C4d Detection Research Articles

Comparison of C4d Detection on Erythrocytes and PTC-C4d to Histological Signs of Antibody-Mediated Rejection in Kidney Transplantation

C4d on erythrocytes (EC4d), C4d peritubular capillary deposition (PTC-C4d) staining and histology were compared in a cross-sectional cohort of 146 renal allograft biopsies (132 patients). EC4d levels paralleled PTC-C4d staining, but were more predictive of peritubular capillaritis (PTC). Donor-specific antibodies (DSA), PTC-C4d, EC4d and PTC were analyzed in an independent longitudinal follow-up cohort (96 biopsies, 76 patients). Seventy-six samples were PTC and EC4d concordant, 11 positive and 65 negative, 7 PTC-EC4d+ and 13 PTC+EC4d-. EC4d levels were related to DSA occurrence. With ABMR defined by PTC and DSA, all apparently discordant patients, EC4d negative, were correctly reassigned comparing EC4d level curves with rejection kinetics, with positive EC4d samples predating biopsy or late biopsies compared with ABMR flare-ups. All EC4d-positive patients without PTC or DSA had permanent high EC4d levels unrelated to rejection. EC4d was more abundant in PTC-positive (mean = 108.5%± 3.4; n = 50) than PTC-negative samples (mean = 88.1%± 1.3; n= 96; p < 0.0001). Sensitivity, specificity, positive predictive value and negative predictive value of PTC-C4d and EC4d for PTC were, respectively, 75%, 79%; 64%, 76% (p < 0.05); 28%, 46% (p < 0.05) and 93%, 94%. Values were similar for DSA. A noninvasive blood test, EC4d, and particularly longitudinally monitoring EC4d levels, may increase surrogate ABMR testing options.

Sinusoidal C4d deposits in liver allografts indicate an antibody-mediated response: Diagnostic considerations in the evaluation of liver allografts

There is a paucity of data concerning the correlation of complement component 4d (C4d) staining in liver allografts and antibody-mediated rejection. Data about the location and character of C4d deposits in native and allograft liver tissues are inconsistent. We performed C4d immunofluorescence (IF) on 141 fresh-frozen liver allograft biopsy samples and native livers, documented the pattern of C4d IF staining, and correlated the findings with the presence of donor-specific alloantibodies (DSAs). A linear/granular sinusoidal pattern of C4d IF was noted in 18 of 28 biopsy samples obtained after transplantation from patients with positive crossmatch and detectable donor-specific alloantibody (pos-XM/DSA) findings. None of the 59 tested biopsy samples from patients with negative crossmatch and detectable donor-specific alloantibody (neg-XM/DSA) findings were C4d-positive (P < 0.001). No significant association was found between pos-XM/DSA and C4d IF staining in other nonsinusoidal liver compartments. To compare the results of sinusoidal C4d staining with IF and 2 immunohistochemistry (IHC) techniques, C4d IHC was performed on 19 liver allograft biopsy samples in which a sinusoidal pattern of C4d IF had been noted. Sinusoidal C4d IHC findings were negative for 17 of the 19 biopsy samples; 2 showed weak and focal staining, and both patients had pos-XM/DSA findings. Portal vein endothelium staining was present in only 1 IF-stained biopsy sample (pos-XM/DSA) but in 11 IHC-stained biopsy samples (2 of the 11 samples had neg-XM/DSA findings). We conclude that sinusoidal C4d deposits detected by IF in frozen tissue samples from liver allograft recipients correlate with the presence of DSAs and an antibody-mediated alloresponse. These observations are similar to findings reported for other solid organ transplants and can provide relevant information for patient management. Further validation of IHC techniques for C4d detection in liver allograft tissue is required.

Underway determination of dissolved inorganic carbon in estuarine waters by gas-diffusion flow analysis with C4D detection

The development and evaluation of a gas diffusion flow analysis system for the underway determination of dissolved inorganic carbon in marine and estuarine waters is described. Carbon dioxide produced when sample is injected into an acidic donor stream, diffuses through an efficient hollow fibre microporous membrane into an acceptor stream of ultrapure water, where the resultant changes in electrical conductivity are detected using a contactless capacitively coupled conductivity detector (C4D). The optimal parameters for the construction and operation of the C4D system are reported. Under field operational conditions, the flow analysis method had a linear calibration range for DIC of 0.2–10 mM, a limit of detection of 0.12 mM, repeatability of 0.46% RSD (n = 9 at 6 mM), a sample throughput of 90 h−1 and excellent correlation with comparative analyses (R2 = 0.9951, n = 16). The system was used to perform >250 determinations of DIC measurements underway during a short cruise on the Yarra River estuary, which demonstrated that DIC was conservative over much of the salinity gradient, with the exception of the low salinity region which exhibited the effects of respiratory CO2 and other DIC inputs. The results illustrate the advantages of the use of rapid flow analysis techniques for chemical mapping in transient environments like estuaries.

C4D Immunostaining in Surveillance Endomyocardial Biopsies From Well-Functioning Heart Allografts

Objective The meaning of biopsy C4d detection in heart allografts without dysfunction or morphologic changes suggesting antibody-mediated rejection (AMR) is not clear. The aim of this study was to search for an association between C4d detection in allograft biopsies of well-functioning hearts without changes suggestive of AMR, and clinical outcomes. Methods Endomyocardial protocol biopsies from 44 heart transplant patients with well-functioning grafts and without changes suggesting AMR were performed at 1 month and 1 year after transplantation and analyze the presence of C4d deposition using immunohistochemistry. Two-year follow-up was based on clinical parameters and echocardiographic information. Heart graft function was categorized as good vs. poor. The presence of C4d, using diverse schemes to graduate the extension of the deposition, was correlated with clinical graft outcomes. Results C4d deposition was observed in the capillary walls of 33 biopsies (37.5%; n = 25 patients; 56.8%). No biopsy had diffuse (>50%) immunostaining. Six patients presented with multifocal capillary C4d immunostaining in at least 1 biopsy. Capillary positivity for C4d (if focal or multifocal) showed no statistical association with cellular rejection or graft function. Perimyocytic C4d detection was neither associated with rejection nor graft outcome. Conclusion Our work failed to demonstrate an association between C4d detection in protocol biopsies of heart grafts and clinical outcomes. The clinical utility of C4d staining in solid organ transplantation may vary by organ. Our results suggest that C4d did not have clinical utility in surveillance biopsies of well-functioning heart grafts without morphological changes suggesting AMR.

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.

34: C4d Detection and Outcome in Long-Term Heart Transplant Recipients

Although the concept of antibody mediated rejection (AMR) has been associated with poor outcome in heart transplant (HT) recipients since long ago, only recently pathological criteria for its diagnosis have been standardized. Nevertheless, clinical correlates of criteria for AMR in endomyocardial biopsies (EMB) are poorly characterized. In this report, we analyzed the association of intravascular C4d detection with morphological criteria for AMR, hemodynamic features, and survival in mid and long-term HT recipients.

Effects of Hypoxia-Inducible Factor-1α Expression and C4d Deposition in Implantation Biopsies on Renal Allograft

Upregulation of hypoxia-inducible factor-1α (HIF-1α) in response to ischemic states has been suggested to have a role in the development of chronic allograft nephropathy. Deposition of C4d in the peritubular capillaries of renal allografts has been reported to be a sensitive marker of acute humoral rejection. The purpose of this study was to determine the effects of HIF-1α expression and C4d deposition in implantation biopsies of renal allografts. Implantation biopsies and 22 rejection proved biopsies were performed in 54 renal transplant recipients between December 1996 and July 1999. The mean follow-up was 82.8 months. Immunohistochemical studies were performed using a mouse monoclonal antibody for HIF-1α expression and a rabbit polyclonal antibody for C4d detection. HIF-1α was demonstrated in 19 of 54 implantation biopsies (35%), and C4d deposition in one (1.9%). The HIF-1α–positive group included a higher percentage of deceased donor organs (66.4% vs 17.1%; P = .002) and longer mean cold ischemia times (261.3 ± 231 vs 103 ± 40 min; P = .008) compared with the HIF-1α–negative group. The relative risks (95% confidence intervals) of expression of HIF-1α for allograft rejection, chronic allograft nephropathy, and graft loss were 1.53 (0.82–2.87), 0.61 (0.06–5.50), and 2.45 (0.62–9.85). The C4d-positive patient developed acute accelerated rejection on postoperative day 4. In the present study, the expression of HIF-1α showed a significant correlation with the use of a deceased donor kidney and with cold ischemia time. However, there were no significant effects on the prognosis for a graft after implantation of a kidney with HIF-1α expression.

C4d Immunohistochemistry in glomerulonephritis with different antibodies

The presence of C4d in the kidney is generally detected particularly for the diagnosis of antibody-mediated rejection in renal transplants. In frozen sections of immunofluorescence (IF) staining with anti-C4d monoclonal antibodies (mAbs), we noted intrinsic C4d deposition even in normal glomeruli though their pathogenic or an intrinsic role is unkown. An anti-C4d polyclonal antibody (C4dpAb), which is suitable for paraffin immunoperoxidase (IP) staining, is less used than mAbs, and it has demonstrated that intrinsic C4d is not evident. To establish a stable and reproducible procedure for C4d detection with the C4dpAb and to determine the staining characteristics of it, the present study aimed to test whether the method was comparable with IF with a mAb. We compared the C4dpAb with the mAb in adjacent sections of human diseased kidneys, and then compared IP with IF of C4dpAb. Two ways of antigen retrieval was examined for IP. On comparing the two antibodies for glomerular staining with IF, we found that the pattern and intensity (C4dpAb showed intrinsic C4d with IF) were similar. In addition, C4dpAb staining with IP and IF demonstrated that the intrinsic staining in the normal glomerulus was mostly undetectable by IP, whereas IF showed distinct staining. Likewise, C4d deposition with IP in some cases was apparently weaker than that on IF, suggesting that this deposition is not intrinsic but indicates pathogenic complement activation. The advantage of the C4dpAb for immunohistochemistry is of value for reconsidering the role of C4d in glomerular diseases.

Chronic Transplant Glomerulopathy

Chronic transplant glomerulopathy (CTG), investigated in a large single-center study by Akalin et al. (1), is an interesting pathologic lesion that develops in the renal allograft. It is characterized by endothelial cell injury or loss, accumulation of flocculent material in a widened subendothelial space, and mesangial interposition with deposition of new matrix along glomerular capillary walls. These progressive changes produce a thickened glomerular capillary profile and the capillary wall “double contours” that are demonstrable on periodic acid-Schiff and silver staining and that define CTG (2). Analogous changes occur in later stages of thrombotic microangiopathy (TMA) in the native kidney, initiated by endothelial injury and/or dysfunction. Development of this lesion in the renal allograft has been associated with worse graft outcome. CTG has been documented in the context of “chronic rejection” with antidonor antibody and may be strongly correlated with other features that are consistent with chronic/persistent/recurrent antibody-mediated rejection (AMR), including positive staining for C4d and multilayering of peritubular capillary basal lamina. Akalin et al. briefly reviewed this literature, including the seminal studies by Mauiyyedi et al. (3) and Regele et al. (4); however, studies of CTG correlation with C4d staining and detection of donor-specific antibody (dsAb) are emerging, and clearly many cases of CTG do not have these features at the time of biopsy. It is likely that the triad of CTG, C4d staining and presence of dsAb …

Incidence of C4d Stain in Protocol Biopsies from Renal Allografts: Results from a Multicenter Trial

C4d staining of renal allografts is regarded as an in situ marker of active humoral rejection. Few data are available about the incidence of C4d deposition in protocol biopsies compared to indication biopsies. To evaluate whether center-specific factors influence the incidence of C4d detection, we performed a multicenter study. From three European centers, 551 protocol and 377 indication biopsies were reclassified according to the updated Banff criteria and stained for C4d. C4d results were recorded as diffuse or focal positive and statistically correlated to clinical parameters, morphology and graft survival. In the protocol biopsies, a diffuse C4d stain was found in 2.0%, and a focal stain in 2.4%. In indication biopsies, 12.2% were diffusely and 8.5% focally C4d positive (protocol:indication p < 0.0001). The incidence of C4d deposition varied significantly between centers, attributable to variable numbers of presensitized patients with more C4d positive indication and protocol biopsies. Diffuse and focal C4d stain correlated with morphology of humoral rejection in protocol as well as in indication biopsies. Protocol biopsies show a significantly lower incidence of C4d deposition than indication biopsies. Subclinical C4d detection in protocol biopsies had no significant impact on allograft survival in our series.