immunofluorescence detection of complement activation products c4d and c3d:

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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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

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