P.Randhawa, A. Girnita, A. Zeevi, R. Shapiro, I. Batal, Departments of Pathology, Surgery, University of Pittsburgh
SIGNIFICANCE OF FOCAL C4d DEPOSTIS IN THE KIDNEY
OUTLINE OF TALK
• Definition of focal C4d
• Clinical significance
• Management issues
• Occurrence of DSA –ve cases
• Association with Dx other than AMR
GUIDELINES FOR C4d INTERPRETATION
• Minimum 5 hpf Cortex or medulla (concordant in 75% graft nephrectomy).
• Necrotic/scarred area exclude ( intensity)
• Linear, circumferential, finely granular
• Intensity at least 1+ intensity on FS
• HCHO weak stain may be significant
BANFF 2007 DEFINITION OF C4d STAINING PATTERNS
% biopsy area Interpretation according to technique
(cortex and medulla) IF IHC
• C4d0 Negative: 0%
• C4d1 Minimal 1-10%
• C4d2 Focal 10-50%
• C4d3 Diffuse >50% Pos Pos
?PosUnknown
UnknownNeg
Neg Neg
: BANFF 2001 MEETING
Only C4d + and – categories recognized.
Positive staining was defined as bright linear staining along capillary basement membranes typically involving OVER HALF OF SAMPLED peritubular capillaries
NUMBER of capillaries expressed as a percentage, rather than SURFACE AREA of biopsy was the defining criterion
Racusen et al. Am J Transplant 2003: 3: 708
% CAPILLARY SCORING
• % PTC score used in many studies >2001
• Difficult to apply IF (dark field evaluation)
• Can not take in account loss of sensitivity of C4d staining on formalin fixed tissue
• Underestimates extent of C4d staining in bxs with IFTA & capillary loss
Kayler et al. Transplantation 2008; 85: 813
PTC C4d STAINING PATTERNS (106 BX WITH AR & C4d STAIN)
Diffuse (16)
Focal
(24)
Neg
(66)
I3 6% 8% 3%
T3 31% 58% 64%
V1 38% 17% 12%
PC >25% 13% 14% 15%
ANTI-HLA ANTIBODIES
Diffuse (16) Focal(24) Neg (66)
ELISA I 38% 30% 15%
ELISA II 83% 52% 29%
I or II
(-1, +12m)86% 57% 32%
DSA+/- 1m 94% 38% 17%
RESPONSE TO STEROIDS
Diffuse (16)
Focal(24) Neg (66)
Incomplete 64% 82% 29%
Creatinine
12m
0.7+/-0.6 0.6+/-0.8 0.3+/-0.6
Graft loss 31% 38% 21%
61% if f/u Diffuse
C4D PATTERNS IN F/U BIOPSIES <1 YR (WORST C4d SCORE)
Diffuse (12) Focal(20) Neg (54)
D 58% 17% 25%
F 17% 45% 20%
Neg 25% 35% 67%
EFFECT OF TISSUE FIXATION :C4D PATTERNS IN DSA + PTS (n=14)
Frozen HCHO
Diffuse 11/14 (79%)
5 /14(36%)
Focal 1 (7%) 6 (43%)
Negative 2 (14%) 3 (21%)
MANAGEMENT OF FOCAL C4d+ BIOPSIES AT PITTSBURGH
• Correlate with presence of DSA
• Pure Acute AMR with DSA, rising creatinine, get IVIG &/or PP
• Treat any concurrent T-cell mediated AR
• Assess degree of histologic chronicity
C4d + DSA –VE CASES:Technical Issues
• Technical problems with C4d staining
-high background, necrotic or scarred area
• Technical problems with antibody testing (a) Date
(b) Rare antigen not present in testing panel
(c) Incorrect HLA Typing of donor HLA
(d) Incomplete donor typing (anti-DP, DQ)
DETECTION OF DSA DEPENDS ON SENSITIVITY OF TECHNIQUE
• 41 biopsies focal C4d, ELISA PRA screening test for anti-HLA antibody -ve
• 11/41= 27% had DSA by Luminex
• 7/41 = 17% antibodies to MICA
BIOLOGIC EXPLANATIONS FOR C4d + DSA –VE CASES:
• Adsorption of DSA to graft
• Non-donor specific antibodies
• Non-HLA antibodies
• C4d deposition in dx other than AMR
NON-DONOR SPECIFIC HLA ABS
• Statistically more AR & worse outcome
• Marker for high immune responsiveness
• DSA may actually be present but absorbed
• Monitor carefully
Hourmant et al. JASN2005;16;2804
NON-HLA ANTIBODIES
• AECA: anti-endothelial antibodies • Anti-GSTT1 Glutathione S-Transferase T1• MICA, MICB• AT1R ab: Angio II type I receptor ab • Anti-VIM/ICAM-1 ab assoc GAX in heart• Anti-AGRIN (GBM) ab associated cg• Anti-HY ab products of Y chromosome
POTENTIAL TARGETS OF AECA
• MHC antigens
• ABO antigens
• AT1R receptors
• MICA (Mhc class I related Chain Ag)
• Other unknown polymorphic ags
PROBLEMS WITH AECA STUDIES
• Most assays do not attempt to define ag.
• Studies cross sectional: cause & effect?
• Some AECA definitely 20 vascular injury
- due to rejection (intimal arteritis)
- viral infection (CMV)
AECA & ANTI-HLA CAN CO-EXIST
• FCM assay XM-ONE Kit PBL endoth progenitors
-35/147 (24%) pre-tx sera had donor reactive ab
-Acute rejection 16/35 (46%) vs 13/112 (12%);
-6/16 C4d +, ALL had confounding HLA ab
Breimer et al. Txn 2008; 87: 549:
SOME AEC ASSAYS DO MEASURE
COMPLEMENT FIXING AB
• EUROIMMUN indirect IF reagent kit and HUVEC deposited on BIOCHIPs
• AECA in 13/47 patients vascular rejection• 6/13 C4d+ (46%); 1/6 anti-HLA +• Plasma cell infiltrate 54% AEC-AR vs 12%no AR• Overall 1 yr graft loss 46% AEC vs 19 % no AEC
Sun et al. CJASN 2008; 3; 1479
ANTI-GLUTATHIONE S-TRANSFERASE T1 ANTIBODIES
• Donor has GSTT1 gene, recipient does not• Incidence of GSTT1 mismatch ~ 20%• Initial associative studies severe liver dysfunction• Ktx: one study reported 4 cases of CHRONIC
AMR with C4d in peritubular capillaries• 1 case report acute AMR is also available
Aguilera et al NDT 2008; 23; 1393
Feucht et al. KI 2001:5934; AJT 2003:3:646
BANFF CATEGORIES OTHER THAN AMR WITH C4d DEPOSITS
• Recurrent antiGBM
• Post-tx IgA 16/66 PTC Cho et al Clin Tx 2007:21:159
• Colvin: USCAP 38% Denovo 17% rMGN
• Feucht 2001: 6/10 GN 11/19 ATN
• Feucht 2003: ATN C3d, not C4d
• Lupus nephritis (31/455, D)
-Li et al. Lupus 2007:16:875- granular, EM immune complex deposits
• 2/2 Bacterial endocarditis GN
• Scleroderma renal crisis-diffuse 1/11, focal 3/11
• Two donor, 1 DIC kidney (F)• C activation multiple paths
C4d DESCRIBED IN NATIVE KIDNEY DISEASES
SUMMARY
• Focal C4d PTC <50% surface area
• Staining pattern affected by tissue fixation
• Significance: correlate histology & DSA
• % patients with DSA intermediate
• DSA–ve: technical issues, non-HLA abs, diseases other than AMR
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