A HF ultrasonic test system, Figure , was developed to collect simultaneous pulse-echo and through-transmission measurements from margins and other tissue specimens following resection from BCS. The data were analyzed with a variety of methods to search for correlations to tissue pathology.
Photograph of aluminum test fixture with instrumentation (a) and surgical margin in plastic re-sealable bag (b).
The ultrasonic testing of tissue specimens obtained during the course of routine breast conservation surgery was approved by the University of Utah Institutional Review Board on October 14, 2009, as a minimal risk study (IRB #00037350). Informed consent was obtained from patients for the use of their tissues for research purposes. Surgeries were performed on 17 patients by the collaborating surgeon and coauthor (LAN) at the Huntsman Cancer Hospital, Salt Lake City, Utah. The surgeries provided 34 resected specimens consisting of margins and other tissues such as lymph nodes and adenomas. The samples ranged from 1-5 cm in length and width, 0.1-1.5 cm in thickness, comprised a spectrum of both benign and malignant tissue pathologies, and did not require any additional procedures or resection that affected the patient or surgical outcome. Table lists the range of pathologies provided by the specimens. For the purposes of this study the pathologies were categorized into 15 classifications. Immediately following resection, the surgeon placed each specimen inside a re-sealable plastic storage bag for ultrasonic testing (Figure ), and labeled the bag with a de-identified specimen number and, if applicable, the orientation of the margin.
Pathology, number of specimens, and number of positions tested with high-frequency ultrasound
During the ultrasonic testing, the outside of the bag was coupled to the ultrasonic transducers with ultrasound scanning gel (Sonotech® Clear Image). The surface moisture of the tissue provided sufficient coupling of the specimen to the inside of the bag for ultrasonic transmission. The bag therefore prevented contamination of the specimen with coupling fluid and additionally provided improved transmission of ultrasound between the transducers and specimen. One to four sites were tested on each specimen depending on the specimen size, resulting in a total of 57 sites tested. Triplicate waveforms were acquired from each test site on a specimen. After ultrasonic testing, routine pathology analyses were performed on the specimens. Ultrasonic results were correlated to pathology reports for each specimen.
Ultrasonic materials and procedure
Ultrasonic pulse-echo and through-transmission data were acquired from breast tissue specimens with the use of two immersion transducers (Olympus NDT, V358-SU, 50 MHz, 0.635-cm diameter active element), a HF square-wave pulser/receiver (UTEX, UT340), and a digital storage oscilloscope (Hewlett-Packard, HP-54522A, 500 MHz, 1Gs/s). Ultrasonic waveforms were averaged in the signal acquisition and downloaded onto a notebook PC using LabVIEW. The data acquisition parameters were pulse voltage = 100 V, pulse width = 10 ns, pulse repetition rate = 5 kHz, and receiver gain = 0-48 dB. An aluminum test fixture, Figure , was used to support the tissue specimen, to position the transducers both above and below the sample for simultaneous pulse-echo and through-transmission measurements, and to lock the transducers into position. The thickness of the specimen was recorded for each ultrasonic measurement. A description was also recorded for each specimen, and photographs were taken of 19 specimens (e.g., Figure ).
Aluminum test fixture (a) and operation modes (b-c) used to position specimens and collect ultrasonic measurements. In the pulse-echo mode (c), the receive transducer functions as a specular reflection surface for the ultrasonic waves.
The ultrasonic transducers each had a center frequency of 50 MHz and were broadband, providing a short pulse length and enhanced signal-to-noise in highly scattering or attenuating materials. The broadband characteristics of the transducers were also desired to obtain the ultrasonic response of the tissue across a wide frequency band.
Ultrasonic data analysis
The HF ultrasonic signals acquired in this study were substantially different from the typical ultrasonic signals used for medical imaging, Doppler flow imaging, or tissue characterization. Whereas typical medical ultrasound signals are comprised of scattered waves from dispersed scattering centers, typically cells or nuclei, and other tissue inhomogeneities such as blood vessel walls, the signals collected in this study were of the transmitted pulse after propagating through the tissue specimen (through-transmission mode, Figure ) or of the specular reflection of the transmitted pulse from the surface of the second transducer (pulse-echo mode, Figure ). Therefore, in contrast to most medical ultrasound signals, the signals in this study had pulse-like characteristics with amplitudes significantly greater than background noise.
For through-transmission measurements, Figure , the ultrasonic data consisted of time-domain waveforms of ultrasonic pulses, Figure , that were transmitted from the top transducer, passed through the specimen only once, and received by the bottom transducer. For the pulse-echo measurements, Figure , the ultrasonic data consisted of time-domain waveforms of ultrasonic pulses, Figure , that were transmitted from the top transducer, passed through the specimen, reflected from the surface of the bottom transducer, passed through the specimen a second time, and received by the top transducer. The ultrasonic signals for both modes of operation therefore provided a convolution of the transducer and tissue responses.
Ultrasonic waveforms from through-transmission (a) and pulse-echo (b) measurements of surgical tissue specimens. Amplitudes have been normalized and offset for comparison.
Tumor progression and other atypical conditions affect the acoustic properties of tissues by altering the cell properties, the extracellular matrix properties, and the tissue microstructure. Measurement of sound speed and attenuation can therefore be used to reveal benign, pre-cancerous, or malignant tissues in breasts [25
]. For calculation of ultrasonic sound speeds and attenuation coefficients, the arrival times and amplitudes of the time-domain waveforms were determined using a Hilbert transform. Arrival times were calibrated using a Plexiglas block as a substitute for the tissue samples. Attenuation coefficients were based on a relative scale by setting the lowest calculated attenuation value for the specimens (a fibroadenoma) to 0.003 Nepers/cm. Attenuation calculations accounted for receiver gain and specimen thickness.
The ultrasonic data were additionally analyzed in the frequency domain since previous numerical studies had indicated that the structure of HF ultrasonic spectra should be sensitive to neoplastic changes in breast tissues. Frequency spectra of the signals, Figures and , were obtained by subtracting background waveforms from the tissue waveforms, windowing the main signals in the waveforms, padding the waveforms to 4000 points to increase the spectral resolution, and performing a fast Fourier transform (FFT). The power spectra were then derived by taking the absolute value of the complex spectra. Analysis of the spectra included correlating specific spectral features, centroid frequencies of peak clusters, and the density of peaks and valleys. The density of peaks and valleys of a spectrum, from hereon referred to as the density of peaks or peak density, was calculated by counting the number of zero crossings of the derivative of the spectrum in the 20-80 MHz band.
Figure 4 Ultrasonic spectra from through-transmission (a) and pulse-echo (b) measurements of surgical tissue specimens. Amplitudes have been normalized and offset for comparison. Note the increase in spectral structure (peaks and valleys) from FA to normal to (more ...)
The cepstrum is the inverse Fourier transform of the log power spectrum, and has been used to provide the mean scatterer spacing from ultrasonic data [20
]. Applications have included measuring tibial cortical thickness and the location of brachytherapy seeds in tissue [40
]. The cepstrum has also been used to obtain the mean scatterer spacing for breast tissue classified as benign, simple carcinoma, infiltrating papillary carcinoma, and fibroadenoma [20
]. However, the low spectral range, 0-10 MHz, limited the measurement of scatterer spacings to greater than 0.15 mm, and the measured mean scatterer spacing varied from 0.82 ± 0.10 mm for normal breast tissue to 1.25 ± 21 mm for simple carcinoma.
The cepstra of waveforms were calculated in this study by computing the spectrum from the unpadded waveform, computing the inverse FFT of the log power spectrum, and then taking the absolute value of the resulting complex function. A modified cepstrum was also used in this study to analyze data. Computation of the modified cepstrum involved using the power spectrum derived from the padded waveform, and were obtained by windowing the power spectrum from 0 to 62.5 MHz, re-padding the spectrum to 4000 points, performing a second forward FFT on the padded spectrum, taking the absolute value of the complex function, and normalizing the curves. The results produced modified cepstra that showed a maximum at 0 μs and that sloped downward with multiple peaks at various positions. The modified cepstra were analyzed by calculating the slope of the log of the modified cepstrum, which was approximately linear in the 0-0.3 μs range. The value of the modified cepstrum at 0.3 μs was also calculated. The intercept at 0.3 μs was chosen as a measurement parameter due to the change in slope of the modified cepstrum at this point in the curve.
The data were evaluated with bar charts using the median for the bar height and the median absolute deviation (MAD) of the analyzed parameters for the error bars. After analyzing the data by the 15 pathology types as shown in Table , the data were reclassified into 5 pathology types: (1) normal breast tissue, (2) FN-FA-TA (fat necrosis, fibroadenoma, and tubular adenoma), (3) benign pathologies (BC, ADH, FC, and PA), (4) ductal carcinomas (DCIS, DCIS-SC, DCIS + IDC, and IDC), and (5) lobular carcinomas (LCIS and ILC). These categories were used to assess the efficacy of the preliminary measurements in this study for differentiating carcinoma in resected margins. Specificities and sensitivities for pathology types (2)-(5) were calculated with respect to normal tissue (1). Specificities and sensitivities for the five pathology types were additionally determined using a two-parameter multivariate analysis. Finally, t-tests and one-way ANOVA tests were performed to evaluate the significance level of the results.