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The aim of the work described here was to evaluate the diagnostic performance of perfluorobutane (PFB)-enhanced ultrasound in differentiating hepatocellular carcinoma (HCC) from non-HCC malignancies and other benign lesions using different acquisition methods. This prospective study included 69 patients with solid liver lesions larger than 1 cm who were scheduled for biopsy or radiofrequency ablation between September 2020 and March 2021. Lesion diagnosis was designated by three blinded radiologists after reviewing three different sets of acquired images selected according to the following presumed acquisition methods: (i) method A, acquisition up to 5 min after contrast injection; (ii) method B, acquisition up to 1 min after contrast injection with additional Kupffer phase; and (iii) method C, acquisition up to 5 min after contrast injection with additional Kupffer phase. After excluding 7 technical failures, 62 patients with liver lesions (mean size: 24.2 ± 14.8 mm), which consisted of 7 benign lesions, 37 non-HCC malignancies and 18 HCCs. For the HCC diagnosis, method C had the highest sensitivity (75.9%), followed by method B (72.2%) and method A (68.5%), but failed to exhibit statistical significance (p = 0.12). There was no significant difference with respect to the pooled specificity between the three methods (p = 0.28). Diagnostic accuracy was the highest with method C (87.1%) but failed to exhibit statistical significance (p = 0.24). Image acquisition up to 5 min after contrast injection with additional Kupffer phase could potentially result in high accuracy and sensitivity without loss of specificity in diagnosing HCC with PFB-enhanced ultrasound. [ABSTRACT FROM AUTHOR]