Immunofluorescence Detection of Complement Activation Products C4d and C3d: Cleveland Clinic Experience Carmela D. Tan August 12, 2009

AMR at the Cleveland Clinic Describe the staining patterns of C4d and C3d in heart transplant biopsies Describe the clinical utility of C4d and C3d staining in the diagnosis of AMR Report the prevalence of AMR in a single high- volume heart transplant center Describe how regulation of complement correlates with clinical presentation

Methods Every single endomyocardial biopsy since November 2006 is routinely stained with C4d & C3d for clinical diagnosis 4 or more pieces frozen for H&E and IF Staining patterns: capillary vs perimyocytic, diffuse vs focal Staining intensity : 0 to 3+ Study period: November 2006 to December 2007 In addition to C4d & C3d, for this study all available biopsies were also stained for the complement regulators CD55 (DAF) and CD59 (Protectin)

Retrospective review of electronic medical records with follow-up until December clinical evidence of allograft dysfunction Cardiac allograft dysfunction was defined as: 1.A decline in left ventricular ejection fraction 2.A decrease in cardiac index 3.Elevation of right side cardiac pressures and 4.A need for inotropic support. Serologic determination of anti-HLA antibodies in all C4d positive patients Methods

Results 1511 EMBs 330 adult patients (age: 20-73) males; 64 females Years after transplant - 1 year or less: years: >5 years:39 Average number of biopsies: 5

C4d, C3d

Cap and perimyocytic

C4d only

Perimyocytic only

Results of 330 patients Capillary diffuse Capillary focalPerimyocytic focal or diffuse C4d and C3d19 patients (6%) 07 patients (2%) C4d only19 patients (6%) 13 patients (4%) 4 patients (1%) C3d only000

Only diffuse capillary staining is relevant when clinical and serologic data are correlated. Tan CD et al, Am J Transplant Epub 2009 Jul 16 Group 1 Group 2

Group 1Group 2Group 3Group 4P value Staining patternC4d+ diffuse/ C3d+ C4d+ diffuse/ C3d - C4d+ focal/ C3d- Perimyocytic # of patients M:F13M : 6F17M : 2F8M : 5F10M : 1 FNS Age (mean, range)51 (27-67)49 (21-65)48 (24-68)52 (23-64)NS Time of biopsy with complement (median, range - months) 21 ( ) 15 ( ) 15 ( ) 41 (2-147) NS Sensitized patients58%37%54%36%NS LVAD15%21%30%45%NS Allograft dysfunction 84%5%0% P < Donor specific Anti-HLA Ab 95%35%56%0%P = Mortality801 (ACR)1 (Sepsis)

Group 1Group 2Group 3Group 4P value Staining patternC4d+ diffuse/ C3d+ C4d+ diffuse/ C3d - C4d+ focal/ C3d- Perimyocytic # of patients M:F13M : 6F17M : 2F8M : 5F10M : 1 FNS Age (mean, range)51 (27-67)49 (21-65)48 (24-68)52 (23-64)NS Time of biopsy with complement (median, range - months) 21 ( ) 15 ( ) 15 ( ) 41 (2-147) NS Sensitized patients58%37%54%36%NS LVAD15%21%30%45%NS Allograft dysfunction 84%5%0% P < Donor specific Anti-HLA Ab 95%35%56%0%P = Mortality801 (ACR)1 (Sepsis)

Correlation of C4d and C3d with DSA, allograft dysfunction and mortality Tan CD et al, Am J Transplant Epub 2009 Jul 16

CD55 negative CD55 positive (1+) CD55 positive (3+) Tan CD et al, Am J Transplant Epub 2009 Jul 16

Control group (n=264)

Tan CD et al, Am J Transplant Epub 2009 Jul 16 Group 1 Group 2 Group 3 Group 4

Tan CD et al, Am J Transplant Epub 2009 Jul 16 Group 1Group 2 C4d+/C3d+ Diffuse DSA + Allograft Dysfunction C4d+ diffuse/C3d – DSA +/ - No Allograft dysfunction

In summary, A panel of C4d and C3d is more useful than C4d alone in the evaluation of AMR in heart transplants. Presence of C4d and C3d correlates with DSA and cardiac allograft dysfunction. Prevalence of AMR in this cohort: 5% AMR can occur months to years after transplantation. Regulators of complement activation CD55 and CD59 may provide a protective mechanism from complement-mediated damage to the allograft.

Antibody Mediated Rejection (AMR) in Heart Transplantation Acknowledgements: Pathology : E Rene Rodriguez Heart and Vascular Institute: Randall C. Starling David O. Taylor Gonzalo Gonzalez-Stawinski Nicholas Smedira Lerner Research Institute: William M. Baldwin III Transplant Center: Diane Pidwell Lynn Klingman Tan CD et al, Am J Transplant Epub 2009 Jul 16