Calculation of Myocardial Percentage Collagen, Mast Cell Count and Presence of Arteriosclerosis in Dogs With Myxomatous Mitral Valve Disease and Control Dogs-A Pilot Study

Objectives:
This study aimed to 1) determine the feasibility of calculating percentage myocardial collagen (PMC) in formalin-fixed, paraffin embedded canine tissue samples, 2) explore relationships be-tween PMC, mast cell (MC) count and myocardial arteriosclerosis/arteriolosclerosis (MA) and 3) calculate the sample size required to compare PMC between dogs with and without myxomatous mitral valve disease (MMVD).

Methods:
Histological sections were prepared from retrospective samples of formalin-fixed, wax-embedded ventricular myocardial tissue from 15 dogs with, and 7 dogs without, MMVD. Sec-tions from each sample were stained with Masson’s trichrome, haematoxylin and eosin (H&E) or toluidine blue. In each Masson’s trichrome stained section, digital images of 12 random fields were captured and the total image area and total collagen area were measured using computer-assisted morphometric analysis. Mean collagen area, mean total image area, and mean PMC [(mean collagen area/mean total area) x 100] were calculated per sample. MC were counted man-ually in toluidine blue stained sections and the mean MC count calculated from 10 fields. MA was identified by visual inspection of transversely-cut vessels in each H&E-stained section.

Results:
No significant difference was detected between MMVD and control dogs in PMC (P=0.063), MC (P=0.476) or MA (P=0.172). A sample size calculation suggests data from at least 26 MMVD dogs and 26 controls are required to detect a difference in PMC. No significant associa-tion was detected between PMC and MC count (P=0.606) in cases of MMVD.

Clinical Significance:
The methods described are feasible and the sample size for a definitive study has been estimated.