The surgical pathology files from two participating institutions (Tisch Hopsital, New York University Langone Medical Center and the Hospital of University of Pennsylvania) were searched for cases on thyroid resection specimens that contained in the gross description and/or diagnosis with key word “decalcification” from 2008 to 2012. Twenty-six cases were recruited, including seventeen cases of papillary thyroid carcinoma, eight cases of nodular hyperplasia, and one case of follicular carcinoma. Hematoxylin and eosin slides from all cases were reviewed to confirm the diagnosis. Clinical information was obtained by reviewing the charts.

Immunohistochemistry was performed on 4 micron formalin fixed, paraffin embedded papillary thyroid carcinomas using mouse antihuman mesothelial cell, tissue culture supernatant clone HBME-1 (Dako Carpentaria, CA USA). In brief, sections were deparaffinized in xylene (3 changes), rehydrated through graded alcohols (3 changes 100% ethanol, 3 changes 95% ethanol) and rinsed in distilled water. Heat induced epitope retrieval was performed in a 1200-Watt microwave oven at 100% power in 10 mM sodium citrate buffer, pH 6.0 for 10 minutes. Sections were allowed to cool for 30 minutes and then rinsed in distilled water. Antibody incubation and detection were carried out at 40°C on a NexES instrument (Ventana Medical Systems Tucson, Arizona USA) using Ventana’s reagent buffer and detection kits unless otherwise noted. Endogenous peroxidase activity was blocked with hydrogen peroxide. Anti-HBME-1 was diluted 1:100 in Dulbecco’s Phosphate Buffered Saline, (Life Technologies Grand Island, New York USA) and incubated overnight at room temperature. Primary antibody was detected with iView biotinylated goat antimouse followed by application of streptavidin-horseradish-peroxidase conjugate. The complex was visualized with 3,3-diaminobenzidene and enhanced with copper sulfate. Slides were washed in distilled water, counterstained with hematoxylin, dehydrated and mounted with permanent media. Appropriate positive and negative controls were included with the study sections.

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