Peritubular Capillary C4d Staining Research Articles

Treatment of Antibody-Mediated Rejection in Kidney Transplant Recipients: A Single-Center Experience With a Bortezomib-Based Regimen

Antibody-mediated rejection after kidney transplant is less responsive to conventional antirejection therapies. The proteasome inhibitor bortezomib has activity against mature plasma cells that produce damaging donor-specific antibodies. We present our experience of using a bortezomib-based regimen in patients with severe antibody-mediated rejection. A retrospective chart review was performed on patients with biopsy-proven antibody-mediated rejection after kidney transplant at our institution over 12 months. Diagnosis of antibody-mediated rejection was made on the basis of positive peritubular capillary C4d staining along with either histologic evidence of acute rejection or positive donor-specific antibody titers. Treatment for antibody-mediated rejection included plasmapheresis, intravenous immunoglobulin, steroids, single-dose rituximab (375 mg/m²) along with bortezomib (1.3 mg/m²) on days 1, 4, 8, and 11. Antibody-mediated rejection was diagnosed in 6 patients. Patients received induction with either alemtuzumab (n=4) or rabbit-antithymocyte globulin (n=2) and were maintained on a tacrolimus/mycophenolate mofetil/early steroid withdrawal protocol. Four of 6 patients responded to treatment. Patients had stable kidney function during followup (median 14 months) after bortezomib therapy. In this series, we demonstrated the effectiveness of a bortezomib-based treatment regimen in achieving reduction of donor-specific antibody titers and stable renal function in patients experiencing severe antibody-mediated rejection.

Glomerular C4d Immunoreactivity in Acute Rejection Biopsies of Renal Transplant Patients

In renal transplant patients, glomerulitis may be present in all types of acute rejection, often accompanied by diffuse C4d staining of peritubular capillaries: C4d3 positivity in more than 50% of peritubular capillaries. It may progress to chronic transplant glomerulopathy, characterized by capillary basement membrane multilayering, proteinuria, and progressive loss of renal function. While C4d3 is a recognized marker of an antibody-mediated reaction, the significance of glomerular C4d (GlC4d) staining is unknown. The aim of this study was to evaluate GlC4d immunoreactivity and its correlation with C4d3 in acute rejection biopsies. Paraffin-embedded acute rejection biopsies from 90 renal transplant patients were evaluated according to the Banff classification. Biopsies showing C4d-positive endothelial cells in more than 50% of glomeruli were considered GlC4d-positive. C4d3-positive staining prevalence was 23%. GlC4d-positive staining showed an 89% concordance rate (r = 0.81, P < .0001; Cohen's k = 0.80, P < .0001). GlC4d detection sensitivity was 0.80 and specificity 0.97. C4d3 and GlC4d immunoreactivity was significantly associated with glomerulitis (P < .006 and P < .03, respectively) and with proteinuria at the time of biopsy (P < .03 and P < .01, respectively). Interestingly, GlC4d positivity correlated better than C4d3 positivity with the presence of posttransplant circulating anti-human leukocyte antigen alloantibodies (P < .04 and P = .7, respectively). Patients with C4d3- or GlC4d-positive acute rejections underwent graft loss due to interstitial fibrosis and tubular atrophy more frequently than those with C4d0- or GlC4d-negative rejections (P < .0001 and P < .005, respectively), whereas no differences were observed in graft loss due to death. In conclusion, C4d3 and GlC4d stains showed a high correlation rate. Compared with C4d3, GlC4d staining demonstrated good sensitivity and excellent specificity. Our results suggested that GlC4d staining may indicate glomerular endothelial damage and be of prognostic value.

The Impact of C4d Staining as a Humoral Injury Marker

PurposeAcute 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. MethodsFrom 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. ResultsBiopsy 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. ConclusionsC4d staining refractory humoral rejection injury was related to poor graft outcomes.

Renal C4D Deposition Is a Marker of Hematopoietic Stem Cell Transplant (SCT)-Associated Thrombotic Microangiopathy (TMA)

C4d is a marker of classical complement activation by tissue specific antibodies (abs) or endothelial damage. Currently, peritubular capillary (PTC) C4d deposition aids in the diagnosis (dx) of ab mediated kidney transplant rejection (AMR). An uncontrolled case series reported PTC and glomerular C4d staining in patients with SCT-TMA (Mii et al, Path Int 2011). Expanding on these findings, we hypothesized that renal tissue from patients with SCT-TMA would more frequently show C4d deposition compared to controls. Retrospective analysis of all renal biopsy and autopsy specimens from pediatric patients undergoing SCT at our institution. Using histological criteria alone, patients were divided into TMA and non-TMA (control) groups. C4d staining was performed by immunohistochemistry on formalin-fixed, paraffin-embedded tissue and was evaluated on arterioles, PTCs, glomeruli, and tubular basement membranes. Specifically, rabbit anti-human C4d polyclonal ab was applied to sections and C4d was independently graded (diffuse: 50-100%; focal: 5-50%; rare: 1-5%; negative: 0%) by two pathologists, blinded to each subject's underlying dx. 20 total specimens were identified (19 subjects). Of these, 8 specimens (7 subjects) had histologic evidence of TMA (5 biopsy, 3 autopsy). 1 TMA subject had both a biopsy and autopsy with similar findings, therefore only the autopsy findings are included. The 12 SCT recipient autopsies without TMA served as controls. Diffuse and focal arteriolar C4d staining was far more common in TMA specimens compared to controls (71.4% versus 8.3%, p = 0.01). PTC C4d staining was present in just under half of TMA samples and was absent in controls (p = 0.04). Glomerular C4d staining was similar in both groups and tubular basement membrane staining was rare.TableC4d Deposition in TMAHistology proven TMA (n = 7)Control (n = 12)p-value∗from Fisher’s exact test for categorical variables and Wilcoxon Rank sum for continuous variables; data expressed as n (%) or median (25th-75th percentile) [range].Pathology Results Arteriolar C4d+5/7 (71.4%)1/12 (8.3%)0.01 PTC C4d+3/7 (42.9%)0/12 (0%)0.04 Glomerular capillary C4d+4/7 (57.1%)10/12 (83.3%)0.30 Tubular basement membrane C4d+2/7 (28.6%)0/12 (0%)0.12Clinical Results Age (years)3.6 (1.7-4.5) [0.6-4.9]3.2 (1.4-17.1) [0.9-28.1]0.55 Gender (male)3/7 (42.9%)8/12 (66.7%)0.38 Specimen day post-SCT237 (43 - 631) [19 - 2281]172 (78 - 253) [9 - 887]0.50 Malignancy5/7 (71.4%)2/12 (16.7%)0.03 Immunodeficiency1/7 (14.3%)9/12 (75.0%)0.03 Bone marrow failure1/7 (14.3%)1/12 (8.3%)0.03 Allogeneic2/7 (28.6%)11/12 (91.7%)0.01 Autologous5/7 (71.4%)1/12 (8.3%)0.01 GVHD in those at risk (allogeneic)1/2 (50%)3/11 (27.3%)1.00 Viral infection4/7 (57.1%)4/12 (33.3%)0.38 Dialysis6/7 (85.7%)5/12 (41.7%)0.15∗ from Fisher’s exact test for categorical variables and Wilcoxon Rank sum for continuous variables; data expressed as n (%) or median (25th-75th percentile) [range]. Open table in a new tab This is the first report of arteriolar C4d deposition in SCT-TMA, implicating localized complement fixation due to as yet unidentified abs or ab-independent direct endothelial damage in the pathogenesis of TMA. PTC C4d staining was found only in TMA, while glomerular staining was non-specific as it was similar in both groups. Although the preferential arteriolar C4d staining is incompletely understood, it may identify a renal site of injury, explaining the dramatic hypertension often seen in TMA. Future research should assess if patients with SCT-TMA and renal C4d deposition will benefit from therapies currently used to treat kidney transplant AMR.

C4d Staining In Renal Allograft Biopsies with Early Acute Rejection and Subsequent Clinical Outcome

Diffuse C4d staining in peritubular capillaries (PTCs) during an acute rejection episode (ARE) is the footprint of antibody-mediated rejection. In current clinical practice, diffuse C4d+ staining during acute rejection is regarded as an inferior prognostic sign. This case-control study investigated the prognostic role of mere C4d staining for graft outcome during an ARE in a well defined cohort of similarly ARE-treated patients. All kidney transplant recipients in the authors' center from January 1, 1995 to December 31, 2005 were reviewed. From these patients, 151 had a clinical ARE. Paraffin and/or frozen material was available for 128 patients showing a histologically proven ARE within the first 6 months after transplantation. All ARE patients were treated similarly with high-dose pulse steroids and in the case of steroid unresponsiveness with anti-thymocyte globulin. Biopsies were scored according to Banff criteria. Frozen and paraffin sections were stained by immunofluorescence (IF) and immunohistochemistry (IHC) for C4d, respectively, and scored for PTC positivity. Diffuse C4d+ staining in PTCs was found in 12.5% and 4.2% sections stained by IF or by IHC, respectively. Four patients showed diffuse positive staining with both methods but showed no different risk profile from other patients. No relation between C4d staining and clinical parameters at baseline was found. C4d staining was not associated with steroid responsiveness, graft, or patient survival. This study shows that C4d staining is not related to clinical outcome in this cohort of histologically proven early AREs.

Significance of Peritubular Capillary, Glomerular, and Arteriolar C4d Staining Patterns in Paraffin Sections of Early Kidney Transplant Biopsies

Although diffuse linear C4d deposition in peritubular capillaries (PTCs) is a well-established criterion of alloantibody-mediated kidney transplant rejection, the actual relevance of focal or granular C4d deposits or staining outside PTC (glomeruli and arterioles) has yet to be established. This study was designed to evaluate the diagnostic significance of such nontypical C4d staining patterns. A total of 539 early indication biopsies (329 kidney transplants) were analyzed by immunohistochemistry using a polyclonal anti-C4d antibody. We found a close interrelationship between diffuse or focal linear C4d deposition in PTC, linear endothelial deposition in glomeruli, and arteriolar C4d. These specific patterns were also related to transplant glomerulitis and recipient presensitization. No such associations, however, were observed for other patterns, such as granular C4d in PTC. Detection of diffuse but not focal linear C4d in PTC was found to be associated with adverse allograft survival (5-year death-censored graft survival: 48% vs. 82%, 89%, or 84% in patients with focal, minimal, or no C4d, respectively; P<0.0001). Univariate analysis also revealed inferior graft survival in recipients with linear C4d in glomeruli (P=0.02). Applying multivariate Cox regression analysis, however, only diffuse linear PTC staining was found to be predictive of graft loss (hazard ratio 3.95 [95% confidence interval 1.62-9.60]; P=0.002). There might be a relationship between humoral alloimmunity and distinct less established staining patterns, such as focal linear C4d in PTC, endothelial C4d in glomeruli, or arteriolar C4d. Nevertheless, our results reemphasize the prognostic value of diffuse linear PTC staining.

A Critical Appraisal of Methods to Grade Transplant Glomerulitis in Renal Allograft Biopsies

Transplant glomerulitis is an increasingly recognized lesion in renal transplant biopsies. To develop a refined grading system, we defined glomerulitis by the presence of ≥5 leukocytes/glomerulus and evaluated 111 biopsies using three different grading systems: (i) percentage of glomerular involvement, (ii) peak inflammation in the most severely affected glomerulus and (iii) presence/absence of endocapillary occlusion by inflammatory cells. Endocapillary occlusion had no impact on graft survival, but was associated with increased serum creatinine, proteinuria and subsequent transplant glomerulopathy. Grading based on either percent or peak glomerular involvement correlated with graft failure and peritubular capillaritis. However, the percent glomerular involvement method had the additional advantage of displaying associations with: concurrent proteinuria, focal or diffuse immunoperoxidase peritubular capillary C4d staining, 1-year postbiopsy serum creatinine, subsequent detection of donor-specific antibody and development of transplant glomerulopathy. Patients with >75% glomerular involvement also revealed persistent high-grade glomerulitis on follow-up biopsies despite antirejection treatment. In conclusion, grading of glomerulitis is a meaningful exercise, and a quantification system based on percentage of glomerular involvement shows the most robust associations with clinical parameters and prognosis.

Treatment of Acute Antibody-Mediated Rejection: A Single-Center Experience

Introduction Acute antibody-mediated rejection (AMR) leads to graft loss. The combination of plasmapheresis (PP), intravenous immunoglobulin (IVIG), and rituximab (RTX) has been reported to be effective therapy. Patients and methods Between October 2005 and September 2009, 8 (4.7%) kidney transplant recipients developed AMR, diagnosed by severe acute rejection and extensive C4d staining in peritubular capillaries. Results All patients were treated with two to six sessions of PP with IVIG added after the last PP. In two patients, RTX was prescribed after PP and IVIG. Baseline immunosuppression was based on steroids, mycophenolate mofetil or azathioprine, and tacrolimus or cyclosporine or everolimus. The presence of subsequent significant decrease in anti-HLA class I antibodies was demonstrated in a highly sensitized patient before and after transplantation with PP treatment. An increase was observed before the diagnosis of AMR. After a mean follow-up of 10 months (range = 1–23), patient and graft survivals were 100% and 50%, respectively. Three patients lost their transplants to AMR refractory to treatment and one patient, due to interstitial fibrosis and tubular atrophy at 23 months after AMR. Finally, four patients recovered renal function, showing a mean serum creatinine of 2.2 ± 0.45 mg/dL. Conclusions Early diagnosis and treatment with PP, IVIG, and RTX may resolve AMR. PP before and after transplantation in high-risk patients may result in anti-HLA class I and class II antibody removal from plasma and prevention of AMR.

C4d-Fixing Capability of Low-Level Donor-Specific HLA Antibodies Is Not Predictive for Early Antibody-Mediated Rejection

Recent studies indicate that not all low-level donor-specific human leukocyte antigen (HLA) antibodies (HLA-DSA) (i.e., positive by solid-phase assays, negative by complement-dependent cytotoxic-crossmatch) have a detrimental clinical impact. The aim of this study was to investigate whether the pretransplant C4d-fixing capability allows distinguishing harmful from presumably clinically irrelevant HLA-DSA. We retrospectively investigated 64 patients with low-level HLA-DSA detected by single-antigen flow beads (SAFB). Thirty-four of 64 patients (53%) experienced early antibody-mediated rejection (AMR), whereas 30 patients (47%) did not. HLA-DSA characteristics (i.e., number, class, and strength), frequency of retransplants, and immunosuppressive regimens were not different in these two groups. Pretransplant sera were reanalyzed using a modified SAFB assay measuring C4d fixation induced by HLA antibodies. C4d-fixing HLA-DSA were observed in 11 of 64 patients (17%). AMR occurred in 6 of 11 patients (55%) with C4d-fixing HLA-DSA and in 28 of 53 patients (53%) without C4d-fixing HLA-DSA (P=1.0). Positive C4d staining in peritubular capillaries was detected in 6 of 11 patients (55%) with C4d-fixing HLA-DSA and in 21 of 53 patients (40%) without C4d-fixing HLA-DSA (P=0.50). The pretransplant capability of low-level HLA-DSA to trigger C4d fixation in vitro is not predictive for early AMR or C4d deposition in peritubular capillaries in vivo. This argues against pretransplant C4d SAFB testing to define the clinical relevance of low-level HLA-DSA.

Focal C4d Staining in Peritubular Capillaries and Kidney Graft Survival: Results of a Retrospective Study

A diffuse positivity (≥50%) of C4d in kidney graft peritubular capillaries (PTC) significantly correlates with the presence of acute or chronic antibody-mediated rejection. In contrast, significance of a “focal” deposit (10%–50%) is not yet completely defined. The purpose of this study was to assess the impact of focal positive C4d staining on graft survival. We retrospectively reviewed 63 renal biopsies in 54 kidney transplant recipients. They were performed between January 2005 and December 2008 because of graft impairement, namely, a significant increase in serum creatinine and/or urinary protein. C4d positivity was assessed by immunohistochemistry on paraffin-embedded sections, in combination with conventional histopathologic evaluation. Biopsies were classified as negative (<10%) versus with focal (10%–50%) or diffuse deposits (>50%). Cumulative survival was calculated by the Kaplan-Meier method, and Cox regression analysis was used for the multivariate analysis. Focal C4d staining in PTC significantly correlated with worse graft survival ( P = .006), similarly to diffuse C4d staining. On multivariate analysis, focal C4d staining prognostically correlated with graft survival, but not recipient or donor age, prior transplantation, number of HLA mismatches or the presence of tubulitis in the sample. Focal C4d staining was associated with worse graft survival.

Proteasome Inhibitor-Based Primary Therapy for Antibody-Mediated Renal Allograft Rejection

Rapid and complete elimination of donor-specific anti-human leukocyte antigen antibodies (DSA) during antibody-mediated rejection (AMR) is rarely achieved with traditional antihumoral therapies. Proteasome inhibitor-based therapy has been shown to effectively treat refractory AMR, but its use as a primary therapy for AMR has not been presented. Our initial experience with proteasome inhibition as a first-line therapy for AMR is presented. Adult kidney transplant recipients with AMR, diagnosed by Banff criteria, received a bortezomib-based regimen as the primary therapy. Bortezomib therapy was administered per package insert with plasmapheresis performed immediately before each bortezomib dose, and a single rituximab dose (375 mg/m2) given with the first bortezomib dose. DSA were quantitated using single-antigen beads on a Luminex platform. Two patients underwent bortezomib-based therapy for acute AMR occurring within the first 2 weeks after transplantation. High DSA levels and positive C4d staining of peritubular or glomerular capillaries were present at the time of diagnosis. Both patients experienced prompt AMR reversal and elimination of detectable DSA within 14 days of bortezomib-based therapy. Renal function remains excellent with normal urinary protein excretion at 5 and 6 months after AMR diagnosis. One patient experienced a repeated elevation of DSA (including two new human leukocyte antigen specificities) 2 months after initial bortezomib therapy, but without C4d deposition or histologic evidence of AMR. Retreatment with bortezomib provided prompt, complete, and durable DSA elimination. Proteasome inhibitor-based combination therapy provides a potential means for rapid DSA elimination in early acute AMR in renal transplant recipients.

Late Acute Kidney Transplant Rejection: Clinicopathological Correlates and Response to Corticosteroid Therapy

Acute rejection is a major cause of kidney allograft dysfunction. It is important to distinguish between cellular and antibody-mediated rejection to guide the treatment strategy. The management of acute antibody-mediated rejection includes aggressive therapy with plasmapheresis and intravenous immunoglobulin. C4d staining of peritubular capillaries has emerged as a valuable tool in identifying antibody-mediated rejection. Late acute rejection has a worse prognosis than early acute rejection. The clinical and pathological features of late acute kidney allograft rejection are not fully understood. We studied the clinicopathological correlates of late acute rejection in our patient population. During an 8-year period, all patients who had late acute rejection (6 months posttransplant) were identified. Patients with severe chronic changes and transplant glomerulopathy were excluded. Patients were divided into C4d− and C4d− groups. Histopathological features and treatment response were evaluated. Nine patients met inclusion criteria (4 C4d+, 5 C4d−). Maintenance therapy consisted of mycophenolate mofetil, calcineurin inhibitors, and low-dose prednisone. All patients received intravenous methlyprednisolone or high-dose oral prednisone as antirejection therapy. Seventy-five percent of patients in the C4d+ group and 80% of patients in the C4d− group had a clinical response to antirejection therapy. The majority of C4d+ patients with late acute rejection who were treated with corticosteroids alone responded to treatment. The study raises the possibility that a subset of C4d+ patients with acute rejection who do not have severe chronic changes might respond to corticosteroid therapy alone.

The histological development of acute antibody-mediated rejection in HLA antibody-incompatible renal transplantation

The aim of this study was to examine the development of acute antibody-mediated rejection in HLA antibody-incompatible renal transplantation in relation to the Banff 07 histological classification. Renal biopsies were scored using the Banff 07 diagnostic criteria, and paraffin-embedded sections were stained with the pan-leucocyte marker CD45. Thirty-six patients had 72 renal biopsies. In biopsies performed 30 min after graft reperfusion, the mean number of CD45+ cells per glomerulus was higher than in control grafts (P < 0.04) and was associated with the donor-specific antibody (DSA) level at transplantation measured by microbeads (P < 0.01), and eight out of nine patients with greater than five CD45+ cells per glomerulus had early post-transplant rejection or oliguria, compared to 11 out of 20 with less than five cells per glomerulus (P < 0.01). In the first 10 days post-transplant, although peritubular capillary (PTC) leucocyte margination grade 3 and C4d deposition were specific for rejection, their sensitivities were low. PTC C4d staining was only seen in two out of 11 biopsies taken in the first 5 days after transplant, even in the presence of rejection, but was present in the majority of later biopsies with rejection. In biopsies stained for CD3, CD68 and CD20, it was notable that CD20+ cells were not seen during acute rejection, the infiltrates comprising CD3+ and CD68+ leucocytes. Glomerular margination of leucocytes occurred early after transplantation and was associated with DSA level and early graft dysfunction. The Banff 07 PTC margination scoring system was easy to apply, especially when CD45 staining was used, and PTC margination grade 3 was always associated with clinical rejection.

The significance of renal C4d staining in patients with BK viruria, viremia, and nephropathy

Peritubular capillary C4d staining in allograft kidney is an important criterion for antibody-mediated rejection. Whether BK virus infection can result in complement activation is not known. We studied 113 renal allograft biopsies from 52 recipients with a history of BK virus activation. The samples were classified into four groups according to the concurrent detection of BK virus DNA in urine, plasma, and/or biopsy: BK-negative (n=37), viruria (n=53), viremia (n=7), and nephropathy (n=16) groups. The histological semiquantitative peritubular capillary C4d scores in the viremia (0.3±0.8) and BK nephropathy (0.6±0.9) groups were lower than those in the BK-negative group (1.2±1.1, P=0.05 and P=0.06, respectively) and the viruria group (1.2±1.1, P=0.04 and P=0.06, respectively). Diffuse or focal peritubular capillary C4d staining was present in 9/76 (12%) and 14/76 (19%) of all samples with concurrent BK virus reactivation (viruria, viremia, and nephropathy). The diagnosis of antibody-mediated rejection could be established in 7/9 (78%) and 5/14 (36%) of these samples, respectively. Diffuse tubular basement membrane C4d staining was restricted to BK nephropathy cases (4/16, 25%). Semiquantitative tubular basement membrane C4d scores were higher in BK nephropathy (1.2±1.3) compared with BK-negative (0.05±0.3, P=0.017) and viruria (0.0±0.0, P=0.008) groups. Bowman's capsule C4d staining was more frequent in BK nephropathy (5/16) compared with the aforementioned groups (2/36 (P=0.023) and 4/51 (P=0.03), respectively). Within the BK nephropathy group, samples with tubular basement membrane stain had more infected tubular epithelial cells (12.1±7.6% vs 4.4±5.0%, P=0.03) and a trend toward higher interstitial inflammation scores. In conclusion, peritubular capillary C4d staining remains a valid marker for the diagnosis of antibody-mediated rejection in the presence of concurrent BK virus infection. A subset of biopsies with BK nephropathy shows tubular basement membrane C4d staining, which correlates with marked viral cytopathic effect.

Combination of Peritubular C4d and Transplant Glomerulopathy Predicts Late Renal Allograft Failure

The histologic associations and clinical implications of peritubular capillary C4d staining from long-term renal allografts are unknown. We identified 99 renal transplant patients who underwent an allograft biopsy for renal dysfunction at least 10 yr after transplantation, 25 of whom were C4d-positive and 74 of whom were C4d-negative. The average time of the index biopsy from transplantation was 14 yr in both groups. Compared with C4d-negative patients, C4d-positive patients were younger at transplantation (29 +/- 13 versus 38 +/- 12 yr; P < 0.05) and were more likely to have received an allograft from a living donor (65 versus 35%; P < 0.001). C4d-positive patients had more inflammation, were more likely to have transplant glomerulopathy, and had worse graft outcome. The combined presence of C4d positivity, transplant glomerulopathy, and serum creatinine of >2.3 mg/dl at biopsy were very strong predictors of rapid graft loss. C4d alone did not independently predict graft loss. Retrospective staining of historical samples from C4d-positive patients demonstrated C4d deposition in the majority of cases. In summary, these data show that in long-term renal allografts, peritubular capillary staining for C4d occurs in approximately 25% of biopsies, can persist for many years after transplantation, and strongly predicts graft loss when combined with transplant glomerulopathy.

Expression of the Decay-Accelerating Factor (CD55) in Renal Transplants—A Possible Prediction Marker of Allograft Survival

Decay-accelerating factor (CD55) accelerates the decay of C3 and C5 convertases, participating in classical and alternative complement activation pathways. Complement activation plays a major role in antibody-mediated rejection of allografts (AMR); C4d is used as a marker of AMR. Emerging evidence suggests an important role of CD55 in the pathogenesis of AMR. The aim of this study was to investigate the expression of CD55 in renal allografts and to correlate it with the expression of C4d, allograft survival, changes in serum creatinine (SC). More than 200 renal allograft biopsies, performed for allograft dysfunction, were assessed for peritubular capillary (PTC) C4d and CD55 expression. We found significant correlation between changes in SC and PTC CD55 staining pattern in patients with no PTC C4d staining. In these patients, SC increased from baseline by 2.2+0.34, 1.7+0.36, and 0.93+0.24 mg/dL in negative, focal, and diffuse PTC CD55 staining subgroups, respectively. Survival of renal allografts was better in diffuse PTC CD55 staining subgroup than in negative PTC CD55 staining subgroup. These data suggest that CD55 expression has a protective effect on PTC C4d negative renal allografts, and the pattern of PTC CD55 expression may be used as a potential marker of renal allograft survival in patients with no evidence of AMR.

Isoagglutinin titre adsorption: Breaking the barrier in major AB0-incompatible organ transplantation

Blood group AB0-incompatible live donor (LD) renal transplantation may provide a significant source of organs. AB0-incompatible LD renal transplantation can be accomplished using specific anti-A/B antibody (Ab) immunoadsorption (IA) and anti-CD20 monoclonal Ab (Rituximab ®) treatment. One dose of anti-CD20 mAb (rituximab, 375 mg/m 2) is given for weeks pre-operatively. Recipients are pre-treated with 3–5 sessions of immunoadsorption, using A or B carbohydrate antigen specific columns, until their anti-A1/B RBC panel indirect antiglobulin test (IAT) titres are <8. Polyclonal immunoglobulins are applied four days before transplantation. Recipients receive preoperative mycophenolic acid, steroids/tacrolimus therapy. No splenectomy was performed. Postoperative IA is applied if RBC panel indirect antiglobulin test (IAT) titres are >8. Postoperative renal biopsies may yield positive C4d staining in peritubular capillaries as sign of humoral immune activation but the clinical course is favourable if IAT titres can be kept below 8 within the first weeks after transplantation. This protocol has excellent short-term and long-term graft survival as well as function although recipient anti-A/B titres after transplantation return to pre-surgical levels within months after transplantation.

Neutrophilic Tubulitis as a Marker for Urinary Tract Infection in Renal Allograft Biopsies With C4d Deposition

Neutrophilic tubulitis accompanied by intratubular neutrophil clusters in the renal allograft is a surrogate marker for urinary tract infection (UTI). Overlapping histologic findings can occur in antibody-mediated rejection, which is characterized by peritubular capillary (PTC) deposition of C4d. This study evaluated the incidence of UTI in biopsies with concurrent neutrophilic tubulitis and PTC C4d staining. Thirty-three allograft biopsies from 27 patients selected for the presence of simultaneous C4d staining and neutrophilic tubulitis were correlated with urine culture (U/C) results. U/C obtained on the same day as the biopsy confirmed UTI in 13 of 33 (39%) biopsies. Among 20 patients with negative U/C; prior culture results within 10 days of the biopsy were available for nine patients, and 5 of 9 (55%) were positive. Thus, UTI was confirmed in 18 of 33 (54%) biopsies. Biopsy interpretation and clinical management was confounded by changes of concurrent acute cellular rejection and antibody-mediated rejection confirmed by demonstration of donor-specific antibodies. Combined therapy with antibiotics and antirejection medications (ART) was administered to 12 of 18 (67%) patients. Neutrophilic tubulitis accompanied by neutrophil clusters in the tubular lumen is a useful marker of UTI, even in the presence of PTC C4d deposition. Therapeutic response to antibiotics is limited by co-existent T-cell or antibody-mediated rejection and underlying chronic allograft nephropathy.

Clinical significance of the distribution of C4d deposits in different anatomic compartments of the allograft kidney

Diffuse C4d deposition in peritubular capillaries is a well-recognized marker of antibody-mediated rejection. The significance of staining patterns that are focal or affect non-peritubular capillary compartments is less well defined. Paired frozen section and paraffin-embedded tissue stains were performed in 52 kidney allograft biopsies, and correlated with clinicopathologic parameters. Diffuse peritubular capillary C4d deposits were more often seen in frozen sections (22/52, 43% frozen tissue vs 10/52, 19% paraffin-embedded tissue), whereas focal staining was observed more frequently within paraffin sections (13/52, 25% paraffin-embedded tissue vs 7/52, 14% frozen tissue). In biopsies taken from patients with a history of donor-specific antibodies, diffuse, focal and negative peritubular capillary C4d staining patterns were seen in 11/14 (79%), 1/14 (7%) and 2/14 (14%) of frozen biopsies vs 5/14 (36%), 6/14 (43%) and 3/14 (21%) of paraffin-embedded biopsies. Transplant glomerulopathy score in paraffin-embedded biopsies was higher in specimens with vs without glomerular basement membrane C4d staining (1.5±0.8 vs 1.0±0.6, P=0.03). Tubular basement membrane staining was present in 4% paraffin-embedded and 48% frozen specimens independent of tubular atrophy. Arteriolar hyalinosis score in paraffin-embedded specimens was higher in biopsies with vs those without arteriolar C4d deposits (1.3±0.9 vs 0.9±0.8, P=0.04). Arterial staining was unrelated to the degree of intimal thickening. In conclusion, peritubular capillary deposits correlate well with circulating donor-specific antibody. For paraffin-embedded tissue, combining the results of focal and diffuse staining allows a diagnostic sensitivity comparable to diffuse staining in frozen tissue. Finally, C4d deposits preferentially in lesions of chronic transplant glomerulopathy and arteriolar hyalinosis.

Renal biopsy findings predicting outcome in scleroderma renal crisis

Scleroderma renal crisis is irreversible in some patients despite aggressive treatment. This study was designed to identify pathologic prognostic features in scleroderma renal crisis. We retrospectively reviewed the pathology and the clinical records of 17 patients who underwent kidney biopsies during scleroderma renal crisis (group A, recovered renal function [n = 7]; group B, remained in renal failure or died [n = 10]). Multiple histologic features were assessed semiquantitatively (0-3) or as percentages. C4d staining of peritubular capillaries and small vessels was assessed semiquantitatively (0-3) in patients with scleroderma (n = 11), normotensive (n = 10), and hypertensive (n = 12) nonscleroderma native kidney controls. The percentage of thrombosed vessels (25.1 +/- 21.0 versus 5.6 +/- 12.3, P = .045) and the severity of glomerular ischemic collapse (2.9 +/- 0.3 versus 1.4 +/- 0.8, P = .001) were significantly higher in group B than in group A. Also, group B patients tended to have more severe acute tubular injury and vascular fibrinoid changes. The peritubular capillary C4d score in patients with scleroderma, normotensive controls, and hypertensive controls were 1.1 +/- 0.9, 0.3 +/- 0.7, and 0.3 +/- 0.5, respectively (P = .018, scleroderma versus other controls). Small vessel C4d score was higher in scleroderma compared to normotensive but not hypertensive controls. Within scleroderma samples, a significantly higher peritubular capillary C4d score (1.6 +/- 0.7 versus 0.3 +/- 0.5, P = .024) but not small vessel score was found in group B compared to group A. This tended to be associated with peritubular capillary leukocyte margination. Vascular thrombosis, severe glomerular ischemic collapse, and peritubular capillary C4d deposits in scleroderma renal crisis kidney biopsies correlated with increased risk of failure to recover renal function.