Transcript Slide 1

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 highvolume 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)
Methods
•
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 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
Results
•
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 patients
Capillary
diffuse
Capillary focal Perimyocytic
focal 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.
Group 1
Group 2
Tan CD et al, Am J Transplant Epub 2009 Jul 16
Group 1
Staining pattern
# of patients
C4d+ diffuse/
C3d+
Group 2
Group 3
Group 4
P value
C4d+ diffuse/ C4d+ focal/ Perimyocytic
C3d C3d-
19
19
13
11
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 specific
Anti-HLA Ab
95%
35%
56%
0%
P = 0.002
8
0
1 (ACR)
1 (Sepsis)
M:F
Mortality
Group 1
Staining pattern
# of patients
C4d+ diffuse/
C3d+
Group 2
Group 3
Group 4
P value
C4d+ diffuse/ C4d+ focal/ Perimyocytic
C3d C3d-
19
19
13
11
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 specific
Anti-HLA Ab
95%
35%
56%
0%
P = 0.002
8
0
1 (ACR)
1 (Sepsis)
M:F
Mortality
Correlation of C4d and C3d with DSA, allograft dysfunction and mortality
Tan CD et al, Am J Transplant Epub 2009 Jul 16
Tan CD et al, Am J Transplant Epub 2009 Jul 16
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 1
C4d+/C3d+ Diffuse
DSA +
Allograft Dysfunction
Group 2
C4d+ diffuse/C3d –
DSA +/ No Allograft dysfunction
Tan CD et al, Am J Transplant Epub 2009 Jul 16
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.
Acknowledgements:
Pathology:
E Rene Rodriguez
Antibody
Mediated
Rejection (AMR) in
Heart and Vascular
Institute:
Randall C.Heart
Starling Transplantation
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