Renal allograft failure can be caused by a large variety

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1 Acute Antibody-Mediated Rejection of Renal Transplant Pathogenetic and Diagnostic Considerations Luan D. Truong, MD; Roberto Barrios, MD; Horacio E. Adrogue, MD; Lillian W. Gaber, MD Context. Acute antibody-mediated rejection (AMR) has emerged recently as an important cause of graft failure. Objective. To review the pathogenetic, clinicopathologic, and diagnostic considerations of AMR. Data Sources. Review of literature and the authors experience. Conclusions. Acute antibody-mediated rejection is mediated by antibodies specific for donor antigens, which bind to target antigens and activate the complement system, culminating in tissue injury. The clinical manifestation of AMR is not specific, and transplant biopsy is needed for diagnosis. The glomeruli show thrombosis or neutrophils or mononuclear leukocytes in capillary lumens. The tubulointerstitial compartment shows edema, hemorrhage, necrosis, mild inflammation, and neutrophils or mononuclear leukocytes in the peritubular capillary lumens. The blood vessels show thrombosis, thrombotic microangiopathy, fibrinoid necrosis, or transmural vasculitis. Strong staining for C4d in the peritubular capillaries is characteristic. A definitive diagnosis of AMR requires (1) morphologic evidence of acute tissue injury, (2) immunopathologic evidence for antibody action, and (3) serologic evidence of circulating donor-specific antibodies. Acute antibody-mediated rejection should be suspected if some but not all 3 criteria are met. Since effective treatment is currently available, accurate and timely diagnosis of AMR is essential. (Arch Pathol Lab Med. 2007;131: ) Renal allograft failure can be caused by a large variety of diseases, most of which can only be accurately diagnosed by transplant biopsies. 1 Among them, acute rejection is both frequent and clinically significant. 1 Acute rejection can be mediated by alloreactive inflammatory cells or allospecific antibodies. 1 Although acute cell-mediated rejection (CMR) has been traditionally recognized, acute antibody-mediated rejection (AMR) has recently emerged as an important cause of graft failure, due largely to the introduction of tissue deposition of C4d, a complement split product, as a sensitive and specific marker for AMR. 2 9 Acute antibody-mediated rejection is increasingly recognized and has a profound adverse effect on the graft outcome. However, effective treatment for AMR is now available, 10 and if it is successfully treated, AMR may not impart a significant long-term adverse effect on the grafts. 5,8,11 Because of these considerations, accurate diagnosis of AMR is essential. This review focuses on the clinical, pathogenetic, and Accepted for publication April 16, From the Department of Pathology, The Methodist Hospital, Houston, Tex (Drs Truong and Barrios); the Departments of Pathology (Dr Truong) and Medicine, Renal Section (Drs Truong and Adrogue), Baylor College of Medicine, Houston, Tex; the Department of Pathology, Weill Medical College of Cornell University, New York, NY (Drs Truong and Barrios); and the Department of Pathology, University of Tennessee Health Science Center, Memphis (Dr Gaber). Dr Gaber is now with the Department of Pathology, The Methodist Hospital, Houston, Tex. The authors have no relevant financial interest in the products or companies described in this article. Reprints: Luan Truong, MD, Department of Pathology, MS 205, The Methodist Hospital, 6565 Fannin St, Houston, TX ( ltruong@tmh.tmc.edu). diagnostic aspects of AMR. Although allospecific antibodies are pathogenetically important in hyper-acute rejection, 1 acute rejection of ABO-incompatible or HLA-incompatible (crossmatch-positive) grafts, 1,12 and chronic allograft nephropathy, 13 these topics are not covered in this review. DEFINITION Acute antibody-mediated rejection is only recently recognized as a distinctive clinicopathologic entity. It was not listed in both the original (1993) and revised (1997) Banff schemes for renal transplant diseases, 14,15 but was officially added to this scheme in In the older literature, AMR was probably diagnosed under a variety of pathogenetically noncommittal but clinicopathogically descriptive names, including vascular, accelerated acute, delayed acute, ordelayed humoral rejection. 1 Several cases previously included in the Banff 1993 or 1997 category 4 type 3 (active/acute rejection, type 3) probably represent AMR or have a component of it, since the then-definition of this category included many morphologic features identical to those of AMR, including arteritis and vascular thrombosis. 14,15 These nosologic limitations are due chiefly to the fact that although alloreactive inflammatory cells infiltrating grafts are well accepted as a marker for CMR, such a marker for AMR was not then known. 14,15 The recent rediscovery of tissue deposition of C4d, a complement spit product originally described by Feucht et al in 1991, 2,3 and its confirmation as a sensitive and specific marker for AMR by several groups 4 9 have lead to better recognition of AMR as well as the pathogenesis, diagnosis, and treatment of this entity Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al

2 Figure 1. Antigen-antibody (Ag-Ab) complexes activate complement cascade. Complement activation also can be mediated by alternate or lectin pathways. Some complement split products are released into the environment, whereas others are bound to the Ag-Ab complexes. C4d and C3d form stable complexes with amino- or hydroxyl-containing molecules adjacent to the antigenic site. ( ), mediating the chemical reaction;,is converted into; *, released into the environment;, bound to the Ag-Ab complexes; and, bound to tissue adjacent to antigenic site. MECHANISM OF INJURY The mechanism of AMR represents a new paradigm in the pathobiology of solid organ transplantation and helps elucidate many clinical and morphologic features of AMR. 7,19 21 It involves donor-specific antigens that can initiate donor-specific antibodies (DSAs), leading to in situ antigen-antibody interaction, complement activation, and donor tissue injury. 20,21 Antigens These antigens may be alloantigens or autoantigens. Among the alloantigens, the HLA class I and II are most frequently pathogenetic. 19,20 These antigens are widely distributed in kidney but are most abundant in endothelial cells, accounting for the observation that the most severe changes of AMR are seen in the renal vasculature. 1 Although they are constitutively expressed in kidney, the increased expression or even de novo expression of some of them 22 in response to environmental factors, including cytokines, accounts for clinical conditions that promote AMR, as well as the frequent association of AMR and CMR. 19,22 Other alloantigens include ABO blood group antigens, accounting for rare cases of AMR in HLA-identical grafts, 17,18 or polymorphic endothelial antigens. 10,19,20,23 25 Since the current serologic testing is designed to detect anti-hla antibodies only, the latter may be reponsible for some cases of AMR with negative DSAs. 10 Autoantigens from the grafts those without genetic variations within the population may be the targets of AMR. 25 These include (1) vimentin, which is abundantly expressed by injured endothelial cells 25,26 ; (2) angiotensin II type I receptor, as reported in a recent well-documented series of unequivocal renal transplant AMRs without DSA 27 ; and (3) other antigens released from damaged transplanted or native kidneys, as evidenced thus far by experiment observations. 19,25 Antibodies How these antigens initiate their specific antibodies is not completely known. It is traditionally suggested that antigens migrate by themselves or are carried by antigenpresenting cells to peripheral lymphoid organs, where they are recognized by B cells. This leads to B-cell proliferation, maturation, and development into plasma cells as the source of DSAs. 21 This pathway remains to be conclusively proven in renal allograft AMR. Recent emphasis was made on antigen presentation within the renal allograft itself, independent from the peripheral lymphoid organs, as an important pathway leading to CMR. 28 Whether this pathway also plays a role in AMR is not clear, but it is relevant to the observation that neogenesis of lymphoid organs and abundance of mature plasma cells are prominent features within some grafts with AMR. 29,30 The antibodies against HLA and probably other donor-specific antibodies are of immunoglobulin (Ig) G type, the synthesis of which requires antigen sensitization and helper T cells, accounting for the frequent association of AMR and CMR. 1,20 The antibodies against ABO blood group antigens and some HLAs are of IgM type and may develop spontaneously in response to cross-reacting exogenous carbohydrate antigens. 1,20 Finally, contaminated xenoantibodies from equine or rabbit anti-human thymocyte antibodies may cross-react with graft endothelial cells and cause AMR, as evidenced by the development of AMR in 3 patients who received antithymocyte globulin for induction therapy and the finding that the xenoantibodies strongly bound endothelial cells. 31 Effector Mechanisms Effector mechanism is due to activation of complement components (Figure 1) and the tissue effects of these complement split products. 1,20,21,32 These products, bound to the antigen-antibody complexes or released into the en- Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al 1201

3 vironment, can induce several tissue lesions, leading to graft injury. These include (1) receptor-mediated neutrophil and macrophage chemotaxis and activation (C5a, C4a, and C3a); (2) cytolysis and apoptosis of target cells, including endothelial cells (C5b-9); (3) phagocytosis of immune complexes through complement receptors on macrophages (C3b and C4b); (4) activation of B cells (C3d); (5) vasospasm through the release of PG2 from macrophages; (6) edema through histamine released from macrophages (C3a); (7) increased endothelial expression of adhesion molecules, including P-selectin, a powerful attractant for neutrophils (C5b-9); and (8) intravascular thrombosis by triggering endothelial synthesis of procoagulants, including tissue factor. It is noteworthy that no significant influence on T cells, the principal effectors of CMR, is reported. The complement components involved in the activation cascade are derived from the circulation, but local sources have recently been proposed, as evidenced by an increased synthesis of C3 by renal tubular cells in the context of AMR. 33,34 Diagnostic Relevance Whereas several complement split products are released into the environment, some others (C1s, C3b, C3d, C4b, C4d, and C3b-9) are bound to the antigen-antibody complexes, and their tissue detection may theoretically serve as a marker for AMR (Figure 1). 21 In reality, only C4d fulfills this diagnostic goal. This reflects the observation that after C1s-mediated cleaving of C4 into C4a and C4b, C4a is released into the environment, but C4b, through its thiol ester group, binds covalently to nearby amino or hydroxyl-containing molecules. Bound C4b then is converted to C4d by plasma factor 1, which maintains the thiol ester group and remains bound covalently to tissue, thus serving as a durable marker for AMR. 2 4,6 C3d is theoretically an even better AMR marker, since it not only binds covalently to tissue as C4d, but is also more abundant, reflecting C3 activation as the central point of all complement pathways. Several studies, however, have shown that C3d tissue deposits are frequent in AMR, and C3d tends to codeposit with C4d. However, it is also often seen in normal renal tissue, tubular basement membrane (not a target of AMR), and not correlated with DSA, negating its role as a tissue marker for AMR. 12,35,36 Other complement split products are not or are only rarely seen in renal transplants with AMR, 1,19,37 an observation with no ready explanation, but perhaps due in part to dissolution of the target tissue, elimination of antigen-antibody complexes, or the action of cell surface inhibitors of complement activation to prevent progressive injury. 6,19,21 CLINICAL FEATURES About 7% of renal transplant recipients develop AMR, and AMR with or without CMR is diagnosed in about 24% of renal transplant biopsies. 1 These frequencies are not affected by immunosuppression regimens, level of HLA matching, ischemic time, or donor status. 1 The risk factors for AMR include presentitization due to blood transfusion, previous organ transplant, or pregnancy, and significant HLA mismatch ( 2/6). 1,10 The clinical manifestation of AMR is nonspecific and, by itself, nondiagnostic. It is characterized by features indicative of severe and acute graft injury, including oliguria, rapid deterioration of renal function, with mild or no proteinuria. 1,4,6,8,37,38 This may in part reflect the fact that Table 1. Light Microscopic Features of Acute Rejection: Correlation With C4d or Donor-Specific Antibodies (DSAs)* Features Frequency in % C4d (DSA ) Frequency in % C4d (DSA ) Mononuclear inflammatory cells in glomeruli 57 (46) 43 (10) Neutrophils in glomeruli 55 (29) 4 (15) Fibrinoid necrosis in glomeruli 20 (25) 0 (5) Fibrin thrombi in glomeruli 20 (46) 0 (15) Neutrophils in tubules 55 (25) 9 (15) Mononuclear inflammatory cells in tubules 70 (79) 100 (100) Acute tubular injury Interstitial inflammation Neutrophils in interstitium (50) (65) Interstitial hemorrhage PTC dilatation (33) (10) Neutrophils in PTCs 65 (46) 9 (5) Mononuclear inflammatory cells in PTCs Fibrinoid necrosis in arteries 25 (25) 0 (0 5) Endarteritis 25 (33) 32 (90) Arteritis 0 0 Cortical infarct 5 (37) 2 (0) * Data from Mauiyyedi et al, 4 Trpkov et al, 11 Haas et al, 12 and Nickeleit et al. 8 PTC indicates peritubular capillary. Percent cortex affected. pure AMR probably accounts for a small percentage of acute rejection episodes (5% 10%), and most of the episodes probably have both CMR and AMR components. 1,4,6,8,38 40 The onset is most common 1 to 3 weeks after transplantation but can be much later, which is often associated with decreased immunosuppression. 1 Most patients with AMR had no DSAs at time of transplantation, but at time of diagnosis DSAs have been detected, albeit in only about 63% to 90% of patients. 1 The absence of DSAs in the context of AMR may be due to several reasons, including low levels not detectable by current techniques, complete adsorption of DSA to the rejected kidney, or AMR due to non-hla antibodies, which are not detectable by the current testing designed to evaluate only HLA antibodies. 1,10,16 18 Acute antibody-mediated rejection may be subclinical, as suggested by the presence of at least some features of AMR in up to 13% of protocol biopsies. 41,42 PATHOLOGIC FEATURES Light Microscopy Several renal changes are characteristic for AMR, but none are pathognomonic. 1,4,11,16 18 Most of these changes are also seen in CMR, albeit much less frequently than in AMR (Table 1). Several changes traditionally considered diagnostic for CMR are often seen, and these suggest the coexistence of AMR and CMR. 1,4,6,8,38 40 Among 67 biopsies with acute rejection reported by Mauiyyedi et al, 4 30% showed AMR only, 15% CMR only, 45% both AMR and CMR, and 10% with acute tubular injury only. Changes of AMR can also be seen against a background of chronic allograft nephropathy, suggesting a pathogenetic relationship or coexistence. 16,39 The glomerular capillary lumens may show accumula Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al

4 Figure 2. Glomerular changes in acute antibody-mediated rejection (AMR). A, Rare mononuclear leukocytes (arrows) or neutrophils (arrowhead) in capillary lumens. There is also mesangial sclerosis, which is unrelated to AMR but probably reflects chronic allograft nephropathy also identified in this biopsy (periodic acid Schiff, original magnification 400). B, Many mononuclear leukocytes or neutrophils in glomerular capillaries (periodic acid Schiff, original magnification 400). C, Glomerular capillary thrombosis (lower), together with mononuclear leukocytes or neutrophils (upper) (hematoxylin-eosin, original magnification 400). D, Cortical necrosis. Thrombosis, not obvious in hematoxylin-eosin stain, is clearly demonstrated (arrows) by the trichome stain (Masson trichome, original magnification 400). E, Both mononuclear leukocytes and neutrophils in glomerular capillary lumens. There is also widening (arrows) of the lamina rara interna (electron microscopy, original magnification ). tion of neutrophils (10% 55%), mononuclear inflammatory cells (19% 90%), or both (Figure 2, A through E). These may be widespread but usually involve only few glomerular capillaries. The mononuclear inflammatory cells are mostly monocytes/macrophages (CD68 ), in contrast to the T-cell predominance in CMR. Fibrin thrombi (Figure 2, C and D) with or without necrosis are also characteristic (about 20%). Features suggestive of acute tubular cell injury (thinning of cytoplasm, tubular dilatation, loss of brush border, cell membrane disruption, apoptosis, increased proliferation) are frequent (75%) and may be the only light microscopic changes (up to 10%; Figure 3, A). 4 Accumulation of neutrophils among tubular cells or within tubular lumens, regardless of tubular segments, is seen in about 55% of cases, 1 especially in severe ones, including nephrectomy specimens (Figure 3, B through D). In pure AMR, the interstitium shows edema, hemorrhage, and scant mononuclear inflammatory cells, not reaching the diagnostic threshold for CMR (Figure 3, A and E). This, however, accounts for less than 10% of cases of AMR. 7,40 Many biopsies also display significant interstitial T-cell infiltration and tubulitis, indicating coexisting CMR. Other inflammatory cell types can be seen. Macrophage infiltration is more pronounced in C4d than in C4d rejection. Neutrophils are rarely seen and often are associated with neutrophils in peritubular capillary lumens. Abundant mature plasma cells may be a characteristic feature of AMR, since 75% of cases with this feature but without other traditional light microscopic changes of AMR are C4d and/or DSA and steroid resistant. 30 B cells are rare, but may form lymphoid aggregates, but these changes are not specific for AMR. The peritubular capillaries (PTCs) are dilated and con- Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al 1203

5 Figure 3. Tubulointerstitial changes in acute antibody-mediated rejection. A, Interstitial hemorrhage and edema, without significant inflammatory cell infiltrates. The tubules display acute nonspecific changes, including dilatation and cytoplasmic flattening, but there are no chronic changes (hematoxylin-eosin, original magnification 100). B, Interstitial edema and hemorrhage. Mononuclear leukocytes and neutrophils seen predominantly in peritubular capillaries (arrows), with focal interstitial extension (arrowheads; hematoxylin-eosin, original magnification 200). C, Mononuclear leukocytes and neutrophils seen predominantly in peritubular capillaries (arrows), with focal extension (arrowheads) into adjacent interstitium (hematoxylin-eosin, original magnification 400). D, Severe interstitial edema, mild interstitial inflammation, inflammatory cells in peritubular capillaries (upper left), and abundant neutrophils in tubular lumens (hematoxylin-eosin, original magnification 400). E, Severe antibodymediated rejection (nephrectomy specimen), with marked predominantly neutrophilic interstitial inflammation and hemorrhage, and tubular necrosis, but without significant glomerular changes (hematoxylin-eosin, original magnification 100). tain neutrophils in 3% to 54% of biopsies, but this usually involves a few neutrophils in a small number of capillaries (Figure 3, B and C). Lymphoid cells or monocytes/macrophages in PTC lumens are often seen (Figure 3, B and C). These are characteristic for AMRs but can be seen in CMRs. Fibrin can be observed in severe AMR (nephrectomy specimens). The small arteries or arterioles may show neutrophil intimal arteritis (Figure 4, A), fibromyxoid intimal thickening (Figure 4, C), thrombosis (Figure 4, D and E), fibrinoid necrosis (Figure 4, F), or combinations of these changes. Transmural arteritis may be seen in rare instances (Figure 4, G). These changes strongly indicate AMR, but they are not pathognomonic, since they are described in both C4d and C4d cases, albeit with significantly higher frequency in the former (Table 1). In contrast, endarteritis, defined as mononuclear leukocytes limited to the intima, is equally reported in AMR and CMR (Table 1). The acute vascular changes often coexist with more chronic vascular changes, suggestive of a background of chronic rejection (Figure 4, B). Immunofluorescence Although several immunoglobulins or complement components may be seen, only strong diffuse C4d staining of PTCs is considered diagnostic for AMR. Glomeruli may show patchy mesangial IgG or IgM. C4d may be seen in mesangial areas (3 ) and along the glomerular basement membrane (1 to 3 ). C3 components (C3c or C3d) have been reported. These are nonspecific and not diagnostic for AMR. 1 C4d staining of glomerular endothelial cells was reported in 35% of biopsies with AMR by immunostain on formalin-fixed, paraffin-embedded tissue sections in one study, 9 but this pattern was not reported in frozen tissue 4 and has not been confirmed. Since endothelial cells are targets of AMR, the absence in AMR of C4d staining in glomerular endothelial cells, in contrast with the strong staining of PTC endothelial cells, is surprising. 4,6,8 The reasons for this discrepancy are not known but may be related to the following: (1) anticomplementary activation pathways are well documented, and these pathways may be more active or abundant in glomerular than in peritubular capillaries 1,6,21 ; or (2) the nature of glomerular capillary extracellular matrix is such that tissue binding of C4d is not effective. Immunoglobulins are not seen in tubules or interstitium. Complement components (C3d, C4d, and C5b-9) are seen focally in tubular basement membrane. These changes are nonspecific and can be seen in other conditions, including CMR. 1,21,37 Immunoglobulin G, IgM, C3, C4d, and fibrin are noted in arteries with fibrinoid necrosis/arteritis and are specific for AMR, 4,6,10,16 18 but the rarity of these vascular lesions limits their diagnostic value. C4d is seen focally in endothelial surface and thickened intima of small arteries and arterioles, regardless of whether AMR is present. 4,6,10,16 18 Immunoglobulins or fibrin are only rarely seen in PTCs 1204 Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al

6 Figure 4. Vascular changes in acute antibody-mediated rejection. A, Neutrophils and mononuclear leukocytes in arterial intima, with intact media (hematoxylin-eosin, original magnification 400). B, Neutrophils and mononuclear leukocytes in arterial intima. There is also intimal fibrosis, medial attenuation, and adventitial fibrosis, suggesting a background of chronic rejection (hematoxylin-eosin, original magnification 400). C, Intimal myxoid thickening, reminiscent of thrombotic microangiopathy (Masson trichome, original magnification 400). D, Acute thrombosis of a small artery, arterioles, and glomerular capillaries, against a background of severe intertstitial neutrophilic infiltration, hemorrhage, and necrosis (hematoxylin-eosin, original magnification 200). E, Acute thrombosis of a small artery with intact media (Masson trichome, original magnification 400). F, Fibrinoid necrosis with partial medial destruction of a small artery, but without inflammation (right), a portion of the media (arrows) is still intact. There is also marked perivascular lymphoid cell infiltration, suggestive of an additional element of acute cell-mediated rejection (Masson trichome, original magnification 400). G, Mild but transmural vasculitis characterized by inflammation involving intima, media, and adventitia. There is also intimal fibrosis, suggestive of additional chronic rejection (hematoxylin-eosin, original magnification 400). (1% 20%) in grafts with negative crossmatch, 1 and these are usually associated with severe AMR (nephrectomy specimens). In contrast, IgM is frequent in AMR due to ABO incompatibility. 1 C4d staining of PTCs is characteristic for AMR. The staining is on the endothelial surface and capillary walls, since it is seen even in capillaries lacking endothelial cell markers and colocalizes with anti type IV collagen. 4,6 The stain is linear, circumferential, and equal in cortex and medulla, or even stronger in medulla (Figure 5, A and B). 43 The staining ranges from weak-focal to strong-diffuse (usually defined as 50% capillaries stained), but only the latter pattern is considered diagnostic for AMR. 1 C4 staining reflects current injury and probably is not affected by previous episodes of AMR, since serial biopsies suggested that C4d appears as early as 4 days after onset of AMR 8,16 18 and disappears as early as 8 days after DSAs are no longer detected. 1,8,16 18 C3d is also observed in PTCs with or without C4d, but this is not specific for AMR and is not correlated with features of AMR, including DSAs. 12,35,36 C5b-9 is not found in PTCs. 1,19,37 Electron Microscopy Electron microscopic findings are not diagnostic for AMR. Electron microscopy corroborates light microscopy findings and further demonstrates widening of the lamina rara interna of the glomerular or peritubular capillary basement membrane, and endothelial or tubular cell injury (lysis, apoptosis, cell fragmentation, detachment from basement membrane, cytoplasmic swelling). 1 Although AMR represents an immune complex disease, electrondense deposits are not seen. DIAGNOSTIC CONSIDERATIONS Diagnostic Criteria Since there is no pathognomonic feature for AMR (Table 1), its diagnosis depends on a combination of changes. The diagnostic criteria originally developed by Colvin et al are now incorporated into the most recent Banff schema for further validation (Table 2). 4,6,10,16 18 A definitive diagnosis of ARM requires the presence of all three criteria, whereas cases with some of these criteria are considered suspicious for AMR. 10,16,18 Acute antibody-mediated rejection is further classified on the basis of light microscopic findings into type 1 (acute tubular injury, minimal interstitial inflammation), type 2 (mononuclear leukocytes or neutrophils and/or thrombosis in glomerular or peritubular capillaries), and type 3 (transmural inflammation/fibrinoid change of arterial blood vessels) Acute antibody-mediated rejection often coexists with CMR and can develop against the background of chronic allograft nephropathy, with the characteristic features of these associated conditions being seen in biopsies together with those of AMR. 13,16 18,39 Performance and Interpretation of C4d Staining Several diagnostic caveats are relevant to the performance and interpretation of C4d staining. C4d staining is Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al 1205

7 Table 2. Criteria for Acute Antibody-Mediated Rejection* 1. Morphologic evidence of acute tissue injury, such as a. Acute tubular injury or b. Neutrophils and/or mononuclear cells in peritubular capillaries and/or glomeruli, and/or capillary thrombosis or c. Fibrinoid necrosis/intramural or transmural inflammation in arteries 2. Immunopathologic evidence for antibody action, such as a. C4d and/or (rarely) immunoglobulin in peritubular capillaries or b. Immunoglobulin and complement in arterial fibrinoid necrosis 3. Serologic evidence of circulating antibodies to donor HLA or other antidonor endothelial antigens. * Table reprinted with permission from Blackwell Publishing, Oxford, United Kingdom, from Racusen et al. 16(p713) Figure 5. C4d staining in acute antibody-mediated rejection. A, Diffuse strong circumferential staining of peritubular capilaries. There is also global diffuse staining of the glomeruli, in both mesangial and peripheral patterns, as a built-in positive control (immunofluorescent, original magnification 100). B, Immunoperoxidase staining of the same biopsy, displaying the same pattern as for immunofluorescent staining. There is also nonspecific staining of arterial intima (left) (original magnification 200). recommended for all diagnostic transplant biopsies. 16 The minimum amount of tissue needed for adequacy has not been established, but two cores, with or without glomeruli, are recommended. 1 C4d can be identified by staining of fresh frozen tissue sections with either of the two available monoclonal antibodies (Biogenesis, Brentwood, Calif, or Quidel Corporation, Santa Clara, Calif) by immunofluorescent techniques or in formalin-fixed, paraffin-embedded tissue section with the single available polyclonal antibody (C4dpAb; Biomedia, Vienna, Austria) by immunoperoxidase technique. 41,42 In frozen tissue, glomeruli with or without AMR show strong mesangial and variable glomerular basement membrane staining, serving as built-in control. 1,4,6,43,44 However, kidney tissue with membranous glomerulonephritis ( C4d in glomerular capillaries) or nephrectomy specimens for rejection ( C4d in PTCs) are excellent extrinsic controls. 44 Freshly cut or stored frozen sections are equally satisfactory. 43 Glomerular staining is not seen in fixed tissue, necessitating an extrinsic control. 43,44 Both the staining intensity and extent are less (36% less area stained) in fixed tissue, compared with frozen tissue, 43 accounting for about 30% of cases with diffuse ( 50% PTCs stained) in frozen tissue being classified as focal in fixed tissue. 19,43 Immunofluorescent staining is crisp with a clean background, against which glomerular basement membrane, mesangium, thickened tubular basement membrane, normal arterial endothelial cells, thicken vascular intima, or arteriolar hyalinosis may show C4d, regardless of whether there is AMR. 4,6,16 18,43 Staining of these structures is considered real (not due to technical reasons) but is not diagnostic for AMR. 4,6,16 18,43 Immunoperoxidase-based methods usually create a heavy background with nonspecific staining of arteriolar hyalinosis, thickened tubular basement membrane, tubular brush border, tubular reabsorption droplets, and plasma protein in vascular lumens, or perivascular connective tissue, making interpretation potentially difficult. 43 It is currently recommended that for immunofluorescence applied to frozen tissue, positivity requires strong linear circumferential widespread (usually defined as at least 50%) staining of sampled PTCs, excluding scarred or necrotic areas, since PTCs may be lost in these areas For immunoperoxidase applied to fixed tissue, the threshold for positivity in term of extent is the same, but the staining intensity may be variable Positive (strong and widespread) C4d staining of PTCs is both a sensitive (78% 95%) and specific marker (63% 96%) for AMR. 4,45 The significance of staining that does not reach the positive threshold is not clear, and there is no consensus on the minimum amount of staining to qualify as focal positivity. 1,17,18 The focal staining may still suggest AMR, since focal staining can precede widespread staining in serial biopsies, 1 3 and in some studies, cases with focal or diffuse C4d staining share the same morphologic and clinical features in terms of AMR. 8,46 Focal C4d staining can also be seen in graft biopsies performed for graft dysfunction, which did not show features of AMR or CMR; treatment, however, improved renal function, suggesting that this C4d pattern may reflect a form of AMR Arch Pathol Lab Med Vol 131, August 2007 AMR of Renal Transplant Truong et al

8 DSAs and the Diagnosis of AMR The presence of circulating DSAs is one of the criteria for the diagnosis of AMR. It is recommended that if features suggestive of AMR (criteria 1 and/or 2) are noted in the biopsies, testing for DSA should be performed. 10,16 18 However, there is no absolute correlation between DSA and AMR or C4d positivity. Thus, DSA is detected in only 63% to 90% of cases with C4d positivity. 1,4,9,45 This discrepancy may be due to non-hla antibodies, not detectable by the current serologic testing, which is designed to detect anti-hla antibodies only. 1,4,10,17 Low levels of antibodies, complete adsorption of antibodies into the graft, or antibody-independent complement activation are also possible causes. 1,10,17 In contrast, DSA also is detected in 2% to 53% of cases without C4d staining. 1,4,45 This wide range probably reflects variable testing techniques for DSA, but it may suggest the presence of antibodies that do activate complements or bind the endothelial antigens. 1,17,19 These discrepancies highlight the need to use combined criteria and emphasize the role of biopsies in the diagnosis of AMR, even in the era of sophisticated serologic testing. TREATMENT AND PROGNOSIS Acute antibody-mediated rejection and/or C4d positivity strongly impair graft survival. The 1-year graft loss after AMR with or without CMR (30%) is significantly worse than after CMR alone (3%). 4 The loss rates for acute rejection with or without C4d positivity are 16% to 50% versus 3% to 7%. 9,45,48 However, grafts successfully treated for AMR do seem to harbor adverse long-term outcomes. 1,4,8 These considerations call for accurate diagnosis of and effective treatment for AMR. Treatment for AMR, as summarized in a recent national consensus conference, is still evolving. 10,17 It reflects current concepts of this disease and aims at different points in the pathogenetic pathway, including vigorous control of the frequently associated CMR, desensitization, inhibition of HLA-specific antibodies, immunomodulation, rapid removal of DSA, ablation of B-cell compartment, and reduction of the plasma cells and their precursors. 10,17 This regimen includes intravenous immune globulin, plamapheresis, low-dose cytomegalovirus hyperimmune globulin, anti-cd20, and splenectomy. 10,17 References 1. Colvin RB, Nickeleit V. Renal transplant pathology. In: Jennette JC, Olson JL, Schwartz MM, Silva FG, eds. Heptinstall s Pathology of the Kidney. 6th ed. Philadelphia, Pa: Lippincott Williams and Wilkins; 2007: Feucht HE, Felber E, Gokel MJ, et al. Vascular deposition of complementsplit products in kidney allografts with cell mediated rejection. Clin Exp Immunol. 1991;86: Feucht HE, Schneeberger H, Hillebrand G, et al. Capillary deposition of C4d complement fragment and early renal graft loss. Kidney Int. 1993;43: Mauiyyedi S, Crespo M, Collins AB, et al. Acute humoral rejection in kidney transplantation, II: morphology, immunopathology, and pathologic classification. J Am Soc Nephrol. 2002;13: Mauiyyedi S, Colvin RB. Humoral rejection in kidney transplantation: new concepts in diagnosis and treatment. Curr Opin Nephrol Hypertens. 2002;11: Collins AB, Schneeberger EE, Pascual MA, et al. Complement activation in acute humoral renal allograft rejection: diagnostic significance of C4d deposits in peritubular capillaries. J Am Soc Nephrol. 1999;10: Nickeleit V, Andreoni K. The classification and treatment of antibody-mediated renal allograft injury: where do we stand? Kidney Int. 2007;71: Nickeleit V, Zeiler M, Gudat F, et al. 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