**7.2. C4d detection pitfalls**

C4 molecule do not show PTC deposition, arguing that what is detected in tissues is primarily

Chronic AMR is now included in the newest update of the Banff 07 classification of renal allograft pathology with the following criteria: [1]morphological changes as glomerular double contours compatible with transplant glomerulopathy (TPG) and severe PTC basement membrane multilayering, interstitial fibrosis and tubular atrophy with or without PTC loss, and fibrous intimal thickening in arteries without internal elastica duplication; [2] diffuse C4d deposition in PTCs; and [3] presence of DSA (Solez et al.,2008). Not all these criteria are always

PTC basement membrane multilayering correlates highly with TPG, and most of TPG have evidence of either C4d-positive staining or DSA. However, the proposed criteria do not apply to all situations of chronic active antibody-mediated rejection. Chronic AMR is distinct from acute AMR in that no acute inflammation (neutrophils, edema, necrosis, thrombosis) is present. However, cellular activity is often reflected by increased mononu‐ clear cells in glomerular capillaries and PTC (Colvin, 2007). The Banff criteria require PTC C4d positivity for diagnosis of ABMR as well as microcirculation injury. However, C4d is not a sensitive marker of chronic ABMR, and in many patients with transplant glomerulopathy, C4d staining is negative in the presence of anti-HLA DSA. Therefore, the recent update of the Banff classification introduced the diagnostic category of "suspi‐ cious for ABMR." It is defined with the presence of morphologic evidence of antibodymediated tissue injury and positive anti-HLA antibody with negative C4d, or PTC C4d

Feucht et al. (Feucht et al., 1993) in Munich showed that peritubular capillary (PTC) C4d deposition in renal transplant biopsies is strongly associated with a poor prognosis and raised the possibility that antibodies were responsible. Currently, C4d has been adopted as a marker of antibody-mediated rejection (Racusen et al., 2003). The justification for the selection of C4d, a split product of C4, as a marker for AMR comes from its position in the cascade of complement activation. C4d, a split product of the classical pathway of complement activation, is present covalently bound on tissue near the sites of complement activation by alloantibody, e.g.,

C4d deposition in renal peritubular capillaries is strongly associated with circulating antibody to donor HLA class I or class II antigens (Bohmig et al., 2002; Haas et al., 2006) and is currently the best single marker of complement-fixing circulating antibodies to the endothelium.

fulfilled in an individual patient at every given time point (Fehr et al., 2009).

positivity in the absence of alloantibody (Solez et al., 2008).

**7. Markers of antibody mediated rejection**

**7.1. Histopathologic detection of C4d**

vascular endothelial cell membrane.

C4d (Seemayer et al., 2006).

**6.2. Chronic antibody mediated rejection**

426 Current Issues and Future Direction in Kidney Transplantation

C4d is not a magic marker for antibody-mediated rejection and in many patients with transplant glomerulopathy. It is negative in the presence of anti-HLA DSA. Another issue with chronic active antibody-mediated rejection is non-HLA antibody induced rejection without complement fixation of C4d. Moreover, it was shown in many studies that focal C4d staining was not a reliable indicator of AMR (Kayler et al., 2008), and it is not a guarantee of AMR: diffuse C4d staining can occur with no morphologic injury or impaired outcome in ABOincompatible allografts (Solez et al., 2008). Another important problem is the significance of positive C4d staining in the peritubular capillaries (PTC) and glomerular capillaries.

There are significant data to show that C4d positivity is usually long-lasting but is not permanent. C4d staining can change from negative to positive and vice versa within days to weeks. The detection of C4d signifies a humoral alloresponse in a subgroup of kidney transplants, which is often associated with signs of cellular rejection (Nickeleit et al., 2002). It is not clear how long C4d deposits persist in the absence of continued DSA production. One study reported that C4d deposits were no longer detectable on repeat biopsy performed 2–3 weeks after DSA (Mauiyyedi et al., 2002). If C4d staining misses some cases of antibodymediated injury, and the presence of alloantibody does not identify which grafts are under‐ going antibody-mediated damage, we need new methods for identifying which kidneys are being damaged by alloantibody.
