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Mark Haas Department of Pathology Cedars-Sinai Medical Center Los Angeles, California, USA

Challenging C4d Staining Results: C4d in Late Renal Allograft Biopsies, Focal C4d Staining, and C4d in ABO-Incompatible Grafts. Mark Haas Department of Pathology Cedars-Sinai Medical Center Los Angeles, California, USA. Outline/Objectives.

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Mark Haas Department of Pathology Cedars-Sinai Medical Center Los Angeles, California, USA

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  1. Challenging C4d Staining Results:C4d in Late Renal Allograft Biopsies, Focal C4d Staining, and C4d in ABO-Incompatible Grafts Mark Haas Department of Pathology Cedars-Sinai Medical Center Los Angeles, California, USA

  2. Outline/Objectives 1. Briefly review the literature on the association of C4d staining with histologic findings of chronic renal allograft rejection and evaluate studies of the clinical significance of C4d staining in late renal allograft biopsies 2. Evaluate the significance of focal (versus diffuse) C4d staining in renal allograft biopsies and how the staining method (IF on frozen sections versus IHC on paraffin sections) may affect these results 3. To appreciate differences in the interpretation of positive C4d staining in ABO-incompatible renal allografts versus conventional and positive cross-match (HLA-incompatible) renal allografts

  3. C4d • Degradation product of complement factor C4, a component of the classical complement pathway that is typically initiated by antibody bound to antigen • After cleavage of remaining C4 domains, C4d remains covalently bound at site of C4 activation • Thus, C4d is a relatively long-lived marker of a humoral response • Easily detected in tissue sections by immunofluorescence or immunohistochemistry

  4. C4d and Early Graft Loss (H.E. Feucht et al, Kidney Int 43: 1333-8, 1993) • 93 renal allografts biopsied for early dysfunction(mean 11 days post-transplant) • 43 biopsies – diffuse PTC C4d 18 graft losses in 1st year (58% graft survival) • 8 biopsies – focal PTC C4d 3 graft losses in 1st year (63% graft survival) • 42 biopsies – C4d negative in PTC 4 graft losses in 1st year (90% graft survival) • 3/4 cases of graft loss in C4d- group were C4d+ on a later biopsy • C4d+ associated with re-transplant, elevated PRA

  5. C4d Staining in Renal Allografts: correlation with donor-specific Ab • Collins et al, JASN 10: 2208-14, 1999 100% of AR with +DSA were C4d+ No C4d in DSA- AR, CSA toxicity • Crespo et al, Transplantation 71: 652-8 2001 18/19 with steroid-resistant AR and DSA C4d+ 2/32 with steroid-resistant AR and no DSA C4d+ C4d positivity 95% sensitive for presence of DSA (IF) • Maueyyedi et al, JASN 13: 779-787, 2002 30% of early AR C4d+ - 90% had anti-donor antibody 2 morphologic subtypes of AMR - capillary, arterial Arterial had worse outcome • Bohmig et al, JASN 13: 1091-9, 2002 21/24 C4d+ cases had DSA by flow cytometric XM 50% of C4d- biopsies had DSA 93% specificity, 31% sensitivity (IHC on paraffin sections)

  6. DiagnosticCriteriaforAcuteAMRinRenalAllograftBiopsies(L.C. Racusen et al, Am J Transplant 3: 1-7, 2003) • 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 • Immunoglobulin and/or complement in arterial fibrinoid necrosis • Serologic evidence • Circulating antibodies to donor HLA or other specific anti-donor antibodies at the time of biopsy

  7. C4d in Late Renal Allograft Biopsies: Association with Chronic Rejection and Clinical Significance

  8. RN Smith et al, AJT 8: 1662-72, 2008

  9. C4d in Late Renal Allograft Biopsies – Clinical Implications (1) Poduval et al (Univ. Chicago), Transplantation 79: 228-35, 2005 • 3-year study period (1999 – 2001) • 55 episodes of late AR (>6 mo post-tx, Banff 1A or greater) in 51 grafts • 39 episodes of early AR (<6 mo post-tx) in 39 grafts • Diffuse C4d staining (>50% of PTC, by IHC on paraffin) in 42% of biopsies with late AR versus 18% of biopsies with early AR (p = 0.014) • PTC C4d in biopsies with late AR was significantly correlated with interstitial plasmacytic infiltrates and hemorrhage but NOT with Banff g or cg scores or PTC neutrophil margination; many C4d+ biopsies did not meet Banff diagnostic criteria for AMR • In biopsies with late AR, diffuse, but not focal, PTC C4d was associated with significantly worse graft survival at 1 and 2 years post-biopsy

  10. Poduval et al, Transplantation 79: 228-35, 2005

  11. C4d in Late Renal Allograft Biopsies – Clinical Implications (2) Sun et al (Nanjing, China), Kidney Int 70: 377-383, 2006 • Compared outcomes in 56 patients with C4d-positive (>50% of PTC; IF on frozen sections), steroid resistant acute rejection (Banff 97 1A or greater, 42 were 2A or greater) based on time of diagnosis post-transplantation: • Very early acute rejection (VER, <14 days post-tx, n = 28) • Early acute rejection (ER, 15 – 180 days post-tx, n = 5) • Late acute rejection (LR, >180 days post-tx, n = 23) • 89% of patients with VER had complete response to treatment (with tacrolimus/MMF + immunoadsorption) compared with 0% patients with ER or LR; 40% and 30%, respectively, of patients with ER and LR had partial response • Graft survival 1 year post-biopsy was 96% in patients with VER, 60% in those with ER, 52% in those with LR (P < 0.001)

  12. C4d in Late Renal Allograft Biopsies – Clinical Implications (3) Issa et al (Mayo Clinic), Transplantation 86: 681-5, 2008 • Found that among 73 patients (negative pre-transplant T cell crossmatch, none ABO-incompatible) diagnosed with transplant glomerulopathy (TG) during a follow-up period of 54 + 19 months: • 12/16 (80%) grafts with PTC C4d at the time of diagnosis of TG failed, compared with 13/52 grafts that were C4d-negative at the time of diagnosis of TG • Presence of PTC C4d (by IF on frozen section or IP on paraffin section) was an independent risk factor for graft loss after TG diagnosis by multivariate analysis • PTC C4d in grafts showing TG was associated with higher levels of anti-HLA class II antibodies

  13. no TG TG, C4d- TG, C4d+ Issa et al, Transplantation 86: 681-5, 2008

  14. C4d in Late Renal Allograft Biopsies – Clinical Implications (4) Satoskar et al (Ohio State Univ.), Clin Transplant 22: 61-67, 2008 • Studied 40 patients whose first acute rejection episode occurred >1 year post-transplant • Two groups of 20 patients each: diffuse (>80%) PTC C4d staining versus focal (<40%) or absent PTC C4d staining, all by immunofluorescence on frozen tissue • Groups were matched for CADI scores; majority of biopsies in each group showed Banff 97 type 1B acute rejection; only 6/40 biopsies showed type 2 acute rejection • Diffuse PTC C4d staining seen in 50% of cases of late acute rejection (>1 year post-transplant) versus 30% of cases of early acute rejection • 6/20 biopsies with diffuse PTC C4d versus 0/20 biopsies with focal/absent C4d had TG on the initial biopsy (n = 1) or a later biopsy • No difference in graft survival (p = 0.57) between the 2 groups with mean post-biopsy follow-up of 20 months

  15. C4d in Late Renal Allograft Biopsies – Summary (1) • Diffuse PTC C4d staining (by IHC or IF) is more commonly seen in biopsies with late versus early acute rejection (Banff 97 1A or greater), although a substantial fraction of C4d+ late acute rejection biopsies do not meet Banff criteria for diagnosis of AMR • C4d+ late acute rejection (>6 mo post-transplant) is likely to be associated with a worse long-term outcome than C4d+ acute rejection in the initial few weeks post-transplant

  16. C4d in Late Renal Allograft Biopsies – Summary (2) • In some but not all studies, diffuse PTC C4d staining in the context of late acute rejection was found to be associated with shortened graft survival; differences between studies may be related to relative proportions of Banff 97 type 1 versus type 2 acute rejection • Diffuse PTC C4d staining with transplant glomerulopathy (TG) is associated with worse graft survival than TG alone, although it is not known if this effect is independent from that of documented higher levels of anti-HLA class II antibodies in patients with C4d+ biopsies

  17. Focal PTC C4d Staining

  18. In interpreting C4d staining, one should consider the method used: Indirect IF on frozen sections of fresh tissue using monoclonal anti-C4d antibody Immunoperoxidase on paraffin sections of formalin-fixed tissue using polyclonal anti-C4d antibody

  19. Seemayer et al (Basel), Nephrol Dial Transplant 22: 568-76, 2007 • Compared C4d staining by indirect IF on frozen sections (F-IF) and immunohisotchemistry on paraffin sections (P-IHC) in the same set of 64 renal allograft biopsies • Inter- and intra-observer concordance rates in interpreting C4d staining by F-IF were excellent (kappa = 0.9), but were lower with C4d staining by P-IHC (kappa < 0.67) • C4d staining by F-IF was more sensitive than staining by P-IHC

  20. Seemayer et al, NDT 22: 568-76, 2007

  21. Seemayer et al, NDT 22: 568-76, 2007

  22. Nadasdy et al (Ohio State), Human Pathology 36: 1178-85, 2005 • Compared C4d staining by indirect IF on frozen sections (F-IF) and immunohisotchemistry on paraffin sections (P-IHC) in the same set of 20 renal allograft biopsies that were C4d+ by F-IF on initial diagnostic evaluation, and had sufficient remaining frozen and paraffin-embedded tissue for repeat analyses • There was a suggestion of greater sensitivity of the F-IF method (15 cases diffuse positive, 5 focal positive) than with the P-IHC method (12 diffuse, 7 focal, 1 negative), although differences were less pronounced than in the study of Seemayer et al

  23. Troxell et al (Stanford/Oregon), CJASN 1: 583-591, 2006 • Examined C4d staining by P-IHC in 26 renal allograft biopsies that were C4d+ (diffuse in 25, focal in 1) by F-IF • All but 3 of these biopsies were C4d+ (diffuse in each case) by P-IHC • Using F-IF staining as the “gold standard”, sensitivity of P-IHC staining was 88%

  24. Batel et al (Pittsburgh), Modern Pathology 21: 1490-1498, 200814 renal allograft biopsies in patients with known DSA

  25. Banff 2007 Scoring of PTC C4d Staining (AJT 8: 753-760, 2008) C4d2 by P-IHC may be equivalent to C4d3 by F-IF; in such cases (as well as C4d1 by P-IHC) restaining by IF is advised if frozen tissue is available.

  26. Focal C4d Staining – Clinical Implications (1) Magil and Tinckam (Vancouver), NDT 21: 1382-8, 2006 • 65 biopsies from 53 patients with acute graft dysfunction, within the first 6 months post-transplantation • C4d staining done by IHC, but using frozen sections and the same monoclonal anti-C4d antibody used for IF in most centers • C4d staining was significantly correlated with several histologic findings of AMR:

  27. Focal C4d Staining – Clinical Implications (2) Magil and Tinckam (Vancouver), NDT 21: 1382-8, 2006 By trend analysis, C4d staining was significantly (P = 0.042) correlated with graft failure (defined as GFR <30 ml/min by MDRD) at 48 months post-transplant:

  28. Focal C4d Staining – Clinical Implications (3) Worthington et al (Manchester, UK), Transplantation 83: 398-403, 2007 • 92 patients undergoing for-cause renal transplant biopsy at median 79 days post-transplant (51 within first 6 months) • C4d staining by IHC on paraffin sections • Diffuse, but not focal, PTC C4d staining was correlated histologic findings of AMR (glomerulitis, transplant glomerulopathy, WBC in PTC, TMA and/or arterial fibrinoid necrosis) and presence of DSA:

  29. Focal C4d Staining – Clinical Implications (4) Worthington et al (Manchester, UK), Transplantation 83: 398-403, 2007 Graft loss between 1 month and 15 years post-transplant was significantly correlated with diffuse PTC C4d staining and presence of DSA. Graft survival with focal C4d was intermediate between diffuse and negative, but difference between focal and negative was not statistically significant:

  30. Focal C4d Staining – Clinical Implications (5) Kedainis et al (St Louis), Am J Transplant 9: 812-819, 2009 • 368 for-cause renal transplant biopsies from 301 patients • C4d staining by IF on frozen sections • C4d staining was significantly correlated with several histologic findings of AMR, with ACR, and with DSA at the time of biopsy (by ELISA):

  31. Focal C4d Staining – Clinical Implications (6) Kedainis et al (St Louis), Am J Transplant 9: 812-819, 2009 Post-biopsy graft survival was significantly worse (P = 0.014) with diffuse PTC C4d staining (mean 22.5 months) than with negative PTC C4d (43.4 months). Graft survival with focal C4d was intermediate between diffuse and negative (35.0 months), but difference between focal and negative was not statistically significant (P = 0.09).

  32. Kedainis et al, AJT, 2009

  33. Poduval et al, Transplantation 79: 228-35, 2005

  34. Focal C4d Staining – Summary (1) 1. With respect to association with histologic findings of AMR, association with the presence of anti-HLA DSA, and implication for graft survival, focal PTC C4d staining appears to be intermediate between diffuse C4d and negative C4d. 2. These findings were reproducible in 2 different studies using frozen tissue and a monoclonal anti-C4d antibody. Studies using paraffin-embedded tissue and a polyclonal anti-C4d antibody yielded some similar findings, although results were less consistent overall.

  35. Focal C4d Staining – Summary (2) 3. Unlike the case with diffuse C4d staining, the association of focal C4d staining with reduced graft survival generally did not reach statistical significance, although there was a trend in this direction. It is quite possible that this lack of significance reflects relatively small numbers of biopsies with focal C4d. 4. Regardless, it is advisable that testing for DSA be done on patients with biopsies showing focal PTC C4d, and that focal C4d together with DSA and histologic findings of AMR should be considered highly consistent with (and probably diagnostic of) AMR.

  36. C4d Staining in ABO-Incompatible Renal Allografts

  37. What is the effect of C4d staining without histologic evidence of AMR in ABO-I grafts? 44 recipients of ABO-I grafts 1/00 – 6/07 37 met the following criteria: • One or more protocol biopsies were done during the first ~3 months post-transplantation, meeting Banff ’97 adequacy criteria • One or more subsequent protocol biopsies were done at 6 and/or 12 months post-transplantation, meeting the same adequacy criteria • C4d staining was done on all protocol biopsies

  38. What is the effect of C4d staining without histologic evidence of AMR in ABO-I grafts? Of these 37 patients: 21 (Group A) had an initial (1 or 3 month) protocol biopsy meeting all of the following criteria: • >1+ diffuse PTC C4d • No ACR (Banff ’97 1a or greater) or histologic evidence of AMR (PTC neutrophil or mononuclear WBC margination [ptc >0], glomerulitis [g>1], TMA, or arterial/arteriolar fibrinoid change) • Presence of circulating antibody against donor blood group antigen(s) Of remaining 16 patients,12 had negative or focal and weak (<1+) PTC C4d on their initial protocol biopsy (Group B), while the other 4 had >1+ diffuse C4d but with PTC WBC margination (SC AMR; omitted). All had circulating anti-blood group antibody. No patient had anti-HLA Class I or Class II antibodies.

  39. Demographic and Serologic Data in Patient Cohorts

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