Post on 01-Apr-2015
Immunofluorescence Detection of Complement Activation Products C4d and C3d:
Cleveland Clinic Experience
Carmela D. TanAugust 12, 2009
AMR at the Cleveland ClinicAMR 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
MethodsMethods• 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 2008 - clinical evidence of allograft dysfunction
Cardiac allograft dysfunction was defined as:1. A decline in left ventricular ejection fraction2. A decrease in cardiac index3. Elevation of right side cardiac pressures and4. A need for inotropic support.
• Serologic determination of anti-HLA antibodies in all C4d positive patients
MethodsMethods
ResultsResults• 1511 EMBs
• 330 adult patients (age: 20-73) - 266 males; 64 females
• Years after transplant- 1 year or less: 50- 1-5 years: 241- >5 years: 39
• Average number of biopsies: 5
C4d, C3d
Cap and perimyocytic
C4d only
Perimyocytic only
Results of 330 patientsResults of 330 patients
Capillary diffuse
Capillary focal Perimyocyticfocal or diffuse
C4d and C3d 19 patients (6%)
0 7 patients(2%)
C4d only 19 patients (6%)
13 patients (4%)
4 patients(1%)
C3d only 0 0 0
Only diffuse capillary staining is relevant when clinical and serologic data are correlated.
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Group 1
Group 2
Group 1 Group 2 Group 3 Group 4 P value
Staining pattern C4d+ diffuse/C3d+
C4d+ diffuse/C3d -
C4d+ focal/C3d-
Perimyocytic
# of patients 19 19 13 11
M:F 13M : 6F 17M : 2F 8M : 5F 10M : 1 F NS
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 (0.25-157)
15 (0.25-56)
15 (0.25-117)
41 (2-147)
NS
Sensitized patients
58% 37% 54% 36% NS
LVAD 15% 21% 30% 45% NS
Allograft dysfunction
84% 5% 0% 0% P < 0.001
Donor specificAnti-HLA Ab
95% 35% 56% 0% P = 0.002
Mortality 8 0 1 (ACR) 1 (Sepsis)
Group 1 Group 2 Group 3 Group 4 P value
Staining pattern C4d+ diffuse/C3d+
C4d+ diffuse/C3d -
C4d+ focal/C3d-
Perimyocytic
# of patients 19 19 13 11
M:F 13M : 6F 17M : 2F 8M : 5F 10M : 1 F NS
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 (0.25-157)
15 (0.25-56)
15 (0.25-117)
41 (2-147)
NS
Sensitized patients
58% 37% 54% 36% NS
LVAD 15% 21% 30% 45% NS
Allograft dysfunction
84% 5% 0% 0% P < 0.001
Donor specificAnti-HLA Ab
95% 35% 56% 0% P = 0.002
Mortality 8 0 1 (ACR) 1 (Sepsis)
Correlation of C4d and C3d with DSA, allograft dysfunction and mortality
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Tan CD et al, Am J Transplant Epub 2009 Jul Tan CD et al, Am J Transplant Epub 2009 Jul 1616
Tan CD et al, Am J Transplant Epub 2009 Jul Tan CD et al, Am J Transplant Epub 2009 Jul 1616
CD55 negative
CD55 positive (1+)
CD55 positive (3+)
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Control group(n=264)
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Group 1 Group 2 Group 3 Group 4
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Group 1 Group 2
C4d+/C3d+ DiffuseDSA +
Allograft Dysfunction
C4d+ diffuse/C3d –DSA +/ -
No Allograft dysfunction
In summary,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 RodriguezHeart and Vascular Institute: Randall C. Starling David O. Taylor Gonzalo Gonzalez-Stawinski Nicholas SmediraLerner Research Institute: William M. Baldwin IIITransplant Center: Diane Pidwell Lynn Klingman
Tan CD et al, Am J Transplant Epub 2009 Jul Tan CD et al, Am J Transplant Epub 2009 Jul 1616