**6.1. Acute antibody mediated rejection**

**5. Stages of antibody-mediated rejection**

424 Current Issues and Future Direction in Kidney Transplantation

reasons, donor-reactive antibodies do not elicit AAMR.

progression is inexorable (Colvin & Smith, 2005).

Group).

**6. Pathology**

At a National Institutes of Health (United States) consensus conference, draft criteria were established for antibody-mediated rejection and for four theoretical stages in the development

**Figure 1.** Proposed stages of antibody-mediated rejection (Reproduced with permission from Nature Publishing

According to this model, the first evidence of an antibody-mediated response is the de novo generation of donor-reactive antibodies (stage I). In many circumstances and for unknown

The next stage (stage II) shows evidence of antibody reactivity and complement activation in the graft, with C4d deposition in peritubular or glomerular capillary endothelium. At this stage, there is no evidence of pathological or clinical injury in the graft. Both stage I and stage II fit the criteria for accommodation and are therefore not necessarily predestined to lead to graft injury. In stage III, in addition to positive staining for C4d, there are identifiable patho‐ logical changes, but graft function is still normal (that is, there is subclinical rejection). Finally, in stage IV, in addition to positive staining for C4d and pathological changes, graft dysfunction occurs. The interval between stages can be long and variable, and it is not known whether

The past 20 years have seen major advances in the understanding of the effects of antidonor antibodies on renal allografts at various stages after transplantation. These advan‐ ces have been due in large part to pathologic examination of both early and late renal

of CAMR (Takemoto et al., 2004) as shown in FIG. 1 (Colvin & Smith, 2005).

As pathologists have become increasingly adept at diagnosing antibody-mediated rejection (AMR) on allograft biopsies, substantial progress has been made in the treatment of AMR and in successful renal transplantation in recipients with pre-existing antibodies against donor blood group (ABO) and/or major histocompatibility (HLA) antigens. It has become critical to develop standardized criteria for the pathological diagnosis of AMR.

The diagnostic criteria for acute humoral rejection (AMR; acute antibody-mediated rejection). Patients with AHR present with an acute loss of graft function that often arises in the first few weeks after transplantation and cannot be distinguished from cell-mediated rejection on clinical grounds (Halloran et al., 1992; Takemoto et al., 2004). AMR can also develop years after transplantation, often triggered by a decrease in immunosuppression (iatrogenic, noncompli‐ ance, or malabsorption). Presensitization is the major risk factor, but most of the patients with AMR had a negative cross-match. AMR has occurred with all immunosuppression regimens, even profoundly depleting therapy (Lorenz et al., 2004). The first clue that circulating anti– class I HLA antibody caused a different pattern of acute rejection came from the studies of Halloran's group in Edmonton (Halloran et al., 1992). These investigators showed that neutrophils in peritubular capillaries (PTC) and glomerular capillaries are strongly associated with circulating anti-donor HLA antibodies. Other features, such as fibrinoid necrosis of arteries and microthrombi, are also more common. However, none of these features is specific.

The pathology of AMR has a wide spectrum and can easily be missed by histologic criteria alone. Renal biopsies may show acute cellular rejection, acute tubular injury, or thrombotic microangiopathy. Neutrophils in capillaries are characteristically but not always found. Macrophages are now recognized as a common intracapillary cell in AMR in kidney (Tinckam et al., 2005) and heart (Lepin et al., 2006) allografts. Typically, the PTC are dilated. Fibrinoid necrosis is found in a minority of cases (approximately 10 to 20%). A component of acute cellular rejection may also be present, as manifested by a prominent mononuclear infiltrate, tubulitis, or endarteritis. These lesions are generally not attributable to antibody alone. Treg cells (FOXP3+) are rarer in the infiltrate than in cell-mediated rejection, perhaps contributing to the poorer prognosis in AMR (Veronese et al., 2007). Microthrombi and interstitial hemor‐ rhage also sometimes occur. The PTC and glomerular endothelium shows a variety of ultrastructural changes, including loss of fenestrations, detachment from the basement membrane, lysis, and apoptosis; complete destruction of capillaries can occur, leaving thickened laminated basement membranes (Liptak et al., 2005). Immunofluorescence (IF) curiously does not often show antibody or C3 deposition in the vessels. However, IF does show C4d in the majority of the PTC as a bright ring pattern, using a mAb in cryostat sections (Collins et al., 1999; Mauiyyedi et al., 2001, 2002). Immunohistochemistry (IHC) works in formalinfixed, paraffin-embedded tissues with a polyclonal antibody (Lorenz et al., 2004). By immu‐ noelectron microscopy, C4d is detected on the surface of the endothelial cells and in intracytoplasmic vesicles (Regele et al., 2002). Antibodies that react to non-C4d portions of the C4 molecule do not show PTC deposition, arguing that what is detected in tissues is primarily C4d (Seemayer et al., 2006).

**7.2. C4d detection pitfalls**

being damaged by alloantibody.

**7.3. Alternative markers (New diagnostic tools)**

microarrays in 173 renal allograft biopsies for cause.

*7.3.1. Endothelial-associated transcripts (ENDATs) as a new marker for CAMR*

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

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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

Recognizing the key role of endothelial changes in AMR, it was postulated by Sis and collea‐ gues (Sis et al., 2009) that altered expression of endothelial genes in biopsies from patients with alloantibody would identify kidneys incurring antibody-mediated damage and at risk for graft loss, whether they were C4d+ or negative. They explored whether expression of endothelial genes was increased in biopsies manifesting antibody-mediated graft injury, and whether such changes could be seen in C4d negative as well as C4d positive biopsies. They identified 119 endothelial-associated transcripts (ENDATs) from literature, and studied their expression by

Mean ENDAT expression was increased in all rejection but was higher in AMR than in T-cell-mediated rejection and correlated with histopathologic lesions of AMR, and alloan‐ tibody. Many individual ENDATs were increased in AMR and predicted graft loss. Kid‐ neys with high ENDATs and antibody showed increased lesions of AMR and worse prognosis in comparison to controls. Only 40% of kidneys with high ENDAT expression and chronic AMR or graft loss were diagnosed by C4d positivity. High ENDAT expres‐ sion with antibody predicts graft loss with higher sensitivity (77% versus. 31%) and slightly lower specificity (71% vs. 94%) than C4d. The results were validated in inde‐ pendent set of 82 kidneys. They concluded that in patients with alloantibodies, abnor‐ malities in expression of endothelial genes identify not only C4d+ AMR but some kidney transplants developing antibody associated graft injury despite negative C4d staining
