

Combined liver-kidney transplantation is considered a low risk for immunologic complication. We report an unusual case of identical ABO liver-kidney recipient without preformed anti-human leukocyte antigen (HLA) antibodies, transplanted across a T- and B-cell-negative cross-match and complicated by early acute humoral and cellular rejection, first in the liver then in the kidney. While analyzing the immunologic complications in our cohort of 12 low-risk combined liver-kidney recipients, only one recipient experienced a rejection episode without detection of anti-HLA antibody over time. Although humoral or cellular rejection is rare after combined kidney-liver transplantation, our data suggest that even in low-risk recipients, the liver does not always systematically protect the kidney from acute rejection. Indeed, the detection of C4d in the liver should be carefully followed after combined liver-kidney transplantation.

Analysis of Renal Transplant Protocol Biopsies in ABO-Incompatible Kidney Transplantation

Numerous studies have shown that protocol biopsies have predictive power. We retrospectively examined the histologic findings and C4d staining in 89 protocol biopsies from 48 ABO-incompatible (ABO-I) transplant recipients, and compared the results with those of 250 controls from 133 ABO-compatible (ABO-C) transplant recipients given equivalent maintenance immunosuppression. Others have shown that subclinical rejection (borderline and grade I) in ABO-C grafts decreased gradually after transplantation. In our study, however, subclinical rejection in the ABO-I grafts was detected in 10%, 14% and 28% at 1, 3 and 6-12 months, respectively. At 6-12 months, mild tubular atrophy was more common in the ABO-C grafts whereas the incidence of transplant glomerulopathy did not differ between the two groups (ABO-C: 7%; ABO-I: 15%; p = 0.57). In the ABO-I transplants, risk factors for transplant glomerulopathy in univariate analysis were positive panel reactivity (relative risk, 45.0; p < 0.01) and a prior history of antibody-mediated rejection (relative risk, 17.9; p = 0.01). Furthermore, C4d deposition in the peritubular capillaries was detected in 94%, with diffuse staining in 66%. This deposition, however, was not linked to antibody-mediated rejection. We conclude that, in the ABO-I kidney transplantation setting, detection of C4d alone in protocol biopsies might not have any diagnostic or therapeutic relevance.

Peritubular capillary C4d deposition in lupus nephritis different from antibody-mediated renal rejection

C4d deposition in peritubular capillaries (PTC) has been used as a marker of antibody-mediated rejection (AMR). However, PTC C4d deposition is not described in patients with lupus nephritis (LN). C4d deposition in PTC was detected in 455 patients with biopsy proven LN in the present study. Renal tissues from 21 cases of acute AMR served as controls. C4d deposition in PTC was found in 31 patients (6.81%) with LN. Patients with PTC C4d positive showed higher SLEDAI score and higher frequency of hypocomplementemia as compared to C4d negative. The prevalence of ANA, anti-dsDNA, anti-Sm and ACA were higher in C4d positive group, and most of these patients showed the diffuse proliferative glomerular lesion. In contrast with acute AMR, the staining pattern of C4d was granular deposition and the detection of C4d along PTC was accompanied by deposition of IgG and C1q or C3. Electron dense deposition on PTC was observed in most of LN patients. In conclusion, C4d deposition in PTC could be found in a small part of patients with LN. Our study suggested for the first time that C4d positive deposition were close relation with the higher disease activity of LN, and that immune complex formation might be involved in PTC C4d deposition in LN patients, Such PTC C4d deposition is distinct from that of AMR.

Significance of C4d staining in ABO-identical/compatible liver transplantation

Complement degradation product C4d has become an important marker of humoral or antibody-mediated rejection in renal and heart allograft biopsies. Although there have been several reports on the detection of C4d in liver allografts, the significance of C4d in liver transplantation and its relationship with humoral rejection are still not clear. We investigated the frequency and pattern of C4d staining in liver allograft biopsies with reference to preoperative lymphocyte crossmatch tests, which detect donor-reactive lymphocyte antibody. Survival rates at 5 years were 77% for crossmatch-negative patients and 53% for crossmatch-positive patients (P=0.009). In crossmatch-negative patients, reproducible positive staining was obtained in 28 of 86 (33%) biopsies taken within 90 days after transplantation and 33 of 96 (34%) biopsies 90 days or after transplantation. Most C4d staining was observed in the portal areas, and no clear correlation was observed between C4d positivity and histological diagnosis. In crossmatch-positive patients, 9 of 11 (82%) biopsies showed positivity for C4d. C4d stained perivenular areas as well as portal areas. Histology of crossmatch-positive patients included acute rejection and cholangitis, but did not include periportal changes that were seen in humoral rejection in ABO-incompatible liver transplantation. In summary, focal C4d deposition was seen in various types of liver allograft injury and had little clinical impact on crossmatch-negative patients, but extensive C4d staining in crossmatch-positive patients may be associated with humoral rejection and poor graft survival.

The classification and treatment of antibody-mediated renal allograft injury: Where do we stand?

Since the acceptance of the detection of C4d in allografts as a reliable tool to mark a humoral alloresponse, de novo antibody-induced graft injury has attracted much attention. Antibodies and B cells are the new frontier in transplantation. At this juncture carefully designed studies are critical in order to gain solid diagnostic, therapeutic, and prognostic knowledge about the role of antibodies in graft injury and to avoid any confusion and misconception. One prerequisite is the strict adherence to refined classification systems of renal transplant rejection that carefully split and categorize different phenotypes of humoral mediated graft damage and ideally also include information on anti-donor antibody specificity and titers. Sun and colleagues follow this concept and provide evidence that mixed cellular and antibody-mediated graft rejection can respond favorably to intensified immunosuppression with tacrolimus and mycophenolate mofetil. What will the future bring to treat rejection episodes with a dominant, co-dominant, or minor antibody response?

C4d staining of renal allograft biopsies: a comparative analysis of different staining techniques

Detection of C4d along peritubular capillaries (PTC) in renal allograft biopsies is an independent prognostic marker of poor long-term graft survival. It is typically associated with circulating donor-specific antibodies. Since only little information is available on the best technique to stain C4d, we compared the two methods most often used for detecting C4d in renal allograft specimens. We investigated the expression of C4d along PTC in 64 renal allograft biopsies using a monoclonal antibody (Quidel) and immunofluorescence for frozen (F-IF) and a polyclonal antibody (Biomedica) and immunohistochemistry for formalin-fixed and paraffin-embedded (P-IHC) tissue samples. We compared the staining extent (diffuse, focal, minimal, no staining) in frozen and paraffin sections and evaluated the intra- and inter-observer concordance rates using kappa statistics. In addition, we determined the inter-observer concordance in 240 paraffin-embedded biopsies of a multi-centre study. The inter- and intra-investigator concordance rate (kappa = 0.9) of analysing the C4d expression by F-IF was excellent. In contrast, the detection of C4d by P-IHC demonstrated a substantially lower prevalence and extent of C4d expression with a lower intra- and inter-observer concordance rate (kappa = 0.3). Only 69% of diffuse and 13% of focal C4d-expressing cases were in line classified by F-IF and P-IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by P-IHC than by F-IF. The inter-observer concordance rate in paraffin of the 64 renal biopsies and the multi-centre study was good, but not perfect (kappa = 0.57 or 0.67). C4d staining determined on frozen tissue samples using F-IF with a monoclonal antibody appears to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor-specific antibodies.

The fate of C4d positive kidney allografts lacking histological signs of acute rejection

C4d deposits in renal transplants are known to be an independent risk factor of graft failure. The current analysis evaluates the impact of C4d deposits on graft function and survival in renal transplants without morphological signs of rejection. We retrospectively analyzed diagnostic transplant biopsies performed due to allograft dysfunction from June 1994 to June 2001 at the University Hospital in Basel. Grafts/patients with focal or diffuse positivity of C4d along peritubular capillaries; absence of morphological signs of acute cellular and/or humoral rejection; up to 3 year follow-up analysis post index biopsy. Patients treated with anti-rejection therapy or an increase in maintenance immunosuppression post biopsy (intervention group = IG) were compared to patients with unaltered immunosuppression (standard group = SG). Study group: 22 biopsies/patients out of 400 biopsies (5%) were included into the study, 17 in the SG and 5 in the IG. Patient survival (1-/3-years): SG: 100/94%, IG: 80/80%; graft survival censored for death (1-/3-years): SG: 82.5/68.8%, IG: 100/100%; serum creatinine (micromol/l) at index biopsy/1-year/3-years: SG: 221 +/- 70/231 +/- 103/245 +/- 124, IG: 217 +/- 100/143 +/- 28/177 +/- 55; acute rejection episodes within 1 year post index biopsy: SG: 4 (4 patients), IG: 1 (1 patient); all differences not significant. Lowest serum creatinine within 4 weeks post index biopsy (IG vs. SG): 108 +/- 25 vs. 181 +/- 61, p = 0.02. C4d positivity in kidney transplants lacking histological evidence of acute rejection is not associated with rapid functional graft deterioration, even in untreated cases. However, anti-rejection therapy results in the improvement of kidney function. Thus, even in grafts with normal histology, the detection of C4d in diagnostic biopsies can be interpreted as a sign of "smoldering" rejection that benefits from therapy.