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The aim of this study was to investigate the accuracy of locating parathyroid adenomas using dual-Isotope subtraction single-photon emission computed tomography–computed tomography (SPECT-CT) in comparison with clinical follow-up and pathology findings from surgery.A retrospective cohort study of dual-isotope subtraction SPECT-CT was carried out on 224 consecutive patients who were diagnosed with primary hyperparathyroidism. All the patients were injected with 20 MBq of iodine-123-iodide, followed 20 min later by 900 MBq of technetium-99m-sestamibi. Planar neck and chest views and SPECT-CT images were acquired 15 min after administration, followed by an additional planar image set at 2 h to view washout; all images were dual energy. In all, 115 out of 224 of the patients imaged underwent parathyroid surgery. The imaging results were compared with pathology findings when available and, in those who did not undergo surgery, and in some complex cases, with clinical measures after a 2-year clinical follow-up period.Out of the 224 patients, 135 patients had complete pathology and/or clinical follow-up data and were included in the analysis. The sensitivity of the subtraction SPECT-CT findings was measured to be 95%, with a specificity of 89% for the detection and localization of parathyroid adenomas. The positive predictive value was found to be 97% and the negative predictive value was found to be 83%. The accuracy of the technique was 94% in detecting parathyroid adenoma and 92% in accurate localization.Dual-isotope subtraction SPECT-CT imaging has a very high sensitivity and specificity in detecting and locating a parathyroid adenoma, showing that it is a very reliable preoperative imaging technique in primary hyperparathyroidism.