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C4d - staining: Comparison of methods C. Seemayer, A. Gaspert, M. Mihatsch. Introduction. Tx-Normal. Detection of C4d: Frozen sections, mAb Quidel Paraffin sections:C4dpAb, Biomedica. Renal allograft biopsies. Cryo-conservation. Formalin fixation. Paraffin sections. Frozen sections.
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C4d - staining: Comparison of methods C. Seemayer, A. Gaspert, M. Mihatsch
Detection of C4d:Frozen sections, mAb QuidelParaffin sections:C4dpAb, Biomedica
Renal allograft biopsies Cryo-conservation Formalin fixation Paraffin sections Frozen sections IHC with ABC method Indirect IF Mouse monoclonal anti-C4d antibody Rabbit polyclonal anti-C4d antibody Comparison of results by 2 investigators
Aim of the study:1.Comparison of frozen to frozen biopsies with respect to time-dependent stability and investigator dependent reproducibility of results 2.Comparison of F-IF and corresponding P-IHC staining patterns 3.Investigation of P-IHC staining patterns focusing on the inter- and intra-observer variability
Materials & Methods (1) • Patients and biopsies • retrospective study • 64 kidney allograft Bx (Basel 1996 - 2004) • parallel Bx cores either frozen or formalin fixed • 240 renal allografts Bx (50 different units) • independent analysis by two renal pathologists
Materials & Methods (2) • Immunofluorescence • snap frozen tissues • indirect IF technique • 1st ab: mouse anti-human anti-C4d antibody • dilution 1:50 • Quidel (San Diego, CA, USA) • 2nd ab: Alexa-Fluor 488-labeled goat anti-mouse IgG • dilution 1:200
Materials & Methods (3) • Immunhistochemistry • formalin fixed and paraffin embedded tissue sections • rabbit polyclonal antibody • anti-C4dpAb (Biomedica, Vienna, Austria) • antigen: split product C4d (aa 1252-1256 of C4) • dilution 1:10 and 1:20 • heat antigen retrieval • ABC Elite complex method
Materials & Methods (4) • Evaluation / Scoring • Diffuse expression: > 50% of PTC positive • Focal expression: 10 PTC < 50% of PTC positive • Focal minimal expression: 3 - 10 PTC positive • Negative: < 3 PTC or completely negative
Results1.Comparison of frozen to frozen biopsies with respect to time-dependent stability and investigator dependent reproducibility of results
Results1. Comparison of F-IF and correspondingP-IHC staining patterns
Overall comparison of C4d expression in frozen (IF) and formalin fixed (IHC) sections No. of cases
Comparison of C4d expression in diffuses cases frozen (IF) versus paraffin embedded (IHC)
Comparison of C4d expression in focal cases frozen (IF) versus paraffin embedded (IHC) No. of cases
Comparison of C4d expression in focal mini cases frozen (IF) versus paraffin embedded (IHC) No. of cases
Comparison of C4d expression in negative cases frozen (IF) versus paraffin embedded (IHC) No. of cases
Number of PTC expressing C4d in diffuse cases: frozen (IF) versus paraffin embedded (IHC) IHC IF % of C4d PTC
Investigator dependent variability : Frozen (IF2) versus paraffin in investigator 1 / 2
Inter- and intra-investigator dependent variability in paraffin: Investigator 1 / 2 and antibody dilutions 1:10 / 1:20
Conclusion C4d staining results reported on frozen tissue samples using IF with a monoclonal antibody appear to be better suited for diagnostic as well as research purposes. Future studies should correlate C4d staining patterns with circulating donor specific antibodies.
Unanswered Questions • Grading: negative, focal and diffuse • Significance of C4d in glomeruli • Co- staining of C4d and other complement factors
Take Home Message C4d in PTC is a footprint of a humoral immune response: Search for donor specific Ab 2. Tx-glomerulitis, Tx endarteritis, leukocytes in PTC are the most important morphological indicators of humoral rejection: Search for C4d 3. Use -whenever possible- frozen sections for the detection of C4d Focal deposits in frozen sections may be lost in paraffin sections due to lower sensitivity of the method.
Open Questions • Clinical significance of focal deposits of C4d in PTC • Clinical and biological significance of late C4d deposits (after years) • Is the „dose“ of antibodies relevant for the morphological sequelae? • Why are donor specific antibodies not always associated with C4d deposits?
Open Questions • Why is the morphology so variable in C4d positive cases? • Why is only C4d present and no other complement components? • Is the effect of antibodies against HLA Class I the same as against Class II? • Pathogenesis of Tx-glomerulitis and Tx-endarteritis without C4d deposits.