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
Carousel slide 15

4. C4d and Early Graft Loss
(H.E. Feucht et al, Kidney Int 43: , 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

7. C4d Staining in Renal Allografts: correlation with donor-specific Ab
Collins et al, JASN 10: , % of AR with +DSA were C4d No C4d in DSA- AR, CSA toxicity
Crespo et al, Transplantation 71: /19 with steroid-resistant AR and DSA C4d /32 with steroid-resistant AR and no DSA C4d C4d positivity 95% sensitive for presence of DSA (IF)
Maueyyedi et al, JASN 13: , % of early AR C4d+ - 90% had anti-donor antibody morphologic subtypes of AMR - capillary, arterial Arterial had worse outcome
Bohmig et al, JASN 13: , /24 C4d+ cases had DSA by flow cytometric XM % of C4d- biopsies had DSA % specificity, 31% sensitivity (IHC on paraffin sections)

8. Diagnostic Criteria for Acute AMR in Renal Allograft Biopsies (L. C
Diagnostic Criteria for Acute AMR in Renal Allograft Biopsies (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

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

11. Carousel slide 22

13. Carousel slide 25

14. Carousel slide 26

15. Carousel slide 27

16. RN Smith et al, AJT 8: , 2008

17. C4d in Late Renal Allograft Biopsies – Clinical Implications (1)
Poduval et al (Univ. Chicago), Transplantation 79: , 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

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

19. C4d in Late Renal Allograft Biopsies – Clinical Implications (2)
Sun et al (Nanjing, China), Kidney Int 70: , 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)

21. 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 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

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

23. 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

24. 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

25. 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

26. Focal PTC C4d Staining

27. 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

28. 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

29. Seemayer et al, NDT 22: , 2007

30. Seemayer et al, NDT 22: , 2007

31. 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

32. 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%

33. C4d Staining IF - Frozen IHC - Paraffin Diffuse 11 (79%) 5 (36%) Focal
Batel et al (Pittsburgh), Modern Pathology 21: , renal allograft biopsies in patients with known DSA
C4d Staining
IF - Frozen
IHC - Paraffin
Diffuse
11 (79%)
5 (36%)
Focal
1 (7%)
6 (43%)
Negative
2 (14%)
3 (21%)

34. Description of Staining % of Bx or 5 HPF C4d0 Negative C4d1 Minimal
Banff 2007 Scoring of PTC C4d Staining (AJT 8: , 2008)
Numerical Score
Description of Staining
% of Bx or 5 HPF
C4d0
Negative
C4d1
Minimal
1 – 9%
C4d2
Focal
10 – 50%
C4d3
Diffuse
>50%
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.

35. Focal C4d Staining – Clinical Implications (1)
Magil and Tinckam (Vancouver), NDT 21: , 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:
Diffuse (23)
Focal (14)
Negative (28)
Monocytes/glomerulus
PMNs/glomerulus
PTC PMNs/hpf
Acute cellular rejection
8 (35%)
13 (93%)
28 (100%)

36. Focal C4d Staining – Clinical Implications (2)
Magil and Tinckam (Vancouver), NDT 21: , 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:
Diffuse
Focal
Negative
Odds ratio of graft failure
1.00
0.86
0.21

37. Focal C4d Staining – Clinical Implications (3)
Worthington et al (Manchester, UK), Transplantation 83: , 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:
Diffuse (14)
Focal (22)
Negative (56)
Findings of AMR
9 (64%)
9 (41%)
22 (39%)
DSA (ELISA, time of bx)
5 (35%) 37

38. Focal C4d Staining – Clinical Implications (4)
Worthington et al (Manchester, UK), Transplantation 83: , 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:
Diffuse (14)
Focal (22)
Negative (56)
Graft loss
5 (36%)
5 (23%)
4 (7%)

39. Focal C4d Staining – Clinical Implications (5)
Kedainis et al (St Louis), Am J Transplant 9: , 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):
Diffuse (35)
Focal (77)
Negative (256)
Glomerulitis
20 (57%)
26 (34%)
46 (18%)
Transpl. glomerulopathy
7 (20%)
4 (5%)
14 (5%)
PTC margination
22 (63%)
45 (58%)
68 (27%)
Acute cellular rejection
12 (40%)
38 (49%)
61 (24%)
DSA
23/29 (79%)
22/32 (69%)
7/71 (10%)

40. Focal C4d Staining – Clinical Implications (6)
Kedainis et al (St Louis), Am J Transplant 9: , 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).

41. Kedainis et al, AJT, 2009

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

43. 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.

44. 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.

45. C4d Staining in ABO-Incompatible Renal Allografts

48. 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

49. 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.

50. Demographic and Serologic Data in Patient Cohorts
Group A (C4d+)
Group B (C4d-)
P Value
Number of patients 21 12
Age (years, mean + SD)
0.25
Males/Females
12/9
8/4
0.72
Race (white/black/Hispanic)
18/3/0
9/2/1
0.58
Donor blood group type:
0.09 A1 14 4 A2 2 1
A1B B 3 6
Initial BG Ab titer (mean + SD)
0.51
Number of PP/IVIG treatments:
Pre-transplant (mean + SD)
0.48
Post-transplant (mean + SD)
0.74
Other pre-transplant treatments
Splenectomy
Anti-CD20
Splenectomy and anti-CD20
None 7

51. Clinical Findings in Patient Cohorts
Group A (n = 21)
Group B (n = 12)
P Value
Post-transplant day of initial
protocol biopsy (days, mean + SD)
0.90
Post-transplant day of most
recent F/U (days, mean + SD)
0.53
Serum creatinine (mg/dL):
At diag./initial biopsy (mean + SD)
0.84
At 6 mo protocol bx (mean + SD)
0.50
At 12 mo protocol bx (mean + SD)
0.65
At most recent F/U (mean + SD)
0.59
Graft losses
1 (5%)
2 (17%)
0.54

52. Renal Findings in Patient Cohorts
Group A (n = 21)
Group B (n = 12)
P Value
Number (%) of Pts. With AMR
(all were clinical)
1 (5%)
3 (25%)
0.12
Number (%) of Pts. With ACR
(including clinical and subclinical)
7 (33%)
4 (33%)
0.99
ACR Type (Banff ’97)
0.82
None or borderline 14 8 1A 1 1B 2 2A 5
Number (%) of Pts. With BKV
5 (24%)
2 (17%)
No. (%) of Pts. with Recurrent FSGS
1 (8%)
(cg + ci + ct + cv):
At diag./initial biopsy (mean + SD)
0.72
At 6 mo protocol bx (mean + SD)
0.22
At 12 mo protocol bx (mean + SD)
0.032

54. Dickenmann et al, Clin Nephrol 65: 173-179, 2006
Examined outcomes in 22 patients having for-cause biopsies of conventional renal allografts that showed focal (n = 21) or diffuse (n = 1) C4d staining in peritubular capillaries by IF, no morphologic evidence of AMR or ACR, and no prior C4d+ biopsy.
5/22 patients received anti-rejection therapy following the biopsy, while 17/22 continued to receive only baseline immunosuppression
Anti-rejection therapy:
Yes No P
SCr (mg/dl) at biopsy ns
Lowest SCr within 4 weeks post-biopsy
0.02
SCr 1 year post-biopsy
3-year graft survival
100%
69%

55. Conclusions (ABO-I)
Although based on a retrospective analysis of a relatively small number of patients, our findings suggest that diffuse PTC C4d deposition and circulating anti-blood group antibodies plus the absence of clinical or histologic evidence of AMR is most likely associated with a decreased risk of scarring in ABO-incompatible renal allografts in the relatively short term.
It will be important to determine if this apparent beneficial effect persists over the long term and in larger cohorts of patients from different centers. If true, this would indicate that, perhaps unlike the case with conventional allografts, C4d staining without other evidence of AMR in ABO-incompatible grafts most likely represents a state of stable accommodation.

56. Department of Surgery
Robert Montgomery
Dorry Segev
Daniel Warren
Chris Simpkins
Diane Lepley
Jayme Locke
Department of Pathology
Lorraine Racusen
Serena Bagnasco
Karen King
Department of Medicine
Edward Kraus
Hamid Rabb
Andrea Zachary
Hafiz Rahman