ANTIBODY MEDIATED REJECTION OF SOLID ORGAN ALLOGRAFTS.

Moderator : Dr M.L. Yadav

Presented by : Dr Tashi Agarwal

Objectives

1. Introduction of Antibody mediated rejection AMR

2. Role of C4d in transplant rejection

3. Donor specific antibodies DSA

4. Presentation of AMR in kidney, liver, lung and heart.

Introduction

Transplantation : The process of taking cells, tissues or organs from one individual and placing them into a different individual or different site of the same individual.

Graft : Transplanted cells, tissues or organs.

Donor: The individual who provides the graft.

Recipient : The individual who receives the graft. Also called the host.

Types of Grafts

• Autologous or autograft (self)

eg. BM, peripheral blood stem cells, skin , bone.

• Syngenic or isograft (identical twin)

• Allogenic or allograft (another human except identical twin)

• Xenogenic or xenograft (one species to another)

Transplant rejection

Cell mediated

(interstitial)

Antibody mediated

(humoral)

Hyperacute

Acute HumoralRejection

Chronic HumoralRejection

Preformed DSA

Preformed/denovo DSA

CHRONIC ALLOGRAFT REJECTION IS DUE TO MANY CAUSES INCLUDING CHRONIC HUMORAL REJECTION

Pathogenesis of AMR

Introduction to

C4d

• Capillary endothelial cells form the boundary between allograft parenchyma and the recepient’s blood, and are the main target in AMR, mediated by DSA’s.

• All nucleated cells in the body express Class I HLA’s on their surface. While, Class II HLA’s are expressed on the surface of antigen presenting cells and EC’s of capillaries, but not large vessels.

• Inflammation upregulates Class II HLA epression by EC’s which express receptors on their surface to complement.

• Antibody binding to antigen activates complement via the classical pathway, generating C4b and C3b which rapidly undergoes proteolytic cleavage to form stable split degradation products C4d and C3d.

• These are bound to EC’s and act as indirect tissue markers of complement activation.

Detection of capillary C4d deposition in allografts has evolved as a specific and sensitive diagnostic tool for AMR.

Complement system

• C4d has a thioester moiety that enables strong covalent bonding with the endothelial cells and basement membrane.

• C4d has been called 'a footprint' of antibody-mediated tissue injury

C4d

Expression of C4d

• C4d is normally expressed in the mesangium and the vascular pole.

• It also involves glomerular capillaries in cases of immune-mediated glomerulopathies .

• In transplant kidney (AMR) peritubular C4d may be noted.

Chethan Puttarajappa, Ron Shapiro and Henkie P. Tan . Antibody-Mediated Rejection in Kidney Transplantation: A Review

Methods of detection

1.Immunofluorescence (IF) on frozen tissue (monovalent antibody against C4d ) “GOLD STANDARD”

2. Immunohistochemistry (IHC) on paraffin-embedded tissue (polyvalent antibody).

– Diffuse C4d implies >50% of PTC staining for C4d

– Focal staining implies 10–50%

– Minimal staining implies <10%

Note: IHC is less sensitive than IF for C4d detection.

Controls for C4d

• Tissue with known hyperacute rejection of AMR.

• C4d deposition in arterial endothelium and artefacts such as non specific staining of arteriolar elastic lamina are useful as internal positive controls.

Limitations of C4d and staining artefacts

1. A scoring system for C4d staining has not been agreed universally for lung and liver allografts as the picture is less clear for lung and liver allografts.

2. Intense serum staining should not be mistaken for a positive result as it lacks the circumferential granular staining typical for endothelial C4d deposition.

3. Capillary C4d deposition may also occur in lesions not relevant to diagnosis of AMR. Such as biopsy site scars in the heart.

4. Other pathologies such as hyaline membranes in diffuse alveolar damage, necrotic myocytes and necrotic hepatocytes may be positive.

PAS to show peri-tubular capillaries

IF showing C4d staining in peri-tubular capillaries (AMR)

IHC-C4d in Peritubular capillaries (tubular membranes are unstained)in AMR

Strong , linear staining along entire circumference of PTCs

DONOR SPECIFIC ANTIBODIESDSA

DSA

• Patient’s exposure to “nonself-” HLA molecules as after blood transfusion, pregnancy, or organ transplantation can lead to the development of anti-HLA antibodies.

• Thus, a transplant candidate may present with preformed anti-HLA antibodies, while being in the waiting list.

• The antibodies that do not preexist but develop after transplantation and are directed against foreign graft HLA are considered as de novo anti-HLA DSA.

• The distinction as donor specific is crucial, as the DSA are for the graft.

Whether detected pre- or post-transplant, the antibodies directed against antigens expressed on donor organs, when not treated clinically

attack the endothelium of the allograft, resulting in subsequent AMR.

This immune attack on the transplanted organ increases risk of graft loss and/or

rejection

• De novo DSA’s develop in 20-30% of transplant recipients.

• De novo non HLA antibodies may develop such as antivimentin against endothelium, and mediate graft damage. Anti MICA (MHC Class 1 related chain A) may be associated with AMR and late allograft failure.

Detection of DSA’s

• Panel reactive antibodies (PRA)

• CDL ( complement dependant lymphotoxicity)

Specific, but not sensitive for detection of anti HLA antibodies.

• Solid Phase Assays

1. Luminex assay: uses flowcytometry to detect antibodies to HLA by the use of single antigens bound to polystyrene beads.

2. C4d, C1q assay: recent addition with luminex assay. C4d or C1q fixing DSA’s are strongly associated with poor allograft survival.

3. MFI (Mean fluorescence intensity) :Used to monitor the impact of DSA depleting therapies.

Clinical evidence

• Large cohort studies of over 5000 transplant recipients indicate that at any given time, approximately 25% of transplant recipients have antibodie(s).

• Moreover, previous data in renal transplantation have shown that up to 96% of rejected allografts develop some level of detectable DSA.

• Monitoring of DSA to predict allograft outcome- Non invasive surrogate method compared to graft biopsy.

• Not only correlation of pre transplantation but also post transplantation DSA.

• Considered a risk factor more than a contraindication.

• Methodology to detect and therapy to remove is evolving.

Desensitization approaches

1. Remove circulating DSA by PLASMAPHERESIS.

2. Block their effect with proteasome inhibitors

3. Reduce production with anti-CD20

• Significant survival benefit

Pathologic features of AMR

Kidney

• The presentation of renal AMR may be acute, often associated with proteinuria or may be more insidious.

• Untreated, partially treated or multiple episodes of acute AMR can result in structural changes in the allograft, called as Chronic AMR.

• The diagnosis of renal AMR is made by Banff criteria.

Cohen, R. B. Colvin, M. R. Daha et al., “Pros and cons for C4d as a biomarker,” Kidney International, vol. 81, no. 7, pp. 628–639, 2012

CLASS I Presence of acute tubular necrosis (ATN) only, with minimal inflammation

CLASS II glomerulitis, peritubular capillaritis, and microthrombosis

CLASS III ArteritisHISTOLOGIC

AL EVIDENCE

DONOR SPECIFIC

ANTIBODIES (DSA)

BANFF

2003

C4d STAINING

Diagnostic criteria for acute AMR in renal allograft biopsies

1. Morphologic evidence

• neutrophils and/or monocytes/macrophages in PTC and/or glomeruli(acute glomerulitis) arterial fibrinoid necrosis thrombi in glomerularcapillaries, arterioles, and/or small arteries acute tubular injury.

2. Immunohistologic evidence

• C4d in PTC Ig and/or complement in arterial fibrinoid necrosis

3. Serologic evidence

• Circulating antibodies to donor HLA or other specific antidonor antibodies at the time of biopsy

• At least one finding in each of the three categories must be present for a biopsy to be diagnostic of acute AMR.

• Biopsies that meet two of the three criteria may be regarded as suspicious for acute AMR on the basis of

criteria established by National Institutes of Health and Banff working groups.

Histopathology

In acute AMR microcirculatory changes occur in peritubularcapillaries and glomerular capillaries.

1. Peritubular capillaritis

2. Transplant glomerulitis

3. Accumulation of macrophges and T lymphocytes

4. Thrombotic microangiopathy (thrombi in arterioles and/or glomeruli)

5. Fibrinoid necrosis in larger vessels

6. Transmural and intimal arteritis

Histopathological Classification of AMR

• Class I:

Presence of acute tubular necrosis (ATN) only, with minimal inflammation.

• Class II:

Glomerulitis, peritubular capillaritis, and microthrombosis.

• Class III:

Arteritis

• Acute tubular necrosis (ATN) in acute ABMR

H & E

• Glomerulitis, infiltration of capillary loops by monocytes[white arrows]

H & E

• Peritubular Capillaritis (dilatation of capillaries and margination of monocytes [white arrows])

H & E

1.Trpkov K, Campbell P, Pazderka F, Cockfield S, Solez K, Halloran PF. Pathologic features of acute renal allograft rejection associated with donor-specific antibody: Analysis using the

Banff grading schema. Transplantation 1996;61(11):1586-92

Fibrinoid necrosis of the glomeruli, focal interstitial hemorrhage and neutrophilic margination

PAS

1.Trpkov K, Campbell P, Pazderka F, Cockfield S, Solez K, Halloran PF. Pathologic features of acute renal allograft rejection associated with donor-specific antibody: Analysis using the Banff grading schema. Transplantation 1996;61(11):1586-92

Fibrin thrombus

H & E

In Chronic AMR

1. Reduplication of basement membrane and peritubularcapillary basement membranes.

2. On light microscopy seen as double contours of GBM

3. Fibromuscular intimal thickening

4. Minimal elastosis

www.pathologyoutlines.com

Renal artery with chronic vascular rejection are markedly thickened and fibrotic. There is interstitial fibrosis and chronic inflammation

H & E

UPMS-TRANSPLANT PATHOLOGY INTERNET SERVICES

Glomerular basement membrane duplication is now recognized as the most specific lesion for the diagnosis of chronic transplant glomerulopathy.

PAS

Immunohistochemistry

• C4d deposition is seen in PTC’s and GC’s but only peritubularcapillary deposition is assessed as part of Banff criteria.

• By IF, Banff criteria states that 50% of PTC’s need to show positive staining to be considered positive.

• However, recent studies suggests that any staining has an impact on long term graft survival.

• The medulla is the most sensitive area in which to detect C4d deposition in PTCs.

C4d staining

Electron microscopy

1. The earliest feature of AMR is swelling of endothelial cells.

2. Increase in organelles in GC and PTC.

3. Electron lucent widening

4. Accumulation of debris on the subendothelial side of GBM.

5. Necrosis of endothelial cells.

6. Deposition of a new layer of GBM, although there are no electron dense deposits.

7. Reduplication of PTC basement membrane.

8. 3-4 layers : Acute AMR.

9. 5 or more : Chronic AMR

Molecular studies

• Endothelial transcripts : marker of endothelial activation.

• NK cell transcripts

AMR transcripts: potentially more sensitive than C4d staining.

Heart

• Incidence of cardiac AMR is estimated as 10-20%

• In 2011, a preliminary grading system for AMR was drawn up and published by members of ISHLT’s pathology council.

Grading

Histopathology

1. Diffuse myocardial microvascular inflammation is the hallmark of cardiac AMR.

2. In pAMR1 (h+) and pAMR2, capillaries are distended, with lumens narrowed by plump activated endothelial cells. And plugs of Intravascular macrophages.

3. In pAMR3 there is oedema, vasculitis, haemorrhage, microvascular thrombosis, capillary damage and myocytenecrosis with neutrophil infilteration.

Immunopathology

• C4d and CD 68 using IHC/ IF

• In pAMR1(i+) and pAMR2, C4d result is multifocal >50% of intact myocardium, or diffuse capillary deposition of any intensity.

• Diffuse weak C4d deposition or focal C4d deposition 10-50% may reflect resolving AMR.

• In pAMR3 C4d deposition may be absent or weak due to endothelial damage.

• CD 68 positive Intravascular macrophages, typical for cardiac AMR predict myocardial capillary C4d deposition.

H & E stained section of myocardium showing diffuse microvascular inflammation with capillary C4d deposition.

Immunopathological surveillance of cardiac AMR

• The recommended schedule for serological and immunopathological studies is 2 weeks.

• Then 1,3,6 and 12 months after transplantation, anually and when AMR is clinically suspected early or late (>1year) after transplantation.

Liver

• The true incidence and impact of AMR in liver are unknown due to the lack of large prospective studies assessing DSAs.

• Preformed DSAs are associated with worst graft survival. Their incidence ranges from 10.5 to 22.2%

• De novo DSAs occur in upto 10% of recipients.

Histopathology

1. Portal tract changes indicative of duct obstruction: portal oedema, ductular reaction, neutrophillic infilterate.

2. Sinusoidal inflammation

3. Microvascular inflammation

4. Perivenulitis

5. Areas with unexplained coagulative necrosis

Immunopathology

1. The interpretation of C4d is still incompletely understood.

2. C4d may be deposited in portal and/ or sinusoidal microvasculature.

3. IF is more sensitive than IHC, with portal vascular staining dominating in IHC and sinusoidal staining in IF.

Lung

• The lung allograft is subject to challenge from both the recipient’s immune system and the environment.

• Therefore, the diagnosis of pulmonary AMR is one of exclusion, based on graft dysfunction, circulating DSAs and histopathological features suggestive of AMR whether or not capillary C4d is detected.

Histopathology

1. Neutrophillic capillaritis: Patchy or diffuse dense neutrophillic septal infiltrates with karyorrhectic debris.

2. Fibrin with platelet thrombi in microvasculature

3. Alveolar haemorrhage

4. Flooding of neutrophils into adjacent alveolar spaces.

Immunopathology

1. Role of C4d deposition in pulmonary AMR is not clear

2. However, it is still advised.

3. A scoring system analogous to that for renal and cardiac biopsies ( >50% = positive ) is suggested.

Conclusion

1. Serological evidence of

DSAs

2. Biopsy findings

3. Capillary deposition of

C4d

A standard approach to diagnosis and management of AMR

• Review Article Late and Chronic Antibody-Mediated Rejection: Main Barrier to Long Term Graft Survival Qiquan Sun1 and Yang Yang2. 2013.

Thank you !