Inter- and Intra-observer Reproducibility of Thyroid Fine Needle Aspiration Cytology: An investigation of Bethesda 2023 Using Immunohistochemical BRAFV600E Antibody

Abstract

Background: The Bethesda System for Reporting Thyroid Cytology (TBSRTC) recommended for the interpretation of needle aspiration cytology of the thyroid, is the most widely used worldwide. Studies have shown that the disagreement between observers, especially in the Bethesda III and IV diagnostic categories, is not insignificant at 10%-40%. In the TBSRTC 2023 version, some definitions were removed and simplified, and molecular pathology was proposed as a complement to cytopathology. The current availability of molecular tests is limited because they can be performed in a few centers and are expensive. Therefore, our study investigated intra- and inter-observer agreement according to TBSRTC 2023 using only immunohistochemically BRAFV600E antibodies.

Materials and methods: The study included 173 cases with aspiration cytology evaluated between 2019 and 2022. The immunohistochemical procedure applied BRAFV600E (RM8) monoclonal antibody to cell block sections. All slides were assessed and categorized by three different observers. Data were interpreted using Cohen's kappa and Fleiss's kappa test in the Statistical Package for Social Sciences Windows 2021 program.

Results: For the applied RM8 antibody, sensitivity was 64.71% and specificity was 87.27%. In terms of diagnostic categories, inter-observer agreement was good for Bethesda II (K = 0.606) and moderate for Bethesda III (K = 0.429), Bethesda IV (K = 0.523), Bethesda V (K = 0,464), and Bethesda VI (K = 0.544), respectively.

Conclusion: In conclusion, the study reveals that the 2023 version of TBSRTC provides improvement, especially in the categories of uncertain diagnosis, but is still insufficient to improve cytological diagnostic accuracy, at which point molecular analyses become even more important.

Keywords: BRAF V600E antibody; inter-observer agreement; reproducibility; thyroid fine needle aspiration.

Figure 1

Thyroid FNA, Bethesda II (20 × 10). Cytospin smear, PAP staining, and thyroid follicle epithelial cell groups with fine vesicular nuclei are observed. There is no positive reaction in the BRAF V600E (MR8) IHC application

Figure 2

Thyroid FNA, Bethesda II, cystic degeneration (20 × 10). Cellblock, hematoxylin and eosin (H&E) staining; in the ground, colloid, some of them containing pigment, macrophage-rich inflammatory cell reaction, and thyroid follicle epithelial cell groups with fine vesicular nuclei are observed. Artefactual staining is present in macrophages, and BRAF V600E (MR8) IHC staining is absent in thyroid follicle cells

Figure 3

Thyroid FNA, Bethesda II, autoimmune thyroiditis (20 × 10). Cytospin smear (PAP staining), cell block (H&E staining): In the background, lymphocyte-rich inflammatory cell reaction and thyroid follicle epithelial cell groups with oncocytic cytoplasm are observed. Thyroid follicle cells have a weak to moderate reaction on BRAF V600E (MR8) IHC staining

Figure 4

Thyroid FNA, Bethesda V (20 × 10). Cytospin smear (PAP staining), cell block (H&E staining): In follicular epithelial cells with follicular structure, the chromatin network’s opening and the nuclei’s enlargement are observed. BRAF V600E (MR8) IHC staining is negative in follicle cells

Figure 5

Thyroid FNA, Bethesda V (20 × 20). Cytospin smear (PAP staining), cell block (H&E staining): In follicular epithelial cells with papillary structure, the opening of chromatin network, enlargement of nuclei, growth, and intra-nuclear incision are observed. BRAF V600E MR8 IHC staining is positive in follicle cells