Bioelectrical impedance phase angle as a diagnostic indicator in thyroid cancer

Background: Bioelectrical impedance spectroscopy (BIS) is a non-invasive and easy-to-use technique to distinguish tissue properties. Phase angle, determined by BIS, detects changes in tissue electrical properties. We aimed to study the feasibility and validity of phase angle in diagnosis of thyroid cancer for the first time. Methods: Department China Sichuan from March November 2013 were collected. According to the location from thyroid cancer, thyroid specimens were divided into four groups: A, B, C and D. All of the groups were analyzed with phase angles respectively. The results were compared with final pathologic diagnosis. Results: Results showed that the phase angle is the characteristic parameter. The rank-sum test showed, the significant difference between the four groups and between two groups (P<0.05), with statistical significance. Our study showed 86% sensitivity and 72% specificity of mean phase angle difference (MPAD) . The corresponding positive and negative predictive values were 78% and 82%, overall accuracy was 80%, the area under the ROC curve is 0.838. Conclusions: The study demonstrated that phase angle can be used to diagnose thyroid cancer. With further research, the phase angle may be a potential diagnostic indicator for the thyroid cancer.


Introduction
Thyroid cancer incidence is increasing the second fastest among solid tumors 1 , and it is already the first most common malignancy in adolescents and young adults (ages 15 to 29 years) in the United States 2 . Therefore, thyroid cancer seriously affects human health. Currently, Ultrasound and Ultrasound-guided fine-needle aspiration (US-FNA) are widely accepted as the primary diagnostic tools for the evaluation of thyroid nodules and diagnosis of thyroid cancers. However, some patients worry that the puncture might cause needle tract implantation metastases 3 . Furthermore, in some primary hospital US-FNA is unavailable and lack of pathologists, this can be a big challenge. Even if the hospital is equipped with US-FNA and pathological diagnosis, 5-20% of US-FNA results are non-diagnostic 4 . Additionally, the false-negative rate of thyroid nodules with a benign FNA result reaches 13.6-56.6% when the thyroid nodules have suspicious features on ultrasound 5 . Therefore, for these nodules with non-diagnostic cytology results, patients are required either close observation or surgical excision for definite diagnosis. Some patients would choose surgery for fear of cancer, but they have to face a series of surgical risks and complications, including recurrent laryngeal nerve injury, hypoparathyroidism, and lifelong thyroid hormone replacement, etc. 6 , which affect the quality of life seriously. Although immunohistochemistry and genetic testing are now available to assist diagnosis, they are not yet universally available in all hospitals. A less common tool to assess thyroid nodules properties and diagnose thyroid cancer, called Electrical Impedance Spectroscopy (EIS), which can overcome some of these challenges and diagnose thyroid cancer in electrophysiological and pathological perspectives. EIS is an easy-to-use, non-invasive, and reproducible technique to diagnose thyroid cancer. Therefore, the paper introduces the diagnostic technique-EIS. EIS measures the ratio of voltage to current in an alternating current signal, or the phase angle of impedance in function of frequency. The body can be considered as a composite volume conductor comprising a number of spatially distributed tissues with differing electrical properties 7 . EIS measurements are based upon tissue-specific electric field distribution on the surface of the body at the region of interest. In biological tissue, the resistance and the related electrical impedance are associated with the electrical properties of the tissues, which depend on the structure of tissues 8 . Biological tissues are complex electrical impedance system, which is a function of frequency of electrical current applied，because tissues contain components that have both resistive and charge storage (capacitive) properties 9 . Both theory 10 and practice 11 have demonstrated that different tissue structures are associated with impedance in different frequency bands 12 .
There are three major relaxation regions, called α, β, and γ, at frequencies of approximately 100 Hz, a few kHz to 1MHz and 1 GHz. These regions are related to extracellular surface polarizations of large cells (α region), to increasing capacitive charging and discharging of cell membranes (β region) and to the relaxation of the water molecules (γ region), respectively. For many applications the α and β dispersion regions are particularly interesting, since most changes between normal and pathological tissue seem to appear in this frequency range 9 . Therefore, it is more practical to design a measuring system dedicated to the low frequencies. At low frequencies, the current pass extracellular space. Because the current has to pass around the cells with path of least resistance, the resistance to flow depends on the cell spacing and how they are arranged, whereas with increasing frequencies current can penetrate the membrane and hence passes through both intracellular and extracellular spaces 9 . Cellular changes alter the flow of electrical current through living tissue, and a number of literatures indicated differences in human electrical impedance between benign and malignant tumors [breast 13,14 , thyroid [15][16][17] , prostate 18 , bladder 19 , colon 20 ]. Previous studies have confirmed the feasibility of EIS in diagnosis of thyroid cancer. However, due to the low specificity [15][16][17] , in order to screen better diagnostic parameters and improve specificity, we found that phase angle is a good diagnostic indicator. To the best of our knowledge, no investigation was reported that phase angle is a diagnostic indicator in thyroid cancer.
Phase angle reflects the relative contributions of fluid (resistance) and cellular membranes (reactance) of tissues. By definition, phase angle is positively associated with reactance and negatively associated with resistance 21 . Lower phase angles suggest cell death or decreased cell integrity, while higher phase angles suggest large quantities of intact cell membranes 22 . The primary objective of the preliminary study is to evaluate the feasibility and significance of phase angle derived EIS in diagnosis of human thyroid cancer.

Materials and Methods
In this study, 226 invitro human thyroid tumors from 45 man and 165 women.    The EIS of different pathological results were measured form 200 patients. The ratio of the measured potential to the amplitude of the imposed current determines a transfer impedance. In order to reduce the polarization effect of low frequency, measurement frequencies ranged from 1MHz to 100Hz with 10 times intervals (1MHz,100kHz,10kHz,1kHz,100Hz) at 40 different frequencies. A current of 10 µA peakto-peak was passed between gold electrodes, and the resulting voltage was measured between the two remaining silver electrodes. Before each measurement, the probe was regularly calibrated using the known conductivity of saline solutions, and ultrasonically cleaned, then removed moisture and surface oxidation film on electrode front-end with non-woven. The fresh specimens were made by size of 2 cm*2 cm* 0.5 cm, of which surface must be uniformly flat to facilitate measurement, hemorrhage, degenerative cyst, and necrotic tissues were avoided. Then, according to the distance from thyroid cancer, the resection specimens were divided into four groups: A,B, C and D, represented 10mm distance from the edge of malignance (normal thyroid tissue proved by pathology), benign tumor(proved by pathology), malignant tumor(proved by pathology) and malignant tumor margin(1mm distance from the edge of malignance) respectively, and four groups were measured, then EIS data were acquired from three separate sites in the same group in order to check the reproducibility. The measured data were recorded in the laptop for further analysis. The entire collection and recording of impedance spectrum of a single fresh specimen took less than 30 minutes. Lastly, all the groups were fixed in formalin separately and sent for pathological examination. Our analysis results were compared with pathologic diagnosis.
All data were analyzed by SPSS 22.0 software, the Kruskal-Wallis test was applied to assess whether difference among four groups, with p <0.05 statistically significant. The receiver operating characteristic curve was used to screen optimal threshold, and obtain the optimal diagnostic value in diagnosis of thyroid cancer, and then yielded corresponding the sensitivity, specificity, positive predictive value, negative predictive value and accuracy.

The pathologic result of groups
A total of 226 thyroid nodules were sampled from 210 sequential patients who underwent surgical thyroidectomy, 1662 valid impedance data were obtained and analyzed.
All the specimens were compared with the pathological results. The four groups were A:  Table   1. In each case, the repeated measurement data of the same group were highly consistent, the dispersion coefficient was less than 10%.

Statistical analysis of four groups
By studying the impedance data, we found that the phase angle between 1K and  of MPAD for thyroid cancer diagnosis was 7.59 degrees, which was optimal diagnostic critical point, this means that MPAD of tumors with greater than or equal to the value was benign, the opposite was malignant tumors. The corresponding sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 86%, 72%, 78%, 82% and 80%, thereby MPAD were analyzed by diagnostic test fourfold table (Table 2).
In addition, the false positive and false negative results (Table 3, 4).In the false negative result, thyroid papillary carcinoma accounts for more than 88%, and in the false positive result, nodular goiter with adenoma nodules constitute more than 52%.    23 , but when disease occurs, functional changes of tissues or organs often are prior to structural and morphological changes, after a certain functional compensatory or latent period, it develops into organic lesions, structural changes of tissues and organs, therefore it is difficult for potential cancers or precancerous lesions to realize early diagnosis and real-time monitoring for ultrasound and FNA. Instead, EIS has these advantages. EIS is based upon tissue-specific conductivity and permittivity, it reflects electrical properties of tissues. Once tissues alter physiologically or pathologically, their electrical properties may change accordingly 24 . The change may reflect the internal information of tissues in a functional perspective.

Figure 6 ROC curve
In this study, electrical properties of four groups were measured by constant current EIS system equipped with four electrode probes. The acquired data were mainly affected by the following two factors, the property of tissue and environmental interference (including ambient temperature, air humidity and electromagnetic radiation). Since each sample were tested under the same condition, so the EIS data primarily reflected the electrical property of thyroid specimens.
Among EIS parameters, such as impedance modulus, the real and imaginary part of the impedance and phase angle), the phase angle-frequency curve demonstrated a potential to distinguish thyroid property, therefore, all the results were analyzed and discussed in phase angle perspective. Between 1kHz and 31.64kHz, phase angles of four groups changed drastically, so we defined MPAD as the test variable to distinguish four groups. MPAD is the absolute value of mean phase angle difference between 31.64kHz and 1kHz ( Figure 5). From the experimental results, MPAD changed with different pathological tissues, which reflected mainly the property of thyroid tissues. extra-cellular space, orientation of cells and the lack of tight junction 4-6 , so that cancerous cells can be related to a resistor with a lower resistance or a higher conductance, respectively, than its surrounding 4 , it attracts current and thus enhances the current density through the cancerous cell, and the low frequency current easily penetrated cancerous cells, so that when frequencies increased, the current across the cell changed inconspicuously, the phase angle and its amplitude decreased, so these principles can be used to analyze