1. The Significance of C4d Staining with Minimal Histologic Abnormalities
Mark Haas
Department of Pathology
Cedars-Sinai Medical Center
Los Angeles, California, USA

2. Outline/Objectives
1. 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
2. To review potential prognostic implications of C4d staining without histologic lesions of antibody- mediated or cellular rejection in ABO-incompatible versus conventional and positive cross-match 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 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)

5. 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

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

7. No TG TG
Cosio et al, AJT 8:492-6,2008

10. 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

11. 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.

12. 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

13. 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

14. 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

16. 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) peritubular capillary C4d staining 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%

17. +CM
3 month protocol bx
SCr 1.0 mg/dl
2 + diffuse C4d, ptc 3
No ACR

18. Conclusions
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 in 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 C4d staining without other evidence of AMR in ABO- incompatible grafts most likely represents a state of stable accommodation.

19. Conclusions (continued)
3. Unlike the case in ABO-incompatible grafts, in positive crossmatch (and conventional) renal allografts C4d deposition in the absence of histologic evidence of AMR appears to be uncommon, and may in fact indicate (potentially reversible) graft injury. However, there may be notable exceptions.

20. 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