PATHOLOGIC DIAGNOSIS OF ANTIBODY-MEDIATED REJECTION (AMR) Histopathologic findings Immunopathologic findings Immunohistochemistry on paraffin sections Immunofluorescence on frozen tissue

MICROVASCULAR INFLAMMATION Endothelial cell activationEndothelial cells with prominent large nuclei and expanded cytoplasmic projections narrowing or occluding the lumens. Intravascular activated mononuclear cellsMore than occasional focal aggregates or scattered isolated foci should trigger investigations Interstitial edemaFound in AMR but may be a nonspecific finding Haemorrhage, necrosis, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis and/or karyorrhexis Typical for severe AMR pending confirmation with immunofluorescence/immunohistochemistry (IF/IHC) Ischemic injury area, healing biopsy site, Quilty lesions and myocardial scars must be excluded from evaluation HISTOPATHOLOGIC FEATURES

IMMUNOHISTOCHEMISTRY ON PARAFFIN SECTIONS

ANTIBODY PANELS FOR PARAFFIN IMMUNOHISTOCHEMISTRY PRIMARY PANEL  C4d  CD68 SECONDARY PANEL (optional)  CD31/CD34 (to assess capillary network)  C3d (recommended)  CD3 (pan-T cells)  CD20 (pan-B cells)  Complement regulatory proteins or others (according to individual preference)

SPECIFIC STAINING  Only interstitial capillaries must be assessed  The staining of small artery and arteriole wall must be considered as internal control to check the quality and intensity of capillary staining. Intensely staining linear-granular endothelial deposits along the entire circumference of capillaries NONSPECIFIC STAINING The staining of interstitium, myocardial scars, necrotic cardiac myocytes and capillaries in Quilty lesions and biopsy site scars are not considered positive. Evaluation criteria of C4d staining on paraffin section

TO BE CONSIDERED Only macrophages within capillaries and small venules. Threshold: focal or multifocal clusters of intravascular CD68 positive cells in >10% capillaries of intact myocardium NOT TO BE CONSIDERED Interstitial macrophages found in various settings such as AMR, acute cellular rejection, ischemic injury and healing biopsy sites Evaluation criteria of CD68 staining

C4d Distribution  0-10%: negative  10-50%: focal staining  > 50%: multifocal/diffuse staining The percentage refers to the surface of evaluable myocardium. Intensity  negative  weak staining  strong staining FINAL RESULTS Positive C4d:  multifocal/diffuse (>50%) weak or strong staining Negative C4d:  C4d negative (0-10%)  focal (10-50%) weak or strong staining (DSAs and close follow-up should be recommended) CD68 Distribution  0-10%: negative  10-50%: focal staining  > 50%: multifocal/diffuse staining FINAL RESULTS Positive CD68:  focal (>10) or multifocal/diffuse (>50%) intravascular macrophages Criteria for immunopathological diagnosis of AMR

IMMUNOFLUORESCENCE ON FROZEN TISSUE

ANTIBODY PANELS FOR IMMUNOFLUORESCENCE PRIMARY PANEL  C4d  C3d  HLA- DR (used by some centres to identify capillary structures) SECONDARY PANEL (optional)  Fibrin  IgG  IgM  Others (according to individual centres’preferences)

SPECIFIC STAINING  Only interstitial capillaries must be assessed  The staining of small artery and arteriole wall must be considered as internal control to check the quality and intensity of capillary staining Intensely staining linear-granular endothelial deposits along the entire circumference of capillaries Evaluation criteria of C4d / C3d staining on frozen sections

C4d / C3d Distribution  0-10%: negative  10-50%: focal staining  > 50%: multifocal/diffuse staining The percentage refers to the surface of evaluable myocardium. Intensity  0 :negative  1: faint / trace (0-1+)  2: strong (2-3+) FINAL RESULTS Positive C4d / C3d :  multifocal/diffuse (>50%) weak or strong staining Negative C4d / C3d :  C4d / C3d negative (0-10%)  focal (10-50%) weak or strong staining (DSAs and close follow-up should be recommended) Criteria for immunopathological diagnosis of AMR on frozen sections

GRADEDEFINITIONSUBSTRATES pAMR 0Negative for Pathological AMR Both histological and immunopathological findings negative pAMR 1: Indicative of possible pathological AMR pAMR 1 (H+) pAMR 1 (I+) Histopathological AMR alone Immunopathological AMR alone Histological findings present and immunopathological findings negative Histological findings negative and immunopathological findings positive (CD68+ and/or C4d+) pAMR 2Pathological AMR Both histological and immunopathological findings present pAMR 3Severe pathological AMR I nterstitial hemorrhage, capillary fragmentation, mixed inflammatory infiltrates, endothelial cell pyknosis and/or karyorrhexis and marked edema + positive IHC/IF The 2012 ISHLT Working Formulation grading for biopsy diagnosis of pathological AMR (pAMR)

INDICATIONS FOR IMMUNOPATHOLOGICAL TESTS  Histopathological findings  Clinical evidence of graft dysfunction in absence of ACR or other cause  Serology: pre-transplant or de novo donor specific antibodies (DSAs) In the case of a positive endomyocardial biopsy (EMB), immunostaining of subsequent biopsies should be continued until C4d is negative. When a patient has been treated for AMR, a repeat EMB should be taken not less than 2 weeks later. INDICATIONS FOR IMMUNOPATHOLOGICAL TESTS  Histopathological findings  Clinical evidence of graft dysfunction in absence of ACR or other cause  Serology: pre-transplant or de novo donor specific antibodies (DSAs) In the case of a positive endomyocardial biopsy (EMB), immunostaining of subsequent biopsies should be continued until C4d is negative. When a patient has been treated for AMR, a repeat EMB should be taken not less than 2 weeks later. EMB SURVEILLANCE FOR AMR Each biopsy must be fully evaluated for AMR at:  2 and 4 weeks after transplantation  3, 6, 12 months after transplantation  subsequently: annually EMB SURVEILLANCE FOR AMR Each biopsy must be fully evaluated for AMR at:  2 and 4 weeks after transplantation  3, 6, 12 months after transplantation  subsequently: annually

MIXED REJECTION  Histological picture including diagnostic criteria for both cellular rejection (ACR) and antibody-mediated rejection (AMR).  ACR must be evaluated according to 2005 with/without 1990 ISHLT-WF (according to individual centres’ routine )  AMR is separately evaluated according to the 2013 ISHLT-WF