Transcript Document

XXVIIth International Congress of
International Academy of Pathology
Athens 12-17 October 2008
IMMUNOPHENOTYPE OF INFLAMMATORY
CELLS OF RENAL ALLOGRAFT BIOPSIES
WITH ACUTE CELLULAR REJECTION
Mosquera Reboredo J. M1.; Vázquez Martul E.1; Fernández Rivera C.2 ;
Filgueira Fernández P.3 ; and Valdés Cañedo F2.
1Pathology
Deparment , 2Nephrology Deparment and 3Investigation Laboratory.
ACUTE CELLULAR REJECTION
Banff 97 diagnostic categories for renal allograft biopsies—Banff’07 update
4. T-cell-mediated rejection (TCMR, may coincide with categories 2 and 5
and 6)
Acute T-cell-mediated rejection (Type/Grade:)
IA. Cases with significant interstitial infiltration (>25% of parenchyma affected, i2
or i3) and foci of moderate tubulitis (t2)
IB. Cases with significant interstitial infiltration (>25% of parenchyma affected, i2
or i3) and foci of severe tubulitis (t3)
IIA. Cases with mild-to-moderate intimal arteritis (v1)
IIB. Cases with severe intimal arteritis comprising >25% of the luminal area (v2)
III. Cases with ‘transmural’ arteritis and/or arterial fibrinoid change and necrosis of
medial smooth muscle cells with accompanying
lymphocytic inflammation (v3)
Chronic active T-cell-mediated rejection
‘chronic allograft arteriopathy’ (arterial intimal fibrosis with mononuclear cell
infiltration in fibrosis, formation of neo-intima)
American Journal of Transplantation 2008; 8: 753–760
ACUTE CELLULAR REJECTION
•The composition of the inflammatory infiltrate in acute rejection is
dominated by T-lymphocytes (CD4 or CD8) but macrophages,
plasma cell, granulocytes or NK cells can be present.
•The role of T-Lymphocytes in this process is well established but
not the involvement and relevance of the other inflammatory cells.
IMMUNOPHENOTYPE OF INFLAMATORY CELLS IN
ACUTE CELLULAR REJECTION
Rejected human renal allografts: recovery and characteristics of infiltrating cells
and antibody.
Tilney NL Transplantation. 1979 Nov;28(5):421-6.
The relation of different inflammatory cell types to the various parenchymal
components of rejecting kidney allografts.
Reitamo S, Histopathology. 1980 Sep;4(5):517-32.
Composition of interstitial cellular infiltrate identified by monoclonal antibodies in
renal biopsies of rejecting human renal allogafts.
Hancock WW et al. Transplantation. 1983 May;35(5):458-63.
Renal allograft cell infiltrates associated with irreversible rejection.
Sanfilippo F Transplantation. 1985 Dec;40(6):679-85.
The location, as well as the number and type of cell infiltrates are
critical in evaluating cellular forms of rejection
T-Lymphocites
PLASMA CELLS AND
AUTE CELLULAR REJECTION
PLASMA CELL RICH REJECTION
POOR PROGNOSIS
Plasma cell-rich acute allograft rejection is associated with poor graft survival
Is not a manifestation of concomitant chronic allograft nephropathy or viral
infection, including posttransplant lymphoproliferative disorder.
Plasma cell-rich acute renal allograft rejection
Charney DA, Nadasdy T, Lo AW, Racusen LC.
Tansplantation 1999: 68; 791-797
- Plasmacytic infiltrates in renal allografts comprise a spectrum of lesions from
acute rejection to PTLD, with a generally poor prognosis for long-term graft survival.
The clinical and pathologic implications of plasmacytic infiltrates in percutaneus
renal allograft biopsies.
Meehan SM et al.
Hum Pathol. 2001 Feb;32(2):205-15.
PLASMA CELLS AND
AUTE CELLULAR REJECTION
- Oedema and plasma cell-rich acute rejections (OPcR) >10% of the
graft infiltrating cells.
- OPcR rejections are highly resistant to therapy and portend a poor
outcome, irrespective of Banff score.
- Suggest that an antibody-mediated mechanism was involved in most
OPcR ( 9 of 12 patients had sings suggesting antibody mediated
rejection).
Desvaux et al. Nephrol Dial Transplant 2004 19 933-9
PLASMA CELLS AND
ACUTE REJECTION
- 52% of AHR (C4d +) showed plasmacytic infiltrates ( 16% Cd4 -) ( p=0,02)
- Evidence of acute cellular with occult humoral rejection is identified in
more than 40% of late AR episodes.
Implications of Immunohistochemical Detection of C4d along Peritubular
Capillaries in Late Acute Renal Allograft Rejection
Poduval, Meehan et al.
Transplantation 2005: 27:79 228-235
- Plasma Cell-rich infiltrates correlated
with C4d (p = 0.0080).
Acute rejection in non-compliant renal allograft
recipients: a distinct morphology
Lerut E. et al
Clin Transplant. 2007 May-Jun;21(3):344-51.
MAST CELLS AND
ACUTE CELLULAR REJECTION
MAST CELLS
FIBROSIS
- Mast cell were associated with allograft fibrosis in chronic rejection and chronic
CsA toxicity. Might play a pathogenic role in fibrotic process.
-No mast cell increase during AR in those case that progressed to chronic rejection.
Close association between renal interstitial fibrosis and mast cells
Important role in the development of CAN
Mast cells in human allografted kidney: correlation with interstitial
fibrosis.
Goto E et al
Clin Transplant. 2002;16 Suppl 8:7-11.
MAST CELLS AND
ACUTE CELLULAR REJECTION
- Correlation between number of mast cells and time since transplantation, severity of
interstitial fibrosis and with interstitial oedema (all P < 0.005).
- Mast cells are increased in moderate and severe A.R. compared with mild and normal kidneys.
Mast cells in acute cellular rejection of human renal allografts.
Lajoie G et al. Mod Pathol. 1996 Dec;9(12):1118-25.,
Biopsy at 100 days: Mast cells were related to decline of long-term graft function and fibrosis
Toshiro Ishidaa et al, Clinical Transplantation Volume 19 Page 817 Dec 2005
Interstitial mast cell are correlated with interstitial volume, and interstitial expression of
alpha-SMA. Role in the development of early interstitial fibrosis
Danilewicz M Med Sci Monit. 2004 May;10(5):BR151-6. Epub 2004 Apr 28.
EOSINOPHILS AND
ACUTE CELLULAR REJECTION
The prognostic value of the eosinophils in acute renal allograft rejection.
Weir MR Transplantation. 1986 Jun;41(6):709-12
- The increased of eosinophils in peripheral blood(p<0.01) and/or renal graft biopsy
specimen (P< 0.02) is an adverse prognostic factor in acute rejection outcome.
The importance of eosinophils in kidney allograft rejection.
Kormendi F Transplantation. 1988 Mar;45(3):537-9.
- More than 4% eosinophils in the tissue inflamatory exsudate is a specific (91%)
and fairly sensitive (78%) indicator of an severe acute rejections.
EOSINOPHILS AND
ACUTE CELLULAR REJECTION
EOSINOPHILS
VASCULAR REJECTION
-Significant interstitial graft eosinophilic infiltrate (>10% of inflamatory infiltrate) is
associated
with vascular rejection in the patients on high-dose immunosupression (p=0,04)
Abundance of interstitial eosinophils in renal allograft is associated with vascular rejection.
Meleg-Smith S et al. Transplantation 2005;79:444-450
-Tissue eosinophilia (>10 eosinophils/mm2) is associated with vascular rejection
(P= 0.02) but is not of independent prognostic significance .
Banff criteria as predictors of outcome following acute renal allograft rejecction.
Macdonald FI et al. Nephrol Dial Transplant. 1999 Jul;14(7):1692-7.
EOSINOPHILS AND
ACUTE CELLULAR REJECTION
- Eosinophilic infiltration is a negative predictor, which can indicate
higher grade, more severe course of ARAR and increased resistance to
an anti-rejection therapy.
- Chronic rejection was seen in 25% patients of EG and in 11% in Control
Group (CG) in the first year after transplantation.
Biopsy eosinophilia as a predictor of renal graft dysfunction
Pol Merkur Lekarski. 2006 Aug;21(122):152-5
MACROPHAGES AND
ACUTE CELLULAR REJECTION
Macrophages
.
worse prognosis
poor graft survival
Intraglomerular monocyte/macrophages
The significance of monocytes in glomeruli of human renal transplant.
Harry et al Transplantation. 37(1):70-72, January 1984.
Prognosis was significantly worse during the six months after the biopsy
The presence and prognostic importance of glomerular macrophage infiltration in renal
allograft.
Ozdemir BH. et al. Nephron 2002 Apr 90(4) 442-6
- Macrophages are associated with steroid resistance (p < 0.01).
High number of interstitial macrophages are significantly related to unfavorable graft outcome(p < 0.01).
Diagnostic and predictive value of an immunohistochemical profile in aymptomatic acute
rejecion of renal allograft. Copin MC. Et al. Transpl Immunol. 1995 Sep;3(3):229-39.
MACROPHAGES AND
ACUTE CELLULAR REJECTION
- In the intimal arteritis (Banff II or III
)the
infiltrating cells were predominantly macrophages.
T-cell were in the minority (P< 0.01)
The macrophage is the predominant inflammatory
cell in renal allograft intimal arteritis Matheson PJ et al
Transplantation. 2005 Jun 27;79(12):1658-62.
Univariate analysis shows a trend for a higher infiltration with CD8+ (P = 0.053) and CD68+(P =
0.06) cells in clinical rejection.
Inflammatory factor-1+-activated macrophages (activation marker) were increased in clinical
rejection (P = 0.014)
Clinical rejection is distinguished from subclinical rejection by increased infiltration by a
population of activated macrophages Grimm PC et al J Am Soc Nephrol. 1999 10(7):1582-9.
MACROPHAGES AND
ACUTE REJECTION
Macrophages are a part of the pathogenesis of acute humoral rejection
Transplant Mac attack:Humor the macrophages
Colvin
K. International 2003; 63:1953
- Glomerular and interstitial monocyte/
macrophages(MO) infiltrations closely
associated with C4d+ (p<0.0001)
-Confirm the correlation of C4d+ and PMN.
-Sensitivity 91% and Specificity 93%
(MO/glomerulus >1 as threshold) Propose
add to the histological criteria of Acute
Humoral Rejection.
Monocytes and peritubular capillary C4d deposition in acute renal
allograft rejection Magil y Tinckam K. International 2003 63: 1888
C4d
MATERIALS AND METHODS
•
Review 6 month post-transplant kidney biopsies with acute cellular
rejection
•
Grade and tabulate the histological findings according Banff score.
•
Measure the average per high power field (hpf) of plasma cell,
macrophages, mast cells, T- lymphocytes (CD4 and CD8) and eosinophils
using inmunohistochemistry.
•
All biopsies were stained with C4d antibody by inmunohistochemistry.
•
Clinical parameters like HLA mismatch, cytotoxic cross-match, type of
immunosupression, creatinine level, response to anti-rejection therapy
and evolution of allograft were reviewed.
•
Identify follow-up biopsies if available.
•
Statistical analysis: we used SPSS statistical software. Comparisons of
different data were performed with t-Student, Man-Whitney and X2 test.
Kaplan-Meier: Logistic regression for survival analysis
IMMUNOPHENOTYPE OF INFLAMATORY CELLS OF
RENAL ALLOGRAFT BIOPSIES WITH ACUTE
CELLULAR REJECTION
The aim of this study is:
-Try to find relation betwen type of cells in acute rejection and:
- Clinical parameters
- Outcome and survival rates of allograft.
- Response to antirejection therapy.
- HLA.
- Evolution to chronic rejection
- Are really useful do it in allograft biopsies with acute rejection
RESULTS
•
•
•
•
•
•
•
76 biopsies from 70 kidney transplants (51 men and 26 women)
Age: 46,34 ( 20-68).
Donor age: 43,46 ±16,91 (2-71)
Ischemic time: 21,7±5,37 (5-33)
Time to biopsy: 37,28± 48,24 days (4-180d)
Creatinine: 6,47± 2,27 (1,70-12,30)
Proteinuria: 2,04±2,36 (0-12)
Cellular Infiltrate
Cells/hpf
Mean ±SD
Plasma cells
4,08±5,37 (0-47,4)
Macrophages
48.53±41,39 (0-250)
Mast-cells
1,22±2,56 (0-16,6)
Lymphocytes (CD4)
19,98±24,1(0-124)
Lymphocytes (CD8)
42,80±35,1(1,1-141)
Eosinophils
0,68±2,88 (0-23,8)
BANFF GRADE
57,9%
40,8%
39,4%
26,3%
17,1%
10,5%
2,6%
2,6%
Banff grade
Vascular rejection
• T-test: More monocyte/macrophages in vacular rejection compared with
•
•
tubulo-interstitial (60,6 vs 40,6) (p=0,039)
No correlation with U-Mann Withney (p=0,09).
No correlation with other type of cells
Glomerulitis
g0
>g1
Log rank= 5,07 p=0,024
• T-test: High number of macrophages in glomerulitis group (>g1) (55,5vs
•
•
35,7) (p=0,04).
No correlation with U-Mann Withney.
No correlation with other type of cells
RESULTS
Fibrosis
T-test: more plasma cells and Cd8 lymphocytes in cases
with fibrosis compared with those without (plama cell 6,2 vs
1,1, p=0,01) (Cd8 38 vs 29,2, p=0,03)
No correlation with U-Mann Withney.
Nº HLA mismaches
Incompatible DR > o < 1
InDR<1
•
•
•
•
•
•
Plasma cells
Macrophages
Mast cells
CD4
CD8
Eosinophils
1,6±3
In DR> 1
p
5,1±10,4
0,03
50,7 ±45
ns
1,5 ±3
ns
24,1±31
17,7 ±20
ns
43,1±39
42,9±34
ns
45,7±33
0,7 ±1,2
1,3 ±5
0,3±0,8
T-Student
U-Mann Whitney no correlations. Plasma cells (p=0,58)
ns
Nº HLA mismaches
RESULTADOS
2
Incompatible > O < 3
•
•
•
•
•
Plasma cells
Macrophages
Mast Cells
CD4
CD8
• Eosinophils
In<3
In > 3
p
3,8±8,1
42,3±36,2
1,3±2,2
23,5±27,5
45,4±35
4,1±9,9
59,1±47
1,1±3,1
14,4±18,3
39,5±35,8
0,9±3,8
0,2±0,4
ns
0,09
ns
ns
ns
T-test
U-Man Withney : No correlation with any type of cell but
macrophages p=0,07
ns
C4d
- Inmunohistochemistry
-Ac. Polyclonal anti-C4d.
Banff 97 diagnostic categories for renal allograft biopsies
Banff’07 update
Antibody-mediated changes (may coincide with categories 3, 4 and 5 and 6)
Due to documentation of circulating antidonor antibody, and C4d3 or allograft pathology
C4d deposition without morphologic evidence of active rejection
C4d+, presence of circulating antidonor antibodies, no signs of acute or chronic TCMR or
ABMR (i.e. g0, cg0, ptc0, no ptc
lamination). Cases with simultaneous borderline changes or ATN are considered as
indeterminate
Acute antibody-mediated rejection
C4d+, presence of circulating antidonor antibodies, morphologic evidence of acute tissue
injury, such as (Type/Grade):
I. ATN-like minimal inflammation
II. Capillary and or glomerular inflammation (ptc/g >0) and/or thromboses
III. Arterial—v3
Chronic active antibody-mediated rejection4
C4d+, presence of circulating antidonor antibodies, morphologic evidence of chronic tissue
injury, such as glomerular double
contours and/or peritubular capillary basement membrane multilayering and/or interstitial
fibrosis/tubular atrophy and/or fibrous
intimal thickening in arteries
American Journal of Transplantation 2008; 8: 753–760
MIXED REJECTION CELULAR
AND HUMORAL
- Evidence of acute cellular with occult humoral rejection is identified in
more than 40% of late AR episodes.
Poduval, Meehan et al. Transplantation 2005: 27:79 228-235
-
Halloran
Ab Antidonor in 23-38% of. A.C R
Transplantation 1992; 53: 550-555. Transplantation 1996; 61: 1586-1592.
- Collins
et al.
20-25% of Acute Rejection have Ac. Antidonor
J. Am Soc. Nephrol. 1999; 10: 2208-2214.
-Terasaki
Ab. in 96% of Rejections
Am. J. Transplant. 2003 3:6; 665-673
-
Recommend C4d in all cases of acute graft failure
C4d
Negative
Positive
Log Rank=1,15
p=0,28
C4d AND CELLULAR INFILTRATE
C4d
NEGATIVE
POSITIVE
U-MANN WITHNEY
p
Plasma cells
36
41
612
0,25
Macrophages
36,6
40,3
652
0,46
Mast cells
37,8
39,1
696
0,78
CD4 Lympho.
29,3
31,7
412
0,59
CD8 Lympho.
32,1
42,9
487
0,049
Eosinophils
38,1
35,9
626
0,64
U-Mann Withney
Steroid Therapy
58,4%
41,6%
Yes
NO
Steroid resistants
Yes
Corticosteroids Resistence
YES
NOT
Mann Whitney
p
Plasma cells
33,2
27,8
343
0,23
Macrophages
35,9
25,9
277
0,027
Mast cells
26,6
32,3
339
0,2
Lymphocytes CD4
28,5
20,9
180
0,06
Lymphocytes CD8
33,5
25
263
0,05
Eosinophils
27,2
30,2
354
0,47
Univariate analysis
•
In multivariate analysis /logistic regression adjusted for macrophages,
CD4 and CD8 lymphocytes, a high number of monocyte/ macrophages
/hpf is associated with steroid resistance (Odds= 1,021 (ci95% 1-1,041)
p=0,04).
ALLOGRAFT SURVIVAL
Alive
Dead
U-Mann
Whitney
p
Plasma Cells
30,1
40,8
437
0,030
Macrophages
31,7
39,4
489
0,11
Mast cells
37,1
35,1
588
0,66
Lympho. CD4
27
28,7
349
0,7
Lympho CD8
29,1
39,9
407
0,04
Eosinophils
34,7
34,1
559
0,89
Univariate Analysis
•In multivariate analysis /logistic regression adjusted for plasma cells, CD4 and
CD8 lymphocytes, a low number of CD8 lymphocytes /hpf is associated with
better allograft survival rates (Odds= 0,96 (ci95% 0,93-0,99) p=0,039).
RESULTS
Time of biopsy (< o 30 days)
• T-test: higher number of macrophages in biopsies < de 30 days ( 54,8
vs 32,9 ) (p=0,035) and higher number of plasma cells in biopsies > de 30
days ( 6,9 vs 2,9)(p=0,071 ) .
• U Mann Whitney higher number of macrophages < 30 days (P=0,043)
(41,7-30,4) (U Mann Whitney 417)
Immunosupression
•U-Mann-Withney: higher nº of CD8 before MMF (p=0,025) (39,4-28,4)
U-Mann Withney 391,
CONCLUSIONS
• CD8 are associated with poor prognosis (univariate and
multivariate analysis), steroid resistance (univariate) and
C4d.
• Macrophages are associated wih steroid resistance
(univariate and multivariate analysis). Number of
macrophages is higher in < 30 days biopsies.
• Plama cell rich rejections are associated with worse allograft
survival (univariate).
• High percentege of humoral rejection associated with
acute cellular rejection
ACKNOWLEDGEMENTS
- Eduardo Vázquez Martul Pathology Department
- Constantino Fernandez Rivera Nephrology Department
- Purificacion Filgueira and
Investigation Unit