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