Amyloid staining for detection of eosinophil granules in cardiac tissue

Background Eosinophil granules are considered cardiotoxic, making their detection crucial in endomyocardial biopsies of patients with suspected eosinophil-related cardiac disorders. However, standard hematoxylin-eosin (HE) staining may fail to distinguish eosinophil granules due to the reddish background staining of cardiomyocytes and connective tissue. Direct Fast Scarlet (DFS) and Congo Red (CR), widely used for amyloid detection, have been reported to stain eosinophil granules. However, no study has definitively confirmed that these stains specifically target eosinophil granules. Objective This study aimed to verify that DFS and CR stain eosinophil granules and to evaluate their advantages over HE staining. Methods Paraffin-embedded endomyocardial biopsy samples from six patients with eosinophil-infiltrating cardiac disorders and six with lymphocytic myocarditis were analyzed. Results 1) In serial sections, DFS- and CR-stained granules showed a distribution pattern similar to HE-stained eosinophil granules (Figure 1). 2) Immunofluorescence pre-scanning confirmed that eosinophil granules identified by major basic protein (MBP) were present in intact eosinophils, identified by galectin-10. These granules were subsequently stained red by HE, DFS, and CR (Figure 2A). Additionally, MBP-positive granules surrounding galectin-10-negative cells with degenerated nuclei, indicative of cytolytic degranulation (ETosis), were also stained by HE, DFS, and CR (Figure 2B). 3) MBP-positive non-granular interstitial areas were not stained red by HE, DFS, or CR, suggesting that these staining methods do not detect free MBP molecules released from disrupted eosinophil granules (Figure 2C). 4) Eosinophil granules were more specifically identified in DFS-stained images than in CR-stained images by extracting the red color using ImageJ software, whereas they were not specifically detected in HE-stained images due to the reddish background staining of cardiomyocytes and fibres. 5) More eosinophils were counted in DFS-stained sections compared to HE-stained serial sections within a standardized observation period by cardiologists. Conclusion Our findings highlight the potential of amyloid stains, particularly DFS, as a superior method for detecting eosinophils and their cell-free granules in cardiac tissue. DFS staining may improve the identification of eosinophils, facilitating better diagnosis and management of eosinophil-related cardiac diseases.