Histologic Changes observed in C4d positive Renal Transplants V. Nickeleit and M. J. Mihatsch Nephro

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Histologic Changes observed in C4d positive Renal
Transplants V. Nickeleit and
M. J. Mihatsch Nephropathology Laboratory,
Department of Pathology, The University of
North Carolina, Chapel Hill, USAandInstitute
for Pathology, University of Basel, Basel,
Switzerland
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The sytematic evaluation of C4d is THE most
significant recent progress in diagnostic (renal)
transplant pathology
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C4d positive negative
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C4d is Not New !C4d pioneers The group of
H.E. Feucht from Munich Kid Int (36)
1069-1077, 1989 Clin Exp Immunol (86) 464-470,
1991 Immunology (80) 162-167, 1993 Kid Int
(50) 1464-1475, 1996
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• C4d
• C4d degradation product of the
• activated complement factor C4
• C1 C4 activation induced by antibody
  binding
• C4d binds covalently
• C4d deposited along the endothelium/
  basement membranes of peritubular
  capillaries in kidney transplants

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Fascination Finally an immunohistochemical
marker for a humoral allo-response !
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C4d significant independent marker for poor
graft survival Feucht et al. Kid Int (43)
1333-1338, 1993 Herzenberg, Magil et al. JASN
(13) 234-241, 2002 Böhmig, Regele et al. JASN
(13) 1091-1099, 2002
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• C4d
• Knowns, Unknowns Confusion
• C4d and circulating Antibodies
• C4d and Histology
• C4d and Terminology

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C4d A Marker for a Humoral
Allo-Response
Flow-cytometric crossmatch (FCXM) C4d
positive C4d negative (n24)
(n89) FCXM neg. 3 (12.5) 42
(47.2) FCXM pos. 21 (87.5) 47
(52.8) X2 Plt0.01 specificity 93
sensitivity 31 pos. predict. value
87 neg. predict. value 47 antibody
specificity against HLA-class I and / or class
II JASN (13) 1091-1099, 2002
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• C4d
• C4d and circulating antibodies
• Strong correlation
• But..
• What antibodies activate the
• complement cascade in renal allografts?
• What is the association between C4d
• and different antibody titers?

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C4d positivity, rejection and antibodies
targeting angiotensin II type 1 receptor Dragun
D et al. NEJM 352 (6) 558, 2005
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• C4d
• Knowns, Unknowns Confusion
• C4d and circulating Antibodies
• C4d and Histology
• C4d and Terminology

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(Diagnostic) C4d is deposited mainly
along the intact endothelial cell layer of
peritubular capillaries in the renal cortex and
/ or the renal medulla
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JASN (13) 2371-2380, 2002
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C4d and Histology Detection 1)
Immunofluorescence (IF) microscopy on frozen
tissue Advantage - strong staining - no
background - internal staining control
(mesangial regions normally positive) Disadvanta
ge - frozen tissue required 2)
Immunohistochemistry (IHC) on formalin fixed
tissue Advantage - tissue
availability - superior morphology Disadvan
tage - weaker staining - no internal
staining control (mesangial regions
negative) - special antigen retrieval
required (e.g. pressure, steam)
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C4d and Histology Evaluation of C4d
positivity - only in areas without necrosis or
fibrosis (cortex and medulla) - only
circumferential capillary staining - many
capillaries in one area
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C4d and Histology Prevalence of C4d in
diagnostic biopsies (ABO compatible)
Basel 30 Chapel Hill 20 Vienna
21 - 41 Boston (MGH) 30 Hannover (mult
icenter study) 21
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C4d and Histology Prevalence of C4d
in protocol biopsies Hannover 3.3
Hannover (multicenter study) 4.4
(range2.4 - 10.7) Winnipeg 25
(overall) 38 (DSA patients)
17 (DSA - patients) Oslo 30
(1 week post tx) Oxford 13 (1 week
post tx) 33 (DSA patients) 3
(DSA patients)
From stable and dysfunctioning grafts
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C4d and Histology C4d Staining (Basel,
diagnostic biopsies) Median 38 days
post tx (7 - 5646) Change of staining (Basel)
negative to positive 64 days
(median) shortest interval 4
days positive to negative 76 days
(median) shortest interval 8 days
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C4d and Histology No specific
histological feature can reliably predict C4d
positivity by light microscopy ! C4d is often
associated with - Transplant glomerulitis
- Cellular rejection (especially
endarteritis) - Peritubular capillary
dilatation and leukocytosis - Interstitial
hemorrhage - Fibrinoid arterial necrosis
and thrombosis and occasionally other
histological changes
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• Histology C4d positive / total X2 p value
• Tx glomerulitis 57 ( 28 / 49 ) lt 0.0001
• Tubular MHC-Class II
• expression 41 ( 102/246 ) lt 0.0001
• Interstitial rejection 43 ( 56 / 131 ) lt
  0.0001
• Tx endarteritis 45 ( 33 / 74 )
  0.004
• Inactive intimal
• sclerosis 33 ( 16 / 48 ) ns
• Tx glomerulopathy 53 ( 9 / 17 ) 0.07
• Cyclosporine tox. 28 ( 40 / 145 ) ns
• BK-Virus nephropathy 0 ( 0 / 9 ) NA
• Fibrinoid vascular necrosis (v3) 50 ( 5 / 10
  ) NA
• Acute tubular necrosis 27 ( 3/11 ) NA
• JASN (13) 242-251, 2002

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• C4d and Banff types I and II rejection
• Type 1 rejection (tubulo-interstitial)
• 24 to 43 of cases C4d positive
• Type 2 rejection (tx endarteritis)
• 45 to 46 of cases C4d positive
• JASN 13 234 (2002) and JASN 13 242 (2002)

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Tubulo-Interstitial Rejection, C4d positive
Tubular MHC-Class II Expression
C4d Positivity
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• C4d and Histology
• - Acute Pure Humoral Rejection -
• - Capillaries (glomeruli / peritubular
  capillaries)
• Dilated
• Filled with polymorphonuclear leukocytes
• and / or monocytes
• Focal fibrin thrombi
• Interstitial edema and focal hemorrhage
• - Acute tubular injury
• - No evidence of cellular rejection
• - No tubular MHC-class II expression
• - No transplant endarteritis / no large thrombi

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Acute Pure Humoral Rejection
C4d
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C4d and Histology Non-diagnostic
C4d deposits 1) Atrophic tubular basement
membranes 2) Endothelium of arteries 3)
Arteriolosclerosis/-hyalinosis 4) Glomerular
endothelium
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C4d and Histology - C4d positivity
indicates active rejection in dysfunctioning
grafts (involvement of antibodies) - C4d
characterizes rare forms of acute pure
humoral rejection ( lt 5 of all graft biopsies)
- C4d positivity noticed with
various histological changes including
cellular rejection or normal histology -
C4d morphologic subclassifier But.
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• C4d and Histology Caveats
• Significance of persistent C4d with normal
  histology ?
• Exhaustive comparative analysis IF versus IHC not
  reported
• Minimal threshold levels for C4d positivity
  undefined
• ( gt 10, gt 25, gt 50 ? )
• Association between focal C4d and circulating
• DSA undetermined
• No knowledge about the changes in C4d staining
  and
• changes in corresponding antibody titers
• Significance of glomerular staining unknown

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C4d Immunofluorescence frozen tissue
C4d IHC fixed tissue
ML Troxell et al. Mod. Path 2005 18 (suppl 1)
270A (abstract)
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• C4d
• Knowns, Unknowns Confusion
• C4d and circulating Antibodies
• C4d and Histology
• C4d and Terminology

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• C4d and Terminology
• How to classify C4d positivity (nomenclature)
• as a subclassifier to standard histological
  terms
• and the new entity acute pure humoral
  rejection ?
• b) as acute humoral or chronic humoral
  rejection ?
• c) according to the updated Banff 97 scheme ?

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C4d and the updated Banff 97 categories of
rejection
Antibody-mediated rejection (category 2) 1)
Serological evidence of anti-donor
antibodies 2) Morphology and
immunohistochemistry A. ATN like minimal
inflammation, C4d positive B. Capillary Tx
glomerulitis, inflammatory cells in peri-
tubular capillaries, thrombi, C4d positive C.
Arterial fibrinoid vascular necrosis (v3), C4d
positive
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• C4d and Terminology
• How to classify C4d positivity (nomenclature)
• as a subclassifier to standard histological
  terms
• and the new entity acute pure humoral
  rejection
• b) as acute humoral or chronic humoral
  rejection
• c) according to the updated Banff 97 scheme ?

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Conclusion

• C4d is significant !
• Knowledge about C4d is still limited !
• C4d staining results can best be added as
  diagnostic
• classifier !