1. P.Randhawa, A. Girnita, A. Zeevi, R. Shapiro, I. Batal, Departments of Pathology, Surgery, University of Pittsburgh SIGNIFICANCE OF FOCAL C4d DEPOSTIS IN THE KIDNEY

2. OUTLINE OF TALK Definition of focal C4d Clinical significance Management issues Occurrence of DSA –ve cases Association with Dx other than AMR

3. GUIDELINES FOR C4d INTERPRETATION Minimum 5 hpf Cortex or medulla (concordant in 75% graft nephrectomy). Necrotic/scarred area exclude ( intensity) Linear, circumferential, finely granular Intensity at least 1+ intensity on FS HCHO weak stain may be significant

4. BANFF 2007 DEFINITION OF C4d STAINING PATTERNS % biopsy area Interpretation according to technique (cortex and medulla) IFIHC C4d0 Negative: 0% C4d1 Minimal 1-10% C4d2 Focal 10-50% C4d3 Diffuse >50% Pos ?PosUnknown Neg

5. : BANFF 2001 MEETING Only C4d + and – categories recognized. Positive staining was defined as bright linear staining along capillary basement membranes typically involving OVER HALF OF SAMPLED peritubular capillaries NUMBER of capillaries expressed as a percentage, rather than SURFACE AREA of biopsy was the defining criterion Racusen et al. Am J Transplant 2003: 3: 708

7. % CAPILLARY SCORING % PTC score used in many studies >2001 Difficult to apply IF (dark field evaluation) Can not take in account loss of sensitivity of C4d staining on formalin fixed tissue Underestimates extent of C4d staining in bxs with IFTA & capillary loss

8. Kayler et al. Transplantation 2008; 85: 813 PTC C4d STAINING PATTERNS (106 BX WITH AR & C4d STAIN) Diffuse (16) Focal (24) Neg (66) I36%8%3% T331% 58%64% V1 38% 17%12% PC >25%13%14%15%

9. ANTI-HLA ANTIBODIES Diffuse (16)Focal(24) Neg (66) ELISA I38%30%15% ELISA II 83% 52%29% I or II (-1, +12m) 86% 57%32% DSA+/- 1m 94%38% 17%

10. RESPONSE TO STEROIDS Diffuse (16) Focal(24) Neg (66) Incomplete64% 82% 29% Creatinine 12m 0.7+/-0.60.6+/-0.80.3+/-0.6 Graft loss 31%38% 21% 61% if f/u Diffuse

11. C4D PATTERNS IN F/U BIOPSIES <1 YR (WORST C4d SCORE) Diffuse (12)Focal(20) Neg (54) D 58% 17%25% F 17% 45%20% Neg25%35% 67%

12. EFFECT OF TISSUE FIXATION : C4D PATTERNS IN DSA + PTS (n=14) FrozenHCHO Diffuse11/14 (79%) 5 /14(36%) Focal1 (7%)6 (43%) Negative2 (14%)3 (21%)

13. MANAGEMENT OF FOCAL C4d+ BIOPSIES AT PITTSBURGH Correlate with presence of DSA Pure Acute AMR with DSA, rising creatinine, get IVIG &/or PP Treat any concurrent T-cell mediated AR Assess degree of histologic chronicity

14. C4d + DSA –VE CASES: Technical Issues Technical problems with C4d staining -high background, necrotic or scarred area Technical problems with antibody testing (a) Date (b) Rare antigen not present in testing panel (c) Incorrect HLA Typing of donor HLA (d) Incomplete donor typing (anti-DP, DQ)

15. DETECTION OF DSA DEPENDS ON SENSITIVITY OF TECHNIQUE 41 biopsies focal C4d, ELISA PRA screening test for anti-HLA antibody -ve 11/41= 27% had DSA by Luminex 7/41 = 17% antibodies to MICA

16. BIOLOGIC EXPLANATIONS FOR C4d + DSA –VE CASES: Adsorption of DSA to graft Non-donor specific antibodies Non-HLA antibodies C4d deposition in dx other than AMR

17. NON-DONOR SPECIFIC HLA ABS Statistically more AR & worse outcome Marker for high immune responsiveness DSA may actually be present but absorbed Monitor carefully Hourmant et al. JASN2005;16;2804

18. NON-HLA ANTIBODIES AECA: anti-endothelial antibodies Anti-GSTT1 Glutathione S-Transferase T1 MICA, MICB AT1R ab: Angio II type I receptor ab Anti-VIM/ICAM-1 ab assoc GAX in heart Anti-AGRIN (GBM) ab associated cg Anti-HY ab products of Y chromosome

19. POTENTIAL TARGETS OF AECA MHC antigens ABO antigens AT1R receptors MICA (Mhc class I related Chain Ag) Other unknown polymorphic ags

20. PROBLEMS WITH AECA STUDIES Most assays do not attempt to define ag. Studies cross sectional: cause & effect? Some AECA definitely 2 0 vascular injury - due to rejection (intimal arteritis) - viral infection (CMV)

21. AECA & ANTI-HLA CAN CO-EXIST FCM assay XM-ONE Kit PBL endoth progenitors -35/147 (24%) pre-tx sera had donor reactive ab -Acute rejection 16/35 (46%) vs 13/112 (12%); -6/16 C4d +, ALL had confounding HLA ab Breimer et al. Txn 2008; 87: 549:

22. SOME AEC ASSAYS DO MEASURE COMPLEMENT FIXING AB EUROIMMUN indirect IF reagent kit and HUVEC deposited on BIOCHIPs AECA in 13/47 patients vascular rejection 6/13 C4d+ (46%); 1/6 anti-HLA + Plasma cell infiltrate 54% AEC-AR vs 12%no AR Overall 1 yr graft loss 46% AEC vs 19 % no AEC Sun et al. CJASN 2008; 3; 1479

23. ANTI-GLUTATHIONE S- TRANSFERASE T1 ANTIBODIES Donor has GSTT1 gene, recipient does not Incidence of GSTT1 mismatch ~ 20% Initial associative studies severe liver dysfunction Ktx: one study reported 4 cases of CHRONIC AMR with C4d in peritubular capillaries 1 case report acute AMR is also available Aguilera et al NDT 2008; 23; 1393

24. Feucht et al. KI 2001:5934; AJT 2003:3:646 BANFF CATEGORIES OTHER THAN AMR WITH C4d DEPOSITS Recurrent antiGBM Post-tx IgA 16/66 PTC Cho et al Clin Tx 2007:21:159 Colvin: USCAP 38% Denovo 17% rMGN Feucht 2001: 6/10 GN 11/19 ATN Feucht 2003: ATN C3d, not C4d

25. Lupus nephritis (31/455, D) - Li et al. Lupus 2007:16:875 - granular, EM immune complex deposits 2/2 Bacterial endocarditis GN Scleroderma renal crisis -diffuse 1/11, focal 3/11 Two donor, 1 DIC kidney (F) C activation multiple paths C4d DESCRIBED IN NATIVE KIDNEY DISEASES

26. SUMMARY Focal C4d PTC <50% surface area Staining pattern affected by tissue fixation Significance: correlate histology & DSA % patients with DSA intermediate DSA–ve: technical issues, non-HLA abs, diseases other than AMR