Antibody-mediated rejection
Diagnostic criteria
Chris Bellamy, University of Edinburgh
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some cases
background
Banff consensus
Early AMR & AMVR
Subclinical/C4d-negative AMR
– Microcirculation inflammation/de novo DSA
• Some chronic lesions
• C4d
Perspective
• Previously undetectable HLA DSA are now recordable, improving
morphological and clinical correlations.
– There remain serologic problems with rating DSA significance, accuracy, nonuniformity and identification of significant non-HLA DSA
Tait, Transplantation 2013
• With modern transplantation practice, even low level preformed HLA-DSA
increase early acute rejection and graft failure, although larger study
numbers are needed to show the difference for low level DSA
Mohan, JASN 2012
Perspective
• BUT… Sensitised patients live longer after prompt transplantation than
remaining on dialysis
• live donor transplantation after desensitisation
• 8-year survival 81%
81%
49%
31%
Montgomery, NEJM 2011
What do we know about HLA-DSA
• de novo DSA consistently precede graft failure, with some dose
effect (MFI), and their absence predicts good outcome
Terasaki, AJT 2008
• Source
– short-lived germinal centre plasmablasts
– long-lived niche-resident plasma cells
• Localisation
– DSA rapidly sink into a new graft, affecting circulating levels
– DSA recoverable from cortex and medulla
– Pathology studies show inflow circulation from arteries to
capillaries (endothelium) to be the prime target
– Failed grafts left in situ reduce DSA detectability
• Class I DSA: 24% vs 77% (nephrectomised)
• Class II DSA: 43% vs 63% (nephrectomised)
Higgins, Txn 2009
Martin 2005, Bocrie
2003,2007
Del Bello, CJASN 2012
– Appear as soon as 5 days after nephrectomy then stable/diminishing MFI
C4d
Nephrectomy @ 18 days
FXCM+
Woman, 1st graft; d 5.
Anti-DP MFIpeak19000
PEx & Campath
New class I DSA
C4d
Early clinico-pathologic studies
• Presensitised patients
– obliterative arteriopathy is rejection not hypertension
Porter KA 1963, Kincaid-Smith 1964
– vascular changes drive very early rejection, not cellular infiltrates
• peritubular capillaritis
• fibrinoid necrosis and thrombi
• obliterative arteriopathy longer term.
Porter KA 1963, 1964, 1965;
Kincaid-Smith 1964
– DSA cause hyperacute rejection in ABO-compatible renal transplants
Kissmeyer-Nielson F 1966
Early clinico-pathologic studies
• Positive crossmatch test:
– efficacy of C-dependent lymphocytotoxic cross match in defining immunologic
risk
Patel & Terasaki 1969
• De novo DSA
• DSA confer a poor prognosis through early vasculitis (presensitised), or (de novo)
later pauci-inflammatory obliterative arteriopathy and perhaps chronic
glomerulopathy.
Jeannet M 1970
Vascular endothelium is the intuitive target.
• “the total effects may not become evident unless observation of recipients extends
over a period of several years”
• Immunopathology
Porter KA 1968, McKenzie IFC 1968, Andres GA
– Glomerular, Intravascular
1970
Light JA 1975
– Anti-HLA IgM in plasma perfusate causes acute allograft TMA
– C4dptc
Feucht HE 1991, 1993
Second phase
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Preformed class I DSA produce morphologically distinct acute AMR:
– Microcirculation injury, inflammation, thrombosis
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Ultrastructural endothelial injury (g, ptc)
Mononuclear glomerulitis (CD68+ Magil, KI 2003) & endothelial swelling
Neutrophil peritubular capillaritis
Severe intimal arteritis in multiple arteries/transmural arteritis/fibrinoid
Fibrin (glomeruli, vessels)
Interstitial haemorrhage & infarcts
Microcirculation C3 without Ig (glom, ptc)
Halloran/Solez 1990, 1992, 1996
C4dptc associates with features of acute AMR
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DSA (90%): class I/II
neutrophilic capillaritis (65%), glomerulitis (55%), tubulitis (55%)
ptc often dilated
glomerular thrombi (20%)
Collins AB 1999, Crespo M 2001,
fibrinoid necrosis (25%)
Mauiyyedi S 2002
Severe ATI with focal necrotic tubules (75%)
• precedes inflammation in 10%
– 1 year graft loss 30% (vs 4% for TCMR)
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Banff AMR consensus
1991 meeting
– Hyperacute rejection
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1997
– AMR
• Hyperacute rejection
• Accelerated acute rejection (pure/combined)
– Features as per Trpkov K 1992
– Confirm with repeat cross-match
– v3 now excludes multi-artery intimal arteritis
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2001
– AMR
• Pure (3 cardinal features: histologic + immunopathology + DSA)
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» (Implied in text: DSA can be substituted by finding Ig deposition)
immunopathology:
» Diffuse ptc C4d or ptc Ig (rare) or Ig and C’ in arterial fibrinoid necrosis
3 histologic patterns:
ATN-like (minimal inflammation)
Capillary: capillary thrombosis or neutrophils+/mononuclear cells in glomeruli or ptc
Arterial: intimal arteritis/fibrinoid necrosis/intramural/transmural inflammation
(table 1 definition is different: lacks intimal arteritis)
• Combined
– acute cellular AMR (=TCMR + diffuse ptc C4d + DSA)
– Borderline & AMR
– AMR and chronic rejection
• Suspicious for AMR
– focal/weak/negative C4d OR DSA not demonstrated
Banff AMR consensus
• 2005
– Chronic active AMR
• triad: [cg/PTCBMML/IFTA/cv] & diffuse C4dptc & DSA+
• “suggestive of”: no DSA or C4d (caveat re activity)
– Arterial AMR now “v3”C4d+
• unequivocally excluding multi-artery intimal arteritis
• 2007: scoring refinements
– C4d positivity without morphologic evidence of active rejection”
• DSA +ve but no rejection-attributable histology
– “Indeterminate” if i1 [text]/borderline [table]
– erratum: “ATN-like minimal inflammation” removed at 2009 meeting report
– ptcmax scoring
– C4dptc scoring
• 2011
• “Broad acceptance” of C4d-negative AMR but “crucial to avoid over-diagnosis”:
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MCI thresholds to balance sensitivity & specificity
Focal C4d?
Mengel, AJT 2012
Intimal arteritis?
Acute vs. Chronic active AMR
Early acute AMR
• Affects 20-100% sensitised patients (vs. 2-5%)
– Activation of platelets and C’ important
Bentall, AJT 2013
Stegall, AJT 2011
• Eculizumab (C5-blocking) reduced acute AMR incidence in highly sensitised patients
with comparable DSA levels (15% vs 100%)
• Lefaucheur 2007: mixed risk cohort
– AMR in 22% presensitised (vs 2%)
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86% early (median 16d);
71% AMR pure on 1st bx
Lefaucheur, AJT 2007
early AMR all C4d+, 71% on last bx (3-6m post AMR)
neutrophilic capillaritis/glomerulitis (1st bx); last bx mononuclear
– Poor outcome predictors are microcirculation activity, not C4d
• 1st bx: neutrophilic g/ptc, oedema, ptc dilation
• Last bx (3-6m): arteritis, mononuclear g/ptc, ptc dilation
Man; 1st graft, d 11.
DSA neg; MICA neg
C4dptc <5% weak (paraffin)
2nd graft, day 3
DSA neg, MICA neg
C4dptc neg (IF, ps)
2nd graft, day 18
C4dptc 1-2% (IF)
Acute antibody-mediated vascular rejection (AMVR)
• Several studies link intimal arteritis with AMR lesions/C4d
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Intimal arteritis lesions contain CD68+ and CD3+ cells
Lefaucheur, AJT 2007
Sis, AJT 2010
Herzenberg, JASN 2002
Shimizu, Clin Txn 2010
CD68 predominant in 53%, but no relation with C4dptc
Kozakowski, NDT 2009
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AMVR
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– CDC XM- patients
Lefaucheur, Lancet 2013
– indication bx
– Validation cohort
– Unsupervised analyses (PCA, hierarchical cluster)
21% of acute rejection is AMVR
occurs early after transplant, with increased graft failure
4 distinct rejection patterns
according to clinical, histological, and immunological variables
Lefaucheur, Lancet 2013
- arteritis +
TCMR (i, t)
AMR (DSA, g, ptc, C4d)
Lefaucheur, Lancet 2013
TCMR v46%, 3m (IQ <12m)
AMR v24%, 7m (IQ <18m)
TCMVR
9%, 2m (IQ <4.4m)
v1 62%;
v2 19%;
v3 19%
AMVR
21%, 1m (IQ <4.4m)
v1 52%,
v2 30%,
v3 19%
Comparing acute AMVR with AMR and TCMR/TCMVR
– AMVR has widespread inflammation
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more microcirculation inflammation (4 vs. 1)
intermediate tubulitis:
AMR (t0.5)
AMVR (t1)
intermediate C4d:
AMR (1.5)
AMVR (1)
marginal glomerulopathy (0.5 vs 0)
– 9x risk of graft failure (vs. TCMR)
• AMR 2.9x,
• TCMVR=TCMR
– Predictors of graft failure after AMVR
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i+t >3
v3 lesion
DSAmax MFI >3000
Ab-targeted treatment protective
TCMR/TCMVR (t2)
TCMR/TCMVR (0ish)
Arteritis
• Margination?
– steroid resistance; 50% had intimal arteritis
• 5 profiles to be confident of its absence?
– 4 gave 75% confidence of detection
– 2 not 3 slides missed 11%
– 1 not 3 slides missed 33%
McCarthy, Transplantation 2002
• Arteriolitis
• Isolated v1
Nickeleit, JASN 1998
Porter KA 1969, 1972
Bellamy, Histopath 2000
Nickeleit, JASN 1998
Woman, 1st graft, for ANCA+ vasculitis.
MMF stopped (low platelets)
Missed 2 evening doses tacrolimus
C4d neg (IF)
Man; day 11; 1st graft
presensitised (PEx, ATG, IVIg)
FXM neg
class I DSA, MFI 300 now 2000
C4dptc 5-10% (IF, medulla)
Subclinical active AMR is common in sensitised patients
• Subclinical diffuse C4d+ AMR on planned biopsies (1-12m)
– 12% of 83 DSA+ patients undergoing desensitisation
• Increased chronicity scores
Haas, AJT 2006, 2007
– Despite controls (24) having more TCMR, including 10 v1, 1v2, 1v3!
• Subclinical AMR in planned biopsies in high risk patients (3,12m)
– non-desensitised ELISA+
– At 3 months
• 31% had C4d+ AMR
• 49% had C4d negative microcirculation inflammation
• 20% lacked activity (g/ptc/C4d)
Loupy, AJT 2009
– C4d-negative AMR
• lower MFImax
• less severe lesions
• intermediate outcome
– C4d status unstable and often focal
• 42% focal vs 9% diffuse @ 1yr
– AMR at 3 months predicts chronic changes & lower GFR at 1 year
C4d-negative AMR
• Status at 3m correlated progressively with chronic rejection at 12m
• Risk starts with C4d-negative microcirculation inflammation
Loupy, AJT 2011
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Molecular data support C4d as an activity marker: 9/13 C4d+AMR were in the top
25 DSA-specific transcript scores
Hidalgo, AJT 2010
C4d-negative AMR
• C4d status fluctuated in half the patients:
40% , 14% 
• Cumulative C4d score over 2 biopsies correlated with sCr rise
latest SCr
Loupy, AJT 2011
cumulative C4d:
C4d-negative definition of AMR
Einecke, AJT 2009
Sis, AJT 2009
In indication biopsies (standard risk), DSA, C4d, MCI and
arteritis cluster together, distinct from chronic lesions &
tubulointerstitial inflammation.
Sis, AJT 2010
C4d-negative “active” AMR
• Multiple studies have shown that microcirculation inflammation in
patients with DSA correlates with increased risk for chronic histological
lesions (including cg) and graft failure
• Standard risk patients with de novo DSA
– planned surveillance biopsies
– unplanned protocol biopsies
– unplanned indication biopsies
• A better predictor of graft failure than C4d
• Absence of MCI approximates to DSA negative risk
de Kort, AJT 2012
Wiebe, AJT 2012
Hidalgo, AJT 2009
Sis, AJT 2010
Microcirculation inflammation (g + ptc ≠ 0)
• Not diagnostically specific
– high risk stable surveillance (1-12m):
Krauss, AJT 2009
• without diffuse C4dptc MCI>2 did not discriminate DSA+ from DSA-
– standard risk early indication biopsies
• 44% were TCMR/borderline
• 24% were ATI
Sis B, AJT 2012
– standard risk late indication biopsies
• 76% were AMR
• Missed 39% of DSA+
• Missed 15% AMR (mostly C4d-ve cg; occasional TMA, C4d+ IFTA)
– high risk patients
• MCI present in 100% AMR, 73% AMVR, 15% TCMVR, 12% TCMR
Lefaucheur, Lancet 2013
• Shown individually for
– Glomerulitis
– Peritubular capillaritis
Batal, AJT 2010
Gibson, AJT 2008
Microcirculation inflammation (g + ptc ≠ 0)
• Immunophenotyping might help
– CD56+ and CD68+ predominate in AMR
– CD3+ characterize TCMR
Magil, AJKD 2005, KI 2003
Fahim, AJT 2007
Hidalgo, AJT 2010
Sis B, AJT 2012
• Or other severity markers
– oedema, haemorrhage, ectasia, endothelial turnover
– but severer means rarer
• Pathologist (dis)agreement
Sellares, AJT 2013
– diagnosis of microcirculation injury
– problem for microarray studies (Sellares 2013)
– correlates well with multi-pathologist “agreement that AMR is present”
(essentially MCI scoring)
• Only VE-cadherin was in the 5 C4d+ AMR>TCMR subset of ENDATs of Sis, along with
2 other non-ENDAT endo transcripts SOX7, MALL exp in AMR>TCMR
Man, 1st graft, d 6.
BFxM +, DSA -, MICA C4dptc 1% (IF)
de novo DSA
Lee PC 2002, Worthington JE 2003,
Everly MJ 2013
• Precede graft failure with close correlation
• Repeated poor compliance or prescribed marginal immunosuppression is
common
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½ de novo DSA patients (probably much greater)
– Dorje C 2013 (56%), Sellares J 2012 (47%), Wiebe C 2012 (49%, OR 8.75)
• all with acute dysfunction,
Wiebe C 2012
• ½ of indolent dysfunction,
• 6% of stable patients
• Marginal immunosuppresion has a mixed AMR/TCMR/chronic phenotype
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C4d+ and TCMR more likely in acute dysfunction group
Borderline/TCMR accompanying AMR increase graft failure rate
Lerut E 2007, Wiebe C 2012, Dorje C 2013 EverlyMJ2013, Matignon 2012
Late but not early int inflam correlates with gx failure [Sellares2011) - ? Role of noncompliance in that finding
• Late MCI is likely to be an excellent marker for non-compliance
– might correlate better with graft failure better than a specific AMR marker
Accelerated intimal neofibrosis in AMR
• Protocol biopsies (3m, 1yr, some later)
– Excluded arteritis, luminal wbc adhesion cases
• Baseline age and progression rate for intimal fibrosis
– from donor biopsies
– found 3x accelerated progression in DSA+ patients c/w DSA• both early and late
• no accelerating role of HT, older donor
• associated current/prior microcirculation inflammation/C4dptc
Hill, JASN 2011
Accelerated intimal neofibrosis in AMR
• Myointimal proliferation (SMA+) (not T/B cells) (3m)
– Uniform in small arteries
– Stratifies within deeper acellular collagen in larger arteries
– Minimal at most in DSA negative patients (no stratification)
• Elastosis by 1 year
Hill, JASN 2011
• Bland intimal fibrosis in some DSA+ and DSA- patients
• no microcirculation inflammation in the DSA+ cases (post-AMR)
De novo membranous nephropathy
Collins, 11th Banff conf 2011
• Usually late onset (>3 years)
• Associated with features of active chronic AMR
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DSA (esp anti-DQ)
C4dptc
transplant glomerulopathy
ptc bm multilamination
• Unlike recurrent MGN
Lim, Transplant Proc 2012
Kearney, Transplant Proc 2011
– IgG1 not IgG4 localisation
– Unrelated to anti-phospholipase A2 receptor Ab
Debiec, AJT 2011
Woman, 1st graft, day 5
SLE; DSA C4dptc neg
Non-HLA DSA
• MICA
• Major histocompatibility complex (MHC) class I-related chain A (MICA) antigens
• Surface ligand for activating immunoreceptor NKG2D (NK, CD8 T cells)
• Endothelium, keratinocytes, monocytes, fibroblasts, dendritic cells, activated B cells
– Do MICA Ab play a role in allograft dysfunction and AMR?
• 5-9% of recipients after transplantation
• 10% decrease in graft survival at 1 year, maintained at 5 years
• High-risk subset who receive kidney from a truncation mutant donor
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59% donors (46% het, 13% hom)
MICA sensitisation is more prevalent, stronger (2-9x MFImax)
Increased proteinuria, correlating with MFImax (r= 0.4)
C4d+ rejection in MICA5.1+/HLA DSA- patient
• AT II type I receptor
Dragun 2005
Zou, NEJM 2007
Terasaki, AJT 2007
Tonnerre, JASN 2013
100 circles
25%
30%
35%
40%
50%
60%
80%
5%
10%
15%
20%
25%
30%
35%
40%
50%
60%
Banff C4d working group (BIFQUIT)
• Distributed unstained slides from a TMA:
Mengel, AJT 2013
– local C4d stain & score, return slide
– panel reviews
• Technical
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Some negative controls read as positive (7/44 labs, C4d1-3)
prompt fixation,
fix 1 hour
harsher pretreatment, pH6-7,
primary incubation >40 mins
High titre primary (>1:80)
Pc C4d low pH
Pc C4d high pH
C4d High pH
C4d High pH
CD68
CD68
TNFa
CD68
IL10
CD68
CD163
Conclusions
• AMR diagnosis on biopsy
– Is a histologic collective whose specificity depends on the severity & setting
• Characteristic aggressive lesions (especially early)
• Non-specific signs of immune activity (early or later) whose specificity mounts as
they escalate
– Immuno or molecular phenotyping is likely useful
• Characteristic remodelling changes (from quite early onwards)
• C4d-ve AMR clearly exists: diagnostic threshold probably best tempered by setting
or exclusion as MCI alone is not a specific lesion when mild
– C’-fixing DSA/IgG1/3 isotypes
– characteristic chronic lesions
– C4d mean/sum score
– In low risk patients often reflects marginal immunosuppression by patient or
doctor choice, is smouldering rather than highly active, often mixed with
TCMR that speeds graft failure
– Is problematic with non-HLA DSA!
Banff working groups
C4d-negative AMR
Working group
C4d-QA (BIFQUIT)
Working group