Renal transplant Rejection

1. C4d POSITIVITY IN RENAL
REJECTION
Dr.Imrana Tanvir

2. OBJECTIVES
• Introduction of Antibody mediated rejection
AMR
• Classification of AMR
• Role of C4d in transplant rejection
• Pros and Cons of C4d marker
• Donor specific antibodies DSA
• Conclusion

3. 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
Is one of the
causes of
chronic allograft
rejection

4. Histological criteria of presumptive
diagnosis of AMR
• endothelial injury with swelling
• Acute glomerulitis
• Peri-tubular capillary margination and
dilatation
• Fibrin thrombi in vessels/Fibrinoid necrosis
• Interstitial edema/haemmorhage
In minority of cases-Acute tubular necrosis only

5. Fraser-Lendrum
stain
highlighting the
presence of a
fibrin thrombus
in this afferent
arteriole

6. 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

7. 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

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

9. UPMS-TRANSPLANT PATHOLOGY INTERNET SERVICES
Glomerular basement membrane duplication is now recognized as the most
specific lesion for the diagnosis of chronic transplant glomerulopathy.

10. 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
HISTOLOGICAL
EVIDENCE
DONOR
SPECIFIC
ANTIBODIES
(DSA)
BANFF
2003
C4d STAINING

11. Introduction to C4d

12. Complement system

13. • 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

14. 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.
• There have been rare reports of C4d presence in PTC of donor
kidneys
Chethan Puttarajappa, Ron Shapiro and Henkie P. Tan . Antibody-Mediated Rejection in Kidney
Transplantation: A Review

15. Methods of detection
1.Immunofluorescence (IF) on frozen tissue (monovalent
antibody against C4d )
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.

16. PAS to show Peri-tubular capillaries

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

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

19. Strong , linear staining along entire circumference of PTCs

20. C4d
• Feucht et al- C4d in PTCs
Associated with poor graft survival
• Collin et al- close correlation of PTC C4d
staining with
– concurrent circulating DSA
– Neutrophils in cappilaries
(peritubular,gloomerular)
– Vascular arterial fibrinoid necrosis

21. C4d positive AMR
• Feucht et al. - 1991
Significant association of C4d with preformed anti
HLA antibodies in patients with acute rejection .
Confirmed in a study of 16 biopsies with DSA and
histopathological evidence of AMR (neutrophilic
capillaritis).
Diffuse C4d-staining with trace or no staining in
the biopsies with acute cellular rejection and 5 of
the 6 biopsies with cyclosporine toxicity.

22. • Crespo et al.
evaluated DSA and C4d in steroid-resistant
rejections and found positive DSA in 37% of the
cases. Among these, 95% had positive PTC C4d-
staining.

23. Association with Chronic AMR
• Mauiyyedi et al. reported presence of C4d in
23 of 38 biopsies with features of chronic AMR
but no C4d in the control group without
features of chronic AMR .
• However, some studies found no correlation
between presence of TG and diffuse C4d, with
many TG patients showing no C4d positivity .
TG(transplant glomerulopathy)

24. • In 2007, a retrospective study of biopsies from
1320 transplanted patients showed that more
than 40% of cases with transplant
glomerulopathy—were C4d negative, despite
the fact that anti-HLA antibodies were
detected in 73% of patients*
33.Sis B, Campbell PM, Mueller T et al. Transplant glomerulopathy, late antibody-
mediated rejection and the ABCD tetrad in kidney allograft biopsies for cause. Am J
Transplant 2007; 7: 1743–1752

25. C4d negative AMR
• In chronic AMR
• as common, if not more than the C4d-positive
AMR and has similar poor prognosis in terms
of graft survival
• Loupy et al. [38,39] showed that C4d or
capillaritis in 3-month protocol biopsies were
risk factors for later transplant
glomerulopathy, and capillaritis was predictive
even in the absence of C4d*
Loupy et al. [38,39] showed that C4d or capillaritis in 3-month protocol
biopsies were risk factors for later transplant glomerulopathy, and
capillaritis was predictive even in the absence of C4d

26. • Haas &Mirocha,who investigated patients
with DSAs who had a biopsy during the first 3
months after transplantation. Patients with a
C4d-negative biopsy who were not treated for
AMR had a higher rate of progression to
transplant glomerulopathy than those who
were treated for AMR post-biopsy
.Haas M, Mirocha J. Early ultrastructural changes in renal allografts: correlation with antibody-
mediated rejection and transplant glomerulopathy. Am J Transplant 2011.

27. • complement independent
• Experimental studies
“allo-antibodies themselves can alter the state of
the endothelium in the absence of complement or
other inflammatory cells”
• Endothelial transcripts combined with DSA show
excellent sensitivities for AMR (although less
specificity than C4d)

28. • Variability of staining over time suggesting a
constant flux between states of positive to
negative C4d .
• In a significant number of these biopsies,
there was presence of microvascular
inflammation (PTC and glomerular capillaritis)
in spite of negative C4d-staining .*
. Loupy, G. S. Hill, C. Suberbielle et al., “Significance of C4d Banff scores in early protocol biopsies of kidney transplant recipients with
preformed donor-specific antibodies (DSA),” The American Journal of Transplantation, vol. 11, no. 1, pp. 56–65, 2011.

29. Results
cases
• Biopsies-132
• Acute rejection-59
• ATN-10
• CAN-41
• Normal-18
• BK nephropathy-4
(morphology)
• Miscellaneous-8
C4d staining
•Overall
prevelance 45%
•P-AbAR-81%
•ACR-20%
•CAN-30%

30. ADVANTAGES OF C4D

31. • C4d is now one of the core diagnostic tools to
identify AMR
• ‘A magic marker‘:because of its stability, its
strong association with antibody-mediated
rejection (AMR), and finally, its major impact
on graft survival and patient treatment*

32. • Emergence of new therapeutics that block
complement activation makes C4d a marker
with potential to identify patients who may
possibly benefit from these drugs

33. LIMITATIONS OF C4D

34. 1. In ABO-incompatible transplantations ,C4d is
present in the majority of grafts but this
seems to point at 'graft accommodation'
rather than antibody-mediated rejection*
2. Potential biomarker in other fields where
antibodies can cause tissue damage, such as
systemic autoimmune diseases
C4d deposition in native kidneys, mainly in the
setting of autoimmunity

35. C4d positivity along the glomerular
basement membrane
Hui M, Uppin MS, Prayaga AK, Raju SB, Rajasekhar L. C4d immunohistochemistry in
membranous nephropathy. J Lab Physicians [serial online] 2014 [cited 2014 Nov 12];6:76-9
Granular
peritubular
capillary PTC
staining has
been rarely
described in
lupus nephritis

36. • C4d staining was investigated in many forms
of glomerulonephritiswhere peritubular
capillary C4d staining was virtually never
observed*
• Glomerular C4d deposition on the other hand
is a relatively common finding
*Chen M, Daha MR, Kallenberg CG. The complement system in systemic autoimmune disease. J Autoimmun 2010; 34: J276–J286.
*Cohen D, Koopmans M, Kremer Hovinga I et al. Potential for glomerular C4d as an indicator of thrombotic microangiopathy in lupus
nephritis. Arthritis Rheum 2008; 58: 2460–2469.
*Kusunoki Y, Itami N, Tochimaru H et al. Glomerular deposition of C4 cleavage fragment (C4d) and C4-binding protein in idiopathic
membranous glomerulonephritis. Nephron 1989; 51: 17–19..
*Xing GQ, Chen M, Liu G et al. Differential deposition of C4d and MBL in glomeruli of patients with ANCA-negative pauci-immune
crescentic glomerulonephritis. J Clin Immunol 2010; 30: 144–156

37. DEVELOPMENTS

38. Certain endothelial transcripts EDNAT appear to be
expressed more often in patients with histological features
of AMR even in the absence of C4d-staining.
Sis et al. (ENDAT) in kidney transplants
• ENDAT expression was higher in all types of rejection but
more so in AMR.
• 40% of patients with ENDAT and chronic AMR features
demonstrated no C4d-staining.
• strong correlation between elevated ENDAT and presence
of anti-HLA antibodies, particularly HLA Class II antibodies
• Biology: endothelial cell activation, repair, and
angiogenesis which are well known mechanisms of AMR .

39. • it is not inconceivable that this or a simpler
derivative method will partly or fully replace
C4d in future

40. DONOR SPECIFIC ANTIBODIES
DSA

41. DSA
• Anti-HLA antibodies generated against donor
cells
• Positivity for DSA before transplantation is a
contraindication

42. HISTORY
• P.A.Gorer 1938- first to introduce role of
antibodies in transplantation
• Terasaki & Ozawa 2004-monitor DSA as
predictor of transplantation outcome.
(DSA presence indicated higher risk for AMR
,both acute and chronic)

43. DETECTION OF DSA
• CDC :complement dependent cytotoxicity test
Golden standard to detect the presence of non
HLA molecules that could be target for anti-HLA
antibodies.
Based on complement fixation,Specific IgG not
fixing complement-false negatives

44. Solid phase assays
• ELISA – detect complement and non complement
fixing antibodies
• Pei et all 1998-microbeads coated with purified
HLA antigens,detected by flow cytometry
• Luminex assay-HLA attached to bead-most
applied
• Extremely sensitive,no accepted standard cutoff
value
• False negatives-IgM antibodies mask

45. Acute Humoral Rejection
• DSA (donor specific antibodies)
– PRA levels (panel reactive antibody)
– Flow cytometry cross match
– Flow PRA bead assays
Do not correlate with morphological as well as
immunopathological evidence

46. False negatives
• IgM antibodies mask
• Prozone phenomenum-C1 cometitively
displaces antibodies

47. Clinical evidence
• 1/3rd patients are sensitized
• Antibody mediated rejection
• 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

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

49. • Considered a risk factor more than a
contraindication
• Methodology to detect and therapy to
remove is evolving

51. ALTERNATIVES TO C4D

52. • •Alternatives for C4d are emerging (genomics,
molecular diagnostics, and endothelial
transcripts) and if proven useful, effort will be
made to transform these techniques or their
progeny to practical tests

54. REFERENCES
1. Chethan Puttarajappa, Ron Shapiro and
Henkie P. Tan . Antibody-Mediated Rejection
in Kidney Transplantation: A Review
2. Danielle Cohen, Robert B Colvin, Mohamed R
Daha, Cinthia B Drachenberg, Mark Haas,
Volker Nickeleit, Jane E Salmon, Banu Sis,
Ming-Hui Zhao, Jan A Bruijn, Ingeborg M
Bajema .Pros and Cons for C4d as a
Biomarker. Kidney Int. 2012;81(7):628-639

55. REFERENCES
• V. Pietroni, A. Toscano, F. Citterio. Donor-
Specific Antibody in Solid Organ
Transplantation: Where are We?

56. Queries???