Indian Journal of Transplantation

: 2020  |  Volume : 14  |  Issue : 2  |  Page : 94--98

The relevance of complement C4d staining in renal allograft biopsies

Anju Khairwa 
 Department of Pathology, ESIC Model Hospital, Gurugram, Haryana, India

Correspondence Address:
Dr. Anju Khairwa
Department of Pathology, ESIC Model Hospital, Gurugram, Haryana


In renal transplant, the allograft is affected by many triggering agents such as innate and adaptive immune mechanisms, either mediated by macrophages and lymphocytes, or by soluble components antibodies and the complement system, which can ultimately lead to graft rejection. Antibody-mediated rejection (AMR) is a predominant cause of allograft failure. Donor-specific antibodies, mostly reactive to human leukocyte antigen antigens, are now considered by pathologists and clinicians as a significant cause of early and late graft dysfunction and failure. Complement 4d (C4d) is a fragment of the classical complement pathway (that is a part of component C4), which is activated by antigen-antibody complexes. The diagnosis of AMR improves by detection of the complement fragment C4d in renal biopsy, and it has included for diagnosis of AMR in the year 2003. There is more development about C4d after inclusion in the diagnostic criteria of AMR. This review aims to evaluate pathogenesis and current relevance of C4d in AMR.

How to cite this article:
Khairwa A. The relevance of complement C4d staining in renal allograft biopsies.Indian J Transplant 2020;14:94-98

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Khairwa A. The relevance of complement C4d staining in renal allograft biopsies. Indian J Transplant [serial online] 2020 [cited 2021 Jan 23 ];14:94-98
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Post kidney transplant, the allograft is affected by many stimulating agents such as antigen- antibody-mediated immunocomplexes and cellular immunity mediated by various cells (macrophages and lymphocytes), which could lead to graft rejection.[1] Based on etiology, the Banff criteria divided the rejection broadly in antibody-mediated rejection (AMR), and cell-mediated rejection.[2] AMR is a predominant cause of allograft failure. The clinicians and pathologists now consider antibodies against human leukocyte antigen (HLA) antigens defined as donor-specific antibodies (DSA) a remarkable cause of early and late graft dysfunction.[3]

Complement 4d (C4d) (that is part of component C4) is a fragment of the classical complement pathway, which is activated by immune complexes.[4] C4 is cleaved by C1s into C4a and C4b, exposing a sulfhydryl group. The active sulfhydryl group of C4b rapidly forms an amide or ester bond with nearby molecules containing hydroxyl or amino groups and forms C4d.[4] C4d makes covalent bond with the tissue. It stays at the site of complement activation for a longer time, in comparison to other complement pieces.[5] The diagnosis of AMR is improved by identification of the complement pieces C4d in renal biopsy, and it has been introduced first under diagnostic criteria of AMR in year 2003.[6] There is more development about C4d after inclusion in diagnostic tests of AMR. This review aims to evaluate pathogenesis and current relevance of C4d in AMR.

 Pathogenesis of Complement 4d (Complement System and Complement 4d)

Complement is an important component of innate and adaptive immunity in our body. Complement is the leading effector constituent of adaptive humoral immunity. There are three different pathways of complement activation and their activator, as shown in [Flow Diagram 1]. This flow diagram [Figure 1] is showing complement activation and C4d deposition in renal allograft biopsy.[7]{Figure 1}[INLINE:1]

In the classical pathway of complement kick over of C1 (which composed of C1q, C1r, and C1s) is begun by an interplay of C1q with immunoglobulin bound to epitopes on the graft endothelium. C1s splits C4 into C4a and C4b and react with exposing a sulfhydryl group. The ester or amide bond forms with interacting between sulfhydryl group of C4b and nearby molecules containing hydroxyl or amino groups in the tissue. C3 convertase (C4bC2a) of classical pathway is formed by combination C4b with enzymatically loose pieces of C2a. C4bC2a (C3 convertase) splits C3 into C3a and C3b. C3b has an active sulfhydryl group which is dative bound and settles in the immediate locality and forms C5 convertase (C4bC2aC3b). C5 convertase split C5 into C5a and C5b. Further, membrane attack complex (membrane-bound C5b-9) formed by the addition of C5b component of C5 convertase, that destroys cells.[8] The lectin pathway of complement activates by mannan-binding lectin (MBL) and H-ficolin or L-ficolin. The MBL attach to the suitable carbohydrate on apoptotic cells or pathogens.[9] MBL, H-ficolin, and is L-ficolin are homologous to C1q and fibrinogen, which are also activate C4 by their associated serine proteases, mannose-associated serine protease 1 (MASP-1 and MASP-2) (analogs to C1r and C1s). The C-reactive protein (CRP) also activates complement (C4) pathway. The CRP binds to carbohydrate and choline phosphate along with C1q.[10] The complement components C3a and C5a (call as anaphylatoxin) formed during this pathway.

 Clinical Relevance of Complement 4d

The conditions where complement 4d is positive

Following conditions are showing C4d positivity:[11],[12]

Acute AMRChronic antibody-mediated rejectionA, B, O blood group ABO incompatible graftsAccommodation.

The state where complement 4d is negative

The following conditions are showing C4d negativity:[10],[11],[12],[13],[14]

T-cell-mediated rejection (TCMR)A technical error like a type of fixatives, immunofluorescence (IF) versus immunohistochemistry (IHC)Fc receptor (FCR) on NK cells (FcRIIA) mediated rejectionAntibodies unable to fix the complementComplement independent pathways of endothelial activationC4d deposition is a very low in quantity for the identification limits of IF/IHCAlloantibodies can direct endothelium injury to interact with major histocompatibility complex (MHC)Increased expression of endothelial transcripts causing endothelium injury.

The C4d-negative AMR occur due to alloantibodies to MHC molecules on endothelial cells, elicit strong responses that include proliferation and activation of intracellular signaling pathways, which leads to endothelium injury.[12] The C4d-negative AMR also seen with high renal endothelial transcript expression in patients with alloantibody and is an indicator of active antibody-mediated allograft damage and poor graft outcome.[13]

 Antibody-Mediated Rejection

The AMR harms allograft kidney, chiefly in more sensitized patients. AMR accounts for up to 30% of all posttransplant rejection events and 20%–30% graft loss at 1 year in untreated cases.[15] AMR can occur in three forms, namely hyperacute, acute, and chronic rejection.[16] The presensitization (blood transfusion, pregnancy, and prior transplant) is a primary risk factor for hyperacute and acute rejection.[17] Acute AMR is most commonly occurs due to expose of MHC Class I and II HLA antigens.[17] Whereas, chronic AMR is largely associated with Class II DSA.[18]

Besides, other antigens which are responsible for AMR included MHC antigen (polymorphic) MICA (MHC Class I-related chain A) and ABO blood group antigens.[19],[20] Pathological findings of AMR- grossly kidneys become swollen due to interstitial edema and hemorrhage.[21] Microscopic findings of AMR- glomerulitis, peritubular capillaritis, fibrinoid necrosis of arteries, and acute tubular injury.[22] Other diagnostic features of AMR requires the simultaneous presence of DSA and C4d deposition in peritubular capillaries (PTCs).[23]

Hyperacute AMR is defined as rejection occurs within minutes or 24 h after transplantation, mostly due to preexisting DSA present in high titres present in a patient. Grossly, the graft rapidly becomes cyanotic and flaccid. Histomorphology characterized by arteritis, interstitial edema, and extensive cortical necrosis.[24] In acute AMR, C4d is deposited in PTC and glomerular capillaries. In chronic AMR, histomorphology characterized by transplant glomerulopathy (TG) in renal biopsies. The TG specified as capillary basement membrane duplication or splitting and mesangial expansion of glomeruli, tubular atrophy and interstitial fibrosis, and fibrous intimal thickening in arteries. Sometimes, electron microscopy also showed peritubular capillary basement membrane multilayering.[24],[25] The effector mechanisms of AMR (1) Direct effects of antibody to MHC, (2) Complement fixation, and (3) Cellular FCRs.[26],[27] The DSA produced by plasma cells bind to the endothelium of donor PTC and glomerular capillaries and start the pathological progression of AMR. The C1q is a component of the complement of the classical pathway which ties to the endothelium-binding DSA, finally leading to graft injury and dysfunction.[28] Diagnosis of acute AMR based on the identification of DSA in plasma, C4 complement component (C4d) in PTC, structural corroboration of AMR, such as kidney injury and graft dysfunction.[1],[29] Mixed cellular and humoral rejection are very hard to examine because it mostly occurs after months and years posttransplantation, and biopsy may show acute and chronic lesion.[30] Here, it is tough to prove that cellular or humoral immune system responsible for the extracellular injury.[30] There suggested that AMR is the chief cause of graft damage, which positive IHC for C4d in PTC in the combination of high levels of DSA in the serum than cellular rejection.[30],[31] If there is minimum or absent of c4d positivity in PTC and absent of DSA in plasma less likely AMR.[30] Some studies had shown by utilizing microarray investigation showed excessive NK cells in biopsies of patients with acute (in the form of PTCs, glomerulitis) and chronic AMR (in the form of TG).[32]

 Transplant Glomerulopathy

TG is a morphological change that is mostly seen in humoral rejection and related to immunological etiology.[33],[34] TG is a variant of chronic AMR and characterized by deposition of C4d. It proposed that TG may be one manifestation of humoral rejection of graft injury.[2],[33] Morphologically, TG consists of diffuse multilayering or duplication of the glomerular basement membrane (GBM). Their lack distinct de novo or repetitive glomerular lesion or evidence of thrombotic microangiopathies (TMA).[35] GBM duplication best seen jones methenamine silver and periodic acid-Schiff stain. In TG double counter of GBM occurs due to accumulation of fluff such as material and mesangial cells are get deposited at subendothelial space.[36]

IF and IHC show C4d deposition along the GBM presented in a minority of cases (6% in one series).[37]

 Transplant Glomerulopathy Also Associated Multilayering of Peritubular Capillarie

Ultra-structure features of TG consist of cytoplasmic vacuolation, broadening of subendothelial space, with the existence of electron-dense floccular material, probably due to stiffening of the lamina rara interna.[38]

Interstitial fibrosis and tubular atrophy existed along with TG. Arterial intimal fibrosis new-onset, with leukocytic infiltration in intima and absence of internal elastic lamellae, favors chronic rejection with TG.[11] TG was connected with the diagnosis of AMR, in the existence of either peritubular capillaritis or glomerulitis or both as well as with C4d expulsion in PTCs.[39]

 Complement 4d detection in Renal Allograft Biopsies

Two techniques are mostly used for identification of C4d IHC and IF in allograft biopsies of the kidney. IF technique applied to frozen sections whereas IHC technique used to paraffin-embedded tissue used routinely in various laboratories. In acute and chronic AMR-positive C4d stain with IF technique is described as “widespread, strong linear circumferential PTC staining in cortex or medulla, excluding scar or necrotic areas,” according to a consensus at 2003 Banff Conference.[6] In IHC, C4d staining is crisp, linear, continuous, diffuse, and lying around in the PTC wall, while the strength typically is slighter and changeable, but it may have a finely granular pattern in high power.[40] The scoring of C4d is significant by IF on frozen section ie C4d2 or C4d3 and by IHC C4d > 0 on paraffin sections.[11] The circulating DSA are most specifically associated with C4d deposition in PTC and its interactivity with endothelial cells in the graft.[41]

 Current Status of Complement 4d

Devadass et al. reported C4d is not very sensitive marker of AMR, as it originally thought. Histologically, moderate to marked microvascular inflammation (MVI) correlated with dispersing C4d positive cases in contrast to focal C4d-positive cases.[42]

C4d is significant staining in 1% or more of the PTC for formalin/IHC-IP, or 10% or more for frozen/frequency IF.[11] Interobserver and interinstitutional reproducibility for C4d IHC staining in renal allografts was poor but improved with a binary scoring system (positive/negative).[43]

Currently, C4d has become an essential investigation for AMR according to Banff update 2017.[44] In this update, both C4d and validated transcripts/classifiers/molecular marker can serve as potential alternatives and complements to DSAs in the diagnosis of ABMR.[44]

 Complement 4d negative Antibody-Mediated Rejection

There are many causes for C4d-negative AMR, as mentioned above. The C4d-negative AMR was first demonstrated Feucht et al., and Edmonton group.[45] The C4d-negative AMR is included in Banff 2013 classification.[11] The morphology of C4d-positive and C4d-negative AMR having following similar features: (1) varying degrees of glomerulitis and peritubular capillaritis, (2) frequent TCMR, (3) both may occur early or late posttransplantation.[11] However, C4d-negative AMR morphologically have higher intrarenal endothelial gene expression, alloantibodies expression, poor graft outcomes, usually occur after 1-year posttransplantation, and associated acute on chronic AMR.[46]

There is another entity of AMR DSA negative AMR, which characterized negative DSA/lack of DSA detection, moderate MVI with (g + ptc) scores of ≥2) as per Banff 2013 with or without C4d positivity.[47]

The accommodation is defined as C4d deposition in PTCs in the absence of active rejection with or without DSA positivity, mostly seen in ABO-incompatible graft transplantation.[48] The TMA (thrombotic microangiopathy) is a complication of AMR in the absence of other causes characterized with fibrin thrombi, fragmented red blood cells, mesangiolysis, and muco-intimal thickening and injury of small vessels.[49] According Banff diagnostic criteria, C4d staining in PTCs (at least 10% PTC positive is considered as significant) by IHC on paraffin sections Banff score C4d > 0 is significant. In contrast, by IFs on frozen sections Banff scores C4d2 or C4d3 is considered significant.[11],[50] Thus, it is indicated that C4d detection by IHC more specific and C4d detection by IF is more sensitive. The IHC method for C4d detection feasible in formalin-fixed, paraffin-embedded tissue, it is used when frozen sections facility not available, IHC also useful when small biopsy tissue or tissue not available for frozen sections.[51] The disadvantage of C4d staining by IHC are lower sensibility, nonspecific background staining, more costly and more time consuming, and it needs external controls.[51] IF method for C4d requires extra tissue and frozen sections facility.[51]


The C4d-positive staining in PTC is a diagnostic marker as well as a prognostic marker for allograft. According to various studies worldwide, C4d appears to be a less sensitive marker than initially thought.[52] The C4d as a biomarker has following PROS (1) provoked an enormous amount of insight in the diagnosis of allograft rejection, (2) core diagnostic tool to identify AMR, and (3) used for vast amount of research into the deposition patterns of C4d in different clinical settings such as pregnancy, thrombotic complications and CONS as follows: (1) difficulties of interpreting focal staining patterns, (2) relatively low sensitivity of C4d as a marker for AMR in late renal allograft biopsies, and (3) its lack of utility as a marker for antibody-mediated injury in biopsies of ABO-incompatible allografts).[52]

However, in which cases C4d not helpful in diagnosis whereas molecular studies have furnished perceptiveness evocative of a complement-independent form of AMR or C4d-negative AMR.[46] According to us, the detection of AMR should be best reported based on morphological features such as tubulointerstitial, vascular, and glomerular histological changes, with a piece of legislation to the presence or absence of C4d. Despite all pitfalls C4d is excellent marker for AMR.

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Conflicts of interest

There are no conflicts of interest.


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