Background Cell-free eosinophil granules, which are considered harmful to the heart, are stained red by haematoxylin-eosin (HE); however, they can be overlooked in cardiac tissue due to the reddish staining of cardiomyocytes and fibres. Direct fast scarlet (DFS) and Congo red (CR), known for staining amyloid deposits, stain eosinophil granules; however, no firm evidence currently exists. This study aimed to confirm that DFS and CR stain eosinophil granules red and evaluate their advantages over HE.

Methods Paraffin-embedded endomyocardial biopsy samples from six patients, each with eosinophil-infiltrating cardiac disorders, and six patients with lymphocytic myocarditis were stained.

Results The distributions of red granules stained with DFS and CR were similar to those of red granules stained with HE in serial sections. Major basic protein (MBP), a marker of eosinophil granules, was detected within cells positive for galectin-10, a marker of eosinophil cytoplasm. These MBP-positive granules, pre-scanned using immunofluorescence, were stained with a reddish hue by HE, DFS, and CR. MBP-positive granules surrounding galectin-10-negative cells with a degenerated nucleus, characteristic of cytolytic eosinophil degranulation (ETosis), were identified by HE, DFS, and CR staining. Non-granular MBP-positive interstitial areas were not identified by HE, DFS, or CR staining, suggesting that these staining methods did not detect deposited granule proteins released from disrupted eosinophil granules. Eosinophil granules were identified by extracting the red colour using Image-J software in DFS-stained images, more specifically than in CR-stained images and not in HE-stained images. Cardiologists counted more eosinophils in DFS-stained sections than in HE-stained serial sections within a certain time without miscounting.

Conclusion Our results demonstrated the potential of DFS as a superior method for identifying eosinophils and their cell-free granules in cardiac tissues. DFS may enhance the detection of eosinophils and improve the treatment of eosinophil-related heart diseases.

KEY MESSAGES

What is already known on this topic – Several case reports have demonstrated that red-stained granules observed in direct fast scarlet (DFS)- or Congo red (CR)-stained sections exhibit distribution patterns similar to those of eosinophil granules in haematoxylin-eosin (HE)-stained serial sections.

What this study adds – This study provides the first direct evidence that red-stained granules observed with DFS and CR staining are eosinophil granules, as shown by the presence of major basic protein (MBP). Further, this study demonstrated that identifying eosinophil granules is most effective with DFS staining, which, compared to HE staining, allowed a more rapid counting of eosinophils by non-pathologist cardiologists without miscounts.

How this study might affect research, practice or policy – This study may enhance the accuracy of eosinophil count and cytolytic degranulation detection, potentially via machine counting, and improve quantitative and qualitative definitions of eosinophil-infiltrating heart diseases.

The authors have declared no competing interest.

This study was supported by JSPS KAKENHI (22K08598 and 24K11593 to SU, 23K07524 to TI), Research Grants on Allergic Disease and Immunology from The Japan Agency for Medical Research and Development (JP22ek0410097 and MHLW 202213003A to SU).

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Preserved samples were used in this study; therefore, informed consent was obtained through an opt-out option provided on the university website. The study protocol was approved by the ethics committee s of Niigata University (2023-0194) and Akita University (2023-174) and was conducted in accordance with the Declaration of Helsinki.

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All data produced in the present study are available upon reasonable request to the authors