Trichrome, Congo red, and thioflavin S stains are comparable for the diagnosis of amyloid deposits in endomyocardial biopsies

Introduction: Despite imaging advancements, proper management for cardiac amyloidosis hinges on accurate diagnosis and subtyping of amyloid in endomyocardial biopsies (EMBx). Nonetheless, the approach toward identifying amyloid in EMBx varies among pathologists. Various staining techniques can be used, each with its own advantages and limitations. There is a lack of evidence-based data to compare different methodologies to detect amyloid in EMBx. In this study, we compared the efficacy of H&E stains only, Congo red (bright field, polarized, and fluorescence), trichrome, and Thioflavin S for the diagnosis of amyloid in EMBx. We also validated a digital imaging workflow for Congo red (brightfield, polarized, and fluorescence microscopy) and Thioflavin S in EMBx. Methods: We retrospectively identified 40 EMBx (21 positive and 19 negative) that were evaluated for amyloid deposition. Each biopsy was stained with H&E, Congo red, trichrome, and Thioflavin S. The slides were separated into three independently randomized and blinded cohorts: HE + Congo red, HE + trichrome, and HE + thioflavin S. Cases were evaluated for amyloid deposition by three cardiothoracic pathologists (CTP) and two pathologists-in-training (PIT) based on (1) H&E only (2) Congo red brightfield (3) Congo red polarized, (4) Congo red fluorescence, (5) trichrome, and (6) Thioflavin S. After a 3-month washout period, the Congo red and Thioflavin S slides were scanned using an Olympus VS200 slide scanner to capture brightfield, polarized, and fluorescent images, which were then reviewed by all reviewers. This validation was exempt from IRB review. Results: Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated for each stain when reviewed by CTP alone as well as CTP+PIT. When glass slides were reviewed by CTP + PIT, sensitivity ranged from 86.7%-99.1%, specificity 93.6%-99.0%, PPV 94.4%-99.1%, and NPV 86.8%-99.0%. Statistical analysis revealed no significant differences in sensitivity, specificity, PPV, or NPV between Congo red polarized and any other studied stain. When digital images were reviewed by CTP + PIT, sensitivity ranged from 88.6%-97.1%, specificity 91.6%-100%, PPV 92.2%-100%, and NPV 88.6%-96.9%. Statistical analysis revealed no significant differences between stains interpreted by digital imaging and their corresponding glass slides. Conclusion: Congo red staining with polarized microscopy is often considered the gold standard for detecting amyloid deposits. We demonstrated that trichrome and Thioflavin S stains show comparable results and can be considered as appropriate methods for diagnosis. In addition, reviewing Congo red brightfield, polarized, and fluorescence as well as Thioflavin S with digital imaging shows a comparable diagnostic yield to glass slides for identification of cardiac amyloid deposition. These findings suggest that a digital workflow can be implemented for diagnosing cardiac amyloid deposition in routine practice.