1. The Value of Zero-Hour Implantation Biopsies Volker Nickeleit Nephropathology Laboratory, Department of Pathology The University of North Carolina, Chapel Hill, USA

2. Baseline Renal Allograft Biopsies Purpose: 1) Organ adequacy (Harvest biopsy) 2) Pre-existing Disease (Protocol Biopsy) a) Post transplant biopsy interpretation (“book-keeping”) b) Prediction of function / management c) Diagnosis of (living) donor disease

3. a) Donor harvest biopsies Harvest biopsies of limited practical value Purpose:1) Organ adequacy : + / - (adjunct tool) 2) Pre-existing Disease : + / - a) Adequate post transplant biopsy interpretation b) Implantation protocol biopsies

4. Harvest biopsies of limited practical value Purpose:1) Organ adequacy : + / - (adjunct tool) 2) Pre-existing Disease : + / - a) Adequate post transplant biopsy interpretation (“book-keeping”) Improvement: a) standardization of technique, i.e. needle cores, deep wedges b) complete tissue evaluation (PAS, trichrome) and material sharing with managing transplant center c) systematic studies of criteria to discard organs

5. a) Donor harvest biopsies b) Implantation protocol biopsies

6. - Implantation protocol biopsies to assess donor disease - Strengths: a) full histological evaluation “no time constraints” b) good assessment of lesions “special stains” - Problems: a) Risk of complications / bleeding (caveat: living donations) b) Sampling: 15 gauge needles, only one core, no frozen tissue, subcapsular wedge biopsies

7. UNC experience with post perfusion biopsies n=175 kidney transplants n=114 post perfusion zero-hour biopsies (65% of all organs) n=1Complication (extended bleeding) 0.9% of all biopsies Biopsy procedure:biopsy gun, 15 gauge needle, 1 or 2 cores Tissue fixation in formalin and fresh frozen collection

8. Baseline Renal Allograft Biopsies Purpose: a) Diagnosis of (living) donor disease b) Identifying “baseline” histological changes c) Prediction of function / management

10. N=114 biopsies n=72 cadaveric, n=42 living origin donor age: median 37 yrs (range: 9 – 61 yrs) N=78 (68%) Banff minimal adequacy (> 6 glomeruli and > 1 artery; caveat: often medulla) N=22 (20%) Normal (no arteriosclerosis, no glomerulosclerosis) N=42 (37%) Immunofluorescence analysis N=0 Immuncomplex mediated GN N=0 C4d positivity N=0 Tubular HLA-DR expression N=0 (0%) Active disease

11. Histological features (total n=114): Glomeruli median: 14 (range: 0 - 52) Sclerosed Glomeruli > 3 sclerosed glomeruli n=1 % Interst. fibrosis 0% n=66, 10% n=13 % Tub. Atrophy 0% n=84, 10% n=6 Arteriolosclerosis (0-4) (0) n=57, (1) n=33, (2) n=10, (3) n=1 Intimal sclerosis (0-4) (0) n=45, (1) n=24, (2) n=17, (3) n=4 ATN (0-4) (0) n=0, (1) n=78, (2) n=48, (3) n=14, (4) n=1

12. Moderate Arteriosclerosis ( Scoring: > 2 / 4 ) 21 / 114 biopsies (18%) 18% of cadaveric organs N= 13 cadaveric organs (mean: 37 yrs, range: 18 – 59 yrs) N= 1 secondary FSGS (cadaveric organ) 19% of organs from living donations N= 8 organs of living origin (mean: 46 yrs, range: 37- 54 yrs)

13. Baseline Renal Allograft Biopsies Purpose: a) Diagnosis of (living) donor disease Unexpected arteriosclerosis (suggestive of hypertension induced damage) in 19% of donors b) Identifying “baseline” histological changes Arteriosclerosis and arteriolosclerosis in 40% of organs

14. Baseline Renal Allograft Biopsies Purpose: a) Diagnosis of (living) donor disease b) Identifying “baseline” histological changes c) Prediction of function / management

15. Acute rejection - graft failure ( 12 months post transplantation) Multi-organ recipients 4/ 114 (4%) Acute rejection 19/ 110 (17%) Graft failure 5/ 110 (5%) BK-Virus nephropathy 8/ 110 (8%) Lost for follow up 4/ 114 (4%) 40 Patients excluded from functional analyses

16. Statistical analysis Histological features: % globally sclerotic glomeruli % interstitial fibrosis % tubular atrophy arteriolosclerosis (0-4) arterial intimal sclerosis (0-4) ATN (0-4) Clinical data (during 12 months post transplantation): S-Creatinine:2 weeks, 1, 3, 6, 12 months post tx Delayed graft function:at least 1 episode of HD post tx Blood-Pressure:3, 6, 12 months post tx Acute rejection episodes Graft loss Demographic data:Recipient age, sex, race, number of tx Donor age, sex, race Type of donor organ

17. Statistical analysis Histological features: - % globally sclerotic glomeruli - % interstitial fibrosis - % tubular atrophy - arteriolosclerosis - arterial intimal sclerosis Significantly correlated to one another Leading variable: arterial intimal sclerosis

18. Statistical analysis: Arterial intimal sclerosis A) Arterial intimal fibrosis is correlated with the age of the donor Age in years ( mean + SD) Scoring 027,9 + 12.0 Scoring 142,7 + 8.3 Scoring 244.7 + 11.9 Scoring 346,7 + 6.3 p< 0.0001 B) Arterial intimal fibrosis is not correlated with donor organ type, donor sex, donor race

19. Statistical analysis **: Arterial intimal sclerosis Arterial intimal sclerosis * Delay. S-Creatinine (mean + SD) Blood pressure ( scoring 0-3 ) Funct. 2 1 3 6 12 3 6 12 % Wks m m m m m m mths 0n=31 13% 1.27 +.32 1.25 +.33 1n=14 14% 1.28 +.46 1.29 +.39 2n=7 0% 1.60 +.43 1.62 +.40 3n=2 0% 1.25 +.07 1.35 +.21 ns ns ns ns p<0.04 ns ns ns ns mild (0-1) n=45 13% 1.27 +.36 1.26 +.34 moderate (2-3) n=9 0% 1.52 +.40 1.55 +.37 ns ns ns p<0,04 p<0.02 ns ns ns ns * Biopsies fulfilling minimal adequacy criteria only ** Evaluation of functioning renal grafts without rejection during 12 months

20. Statistical analysis: ATN A) Acute tubular injury (ATN) is correlated with donor organ type Cadaveric Living donation Scoring 0 4 9 Scoring 11418 Scoring 23810 Scoring 311 3 Scoring 4 0 1 p<0.001 B) Acute tubular injury (ATN) is not correlated with delayed graft function, acute rejection episodes, arterial hypertension

21. Statistical analysis **: ATN Acute tub. injury Delay. Function S-Creatinine (mean + SD) ( scoring 0 - 4 ) 2 1 3 6 12 Wks m m m m 0 n=7 1.13 + 0.29 1 n=16 1.66 +.79 2 n=27 1.82 + 1.31 (3 and 4) n=6 2.17 + 1.35 ns p<0.05 ns ns ns ns ** Evaluation of functioning renal grafts without rejection during 12 months (total n=54)

22. Baseline Renal Allograft Biopsies Purpose: c) Prediction of function / management Arterial intimal sclerosis: - associated with increased S-Cr 3 and 6 months post tx - associated with donor age ATN: - associated with increased S-Cr 2 weeks post tx

23. Baseline Renal Allograft Biopsies Purpose: a) Diagnosis of (living) donor disease b) Identifying “baseline” histological changes Impact on diagnoses in post transplant allograft biopsies c) Prediction of function / management

24. Chronic vascular rejection – versus - pre-existing donor disease Chronic inactive vascular rejection Zero-Hour Biopsy: Intimal sclerosis

25. Pre-existing donor disease and superimposed vascular rejection: 12 days post transplantation Media Donor disease: intimal sclerosis Banff type II rejection: Endothelialitis

26. Pre-existing donor disease and superimposed rejection 5 months post transplantation: Arterial intimal sclerosis and chronic active vascular rejection

27. Calcineurin-inhibitor induced arteriolopathy -versus – pre-existing arteriolosclerosis Cyclosporine arteriolopathy Zero-Hour Biopsy: arteriolosclerosis

28. Baseline (Zero-Hour) Biopsies Important for adequate classification of transplant pathology caveat: fibrosis and atrophy may be donor disease! active and scarred rejection may be superimposed on donor disease! Prediction on graft function Some help to detect (living) donor disease Help for scientific projects (e.g. gene expression analysis post tx, latent viral load measurements etc) Specific diagnoses

29. Back to the Basics of the “Banff Idea”…. …..Bean Counting.

30. Banff “Edition” for reporting Donor Disease 1) Strongly recommend adequate baseline implantation biopsies 2) Separately score and report donor disease (“D”) Dcv (0-3): arterial intimal fibroelastosis with marked multilayering of elastic lamellae Dah (0-3), Dci (0-3), Dct (0-3) Dcg: percentage of globally sclerotic glomeruli 3) Post transplantation: - score Banff lesions as usual - specifically comment on previous “D” scores