1. C4d – The Birmingham UK Experience Desley Neil, Majid Mukadam, David Briggs* UHBNHSFT, NHSBT* Birmingham

2. Method C4d staining performed routinely on all EMBs from May 2004. May 2004 – May 2008 – 1443 biopsies in 163 patients with 166 transplants – reports for Grade of C4d staining 71 (4.9%) unreported – 14 missing, rest graded Presence or absence DSA (luminex bead) – not routine C3d staining on strong C4d cases Pattern of inflammation reassessed in Strong C4d bx V highest C4d neg/weak Busy, no, focal or diffuse infiltrate, Cells in / around capillary

3. Biopsy protocol Protocol – Weekly x 6 – Fortnightly x 2 – Monthly x 2 – 6 weekly x 21year – 6 monthly x22 years – Yearly x13 years Indication – Symptomatic or change in echo Change of medication – 2-3 biopsy during transition and once established

4. C4d (& C3d) grading system 0Negative 1Weak patchy staining 2Moderate staining 3Diffuse strong staining (Looks like CD31 at low power) Immunoperoxidase Polyclonal Ab Biomedica

5. C4d

6. C3d

7. Demographics 49 (14-65)49 (14-63)44 (19-65)Age 13231 163

8. Biopsies Timing of biopsies – 171 days (0-5806 days) post-tx Number of biopsies / patient – 4 (1-40) biopsies

9. C4d staining 72% 15% 60%

10. Highest C4d grade / patient 12.3% 35.6% 22.1% 29.5%

11. Death related to highest C4d 6.3%11.1%20.7%35% Kruskal Wallis p<0.02

13. Follow up time v highest C4d grade Follow up 0123 missing N=483658201 Median (range) 8 yrs (0.4-16.8) 4.4 yrs (0.5-15.5) 3.6yrs (0.3-14.8) 6 yrs (0.6-15.6) 12.4yr Kruskal Wallis p=0

14. Strong C4d 20 patients 7 had DSA found 6/7 (85.7%) with DSA died 1 with lot of consecutive strong C4d – serum not sent till after Plasma exchange = negative – Retransplanted Others only HLA Ab tested inconsistently

15. DSA 19 (11.7%) patients Found 7.1 (0-12.9) years post-tx 13 (68.6%) class II and 6 (31.4%) class I and II

16. Demographics of DSA +/- Age: DSA + 46 (14-59) DSA – 49 (17-65) NS

17. Symptoms 5 (26.3%) asymptomatic 14 (73.3%) symptomatic – 3 IHD/graft vasculopathy – 11 syncope, heart failure 10 evidence of graft vasculopathy 8 no evidence of graft vasculopathy 1 don’t know

18. Death related to DSA 47.4%18.8%10.5% Death v DSA Wilcoxin p=0.000

19. Follow up time v DSA Not testedNegPos N=1912519 Median (range) 6.1 yrs (0.4-15.7) 5.2 yrs (0.3-15.8) 8.6yrs (0.9-13.5) Kruskal Wallis NS Death 8.8 (3-12.9) yrs post tx 215 (7-1188) days post DSA found

20. DSA v highest C4d grade Kruskal Wallis p=0 35%17.2%2.8%2.1%

21. DSA v Strong C4d +C3d grade

22. DSA /Patient with strong C4d +/- mod/strong C3d 60% P<0.05

23. DSA in relation to C4d persistence Persistent strong4/6 (66.7%) Intermittent mod/strong4/17 (23.5%) Single strong (others neg/weak)4/18 (22.2%) Single bx = strong (no others bx)3/7 (42.9%) Nothing much3/111 (2.7%) Kruskal-Wallis p<0.02

24. C3d staining in relation to persistence of C4d

25. N=357 neg/weak N=42 strong

26. BUSY “B” PATTERN

27. Summary C4d staining relatively uncommon Both DSA and highest grade of C4d correlates with death Correlation between C4d and DSA –? improved by C3d Neither C4d or DSA in isolation is sensitive at a single time point – C4d - comes and goes, precede inflam and symptoms – Repeat DSAs (- to + in 4/7)

28. Using ISHLT criteria will miss 2/3 C4d positive cases same also if C3d mod/strong Busy “B” with cells in or around vessels C4d/C3d needs to be routine C4d grade