Direct Fast Scarlet and Congo Red Staining in Identification of Eosinophils and Their Cell-Free Granules in Cardiac Tissue

Cell-free eosinophil granules, harmful to the heart, are stained red by haematoxylin-eosin (HE); however, they can be overlooked in cardiac tissues. Direct fast scarlet (DFS) and Congo red (CR), known for staining amyloids, may offer clearer detection of eosinophil granules; however, no firm evidence exists. This study aimed to confirm that DFS and CR stain eosinophil granules and evaluate their advantages over HE.Paraffin-embedded endomyocardial biopsy samples from 6 patients with eosinophil-infiltrating cardiac disorders and 6 with lymphocytic myocarditis were stained.The distributions of red granules stained with DFS and CR resembled those stained with HE in serial sections. Eosinophil granules, identified by major basic protein (MBP), were detected in intact eosinophils, identified by galectin-10, using immunofluorescence pre-scanning and were subsequently stained red by HE, DFS, and CR. MBP-positive granules surrounding galectin-10-negative cells with degenerated nuclei, indicative of cytolytic degranulation (ETosis), were also stained by HE, DFS, and CR. Non-granular MBP-positive interstitial areas were not visualised by HE, DFS, or CR, suggesting that they did not detect the deposition of granule proteins released from disrupted granules. Eosinophil granules were identified by extracting the red colour using ImageJ software in DFS-stained images, more specifically than in CR-stained or HE-stained images. Cardiologists counted more eosinophils in DFS-stained sections than in HE-stained serial sections within a certain time without miscounting.DFS staining effectively identifies eosinophils and their cell-free granules in cardiac tissues, outperforming HE and CR. DFS may advance the diagnosis and management of eosinophil-related heart diseases.