Objectives: Introduction of Antibody mediated rejection AMR Role of C4d in transplant rejection Donor specific antibodies DSA Presentation of AMR in kidney, liver, lung and heart.

1. ANTIBODY MEDIATED REJECTION
OF SOLID ORGAN ALLOGRAFTS.
Moderator : Dr M.L. Yadav
Presented by : Dr Tashi Agarwal

2. 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.

3. 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.

4. 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)

8. Transplant
rejection
Cell
mediated
(interstitial)
Antibody
mediated
(humoral)
Hyperacute
Acute Humoral
Rejection
Chronic Humoral
Rejection
Preformed DSA
Preformed/de
novo DSA
CHRONIC ALLOGRAFT
REJECTION IS DUE TO
MANY CAUSES
INCLUDING CHRONIC
HUMORAL REJECTION

9. Pathogenesis of
AMR

10. Introduction to
C4d

11. • 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.

12. • 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.

13. Complement system

14. • 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

15. 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

16. 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.

17. 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.

18. 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.

19. PAS to show peri-tubular capillaries

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

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

22. Strong , linear staining along entire circumference of
PTCs

23. DONOR SPECIFIC
ANTIBODIES
DSA

24. 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.

25. 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

26. • 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.

27. 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.

28. 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.

29. • 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.

30. Desensitization approaches
1. Remove circulating DSA by PLASMAPHERESIS.
2. Block their effect with proteasome inhibitors
3. Reduce production with anti-CD20
• Significant survival benefit

31. Pathologic features of AMR

32. 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.

33. 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 Arteritis
HISTOLOGIC
AL
EVIDENCE
DONOR
SPECIFIC
ANTIBODIES
(DSA)
BANFF
2003
C4d
STAINING

34. 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 glomerular
capillaries, 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.

35. Histopathology
In acute AMR microcirculatory changes occur in peritubular
capillaries 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

36. 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

37. • Acute tubular necrosis (ATN) in acute ABMR
H & E

38. • Glomerulitis, infiltration of capillary loops by monocytes
[white arrows]
H & E

39. • Peritubular Capillaritis (dilatation of capillaries and
margination of monocytes [white arrows])
H & E

40. 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

41. 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

42. In Chronic AMR
1. Reduplication of basement membrane and peritubular
capillary basement membranes.
2. On light microscopy seen as double contours of GBM
3. Fibromuscular intimal thickening
4. Minimal elastosis

43. www.pathologyoutlines.com
Renal artery with chronic vascular rejection are markedly thickened and
fibrotic. There is interstitial fibrosis and chronic inflammation
H & E

44. 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

45. Immunohistochemistry
• C4d deposition is seen in PTC’s and GC’s but only peritubular
capillary 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.

46. C4d staining

47. 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

49. Molecular studies
• Endothelial transcripts : marker of endothelial activation.
• NK cell transcripts
AMR transcripts: potentially more sensitive than C4d staining.

51. 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.

52. Grading

53. 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 myocyte
necrosis with neutrophil infilteration.

54. 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.

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

56. 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.

57. 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.

58. 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

59. 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.

61. 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.

62. 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.

63. 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.

65. Conclusion
1. Serological
evidence of
DSAs
2. Biopsy
findings
3. Capillary
deposition of
C4d
A standard approach to diagnosis and management of
AMR

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

67. Thank you !