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Saudi Journal of Kidney Diseases and Transplantation
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Year : 2018  |  Volume : 29  |  Issue : 1  |  Page : 39-49
C4d-negative antibody-mediated rejection: A pathologist's perspective and clinical outcome

1 Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology, G. R. Doshi and K. M. Mehta Institute of Kidney Diseases and Research Center and Dr. H. L. Trivedi Institute of Transplantation Sciences, Civil Hospital Campus, Ahmedabad, Gujarat, India
2 Department of Nephrology and Transplantation Medicine, G. R. Doshi and K. M. Mehta Institute of Kidney Diseases and Research Center and Dr. H. L. Trivedi Institute of Transplantation Sciences, Civil Hospital Campus, Ahmedabad, Gujarat, India

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Date of Web Publication15-Feb-2018


Banff'13 update included C4d-antibody-mediated rejection (ABMR) as a separate entity responsible for graft dysfunction with limited clinical/prognostic implications. We present a retrospective study to determine the incidence and outcome of C4d-negative ABMR. A total of 987 renal allograft (RA) biopsies obtained from 987 RA recipients were studied from January 2013 to January 2016. All samples were subjected to light microscopy using standard stains and C4d immunohistochemistry on paraffin sections and reported according to modified Banff’s criteria. Adequate biopsies with immunological injuries were categorized as Group 1: pure ABMR, Group 2: combined ABMR with concurrent T-cell-mediated rejection (TCR), and Group 3: pure TCR. Groups 1 and 2 were further subgrouped as C4d positive (Group 1a and 2a) or C4d negative (Group 1b and 2b). Graft function was measured by serum creatinine (SCr) level (mg/dL). Of the 987 biopsies, 43.3% (404) biopsies revealed immunological injury. Of these, 27.7% of the biopsies revealed pure ABMR (Group 1), 60.6% revealed combined ABMR with TCR (Group 2), and 11.3% revealed pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%, of which C4d-negative rejections were 18.48% and 18.7% in Group 1 and Group 2, respectively. The mean SCr at the end of three years follow-up in patients with C4d-negative rejections was comparatively higher. C4d-negative ABMR, recently included in Banff’13, has a low incidence, usually presents early after transplantation but carries better outcome than C4d-positive ABMR. However, further long-term studies are still required for knowing the clinical course over years.

How to cite this article:
Nigam LK, Vanikar AV, Kanodia KV, Patel RD, Suthar KS, Patel HV. C4d-negative antibody-mediated rejection: A pathologist's perspective and clinical outcome. Saudi J Kidney Dis Transpl 2018;29:39-49

How to cite this URL:
Nigam LK, Vanikar AV, Kanodia KV, Patel RD, Suthar KS, Patel HV. C4d-negative antibody-mediated rejection: A pathologist's perspective and clinical outcome. Saudi J Kidney Dis Transpl [serial online] 2018 [cited 2023 Jan 29];29:39-49. Available from: https://www.sjkdt.org/text.asp?2018/29/1/39/225206

   Introduction Top

Renal transplantation (RT) is a well-established therapy for patients with end-stage renal disease. Renal allograft dysfunction (RAD) is a common and complex problem of RT. Appropriate management is critical for long-term graft function and survival. Various causes of immunological and nonimmunological RAD have been identified. In addition, there are several types of rejection that can occur in a RA including hyperacute, accelerated acute, acute, and chronic active rejections.[1] The recognition of the transplanted organ as foreign is mediated by complex immunologic pathways, which in general terms can be divided into cellular (T-cell mediated) and humoral (antibody/B-cell mediated) pathways. In spite of significant developments in the diagnostic methodologies for diagnosis of RAD, biopsy still remains the gold standard and provides valuable insights into the pathogenesis of early and late allograft injury.[2],[3],[4] Acute rejection (AR) episodes are a major determinant of RA survival and long-term outcome of RT.[5] Acute antibody-mediated rejection (ABMR) occurs in 5%–7% of all RTs and is responsible for 20%–48% of AR episodes. The prevalence of chronic ABMR varies from 5% at one year to 20% at five years.[6] ABMR generally has a worse prognosis and requires a different form of therapy than T-cell-mediated rejection (TCR).[7] The detrimental effect of preformed donor-specific antibodies (DSAs) became apparent in the 1960s, but the possible pathogenic role of de novo DSAs production against allografts after RT remained controversial for many years until 1970, when it was suggested that de novo production of DSA against a transplanted donor organ can lead to severe graft injury. Alloantibodies are now appreciated as important mediators of acute and chronic rejection, differing in pathogenesis or “nature,” from TCR. Alloantibodies preferentially attack a different “location,” namely the peritubular and glomerular capillaries in contrast to T-cells which characteristically infiltrate tubules and arterial endothelium. ABMR is caused by antibodies directed against donor-specific human leukocyte antigen (HLA) molecules or other cell antigens. The most common mechanism underlying ABMR is an amnestic robust antibody response that originates from previous antigenic exposure or de novo development of DSA.[8] Other implicated causes could be the presence of antibodies that are directed against HLA/major histocompati-bility complex (MHC) Class I and II antigens. Antibodies can be directed against other donor-specific antigens such as MHC Class I-related chain A antigens, MHC Class I-related chain B antigens, platelet-specific antigens, molecules of the renin–angiotensin pathway, and polymorphisms involving chemokines and their receptors.[9],[10] Halloran et al in the early 1990s established that AR associated with the development of de novo anti-HLA DSA in recipient’s serum is a defined clinicopatho-logic entity carrying a poor prognosis and postulated that the “complement-neutrophil pathway is probably the major mechanism of antibody injury.” They were the first to show that neutrophils in peritubular and glomerular capillaries are strongly associated with circulating anti-donor HLA antibodies.[11],[12] Feucht et al showed that peritubular capillary (PTC) C4d deposition in RT biopsies is strongly associated with a poor prognosis and raises the possibility that antibodies are responsible for graft loss.[13]

C4d is a fragment of complement C4b, an activation product of the classic complement pathway. C4b (and C4d) contains an occult sulfhydryl group that forms a covalent, thio-ester bond with nearby proteins on activation by antibody and C1. C4b/C4d remains bound in the tissue for several days after Ig and C1 have been released. Following activation and degradation of the C4 molecule, thioester groups are exposed which allow transient, covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activation. C4d is also found in intracytoplasmic vacuoles of endothelial cells. Covalent binding renders C4d, a stable molecule to be easily detected by immunohistochemistry. Detection of C4d is regarded as an indirect sign, a “footprint” of an antibody response.[13] No functional role of C4d per se has been reported. C4d deposition is strongly associated with circulating antibody to donor HLA Class I/II antigens and is currently the best single marker of complement-fixing circulating antibodies to the endothelium.[7] Feucht et al suggested that capillary C4d deposition was an evidence for humoral alloreactivity against the graft.[13] Valenzuela et al demonstrated that staining of allograft biopsies for the fragment C4d is a specific and reliable method for identifying lesions due to allo-antibodies against HLA Class I and/or Class II antigens.[14] The specificity of capillary C4d staining for anti-HLA alloantibody-dependent graft injury was further demonstrated recently by Böhmig et al.[15] ABMR presents most of the time as severe allograft dysfunction and its prompt diagnosis and optimal treatment are essential. Nearly 20%–30% of all AR episodes have a humoral component. Taken together, C4d staining of RA biopsies has emerged as an important tool for the diagnosis of antibody-dependent allograft injury independent from its occurrence early or late after transplantation.[16] Antibodies can mediate endothelial injury through complement-dependent and independent mechanisms. On the other hand, under certain conditions, antibodies can lead to graft accommodation through upregulation of complement regulatory or cytoprotective proteins. It depends on the specificity and concentration of the antibodies: high-titer DSAs may cause ABMR, while recipients with low-titer DSAs can develop accommodation. Endothelial cells are targets for immunemediated assaults through antiendothelial cell anti-bodies.[15],[17] Antibodies against intracellular adhesion molecule-1, vimentin, actin, tubulin, and cytokeratin have been implicated in RA loss. The presence of DSAs within the graft does not necessarily mean activation of the complement, pathological changes, and graft dysfunction, since accommodation may occur.[18]

We decided to undertake a review of RABs performed in our center with a view to determine the incidence of recently devised C4d-negative ABMR and evaluate their outcome in terms of graft function and survival. Patients of this group were compared to those having C4d-positive ABMR or TCR.

   Materials and Methods Top

This was an Institutional Review Board approved retrospective study of indicated diagnostic RABs performed from January 2013 to December 2015. A panel of five pathologists independently reported and consensus diagnosis generated was finally reported. The demographic data regarding patient age, gender, indication for RA biopsy, and serum creatinine were collected. Graft function was measured by SCr levels (mg/dL).

Formalin-fixed biopsy specimens were processed for light microscopy and C4d immuno-histochemistry as per manufacturer’s protocol. For light microscopy, 3 μm thick sections were stained with hematoxylin and Eosin, Gomori’s trichrome, periodic acid–Schiff, and Jones silver methenamine stains. The C4d staining was performed on 3 μm thick paraffin sections using “Novolink™ Polymer Detection System” (Leica Biosystems, Germany) with rabbit antihuman C4d monoclonal antibody (clone SP91, Spring Bioscience, USA) and Novolink Polymer Anti-rabbit Poly-HRP-IgG. Biopsies negative for C4d were re-stained to rule out analytical errors associated with staining procedure. Membranous/lupus nephropathy slides were used as positive controls. Endothelial cells lining the medium caliber blood vessels were taken as internal control.

Inclusion and exclusion criteria

Patients whose biopsies revealed immuno-logical rejection were included in the study. Patients who underwent ABO incompatible transplants and those who were lost for follow-up were excluded from the study.

Reporting of renal allograft biopsy


Adequacy–Optimal biopsy was defined as a specimen with at least 10 nonsclerotic glomeruli and two arteries, a marginal biopsy having seven to nine glomeruli and one artery and a minimally acceptable biopsy having seven glomeruli and one artery. Specimens with <7 glomeruli or no arteries or with only medulla were considered as inadequate for interpretation. Histological categories were classified as per Revised Banff’13 diagnostic categories for RA biopsies.

Definition of C4d-positive ABMR – ABMR was defined if there was the presence of circulating DSA along with glomerulitis and peritubular capillaritis on histopathology with the presence of C4d deposition across the PTC membranes with an intensity of ≥2+ on immunofluorescence and C4d >0 by immuno-histochemistry on paraffin-embedded sections. However, we mainly performed immunohisto-chemistry due to lack of availability of tissue for the frozen section in all biopsies. Acute thrombotic microangiopathy (TMA) in the absence of any other cause was also reported as ABMR.

Glomerulitis (g>0) – Glomerulitis was defined as the percentage of glomeruli that revealed infiltration by leukocytes in the capillaries. Score g1, g2, and g3 were offered if <25%, 26%–50%, and >50% of glomeruli showed leukocytic infiltration, respectively.

Peritubular capillaritis – The percentage of PTCs in the renal cortex that contained neutrophils or mononuclear cells was defined as peritubular capillaritis. A score of ptc1 was offered if <10% PTCs revealed cells, ptc2 when 10% of PTCs were infiltrated by <5 cells, and ptc3 if more than 10% of PTCs revealed >10 cells/PTC.

C4d scoring – Demonstration of PTC C4d deposition by immunohistochemistry was considered positive if grade was >C4d0. Score of C4d1, C4d2, and C4d3 was given if 1–9%, 10%–50%, and >50% PTCs were involved, respectively.

Definition of C4d-negative ABMR – C4d-negative ABMR was defined as biopsies with C4d <2 on immunofluorescence or C4d >0 on IHC, ptc> 0 and g >0, g+ptc ≥2, or g>0 or ptc >0 and acute TMA in the absence of any other cause of TMA.

Definition of TCR – TCR was defined as biopsies with tubulitis (t> 1) along with vasculitis (v>0) and/or interstitial inflammation (i>2).

Definition of ABMR with TCR – Biopsies with C4d-negative ABMR which also had histological evidence of TCR also had g ≥1 so as to qualify the criteria of ABMR.

Quantitative estimation of DSA by single antigen (SA) testing – HLA Class I and Class II antibody specificity screening was performed with Single Antigen Beads (One Lambda, Canoga Park, CA, USA). Screening tests for anti-HLA-specific IgG antibodies were performed using LABScreen® Single Antigen beads, Class I and II (One Lambda Inc., Canoga Park, CA, USA). The assays were performed on Luminex platform following the manufacturer’s protocol. Trimmed mean fluorescence intensity (MFI) values were obtained from the output file generated by the flow analyzer and normalized using the formula: [(Sample #N beads – Sample negative control (NC) beads) – (NC serum #N beads – NC serum NC beads)]. Any normalized MFI value over 1000 was considered positive.

Study design

All the biopsies were compared for mean posttransplant and mean follow-up time period in months. SCr levels (mg/dL) at the time of biopsy and at last follow-up were compared. Patient and graft loss data along with SCr levels were used to determine patient and graft survival. After analyzing the light microscopic features, the biopsies were categorized into three main groups. Group 1 comprised of biopsies that revealed ABMR, Group 2 combined ACR +ABMR, and Group 3 with ACR. Group 1 and Group 2 were further subdivided on the basis of presence (C4d positive) and absence (C4d negative) of C4d deposits on PTC membranes. Group 1a and Group 2a included biopsies with PTCs that demonstrated C4d deposition (C4d positive) and Group 1b and Group 2b included biopsies that revealed the absence of C4d (C4d negative) on PTC membranes.

Anti-rejection regimen

All the patients who developed rejection were subjected to antirejection regimen which was decided according to the type of rejection reported on biopsies. Patients who developed cellular rejection were treated with intravenous methyl prednisolone 500 mg/day for 3–5 days; rabbit anti-thymoglobulin (r-ATG) 1.5 mg/kg BW was added for resistant cases. C4d-positive ABMR was treated with three sessions of plasmapheresis (with 40 mL/kg BW/session) followed by i.v. Ig 5 g on alternate days and subsequent bortezomib, 1.3 mg/m2 on days 1, 4, 7. and 11 or rituximab, 50–300 mg single dose. For combined TCR + ABMR, methylprednisolone and rATG (in doses mentioned above) were administered followed by plasmapheresis. Patients with C4d-negative rejection were offered monotherapy with either bortezomib or rituximab. None of these patients underwent therapeutic plasma exchange.


The death-censored graft loss was determined by the time between the date of transplantation and either date of graft failure indicated by need for dialysis, date of death, or last date of follow-up with a functioning graft. Patient death or dialysis dependency was the proposed end points of this study. We compared the death-censored graft loss against each subgroup.

   Statistical Analysis Top

All continuous parameters were expressed as mean and one standard deviation and all qualitative variables as a proportion. The data were subjected to Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS Inc., Chicago, IL, USA) and analyzed. Continuous data follow nonnormal distribution. Mann–Whitney U-test was used to calculate the significant value. P <0.05 was considered statistically significant.

   Results Top

A total of 987 RA biopsies from 987 patients were analyzed; 5.3% biopsies were excluded from the study. Of the remaining 934 allograft biopsies, 43.3% (404) biopsies revealed immunological injury on histology. Out of these, 27.7% biopsies revealed pure ABMR (Group 1), 60.6% mixed ABMR + TCR (Group 2), and 11.3% pure TCR (Group 3). The overall incidence of ABMR (pure ABMR + ABMR with TCR) was 36.27%. The incidence of C4d-negative rejections was 18.48% and 18.7% in Group 1 and Group 2, respectively. The incidence of C4d-negative ABMR was less than that of C4d-positive ABMR.


Of the 404 patients having biopsy-proven immunological rejection, 80.4% were males and 19.5% were females. The incidence of only C4d-positive ABMR (Group 1a) in males was 19.05%, and in females, it was 3.7%. The incidence of C4d-negative ABMR (Group 1b) was 4.7% and 0.49% in males and females, respectively. In Group 2 (n = 245), 11.6% females and 37.6% males developed C4d–positive ABMR with TCR, and 1.98 % females and 9.4% males developed C4d-negative ABMR with TCR. The mean age of patients was comparable in all the groups and statistically insignificant (P >0.5) [Table 1].
Table 1: Demographics of the study population.

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Mean posttransplant period

The time of allograft biopsies ranged from five days to eight years posttransplant. The mean posttransplant period (in months) varied in all groups. C4d-positive ABMR (Group 1a) had the shortest period of presentation of 1.32 ± 0.28 months followed by C4d-negative ABMR-positive TCR (Group 2b), who presented at 1.33 ± 0.15 months. Patients with C4d-negative ABMR (Group 1b) presented at 1.51 ± 0.27 months posttransplant. The Group 3 patients (TCR) presented at 1.61 ± 0.66 months posttransplant in our study.

When biopsies were grouped into those that presented within one year and those that presented after one year, the incidence of C4d-negative ABMR (pure + ABMR with TCR) was 20.2% within one year, whereas nearly 75.5% were C4d positive. Thus, in our study, we had more C4d-positive rejections than C4d–negative ABMR [Table 2].
Table 2: Incidence of C4d-positive and C4d-negative antibody-mediated rejection.

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Prebiopsy serum creatinine levels

The mean SCr (mg/dL) at the time of biopsy in patients with C4d-positive ABMR (pure ABMR with ABMR+TCR) was 2.5 ± 0.24 mg/dL which was higher than 2.11 ±0.32 mg/ dL in patients who had C4d-negative ABMR (pure ABMR with ABMR+TCR). However, this difference was statistically not significant (P = 0.05). On comparing the serum creatinine levels of each subgroup, it was observed that patients with pure C4d positive had a higher SCr level (2.74 ± 0.35) than any other subgroup [Table 3].
Table 3: Comparison of serum creatinine levels at the time of biopsy.

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Qualitative donor-specific antibodies estimation

All the patients who revealed immunological injury were subjected to qualitative DSA estimation and we found a 100% positivity in patients having ABMR. DSA was absent in all the cases with TCR[Table 4].
Table 4: Comparison of mean HLA match and graft loss in each group.

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Human leukocyte antigen match

The mean HLA match in the various groups was compared. The mean HLA match in patients with pure AMR with C4d positivity (group 1a) was 2.2. In this group, 32 patients were transplanted across the HLA match. The mean HLA match in C4d-negative ABMR group was 1.5. We observed that better the HLA match, better was the graft survival in all the groups.

Present serum creatinine levels

The mean SCr levels (mg/dL) were compared at the end of mean follow-up period. The mean SCr level in the C4d-negative group was 1.85 ± 0.56 mg/dL as compared to 1.93 ± 0.21 mg/dL in patients having C4d-positive ABMR (pure as well as those with concurrent TCR). However, the mean SCr level in C4d-negative ABMR (pure) was higher as compared to the control and other groups. Since the C4d- positive group was treated aggressively with plasmapheresis as compared to the C4d– negative group, the SCr levels tended to be less than in patients with C4d-negative ABMR. This shows that C4d-negative injury does affect the graft function if not treated aggressively and causes graft attrition in the long-term [Table 5].
Table 5: Comparison of present serum creatinine levels.

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Graft survival

The graft survival of all the groups is depicted in the Kaplan–Meier survival plot. The overall graft survival appears to be better in the C4d-negative group as compared to the C4d-positive ABMR group in spite of the higher mean SCr levels in this group. The graft loss at mean follow-up period was maximum in C4d positive ABMR (pure-Group-1a). Nearly 6.4% of the grafts were lost in this group. There was no graft loss reported in the C4d-negative ABMR (pure-Group 1b). With concurrent TCR, the graft loss in patients with C4d-positive ABMR (Group 2a) was 4.6% as against with C4d-negative ABMR (Group 2b) where we lost two grafts (2.5%). Thus, broadly considered, graft loss appears to be higher in the C4d-positive group as compared to the C4d-negative group (5.2% vs. 2.5%) [Figure 1].
Figure 1:

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   Discussion Top

Consistent and robust staining procedures for detection of C4d deposition in allograft biopsies have become an important part of the diagnostic workup of renal RAD since treatment protocols including combination of plasmapheresis, IV Ig, immunoadsorption, tacrolimus/mycophenolate mofetil rescue, and rituximab among others, which are targeted at antibody-mediated allograft damage, are being developed for suspected cases of ABMR.[10],[19],[20] The most recent Banff meeting update highlights two major phenotypes of ABMR. The first type appears early in the posttransplant period in a presensitized patient and is more likely to be C4d positive.[10],[19],[20],[21] The second type develops late posttransplant and is due to de novo DSA development and is likely to be C4d negative. The second phenotype is an important factor in late graft loss; however, not many studies have been published regarding the clinical significance of this subset of patients. It appears that Class II HLA molecules may be responsible and that much of the endothelial damage is mediated by NK cells and, to a lesser extent, monocytes and neutrophils; antibody-dependent cell-mediated cytooxicity.[22] C4d-negative ABMR usually occurs >12 months after transplantation but can occur acutely in highly sensitized patients with persistent DSAs (even after desensitization). Technical issues related to type of fixative used, different methods of C4d detection, poor complement fixation by some DSAs, complement-independent pathway of ABMR are some causes of C4d negativity. Autoantibodies to the angiotensin II type 1 receptor have also been associated with graft loss and fibrinoid necrosis that resembles alloantibody-mediated rejection except for the common absence of PTC C4d. Fc receptors on NK cells (FcRIIA) may also play a role in AR, and it is possible that some examples of AHR in biopsies that lack C4d are due to this mechanism.[7],[23],[24],[25],[26],[27],[28],[29],[30],[31],[32] Few studies have described the relationship of C4d-negative ABMR on protocol biopsies with worse graft survival. Loupy et al reported that significant proportion of patients with ABMR transit between negative C4d staining and positive C4d deposition in the peritubular capillaries.[33] Studies have concluded that C4d– negative ABMR tends to occur slightly later posttransplant and has a subclinical presentation with better graft outcome than C4d– positive ABMR.[21],[34],[35],[36]

The overall incidence of ABMR was higher in our study. This could be attributed to various causes such as transplantation across HLA barriers, multiple transfusions, presensitized patients, and multiple pregnancies in women apart from low hygienic conditions prevailing in this country. We demonstrated that there is no difference in demographic data of patients meeting with different immune injuries. Sis et al reported that expression of endothelial cell-associated transcripts (ENDATs) is present in all types of rejection but is significantly higher in ABMR. Only 13/50 kidneys (26%) with high ENDATs and DSA were C4d positive. Only 38% of kidneys with high ENDATs and DSA that subsequently developed chronic ABMR were C4d positive.[17],[18],[19],[20],[21],[22],[23],[24],[25],[26],[27]

The overall incidence of C4d-negative ABMR in our study was 18.2%, which is much less than C4d-positive ABMR (72.3%). C4d–negative ABMR when occurs alone, usually presents later than C4d-positive ABMR (1.51 vs. 1.32 months); however, if concurrent TCR is also seen then it usually presents earlier than C4d-positive ABMR (1.33 vs. 1.98 months).

C4d-positive ABMR, when it occurs along with TCR, presents late (median: 1.98 months) when compared to any other group. Our observations are in concordance with the observations of Orandi et al, who reported an incidence of C4d-negative ABMR of 2.5%, which was less than the incidence of C4d-positive ABMR cohort.[21] In a study conducted by Loupy et al,[33] the prevalence of C4d-negative ABMR was 49% versus 31% cases that showed C4d-positive ABMR. Mark Haas also concluded that C4d-positive and C4d-negative ABMR show similar frequencies of concurrent cell-mediated rejection and both can occur early or late posttransplant. Our observations were also similar to the study of Haas.[34]

The mean SCr level at the time of biopsy in Group 1b (C4d-ABMR pure) was 1.88 ± 0.29 mg/dL, followed by Group 2a patients having combined ACR + ABMR with C4d positivity (mean SCr: 2.05 ± 0.47 mg/dL). The mean SCr levels at the time of presentation was low in the C4d-negative ABMR groups. The cellular rejection group usually presents with lower SCr levels and presents early in the posttransplant period. However, the mean SCr over the mean follow-up period appears to be higher in Group 1b (C4d-negative ABMR) when compared to other groups. Similar observations were made by Orandi et al,[21] with mean SCr levels at the end of follow-up being 2.1 mg/dL in C4d-negative group against 1.6 mg/dL in C4d-positive ABMR group. The reason for high SCr levels in the C4d-negative group may be attributed to not subjecting these patients to aggressive management.[6],[26]

The overall graft survival of patients with C4d-negative ABMR was 97.5% as against C4d-positive ABMR which was 95.5%. Our results are similar to those proposed by Orandi et al,[21] where the graft survival was better in C4d-negative ABMR group than the cohort of C4d-positive ABMR (90.2% vs. 77.7%).

Although associated with better graft survival, the C4d-negative ABMR does have a poorer graft function as reflected by the higher serum creatinine levels. This proves that although negative for C4d, this immune injury is responsible for graft attrition over time and can contribute considerably to the deleterious outcome as observed by Sis et al, Haas, Loupy et al, and Orandi et al. Thus, this subset of patients also needs to be treated aggressively like those with C4d-positive ABMR irrespective of concurrent or absence of TCR.

   Limitations Top

Our study cohort with C4d-negative ABMR was not re-biopsied during follow-up. Hence, the possibility of C4d negative converting to C4d positive that was demonstrated by Orandi et al and others could not be determined. We did not perform quantitative DSA estimation due to financial constraints and compare with biopsy results.

   Conclusion Top

The incidence of C4d-negative ABMR is strikingly low in our study. The demographic profile or clinical symptoms are not helpful in determining the risk factors for the development of C4d-negative ABMR. In our study, C4d-positive and C4d-negative ABMR shows similar frequencies of concurrent cellmediated rejection and both can occur early or late posttransplant. Graft outcomes were comparatively better in the cohort of patients who developed C4d-negative ABMR as compared to those who have C4d-positive ABMR. However, longitudinal prospective studies are required to know the clinical significance and long-term outcome of C4d-negative ABMR. Molecular studies would be helpful to determine antibodies responsible for C4d-negative ABMR to design targeted therapy approach to this group so as to prevent the morbidity associated with this type of injury and improving graft survival in this cohort of patients.

Conflict of interest: None declared.

   References Top

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Troxell ML, Weintraub LA, Higgins JP, Kambham N. Comparison of C4d immuno-staining methods in renal allograft biopsies. Clin J Am Soc Nephrol 2006;1:583-91.  Back to cited text no. 11
Troxell ML, Higgins JP, Kambham N. Evaluation of C4d staining in liver and small intestine allografts. Arch Pathol Lab Med 2006;130:1489-96.  Back to cited text no. 12
Feucht HE, Schneeberger H, Hillebrand G, et al. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int 1993;43:1333-8.  Back to cited text no. 13
Valenzuela NM, Reed EF. Antibodies in transplantation: The effects of HLA and non-HLA antibody binding and mechanisms of injury. Methods Mol Biol 2013;1034:41-70.  Back to cited text no. 14
Böhmig GA, Exner M, Habicht A, et al. Capillary C4d deposition in kidney allografts: A specific marker of alloantibody-dependent graft injury. J Am Soc Nephrol 2002;13:1091-9.  Back to cited text no. 15
Watschinger B, Pascual M. Capillary C4d deposition as a marker of humoral immunity in renal allograft rejection. J Am Soc Nephrol 2002;13:2420-3.  Back to cited text no. 16
Sis B, Halloran PF. Endothelial transcripts uncover a previously unknown phenotype: C4d-negative antibody-mediated rejection. Curr Opin Organ Transplant 2010;15:42-8.  Back to cited text no. 17
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Correspondence Address:
Dr. Lovelesh Kumar Nigam
Department of Pathology, Laboratory Medicine, Transfusion Services and Immunohematology, G. R. Doshi and K. M. Mehta Institute of Kidney Diseases and Research Center and Dr. H. L. Trivedi Institute of Transplantation Sciences, Civil Hospital Campus, Asarwa, Ahmedabad, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1319-2442.225206

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  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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