PATHOLOGIC DIAGNOSIS

OF ANTIBODY-MEDIATED REJECTION (AMR)

Histopathologic findings

Immunopathologicfindings

Immunohistochemist

ry on paraffin sections

Immunofluorescence

on frozen tissue

MICROVASCULAR INFLAMMATION

Endothelial cell activation Endothelial cells with prominent large nuclei and expanded cytoplasmic projections narrowing or occluding the lumens.

Intravascular activated mononuclear cells

More than occasional focal aggregates or scattered isolated foci should trigger investigations

Interstitial edema Found 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

C4dDistribution0-10%: negative10-50%: focal staining> 50%: multifocal/diffuse stainingThe percentage refers to the surface of evaluable myocardium.Intensity negativeweak stainingstrong staining

FINAL RESULTSPositive C4d: multifocal/diffuse (>50%) weak or strong stainingNegative C4d: C4d negative (0-10%)focal (10-50%) weak or strong staining (DSAs and close follow-up should be recommended)

CD68Distribution0-10%: negative10-50%: focal staining> 50%: multifocal/diffuse staining

FINAL RESULTSPositive 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

Distribution0-10%: negative10-50%: focal staining> 50%: multifocal/diffuse stainingThe percentage refers to the surface of evaluable myocardium.

Intensity 0 :negative1: faint / trace (0-1+)2: strong (2-3+)

FINAL RESULTSPositive 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

GRADE DEFINITION SUBSTRATES

pAMR 0 Negative 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 2 Pathological AMR Both histological and immunopathological findings present

pAMR 3 Severe pathological AMR Interstitial 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 findingsClinical evidence of graft dysfunction in absence of ACR or other causeSerology: 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 findingsClinical evidence of graft dysfunction in absence of ACR or other causeSerology: 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 REJECTIONMIXED 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