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COMMENTARY
Year : 2020  |  Volume : 14  |  Issue : 3  |  Page : 197-201

C4d staining and antibody-mediated rejection in renal transplantation: Current status


Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad, Telangana, India

Date of Submission26-Mar-2020
Date of Acceptance31-May-2020
Date of Web Publication30-Sep-2020

Correspondence Address:
Dr. Praveen Kumar Etta
Department of Nephrology and Renal Transplantation, Virinchi Hospitals and Max Superspeciality Medical Centre, Hyderabad - 500 034, Telangana
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijot.ijot_22_20

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How to cite this article:
Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant 2020;14:197-201

How to cite this URL:
Etta PK. C4d staining and antibody-mediated rejection in renal transplantation: Current status. Indian J Transplant [serial online] 2020 [cited 2020 Nov 1];14:197-201. Available from: https://www.ijtonline.in/text.asp?2020/14/3/197/296882




  Introduction Top


Renal transplantation (RT) is one of the best modalities of renal replacement therapy for patients with end-stage renal disease as it offers survival and quality of life benefit over dialysis.[1] The antibody-mediated rejection (ABMR) plays an important role in both short- and long-term graft loss. Almost a third of patients who are waitlisted for RT may have a degree of anti-human leukocyte antigen (HLA) antibodies detected. The presence and development of anti-donor antibodies (anti-HLA, anti- ABO, or non-HLA antibodies) are associated with the risk of rejection after transplantation. The recipients with a history of blood transfusions, multiple pregnancies, and previous transplants are at higher risk for the development of donor-specific antibodies (DSAs).


  Anti-Human Leukocyte Antigen Antibodies Top


The degree of HLA mismatch influences long-term graft survival and risk of rejection. Among DSA, anti-HLA antibodies play a major role in graft rejection and survival. HLA typing and matching in relation to transplant immunology traditionally consider only antigens in HLA-A, B, and DR loci. Recent evidence has shown that antibodies against other HLA loci (especially HLA-DP and DQ antibodies, which mostly arise de novo after RT) also play a role in the rejection.[2] The “active ABMR” (previously termed “acute/active ABMR”) can occur due to alloantibody responses against both major histocompatibility complex (MHC) Class I and II HLA antigens, whereas, “chronic ABMR” is largely associated with DSA against Class II HLA antigens. The “epitope” matching may be superior over HLA antigen matching with respect to the prevention of de novo DSA formation and will enhance the prediction of acceptable HLA mismatches for sensitized patients.[3]


  Complement-Binding Donor-Specific Antibody Top


Serum immunoglobulin (IgG) molecules can be divided into four subclasses (IgG1–IgG4) with varying capacity to activate complement and recruit effector cells through the Fc receptor (IgG3 >IgG1 >IgG2 >IgG4). The complement activation contributes to allograft damage in most cases of ABMR. If complement-fixing DSAs (IgG1 or IgG3) are present and bind to donor cell antigens, the complement cascade will be activated via the classical pathway resulting in ABMR with complement C4d (C4d) deposition.[4] Recent evidence showed that complement-binding IgG3 subclass of DSA was more pathogenic and was associated with active (acute) ABMR, shorter time to rejection, increased microcirculation injury, and C4d deposition. The complement-binding antibodies can be identified by modified solid-phase assays (C1q, C3d, and C4d-binding assays) in addition to standard cell-based assays (complement-dependent cytotoxicity assay).[5] Noncomplement-binding IgG4 DSA was associated with subclinical and chronic ABMR (C4d negative), late allograft injury with increased transplant glomerulopathy (TG), and immunofluorescence (IF)/tubular atrophy lesions.[6]


  Complement System and C4d Top


C4d is a fragment of the classical complement pathway, which is activated by immune complexes. During complement activation, a cascade of events follows, with activation of several complement proteins, and C4 is cleaved into C4a and C4b, exposing a sulfhydryl group. The reactive sulfhydryl group of C4b rapidly forms as an ester or amide bond with nearby molecules containing hydroxyl or amino groups and forms C4d. The ability of C4d to crosslink to nearby proteins at the site of complement activation may explain why C4d remains for several days after alloantibody disappears since antibody binds to cell surface antigens that can be lost by modulation, shedding, or cell death. C3d, produced during complement activation, has been suggested as an indicator of more complete complement activation. However, the normally high background of C3 deposition in tubular basement membranes makes C3d much harder to interpret than C4d [Figure 1]. Although C4d is mainly interpreted as a product of classical pathway activation, C4 can also be generated via the lectin pathway. Consequently, C4d may be generated without prior antibody binding.
Figure 1: Complement pathways leading to C4 activation. CRP: c-reactive protein, MBL: mannose-binding lectin, MASP: Mannose-binding lectin-associated serine proteases

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  C4d Staining of Renal Allograft Biopsies Top


The central diagnostic criterion for humoral rejection/ABMR is the demonstration of C4d in peritubular capillaries (PTCs) and vasa recta. C4d deposition in PTC is the most specific indicator of the presence of circulating DSA and its interaction with endothelial cells in the graft. There is a strong correlation between the presence of C4d deposition and circulating anti-donor HLA antibodies. There are three methods used for C4d detection which use either frozen tissue for immunofluorescence (IF) (monoclonal antibodies are more commonly used than polyclonal antibodies) or formalin-fixed paraffin-embedded (FFPE) tissue for immunohistochemistry (IHC) (using polyclonal antibodies).[7]

The sensitivity of IF using a monoclonal antibody to C4d in frozen sections is greater than immunoperoxidase stains using the polyclonal antibody in FFPE tissue. Staining for C4d by IF was described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to the 2003 Banff conference. Medullary vessels are typically positive and can be the only place of C4d positivity in some cases with marked edema and cortical injury. In IHC, strong staining is usually not seen as tissue pretreatment influences staining intensity. IHC demonstrated a substantially lower prevalence and extent of C4d deposition in PTC and had a lower reproducibility than IF. In one study, only 69% of diffuse and 13% of focal C4d expressing cases were in line classified by IF and IHC. On average, the estimated area of C4d-positive PTC in the diffuse group was 36% lower by IHC than by IF.[8] Another study demonstrated that IHC with anti-C4d polyclonal antibody has acceptable sensitivity and specificity, as compared with IF staining with the monoclonal antibody (the overall specificity of the IHC method compared with IF was 98%, and sensitivity was 87.5%).[9] Sometimes, the plasma in the capillaries is fixed by the formalin processing and also stains for C4d by IHC, which interferes with interpretation. Extravasation of C4d into the connective tissue is also common and should not be mistaken for capillary wall deposition. Hence, intraluminal and interstitial C4d may also be seen and is an artifact of fixation. Nonspecific background staining is also common in IHC. The most sensitive method for C4d is the 3-step indirect IF on frozen sections using one of the monoclonal antibodies. However, many prefer to use the 2-step indirect IF method with the monoclonal antibody because of its simplicity, quick turnaround time, and relatively low cost.[10] Although more sensitive, IF requires extra biopsy sample (other than FFPE tissue) and frozen sections facility. As IHC is feasible in FFPE tissue, it can be easily done from biopsy tissue submitted for light microscopy study when frozen section facility (for IF) is not available or when extra biopsy tissue is not available for frozen sections. C4d can be detected in mesangial regions by IF (not IHC) in patients with no rejection. In the absence of PTC C4d staining, these isolated glomerular deposits are nondiagnostic for ABMR. The various conditions associated with C4d positivity are mentioned in [Table 1].
Table 1: Conditions associated with C4d positivity

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  Banff Schema and C4d Top


The Banff classification schema for the diagnosis and categorization of renal allograft pathology has included C4d deposition as an indicator of active (acute and chronic active) ABMR.[11] The presence of linear staining for C4d, a degradation product of the complement pathway that binds covalently to the endothelium, is highly suggestive of active ABMR. C4d serves as an “immunologic footprint” of complement activation and ABMR.[12]

In the 2001 Banff meeting, it was recognized that antibodies play an important role in causing rejection. Studies presented at this Banff conference showed that biopsies with C4d deposition had distinctly lower graft survival. Tubular HLA-DR expression was the morphologic feature most closely linked to the deposition of C4d. Hence, C4d staining of the PTC was accepted as the marker of ABMR and C4d as a marker was incorporated in Banff criteria published in 2003 (Banff '97 Update).[13] The Banff 2007 conference has suggested that C4d staining is indispensable and every renal allograft biopsy should be stained for C4d, and a grading system was proposed.[14] Recently, C4d scoring in medullary vasa recta was also included in addition to the extent of staining for C4d on endothelial cells of PTC, but the thresholds remain unchanged. Scoring of C4d staining is based on the percentage of PTC and vasa recta that has a linear, circumferential staining pattern. The minimal sample for evaluation is five high-power fields of the cortex and/or medulla without scarring or infarction. C4d must not be scored in the areas of infarction. The scoring of C4d is as follows: C4d0 – negative (no staining of PTC and medullary vasa recta [0%]), C4d1 – minimal (>0 but <10% of PTC and medullary vasa recta), C4d2 – focal (10%–50% of PTC and medullary vasa recta), and C4d3 diffuse (>50% of PTC and medullary vasa recta).[15] The Banff 2013 conference has proposed that C4d staining in at least 10% of PTC (>C4d1) by IF on frozen sections or in any of the PTC (>C4d0) by IHC on FFPE sections should be regarded as sufficient for the diagnosis of ABMR (minimum threshold for C4d positivity) in the presence of tissue injury, regardless of whether detectable DSAs are present.[16]


  C4d as a Surrogate Marker for Donor-Specific Antibody Top


In the olden literature, about 85%–90% of the patients with C4d positivity had circulating DSA, but these assays were less sensitive than currently available solid-phase assays. Cases in which C4d staining are positive but DSA cannot be detected may result from DSA being below the level of detection due to immunoadsorption by the graft or it can also be due to the presence of non-HLA antibodies (directed against non-MHC endothelial target antigens). The 2017 Banff conference has modified the diagnostic criteria for ABMR by stating that both C4d staining and validated molecular assays could serve as potential alternatives to DSAs in the diagnosis of ABMR and it also recognized C4d and molecular classifiers as surrogate markers for DSA. Hence, the recent update of the Banff classification introduced the diagnostic category “suspicious for ABMR” if C4d (in the presence of antibody) or alloantibody (in the presence of C4d) cannot be demonstrated, but morphologic evidence of ABMR is present.[17]


  Other Potential Uses of C4d Top


The emergence of new complement targeted therapeutics (e.g., eculizumab) makes C4d a marker with the potential to identify patients who may possibly benefit from these drugs. There is increasing evidence for the prognostic role of C4d deposition (glomerular >PTC C4d) in various native kidney diseases.[18] In relation to RT, this needs to be kept in mind as C4d may rarely indicate recurrence of the original disease (e.g., lupus nephritis), though the intensity, distribution, and staining pattern differ from ABMR.


  Limitations of C4d Top


In addition to the presence of circulating DSA, the diagnosis of ABMR requires the presence of immunopathologic staining for the C4d and histologic evidence of acute or chronic tissue injury [Table 2]. With the increasing use of C4d, several shortcomings of C4d have been identified and C4d appears to be a less sensitive marker than initially thought. C4d has a relatively low sensitivity as a marker for ABMR in late renal allograft biopsies. Some patients have morphologic evidence of ABMR and a positive DSA with little or no C4d staining. The prevalence of C4d-negative chronic ABMR ranges from 30% to 60%, as observed in patients who have other evidences of ABMR such as DSA with capillaritis or TG or increased levels of endothelial gene transcripts. The concept of C4d-negative ABMR was introduced in a retrospective study of 1036 allograft biopsies from 1320 RT recipients; only 36% of cases with TG had C4d-positive staining despite the presence of DSA in 73% of cases.[19] Additional evidence for C4d-negative ABMR came from a molecular study in which gene expression microarrays were performed in 173 indication biopsies to examine endothelial activation and injury transcripts (ENDATs). High expression of ENDATs correlated with histologic lesions of ABMR but not T-cell-mediated rejection. Among renal allografts with high ENDATs, positive DSA, and morphologic evidence of chronic ABMR, 60% were C4d negative.[20] In one Indian study, authors have noted the sensitivity of C4d by IHC in detecting acute ABMR as 55% and chronic ABMR as 23.5%, and concluded that C4d was not as sensitive a marker of ABMR, as was initially thought.[21] The heterogeneity of findings could be due to the variations in threshold of “C4d-negativity” used, the technique (IF vs. IHC) and the nature of biopsy (protocol vs. indication; early vs. late). Diagnostic criteria for C4d-negative ABMR were incorporated into the 2013 Banff update, as moderate microvascular inflammation (glomerulitis [g] + peritubular capillaritis [ptc] ≥2) or presence of ENDATs, in the absence of C4d (≤C4d1 by IF and C4d0 by IHC), with coexistent histologic evidence of tissue injury and presence of DSA.[22] Capillaritis with DSA and little or no C4d is more common in smoldering and chronic ABMR than active (acute) ABMR. Lack of C4d positivity can be attributed to decreased antigen expression, endothelial complement inactivation, or complement regulation at the level of the endothelium and lack of complement fixation by DSA and/or loss of PTC. Smoldering/indolent ABMR lacks features of active (acute) AMR (neutrophils, necrosis, and thrombosis) or chronic ABMR (increased matrix accumulation) but does have signs of ABMR, namely mild capillaritis (g or PTC) with or without C4d deposition/DSA. This has been shown to be a precursor for chronic ABMR over months to years in protocol biopsies. However, this subtype has not been incorporated into Banff schema. In C4d-negative ABMR, DSA binding to endothelial cells may cause injury through complement independent mechanisms – the innate immune cells such as neutrophils, macrophages, and natural killer cells can bind to Fc fragments of DSAs, trigger degranulation, and release lytic enzymes, which cause tissue injury and cell death (antibody-dependent cell cytotoxicity); DSAs can also cause graft injury by direct activation of endothelial proliferation through increasing vascular endothelial growth factor production, upregulating fibroblast growth factor receptor, and increasing its ligand binding as well as other signaling pathways for cellular recruitment.[23],[24]
Table 2: Revised Banff 2017 classification of antibody-mediated changes in renal allograft pathology

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C4d deposition in PTC without evidence of rejection is commonly seen in biopsies of ABO-incompatible allografts, in which it may represent graft accommodation. Hence, C4d lacks its utility as a marker for ABMR in ABOi allografts. ABO-compatible, nonsensitized patients show C4d deposition in 2%–4% of protocol biopsies in the 1st year with no immediate effect on graft survival. It could be an early stage of chronic ABMR, at least in the setting of HLA-incompatible grafts, and Banff consensus recommends careful monitoring of patients with this pattern.


  Conclusions Top


The central diagnostic criterion for humoral rejection is the demonstration of C4d in PTC and vasa recta. There is a strong correlation between the presence of C4d deposition and circulating DSA. Banff group has considered that C4d staining is indispensable and advised to stain all renal allograft biopsies for C4d. IF in frozen sections remains the technique of choice, with triple-layer IF probably the most sensitive. C4d has a relatively low sensitivity as a marker for ABMR in late renal allograft biopsies. The 2013 Banff conference has accepted the entity of C4d-negative ABMR. The 2017 Banff conference has recognized C4d and molecular classifiers as surrogate markers for DSA.



 
  References Top

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2.
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  In this article
Introduction
Anti-Human Leuko...
Complement-Bindi...
Complement Syste...
C4d Staining of ...
Banff Schema and C4d
C4d as a Surroga...
Other Potential ...
Limitations of C4d
Conclusions
References
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