Immunohistochemical Staining of Lymphocytes for the Reliable Diagnosis of Myocarditis in Endomyocardial Biopsies

Abstract

Interobserver variability in the interpretation of pathologic endomyocardial biopsies for detecting myocarditis has been widely reported. Thus, conflicting reports about the therapeutic benefit of immunosuppressive treatment in myocarditis may be due to differences in the interpretation of the biopsy findings. In doubtful cases, scattered interstitial cells may be present between myocytes and can be misinterpreted as true lymphocytes. In our study, a further characterization of interstitial cells in endomyocardial biopsies previously diagnosed as showing ‘myocarditis’ was performed by lymphocyte immunophenotyping with immunocytochemical techniques for membrane and cytoplasmic antigens. Common leukocyte antigen (CLA), k and λ light immunoglobulin chains and T lymphocyte antigens were made visible by an indirect immunoperoxidase technique. A previous diagnosis of ‘myocarditis’ had been established histologically in 27 patients by the presence of an inflammatory cell infiltrate associated with focal acute cellular damage. These specimens were selected for further study using an immunoperoxidase technique. The number of negative and positive mononuclear cells for each marker was counted on all fields at a magnification of × 400. These numbers were correlated with the extent of interstitial fibrosis and/or myocyte damage on each sample. According to previous studies, 5.0 lymphocytes/high-power field were considered as the lower limit of myocarditis if they were associated with myocyte injury. From the 27 samples previously diagnosed histologically as ‘myocarditis’ only 14 showed 5 or more CLA-positive mononuclear cells/ × 400 field. In 6 out of 8 selected cases having less than 5 CLA-positive cells, no T-antigen-positive cells could be detected. The remaining samples showed T lymphocytes localized in acute infiltrated areas. From these results, only 14 of the 27 patients showed ‘true myocarditis’. Accordingly, when total cell infiltrates and lymphocytes were evaluated, it was shown that although a high mean of apparently mononuclear cells may be found in a high-power microscopic field, only a small percentage of them may result in true lymphocytes. The present approach facilitates the subclassification and quantification of the different subsets of inflammatory cells and helps to define adequate therapeutic and/or prognostic implications, thus avoiding interobserver variability.