ANTIBODY-MEDIATED REJECTION AFTER PANCREAS TRANSPLANTATION: SÃO PAULO (BRAZIL) EXPERIENCE Érika B...
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ANTIBODY-MEDIATED REJECTION AFTER
PANCREAS TRANSPLANTATION: SÃO
PAULO (BRAZIL) EXPERIENCE
Érika B RangelHospital Israelita Albert EinsteinFederal University of São Paulo
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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.
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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%)
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Results
Time post transplantation:• Kidney biopsies: 293.3 ± 535 days (median 64 days).
• Pancreas biopsies: 566 ± 682.3 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)
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Patients and Methods
• Initial immunosuppression:
a) Tacrolimus 0.15 mg/kg/dose, adjusted according to the period after transplantation (serum levels of 10-15 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
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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.
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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.
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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
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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
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Patients and Methods
TCMR - Kidney allografta) 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 1-1.5 mg/kg/day or OKT3 2.5-5 mg/day (10 days): non responsive acute rejections and grades II and III
Antibody Mediated Rejection• Plasmapheresis and intravenous Immunoglobulin
(1g/kg)
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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)
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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
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Histology C4d
Negative Focal Diffuse
Antibody detection
Negative HLA MICA N/A
Outcome
TR PR Lost
Normal (n = 2) 2 0 0 0 0 0 2 2 0 0
ATN (n = 7) 7 0 0 1 0 0 6 7 0 0
Acute AMR (n = 5) ATN + capillaritis: 3; ATN: 2
1 0 4 0 3 2 0 3 2 0
Suspicious for acute AMR (n = 3) ATN: 1; Borderline: 1; IA: 1
0 3 0 0 0 0 3
2 1 0
Acute TCMR (n = 7) IA: 5; IB: 1; IIA: 1
6 1 0
1 0 0 6 6 1 0
IF/AT (n = 3) Grade I: 2; grade II: 1
3 0 0 0 0 0 3 0 3 0
Pyelonephritis (n = 2) 1 0 1 0 0 0 2 1 0 1
Other ( n = 4) 3 0 1 0 0 0 4 3 1 0
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)
Table 1: Kidney allograft biopsies (n=33)
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HistologyC4d labeling
Negative Focal Diffuse
Antibody detection
Negative HLA MICA N/A
Outcome Exocrine
Normal Reduced Amylasuria NE IE
Outcome Endocrine
Normal Partial Lost
Acute cellular rejection (n = 8)- All of them grade I
8 0 0 1 0 0 7 7 0 1 7 0 1
Acute TCMR + AMR (n = 9)-grade I: 3-grade II: 4-grade III: 2*
2 2 5
1 0 5 3
2 3 4
5 1 3
Suspicious for acute AMR (n = 6)-normal: 1-grade I: 3-grade II: 1-other: 1
0 5 1
2 0 0 4
6 0 0
4 1 1
Chronic active AMR (n = 2) 0 0 2 1 0 0 1 1 0 1 ** 1 1 0
Other (n = 10)-Indeterminate: 4-Chronic rejection grade I: 3-Degenerative tubular alterations: 2-Normal: 1
10 0 0
1 0 0 9
10 0 0
9 1 0
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%)
Table 2: Pancreas allograft biopsies (n = 35)
Rejection: 71.4% (25/35):
AMR: 50% (17/35) From all rejections: 68% (17/25)
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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 1
Acute TCMR + AMR (n = 9) 3 5 0 1
Suspicious for AMR (n =6) 3 1 1 1
Chronic active AMR (n =2) 0 1 1 0
Other (n = 10) 7 0 3 0
65% AMR: SPKT-V, PAKT
and PTA
SPKT: 4/28 (14.3%): synchronous
pancreas and kidney rejection
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Table 4: Histological analyses and pancreas allograft
dysfunction
Exocrine dysfunction
Exocrine dysfunction + Hyperglicemia
Hyperglicemia
Acute TCMR (n = 8) 6 2 0
Acute TCMR + AMR (n = 9) 4 4 1
Suspicious for acute AMR (n = 6) 3 3 0
Chronic active AMR (n =2) 1 0 1
Other (n = 10) 5 4 1
19 (54.3%)
13 (37.1%) 3 (8.6%)
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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 )
1288.5 ± 1553.6 (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) 1509.3 ± 1311.5 (median 1137)
1395.2 ± 1484.7 (median 767.5)
P = 0.87
Amylasuria post (U/L) 2153.7 ± 1277.8 (median: 1860.5)
2201.3 ± 1926.7 (median 1558.5)
P = 0.95
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(median 69)
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
Table 5: Laboratorial parameters
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ROC curves and RejectionDiagnosis
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
0.0
0.2
0.4
0.6
0.8
1.0
Se
ns
itiv
ity
ROC Curve
AmylaseAUC = 0.55 (P = 0.62, 95% CI 0.33-0.77)
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
0.0
0.2
0.4
0.6
0.8
1.0
Se
ns
itiv
ity
ROC Curve
LipaseAUC = 0.73 (P = 0.025, 95% CI 0.55-0.91)
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
0.0
0.2
0.4
0.6
0.8
1.0
Se
ns
itiv
ity
ROC Curve
AmylasuriaAUC = 0.24 (P = 0.036, 95% CI 0.04-0.44)
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
0.0
0.2
0.4
0.6
0.8
1.0S
en
sitivity
ROC Curve
Amylasuria VariationAUC = 0.72 (P = 0.06, 95% CI 0.53-0.91)
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ROC curve: Amylasuria Post Treatment and Graft Loss
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
0.0
0.2
0.4
0.6
0.8
1.0S
en
sit
ivit
y
ROC Curve
AUC = 0.17 (P = 0.015, 95% CI 0.03-0.32)
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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)
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Case 1: male, 36 yrs, SPKT, pancreas (endocrine + exocrine) and kidney dysfunctions, diffuse C4d, MICA
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Case -1: Pancreas and kidney recoveries: Methylprednisolone pulse, Thymoglobuline, plasmapheresis and
intravenous Immunoglobulin
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BCJ
PTA, 14 yrs, male, exocrine dysfunction, diffuse C4d, antibody
N/A: outcome with euglycemia, normalized serum enzymes,
amylasuria < 150 U/h
Treatment: pulse Methylprednisolone, Thymoglobuline and OKT3
CASE 2
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SPKT-V, male, 33 yrs, exocrine dysfunction, diffuse C4d, MICA antibody: outcome with euglycemia, persistently increased serum
enzymes, amylasuria < 150 U/h
Treatment: Methylprednisolone,
Thymoglobuline,Plasmapheresis (11
sessions), intravenous Immunoglobuline (1g/kg)
4 doses
CASE 3
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Case 4: SPKT, female, 40 yrs, > 20 blood transfusions, 3 pregnancies
PRA Class I
PRAClass II
01.28.05 0.7% 92%
01.28.06 39% 100%
07.19.06 32% 83%
06.28.07 76%* 97%*
8.22.07 22%* 77%*
Methods: ELISA; * Luminex
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Case 4: Pancreas exocrine dysfunctionnegative C4d
Bx (1): 1m7d – degenerative changes
Bx (2): 5m – Indeterminate + grade I CR
DR1 = 2333 MFIA11 = 559 MFI
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Case 4: Kidney dysfunction
Bx (1): 1m2d – mild ATN, diffuse C4d
Bx (2):1m9d – normal; diffuse C4d
Bx (3): 1m 23d – moderate ATN, negative C4d
Bx (4): 4m21dMild tubulitis, negative C4d
Bx (5): 5mNormal, negative C4d
Bx (6): 8m5dATN, negative C4d , Pulmonary sepsis CVV-HDF
DR1 = 2333 MFIA11 = 559 MFI
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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)
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Conclusions-I
1. C4d detection was frequently detected in kidney and
pancreas grafts with dysfunction: 27.3 % (diffuse
67%) and 43% (diffuse 53%), respectively
2. 68% of pancreas with rejection were classified as
acute or chronic AMR and suspicious for acute AMR
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Conclusions-II
3. Exocrine and endocrine dysfunctions were comparable
between TCMR and AMR
4. Amylasuria values after the treatment of rejection are
associated with poor prognosis
5. 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.
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Caveats
• Short follow-up• Small number of cases• DSA not available for all patients• MICA: donor specific?
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Acknowledgments
Pathology Denise MAC Malheiros
HLA Laboratory Margareth Torres
Transplant group Irina Antunes Fábio Crescentini Maria Cristina Ribeiro de Castro Tércio Genzini Marcelo Perosa-Miranda