1. ANTIBODY-MEDIATED REJECTION AFTER PANCREAS TRANSPLANTATION: SÃO PAULO (BRAZIL) EXPERIENCE 
Érika B Rangel
Hospital Israelita Albert Einstein
Federal University of São Paulo

2. Aims
Report the incidence of AMR after pancreas-kidney and pancreas transplantation in biopsies prospectively screened for C4d,
Describe grafts outcome and the pattern of deposition of C4d in kidney and pancreas allografts, and
Correlate both AMR and TCMR to laboratorial parameters, such as serum amylase and lipase and amylasuria.

3. Patients and Methods August 2006/December 2008
38 patients submitted to pancreas transplantation
SPKT (n = 21), SPKT-V (n = 7), PAKT (n = 7), PTA (n = 3)
68 biopsies for cause:
Kidney: 33 biopsies in 21 patients ( Screat)
Pancreas: 35 biopsies in 27 patients ( serum enzymes and/or amylasuria by 50%)

4. Results
Time post transplantation:
Kidney biopsies: ± 535 days (median 64 days).
Pancreas biopsies: 566 ± days (median 192 days).
Average follow-up:
Kidney: 12.7 ± 9 months (median 12.7 months)
Pancreas: 12.7 ± 8.5 months (median 10.2 months)

5. Patients and Methods Initial immunosuppression: Induction:
Tacrolimus 0.15 mg/kg/dose, adjusted according to the period after transplantation (serum levels of ng/mL in the first 30 days and subsequently 5-10 ng/mL)
b) Methylprednisolone (500 mg intraoperative, 250 mg in the first day and 125 mg in the second day) followed by Prednisone 1 mg/day with tapering
c) Mycophenolate Mofetil 2 g/day or Mycophenolate Sodium 1.44 g/day.
Induction:
a) SPKT: re-transplantation, panel reactive antibody greater than 20% or DGF/SGF
b ) all cases of SPKT-V, PAKT and PTA

6. Patients and Methods
Surgical aspects
In SPKT, exocrine pancreatic drainage was enteric (n = 13) or in the bladder (n = 15).
In PAKT and PTA bladder drainage was exclusive.
Iliac vein or vena cava anastomosis was performed in all cases, except 2 SPKT patients that have portal drainage.

7. Patients and Methods
Kidney biopsies were scored according to Banff 2005 (updated in 2007)
Pancreas biopsies were scored according to Drachenberg et al (2008)
If there were no DSA or these data were unknown, identification of histological features of AMR was considered as suspicious for acute or chronic AMR, particularly if there was graft dysfunction.

8. C4d screening was the inclusion criterion
Patients and Methods
C4d screening was the inclusion criterion
C4d: indirect immunofluorescence; frozen samples; mouse monoclonal anti-human C4d antibody 1:40 dilution; fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG
Diffuse C4d: > 50% peritubular/interacinar capillaries
Focal C4d: < 50% peritubular/interacinar capillaries

9. Patients and Methods
Antibodies
HLA (Human Leucocyte Antigens): The Luminex® (The LABScan™ 100 flow analyzer)
MICA (Major-histocompatibility-complex class I-related A): single-antigen bead assay

10. Patients and Methods TCMR - Kidney allograft TCMR - Pancreas allograft
a) Methylprednisolone pulse (500 mg/day for 3 days): grades IA to IIA
b) Thymoglobuline/OKT3: grades IIB and III (7-10 days).
TCMR - Pancreas allograft
a) Methylprednisolone pulse (500 mg/day for 3 days): grade I
b) Thymoglobuline mg/kg/day or OKT mg/day (10 days): non responsive acute rejections and grades II and III
Antibody Mediated Rejection
Plasmapheresis and intravenous Immunoglobulin (1g/kg)

11. Patients and Methods Kidney allograft outcome
a) total recovery (creatinine < 20% in comparison to baseline values)
b) partial recovery (creatinine > 20% than baseline values)
c) graft loss (return to dialysis)
Pancreas allograft outcome
a) improvement or no improvement of serum enzymes and amylasuria and euglycemia
b) partial function (hyperglicemia and normal C-peptide)
c) graft loss (hyperglicemia and low C-peptide)

12. Results
Demographic data
female (39.5%) and male (60.5%)
median age 33 years
median time on dialysis 31 months
median time of diabetes history: 20 years
Induction 63.2% (Thymo/OKT3)
PRA (ELISA) pre transplant
0%: 92.1% patients;
10-50%: 2.6% patients;
> 80%: 5.3% patients

13. Table 1: Kidney allograft biopsies (n=33)
Histology
C4d
Negative Focal Diffuse
Antibody detection
Negative HLA MICA N/A
Outcome
TR PR Lost
Normal (n = 2)
ATN (n = 7)
Acute AMR (n = 5)
ATN + capillaritis: 3; ATN: 2
Suspicious for acute AMR (n = 3)
ATN: 1; Borderline: 1; IA: 1
Acute TCMR (n = 7)
IA: 5; IB: 1; IIA: 1
IF/AT (n = 3)
Grade I: 2; grade II: 1
Pyelonephritis (n = 2)
Other ( n = 4)
Negative: 24 (72.7%)
Positive: 9 (27.3%)
- Focal: 3 (33.3%)
- Diffuse: 6 (66.7%)
Negative: 2 (6.1%)
HLA: 3 (9.1%)
MICA: 2 (6.1%)
N/A: 26 (78.7%)
PR: 8 (24.2%)
TR: 24 (72.7%)
Graft loss: 1 (3.3%)
Rejection: 45.5% (15/33)
AMR or suspicious: 24.2% (8/33)
From all rejections: 53.3% (8/15)

14. Table 2: Pancreas allograft biopsies (n = 35)
Histology
C4d labeling
Negative Focal Diffuse
Antibody detection
Negative HLA MICA N/A
Outcome
Exocrine
Normal Reduced Amylasuria
NE IE
Endocrine
Normal Partial Lost
Acute cellular rejection (n = 8)
- All of them grade I
Acute TCMR + AMR (n = 9)
grade I: 3
grade II: 4
grade III: 2*
Suspicious for acute AMR (n = 6)
normal: 1
grade II: 1
other: 1
Chronic active AMR (n = 2) **
Other (n = 10)
Indeterminate: 4
Chronic rejection grade I: 3
Degenerative tubular alterations: 2
Normal: 1
Negative: 20 (57.1%)
Positive: 15 (42.9%)
- Focal: 7 (46.7%)
- Diffuse: 8 (53.3%)
HLA: 0
MICA: 5 (14.3%)
Negative: 6 (17.1%)
N/A: 24 (68.6%)
Normalized Amylasuria: 26 (74.3%)
Reduced Amylasuria: 9 (25.7%)
- Normalized enzymes: 3 (8.6%)
- Increased enzymes: 6 (17.1%)
Normal: 26 (74.3%)
Partial: 4 (11.4%)
Graft Loss: 5 (14.3%)
Rejection: 71.4% (25/35):
AMR: 50% (17/35)
From all rejections:
68% (17/25)

15. 65% AMR: SPKT-V, PAKT and PTA
Table 3: Histological analysis (n=35 biopsies) according to the pancreas transplant modality (n=27 patients)
SPKT
(n = 13)
SPKT, V
(n = 5)
PAKT
(n = 6)
PTA
(n = 3)
Acute TCMR (n =8) 4 2 1
Acute TCMR + AMR (n = 9) 3 5
Suspicious for AMR (n =6)
Chronic active AMR (n =2)
Other (n = 10) 7
65% AMR: SPKT-V, PAKT and PTA
SPKT: 4/28 (14.3%): synchronous pancreas and kidney rejection
SPKT: 4/28 (14.3%): synchronous pancreas and kidney rejection

16. Table 4: Histological analyses and pancreas allograft dysfunction
Exocrine dysfunction
Exocrine dysfunction + Hyperglicemia
Hyperglicemia
Acute TCMR (n = 8) 6 2
Acute TCMR + AMR (n = 9) 4 1
Suspicious for acute AMR (n = 6) 3
Chronic active AMR (n =2)
Other (n = 10) 5
19 (54.3%)
13 (37.1%)
3 (8.6%)

17. Table 5: Laboratorial parameters
TCMR
(n = 8)
AMR
(n = 17) P
Amylase pre (U/L)
178.9 ± 95
(median 151.5)
338.4 ± 651.7
(median 149)
P = 0.075
Amylase post (U/L)
90.9 ± 40.6
(median 80 )
92.6 ± 67.2
(median 69)
P = 0.95
Lipase pre (U/L)
1169 ± 670.8
(median 1120 ) ±
(median 721)
P = 0.83
Lipase post (U/L)
355.5 ± 213.6
(median: 296.5)
284.8 ± 228.4
(median 258)
P = 0.47
Amylasuria pre (U/L) ±
(median 1137) ±
(median 767.5)
P = 0.87
Amylasuria post (U/L) ±
(median: ) ±
(median )
Amylasuria variation pre (%)
45 ± 41.1
(median 46.5)
44.1 ± 49.6
(median 61)
P = 0.97
Fasting plasma glucose (mg/dL)
96.6 ± 66.7
143 ± 88.4
(median 97)
P = 0.20
2-hour capillary glucose (mg/dL): Minimum
105.5 ± 28.5
(median 99)
136.1 ± 73.5
(median 109)
P = 0.27
2-hour capillary glucose (mg/dL): Maximum
182.4 ± 91.8
(median 149.5)
213.7 ± 106.5
(median 197.5)
P = 0.49

18. ROC curves and Rejection Diagnosis
Amylasuria
AUC = 0.24 (P = 0.036, 95% CI )
Lipase
AUC = 0.73 (P = 0.025, 95% CI )
Amylase
AUC = 0.55 (P = 0.62, 95% CI )
Amylasuria Variation
AUC = 0.72 (P = 0.06, 95% CI )

19. ROC curve: Amylasuria Post Treatment and Graft Loss
AUC = 0.17 (P = 0.015, 95% CI )

20. Uni- and multivariate analyses
Multivariate analysis and C4d:
amylase and lipase before treatment (P = 0.68 and P = 0.39)
amylase and lipase after treatment (P = 0.96 and P = 0.97)
amylasuria before treatment (P = 0.42)
amylasuria variation (P = 0.41)
pancreas allograft loss (P = 0.23)
pancreas transplantation alone (P = 0.2)

21. Case 1: male, 36 yrs, SPKT, pancreas (endocrine + exocrine) and kidney dysfunctions, diffuse C4d, MICA

22. Case -1: Pancreas and kidney recoveries: Methylprednisolone pulse, Thymoglobuline, plasmapheresis and intravenous Immunoglobulin

23. Treatment: pulse Methylprednisolone, Thymoglobuline and OKT3
PTA, 14 yrs, male, exocrine dysfunction, diffuse C4d, antibody N/A: outcome with euglycemia, normalized serum enzymes, amylasuria < 150 U/h
CASE 2
Treatment: pulse Methylprednisolone, Thymoglobuline and OKT3
BCJ

24. SPKT-V, male, 33 yrs, exocrine dysfunction, diffuse C4d, MICA antibody: outcome with euglycemia, persistently increased serum enzymes, amylasuria < 150 U/h
CASE 3
Treatment: Methylprednisolone, Thymoglobuline,
Plasmapheresis (11 sessions), intravenous Immunoglobuline (1g/kg)
4 doses

25. Methods: ELISA; * Luminex
Case 4: SPKT, female, 40 yrs, > 20 blood transfusions, 3 pregnancies
PRA
Class I
Class II
0.7%
92%
39%
100%
32%
83%
76%*
97%*
22%*
77%*
Methods: ELISA; * Luminex

26. Case 4: Pancreas exocrine dysfunction negative C4d
DR1 = 2333 MFI
A11 = 559 MFI
Bx (2): 5m – Indeterminate + grade I CR
Bx (1): 1m7d – degenerative changes

27. Case 4: Kidney dysfunction
Bx (3): 1m 23d – moderate ATN, negative C4d
Bx (1): 1m2d – mild ATN, diffuse C4d
Bx (2):
1m9d – normal; diffuse C4d
Bx (6): 8m5d
ATN, negative C4d ,
Pulmonary sepsis CVV-HDF
Bx (4): 4m21d
Mild tubulitis, negative C4d
Bx (5): 5m
Normal, negative C4d
DR1 = 2333 MFI
A11 = 559 MFI

28. Treatment
Treatment of acute AMR (n = 5)
On average, acute AMR of either pancreas or kidney allograft was treated with a mean of 6.8 sessions of plasmapheresis (range 3 to 11 sessions) and 2.2 doses of intravenous Immunoglobulin 1g/kg (range 1 to 4 doses)

29. Conclusions-I
C4d detection was frequently detected in kidney and pancreas grafts with dysfunction: 27.3 % (diffuse 67%) and 43% (diffuse 53%), respectively
68% of pancreas with rejection were classified as acute or chronic AMR and suspicious for acute AMR

30. Conclusions-II
Exocrine and endocrine dysfunctions were comparable between TCMR and AMR
Amylasuria values after the treatment of rejection are associated with poor prognosis
The high frequency of C4d staining in pancreas allograft claims its investigation in all cases of pancreas rejection, since it requires specific treatment that may predict graft survival.

31. Caveats
Short follow-up
Small number of cases
DSA not available for all patients
MICA: donor specific?

32. Acknowledgments Pathology HLA Laboratory Transplant group
Denise MAC Malheiros
HLA Laboratory
Margareth Torres
Transplant group
Irina Antunes
Fábio Crescentini
Maria Cristina Ribeiro de Castro
Tércio Genzini
Marcelo Perosa-Miranda