Detection of C4d deposition in cardiac allografts: a comparative study of immunofluorescence and immunoperoxidase methods.
BACKGROUND
Complement activation, evidenced by deposition of C4d, is important in the diagnosis of antibody-mediated rejection of cardiac allografts. C4d deposition can be assessed by either immunofluorescence (IF)- or immunoperoxidase (IP)-based methods. The use of methods varies considerably among institutions, but there are few data addressing their diagnostic equivalence.


OBJECTIVE
To compare IF and IP C4d staining on paired endomyocardial biopsy samples from a large number of heart transplant patients.


METHODS
Retrospectively selected paired frozen and paraffin-embedded samples from the same biopsy were stained for C4d by IF and IP methods. Capillary staining was scored by using a 0, 1+, 2+, 3+ scale.


RESULTS
A total of 296 biopsy pairs from 70 patients were studied. There were two hundred forty-three cases that were scored 0, twenty-four scored 1+, sixteen scored 2+, and thirteen scored 3+ by IF. Two hundred thirty-one cases scored 0, forty scored 1+, ten scored 2+, and fifteen scored 3+ by IP. Complete agreement was seen in 81% of cases. Among discrepant cases, 89% (n  =  51) were minor (±1) and 11% (n  =  6) were major (±2). Five of the 6 major discrepancy biopsies came from 2 patients, both of whom had concordant (IF and IP) 3+ results on prior biopsies. The weighted κ value for the entire sample set was 0.78 and for the first biopsy only set (to correct for bias introduced by multiple biopsies from the same patient) the weighted κ value was 0.88.


CONCLUSIONS
Immunofluorescence and IP C4d staining methods are highly comparable and are both viable options for antibody-mediated rejection surveillance in transplant heart biopsies.
ntermountain Central Laboratory, Murray, UT 84157, USA. dylan.miller@imail.org
Dylan V
DV
Miller
Anja C
AC
Roden
Jeffrey D
JD
Gamez
Henry D
HD
Tazelaar
D000328:Adult; D003167:Complement Activation; D015935:Complement C4b / Q000378:metabolism*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006084:Graft Rejection / Q000378:metabolism* / Q000473:pathology; D016027:Heart Transplantation / Q000473:pathology; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D009206:Myocardium / Q000378:metabolism* / Q000473:pathology; D010446:Peptide Fragments / Q000378:metabolism*; D012189:Retrospective Studies
D010446:Peptide Fragments; D015935:Complement C4b; C032261:complement C4d