Purpose: The sentinel lymph node (SLN) is biopsied during breast conservation surgery (BCS) since it is the first site of metastasis for breast cancer. If malignant, axillary lymph node dissection (ALND) may be performed to remove additional nodes in and around the affected breast, limiting the spread of the malignancy. Often, ALND results in hardship for the patient in the form of further surgeries, having nodes removed that may not be malignant, and in debilitating side effects. Developing a method to analyze SLNs intraoperatively would eliminate additional surgeries and the patient's associated suffering. This research aims to develop the use of high-frequency (HF) ultrasound (20-80 MHz) as a real-time analysis method to determine SLN status during BCS. Background: HF ultrasonic data from BCS tissue specimens were gathered from 73 patients at the Huntsman Cancer Institute, Salt Lake City, UT, immediately following surgery. In addition to 349 margin specimens, data were collected from 78 SLNs, with initial results displaying high statistical measures. A limitation of the SLN analyses, however, was that pathology results were provided only on a per sample basis, whereas the ultrasonic method often tested multiple positions on a node (max = 4, avg = 1.26). Because of the mismatch between the ultrasonic measurements (per position basis) and pathology results (per specimen basis), an ambiguity existed in how to best analyze the data. The aim of this study was to examine the scope of this ambiguity by determining the differences in statistical measures obtained by analyzing the SLN data on a per position basis versus a per specimen basis given the current data available. Method: HF ultrasound parameters extracted from the data were peak density (the number of peaks in the ultrasonic spectra) and attenuation. Both parameters correlate to tissue malignancy, and were used in a multivariate analysis to provide the final results. The statistical measures for the ultrasonic test results were calculated as follows: (1) per position basis: the pathology of each position was determined by the pathology results for the entire specimen; (2) per specimen basis: only one measurement position on each node was selected, based on the highest peak density value, to correlate to the specimen pathology. Results: The analyses revealed that the HF ultrasonic data yielded an accuracy, sensitivity, and specificity of 79.6%, 76.9%, and 80.0%, respectively, for the per position basis, and 84.6%, 87.5%, and 84.3%, respectively, for the per specimen basis. The results indicate that HF ultrasound provides intraoperative detection capabilities competitive with many SLN evaluation methods currently in use, including imprint cytology, frozen-section analysis, and qRT-PCR. Detailed analyses of the SLN pathology slides from the 73-patient study are currently being conducted to improve the correlations between the ultrasound results and histopathology. Image analysis methods are being used to quantify the extent of the malignant tissue in each SLN. This will provide pathology results on a per position basis, and thus more accurate, one-to-one correlations. These correlations would significantly further the development of HF ultrasound for real-time SLN evaluation. Citation Format: Khelfa S, Factor RE, Sanjinez DA, Zambrana AF, Doyle TE. Evaluation of sentinel lymph nodes with high-frequency ultrasound: Correlations to histopathology [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 PD2-05.