1. C4d staining and morphology in protocol biopsies Michael Mengel* For the ESPRIT-Group European Study Group For Protocol Biopsies In Renal Transplantation *Disclosure: M.M. received travel grants form Wyeth and Roche

2. Multicentric Study 3 European TX-Centers Protocol- und Indication Bx Classification according up-dated Banff unstained paraffin slides from all centers C4d positive > 25% of PTC stained –25-50% focal positive –>50% diffuse positive C4d negative < 25% of PTC stained correlation with morphological and clinical parameters

3. Incidence of C4d TX-center type of biopsy Bx n BxC4d (%) >50%PTC C4d (%) 25-50%PTC C4d (%) negative center 1 indication Bx 485 (10.4)7 (14.6)36 (75.0) center 2 indication Bx 9915 (15.2)8 (8.1)76 (76.7) center 3 indication Bx 23026 (11.3)17 (7.4)187 (81.3) total indication Bx377 46 (12.2)32 (8.5) 299 (79.3) center 1 protocol Bx 1281 (0.8)2 (1.6)125 (97.6) center 2 protocol Bx 946 (6.4)4 (4.3)84 (89.3) center 3 protocol Bx 3294 (1.2)7 (2.1)318 (96.7) total protocol Bx551 11 (2.0)13 (2.4) 527 (95.6) total92857 (6.1)45 (4.8)826 (89.1)

5. Indication biopsies: C4d clinico-pathological correlation clinic C4d (%) >50%PTC C4d (%) 25-50% C4d (%) <25% univariate p value multivariate p value Sex(%females)24 (52.2)12 (37.5)106 (35.5)ns recipient age >60 years6 (13.0)4 (12.5)46 (15.4)ns Bx < 3 months after TX34 (73.9)17 (53.1)202 (67.6)ns prior cellular rejection to Bx6 (13.0)8 (25.0)51 (17.1)ns no induction therapy3 (23.1)5 (33.3)49 (40.8)ns induction therapy with anti-IL26 (46.2)6 (40.0)56 (46.7)ns induction therapy with ATG/OKT4 (30.8)4 (26.7)15 (12.5)ns prior TX11 (32.4)7 (23.3)37 (14.5)0.02 ns PRA >30%12 (26.1)5 (15.6)22 (7.4)<0.00010.02 morphology no cellular rejection 8 (24.2) 11 (36.7)107 (43.1)ns borderline7 (21.2)4 (13.3)44 (17.7)ns cellular tubular interstitial rejection (Banff IA+IB) 7 (21.2)9 (30.0)54 (21.8)ns cellular vascular rejection (Banff IIA+IIB) 10 (30.3)6 (20.0)40 (16.1)ns vascular rejection (Banff III) 1 (3.0)03 (1.2)ns ptc inflammation42 (91.3)20 (62.5)123 (41.3)<0.0001 glomerular inflammation24 (53.3)7 (22.6)50 (16.8)<0.0001 ns acute tubular damage42 (91.3)25 (78.1)158 (53.0)<0.0001 ns glomerular thrombosis1 (2.2)1 (3.1)6 (2.6)ns arteriolar necrosis1 (2.2)05 (1.7)ns

6. protocol biopsies: C4d clinico-pathological correlation clinic C4d (%) >50%PTC C4d (%) 25-50% C4d (%) <25% univariate p value multivariate p value Sex(%females)3 (37.5)1 (10.0)190 (40.3)ns recipient age >60 years6 (13.0)4 (12.5)46 (15.4)ns Bx < 3 months after TX3 (27.3)4 (30.8)219 (41.6)ns prior cellular rejection to Bx2 (25.0)2 (20.0)98 (20.2)ns no induction therapy3 (60.0)2 (22.2)96 (22.0)ns induction therapy with anti-IL22 (40.0)7 (77.8)281 (64.3)ns induction therapy with ATG/OKT0060 (13.7)ns prior TX4 (80.6)1 (11.1)53 (12.4)<0.0001 ns PRA >30%1 (12.5)1 (10.0)26 (5.5)ns morphology no cellular rejection 6 (60.0) 7 (63.6)286 (71.7)ns borderline3 (30.0)3 (27.3)89 (22.3)ns cellular tubular interstitial rejection (Banff IA+IB) 1 (10.0)1 (9.1)23 (5.8)ns cellular vascular rejection (Banff IIA+IIB) 001 (0.3)ns vascular rejection (Banff III) 000-ns ptc inflammation6 (85.7)3 (37.5)12 (3.3)<0.0001 glomerular inflammation2 (28.6)2 (25.0)10 (2.8)<0.0001 ns acute tubular damage4 (57.1)3 (37.5)157 (43.5)ns glomerular thrombosis000-ns arteriolar necrosis000-ns

7. 24 C4d+ protocol Bx matched with 72 C4d- protocol Bx for: Age, Sex, time point of Bx, Banff grade

9. F.R., male, born 1944 Renal-Transplantation 31.08.01 Analgesic nephropathy, stone kidneys Hemodialysis 11/1988 1. Transplantation 5/1990 Transplant failure 1/1999 with CAN 2. Transplantation8/2001 Relapsing peptic ulcers (bleeding), Hypertension (4 antihypertensive drugs), Hyperlipoproteinemia, Coronary heart disease

10. K 1

11. K 1-C4d+

12. K 2

13. K 2-C4d+

14. Indication

15. K 3

16. F.R., male, born 1944 Renal-Transplantation 31.08.01 0,5 Biopsies Predni CSA MMF RR Med Pravastatin ASS U- Protein (g/L) S-Creatinine (µmol/L) 1,3 0,9 0,08 0,2 0,4 0,3 (4)

17. Summary Incidence for C4d in protocol bx significantly lower Incidence varies between centers Incidence is higher with presensitized patients focal C4d stain correlates with AHR morphology C4d in protocol bx less often with cellular rejection cases of “subclinical” humoral rejection  prognostic relevance:  No acute graft losses  Harbinger of chronic humoral rejection??

18. Hannover-Germany Hermann Haller Anke Schwarz Wilfried Gwinner Michael Mengel ESPRIT-Group European Study Group For Protocol Biopsies In Renal Transplantation Antwerp-Belgium Marc De Broe Jean-Louis Bosmans Johannes Bogers Barcelona-Spain Daniel Seron Francesco Moreso Marta Carrera