FNA samples were stored in preservative solution (CytoRich™ Red Preservative, Japan Becton Dickinson Corp., Tokyo, Japan) for BD SurePath™-LBC preparation (Japan Becton Dickinson Corp.). Preserved cells were manually prepared for LBC slides according to the manufacturer’s instruction. After washing with 0.01 M PBS (pH 7.4), LBC slides were preincubated with 10% (v/v) normal goat serum. Samples were then incubated with anti-53BP1 rabbit polyclonal antibody (Bethyl Labs, Montgomery, TX, USA) at 1:1000 dilution for 1 h at 24°C. The slides were subsequently incubated with Alexa Fluor 488-conjugated goat anti-rabbit antibody (Molecular Probes Inc.). Specimens were counter-stained with DAPI dihydrochloride (Vysis Inc., Downers Grove, IL, USA), analyzed, and photographed using a High Standard All-in-One Fluorescence Microscope (Biorevo BZ-X710; KEYENCE Japan, Osaka, Japan) with the Z-stack mode, which accumulated images from 20 to 30 slices. IF analyses focused on parts of sheet-like arranged cells in both small and large clusters, which are considered as being originated from follicular epithelium. Signals were analyzed in more than ten viewing areas per case at a 1000-fold magnification. All signals for 53BP1 expression were measured using the image analysis software (BZ-X analyzer) provided with the Biorevo BZ-X710 microscope. Expression of 53BP1 was classified into three types as shown in Fig. 3: (i) low DDR type: none, one or two discrete NF, (ii) high DDR type: three or more discrete NF, or discrete NF that are larger than 1.0 μm in the minor axis, (iii) abnormal type: intense and heterogenous expression. The percentage of follicular cells displaying each pattern of 53BP1 expression was calculated in each case.

Pattern of p53-binding protein 1 (53BP1) expression in liquid-based cytology samples determined via immunofluorescence. Low DNA damage response (DDR) type, faint nuclear staining or one or two discrete nuclear foci (NF); high DDR type, three or more discrete NF or NF larger than 1.0 μm; abnormal type, intense, and heterogeneous nuclear staining. The scale bars indicate 2 μm.

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