Development of Quantitative PCR for BK Virus : Role In Management of Allograft Infection Masquerading as Acute Rejection

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SIGNIFICANCE OF FOCAL C4d DEPOSTIS IN THE KIDNEY
P.Randhawa, A. Girnita, A. Zeevi, R. Shapiro, I.
Batal, Departments of Pathology, Surgery,
University of Pittsburgh
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OUTLINE OF TALK

• Definition of focal C4d
• Clinical significance
• Management issues
• Occurrence of DSA ve cases
• Association with Dx other than AMR

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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

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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 gt50

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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

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CAPILLARY SCORING

• PTC score used in many studies gt2001
• 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

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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 gt25 13 14 15
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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
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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
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C4D PATTERNS IN F/U BIOPSIES lt1 YR (WORST C4d
SCORE)
Diffuse (12) Focal(20) Neg (54)
D 58 17 25
F 17 45 20
Neg 25 35 67

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EFFECT OF TISSUE FIXATION C4D PATTERNS IN DSA
PTS (n14)
Frozen HCHO
Diffuse 11/14 (79) 5 /14(36)
Focal 1 (7) 6 (43)
Negative 2 (14) 3 (21)
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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

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C4d DSA VE CASESTechnical 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)

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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

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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

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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. JASN2005162804

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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

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POTENTIAL TARGETS OF AECA

• MHC antigens
• ABO antigens
• AT1R receptors
• MICA (Mhc class I related Chain Ag)
• Other unknown polymorphic ags

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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)

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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

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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 12no AR
• Overall 1 yr graft loss 46 AEC vs 19 no AEC
• Sun et al. CJASN 2008 3 1479

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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

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BANFF CATEGORIES OTHER THAN AMR WITH C4d DEPOSITS

• Recurrent antiGBM
• Post-tx IgA 16/66 PTC Cho et al Clin Tx
  200721159
• Colvin USCAP 38 Denovo 17 rMGN
• Feucht 2001 6/10 GN 11/19 ATN
• Feucht 2003 ATN C3d, not C4d

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C4d DESCRIBED IN NATIVE KIDNEY DISEASES

• Lupus nephritis (31/455, D)
• -Li et al. Lupus 200716875
• - 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

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SUMMARY

• Focal C4d PTC lt50 surface area
• Staining pattern affected by tissue fixation
• Significance correlate histology DSA
• patients with DSA intermediate
• DSAve technical issues, non-HLA abs, diseases
  other than AMR