Digital image analysis system for the quantification of infiltrates and cell adhesion molecules in inflammatory...

We attempted to develop a digital image analysis (DIA) system for endomyocardial biopsies (EMBs) to reliably quantify a) biopsy quality, b) immunohistochemically-marked infiltrates, and c) cell adhesion molecules (CAMs) in relation to net heart area (HA) for the semi-automated diagnosis of inflammatory cardiomyopathy (InfCM).

140 EMBs from dilated cardiomyopathy (DCM) patients and 14 autopsy heart samples (controls) were immunostained for T-lymphocytes (CD2, CD3, CD4, CD8), beta(2)-integrin+ infiltrates (CD18, LFA-1, Mac-1) and CAMs (immunoglobulin superfamily: ICAM-1, HLA class I, HLA DR, VCAM-1, CD58; selectins: CD62E and CD62P; and the beta(1)-integrin chain CD29). EMB quality was assessed visually on a three-point scale. Infiltrates were quantified visually (per hpf) and by DIA (per mm2 HA). CAM expression was evaluated semiquantitatively and by DIA (area fraction [AF]: stained area relative to HA).

DIA-evaluated HA correlated significantly with the visual assessment of EMB quality. The visual evaluation of both infiltrates and CAMs correlated significantly with the respective DIA-based quantification. DIA-quantified CAM-AF and infiltrates were discriminated by the CAM classification (CAMs+: n=87; 62%) compared to controls. DIA-quantified CAM immunoreactivity correlated significantly with the DIA-quantified counter-receptor+ infiltrates. DIA evaluation of biopsy quality, infiltrates, and CAMs was devoid of inter- and intraobserver variability.

The DIA system presented here enables standardized and observer-independent assessment of EMB quality and intramyocardial inflammation (density of infiltrates and CAM expression) in DCM biopsies related to HA. Our data confirm that endothelial CAM count and counter-receptor+ immunocompetent infiltration are interdependent pathogenic and diagnostic hallmarks of InfCM.