The use of high-frequency (HF) ultrasound (20-80 MHz) to determine tissue pathology is an inexpensive and real-time tool for differentiation of surgical margin specimens from breast conservation surgery (BCS). The development of this method as an intraoperative tool for BCS would greatly reduce the rate of re-excisions due to positive breast tissue margins. HF ultrasound was previously used in a 17-patient study to determine differences in breast tissue pathology of 34 surgical margins. Results from this pilot study demonstrated high accuracy, specificity, and sensitivity in the HF ultrasound data, and showed the immense potential of this method as a breast cancer detection tool. A large-scale validation study of this method was subsequently conducted using 349 margin specimens taken from 73 patients during BCS. Specimens ranged from 1-5 cm in length and width, 0.2-1.6 cm in thickness, and did not require any additional procedures or resection that affected the patient or surgical outcome. Each specimen was tested with HF ultrasound immediately following resection and then forwarded to pathology. Through-transmission data were collected from the specimens using two broadband, single-element transducers with a 50-MHz peak frequency (Olympus NDT, V358-SU), a HF square-wave pulser/receiver (UTEX, UT340), and a 1-GHz digital oscilloscope (Agilent, DSOX3104A). Peak density (the number of peaks and valleys in the 20-80 MHz frequency spectra range) and attenuation data were calculated from the ultrasonic spectra and waveforms, respectively. Peak density and attenuation correlate directly to tissue malignancy, and thresholds for these two parameters for differentiating benign vs. malignant tissue were determined using Fisher's Exact Test applied to the previously collected pilot study data. The peak density and attenuation results were then combined using a multivariate analysis. Since the ultrasonic measurements were collected on a per position basis (1-5 positions per margin), but the histopathology results were reported on a per specimen basis, the statistical measures for the ultrasonic results were calculated using two methods. First, the statistical measures were calculated based on a per position basis, where the pathology of each position was determined by the pathology results for the entire specimen. Second, the statistical measures were calculated based on a per specimen basis, where only one measurement position on each margin was selected (based on the highest peak density value) to correlate to the specimen pathology. The results of the first approach (per position basis) showed a sensitivity of 82.6%, a specificity of 72.3%, and an accuracy of 72.7%. The results of the second approach (per specimen basis) showed a sensitivity of 82.6%, a specificity of 61.7%, and an accuracy of 63.0%. The results of this study show potential for a rapid and inexpensive method of determining pathology of breast tissue surgical margins during BCS. This work was supported by funds from the Elsa U. Pardee Foundation, the Eppley Foundation for Research, the Western Alliance for Expanding Student Opportunities, theGovernor's Office of Economic Development of the State of Utah, the University of Utah, and Utah Valley University. Citation Format: Carter C, Neumayer LA, Factor RE, Doyle TE. Using high-frequency ultrasound (20-80 MHz) to differentiate malignant vs benign breast tissue in surgical margins [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-03-10.