**5. Complexities in the diagnosis of antibody mediated rejection (Table 3)**

**i.** Accomodation

**ii.** C4d negative AMR

in the context of tissue morphology [47, 48]

**iii.** Focal versus diffuse C4d staining

glomerulitis and/or peritubular dilatation [51].

**iv.** Non-PTC C4d staining

The presence of C4d deposition in PTC does not always denote the presence of AMR or tissue injury. In ABO-incompatible renal transplant, the presence of PTC C4d staining often occurs in the absence of tissue injury or AMR, a process known as accommodation and may be observed in >70% of ABO-incompatible transplants; whereas the presence of PTC C4d staining

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AMR in the absence of PTC C4d staining has been reported more frequently. In an analysis of 173 indication kidney biopsies, *Sis et al* demonstrated that a combination of high expression of endothelial-associated transcripts (ENDAT) detected using microarray on tissue biopsy, suggesting endothelial damage from alloantibody, plus the presence of DSA was strongly associated with morphological evidence of AMR but only 38% of these biopsies had evidence of PTC C4d positivity [46]. Other studies have corroborated this initial finding suggesting that over reliance of C4d positivity to diagnose acute or chronic AMR could miss up to 60% of patients with morphological evidence of AMR and C4d staining should always be interpreted

It is generally accepted that the detection of C4d in renal allograft biopsies using immuno‐ fluorescence staining is more sensitive than immunohistochemical staining [42, 49]. The level of C4d staining appears to have prognostic significance and it is widely accepted that diffuse C4d staining involving >50% of PTC by either technique is considered positive and correlates much more strongly with adverse graft outcome compared to focal C4d staining involving <50% of PTC, but this remains controversial [50]. However, there are other studies suggesting that focal C4d staining is also associated with histological evidence of AMR including

Glomerular, arteriolar and/or erythrocyte C4d positivity often occurs in the absence of PTC C4d staining but the clinical significance of these patterns remains unclear. In a retrospective study of 539 indication renal allograft biopsies, *Kikic et al* demonstrated a poor correlation between arteriolar C4d staining and graft survival, whereas linear glomerular C4d staining was strongly associated with graft failure [52]. There has been considerable interest in the detection of erythrocyte C4d deposition (eC4d) by indirect immunofluorescence as a potential surrogate marker of disease activity in patients with systemic lupus erythematosus and may be useful for the monitoring of disease activity and/or response to treatment in these patients [53, 54]. In kidney transplantation, *Haidar et al* showed a greater amount of eC4d in PTC C4d positive samples compared to PTC C4d negative samples. The authors reported that the positive (PPV) and negative predictive value (NPV) of PTC C4d and eC4d for peritubular capillaritis were 28% and 46% for PPV and 93% and 94% for NPV respectively suggesting that

monitoring of eC4d may be an useful non-invasive marker of AMR [55].

in HLA-incompatible grafts correlates strongly with the presence of AMR [45].

The diagnosis of AMR has improved dramatically with the advent of C4d staining and the ability to detect DSA [41]. The diagnosis of acute AMR according to BANFF criteria requires a triad of [1] histological evidence of graft damage including acute-tubular necrosis-like minimal inflammation, capillaritis and/or glomerulitis and/or thromboses and arteritis, [2] immunological evidence of complement activation inferred by C4d positivity in the peritub‐ ular capillaries (PTC), and [3] presence of DSA; whereas the diagnostic criteria for chronic AMR requires [1] morphological evidence of chronic damage of the allograft including duplication of glomerular basement membrane, lamination of peritubular capillaries, arterial intimal fibrosis or interstitial fibrosis/tubular atrophy, [2] diffuse C4d deposition in PTC, and [3] presence of DSA [42]. C4d, a complement split product, is formed by the binding and activation of the classical complement pathway by DSA, which then binds covalently to specific target molecules on the endothelium of PTC and is therefore considered a footprint of AMR [43]. The sensitivity and specificity of diffuse PTC C4d staining for the presence of DSA is >95% [44].


**Table 3.** Histological criteria for acute and chronic antibody mediated rejection and corresponding table of controversies of relying on peritubular capillary C4d deposition as a marker for antibody mediated rejection.

However, there are concerns regarding whether the presence of C4d within peritubular capillaries is essential for the diagnosis of AMR with reports of C4d-negative AMR being identified. There have been a few studies that have demonstrated an association between glomerular or erythrocyte C4d deposition and the presence of acute and chronic AMR but the clinical significance of these deposits remain debatable.

Problems with C4d staining:
