Ranade AA, Joshi MM, Deore RS and Mehrotra SC. In vivo dielectric measuring instrument using picosecond pulse for detection of oral cancer. Med Instrum. 2014; 2:1. http://dx.doi.org/10.7243/2052-6962-2-1
A.A.Ranade1, M.M. Joshi2, R.S.Deore3 and S.C. Mehrotra3*
*Correspondence: S. C. Mehrotra firstname.lastname@example.org
1. Department of Maxillofacial Pathology and Microbiology; Institute of Dental Sciences, Sehora, Jammu, India.
2. Dept of surgery, Byramjee Jeejeebhoy (BJ) Medical college, Near. Pune railway station, bjmedical road, Pune,India.
3. Department of Computer Science and IT, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad (Maharashtra), India.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: Interaction of microwaves with water molecules has been found to be a very sensitive probe of molecular surroundings. It has been shown that the dielectric properties of saliva extracted from the mouth contain information about the health of tissues present in the mouth. Based on these principles, the present paper reports instruments used in vivo to derive information related to dielectric properties of saliva in a subject's mouth.
Methods: The instrument consisted of a pulse generator, transmission line and probe. The probe was made from a microstrip line that could be placed easily in the subject's mouth. The reflected pulses were acquired by placing the probe in the mouth and also again outside the mouth. The difference in reflected pulses in and outside the mouth provided information about impedance of saliva. Pulse features were extracted to determine the status of oral cancer.
Results: The experiments have been carried out on 125 subjects with healthy condition and with different stages of oral cancer. The results have been classified into five groups. The features extracted from the data have been classified using Linear Discriminant Analysis (LDA) using the Matlab toolbox. The clustering patterns for different cases were found to be distinct.
Conclusion: The technique may be used to suggest the presence of oral cancer. It was also concluded that more cases under different backgrounds need to be studied before it will be possible to adopt the equipment in actual practice.
Keywords: Microwaves, time domain reflectometry, saliva, oral squamous cell carcinoma, tobacco, linear discriminant analysis
The microwave group at Dr. Babasaheb Ambedkar Marathwada University (BAMU) has conducted extensive studies on liquid structure using the dielectric relaxation approach . Dielectric parameters provide information regarding pairing of dipoles and their rotation in liquids. The experimental method used for the measurement of dielectric parameters along with conductivity was Time Domain Reflectometry (TDR) with sampling time in picosecond range. The experiments were conducted in vitro, i.e., samples were placed in a sampling cell. It was found that useful information in the frequency range of 100 MHz to 10 GHz can readily be obtained.
The above experimental method has also been carried out on many materials of biological significance. The multimers formed due to interaction of amino group and hydroxyl groups have been studied by the method .
As response of microwaves with systems is very sensitive to the interaction of water with other molecules present in the system, the technique has been used to diagnose clinically the status of healthy tissues in the human body. Water molecules in healthy tissues will behave differently than in infected tissues. It has been shown that dielectric parameters of saliva are significantly different for groups of healthy persons compared to persons with squamous cell carcinoma (SCC), and it is possible to identify these groups using permittivity data . Similar results have been obtained on tissues extracted from the patient's mouth . These studies were done in vitro placing extracted sample in the transmission line.
The objective of the present work was to report the study done in vivo to obtain information related to dielectric properties of saliva by placing a probe directly in subject's mouth. The paper gives the description of the equipment along with the method of analysis, which differs from the experiments done in vitro. The test of the equipment has been carried out on 125 subjects and it was found that the instrument has the potential capability to diagnose oral cancer.
The experiments have been performed in human beings as per permission and directives from the ethical committee of BJ Medical College, Pune, India. Before taking observations, the written informed consent of the subject was taken and their case study was recorded on the prescribed Performa.
The patients in the wards of BJMC, Pune, Dept. of Surgery were examined and clinically diagnosed cases of oral squamous cell carcinoma were grouped into squamous cell carcinoma (SCC). The relevant history of each patient with SCC was recorded thoroughly. All these patients were evaluated for routine haemogram. Incisional biopsy was taken from the oral lesion under local anesthesia, for confirmation of diagnosis. Only those squamous cell carcinoma patients who had not received any treatment before the study were selected. In vivo tissue readings of patients and controls were taken by probes directly from oral cavity by placing the probe on the lesion and/or mucosa. Clinical staging of each patient and histopathological grading were also done.
After screening and thorough clinical examination, those who did not have any renal and liver disorders, allergic conditions, autoimmune diseases and any other systemic disease or previous history of any major disease were selected for the group control C. These individuals did not have tobacco or any other habits and no obvious oral lesion.
The procedure to perform the experiment on a given subject (person) was as follows:
A total number of 48 cases of oral squamous cell carcinoma (OSCC) were screened and all patients consented to biopsy. The written consent was obtained. The routine hematological examination and medical check-ups were done on all the selected cases, and all were found fit to undergo surgical intervention. Clinical photographs of the lesions were taken prior to the biopsy procedure. Biopsies were then taken from the representative sites using 5 mm punch, after achieving anesthesia by 2% lidococaine with 1:80000 adrenaline. The local anesthetic solution was injected well away from the biopsy site.
Specimens of sufficient depth were taken so as to include intact covering epithelium, subepithelial connective tissue, submucosa and muscle. Then the surgical sites were sutured by ethicon suture and hemostasis was achieved. The tissue specimens were labeled and immediately fixed in 10% formaline for 24 hrs. The specimens were processed as per the procedure laid down by Bancroft and Stevens.
The processed tissues were embedded in paraffin wax using Leuckhart's 'L' blocks  as molds. The wax blocks were labeled accordingly. 6 sections out of each wax block of 5 µm were made for staining with haematoxylin and eosin (H & E) stain. The sections were stained by haematoxylin and eosin as per the procedure described by Bancroft and Stevens.
The detailed observations of all slides were made under a light microscope to see changes in epithelium, basement membrane, connective tissue and submucosa in order to histopathologically grade the squamous cell carcinoma according to Broder's grading system.
Accordingly three grades of oral squamous cell carcinoma were found: namely grade I (32 patients), Grade II (13 patients), and Grade III (3 patients).
Histopathological grading was done according to the Broder's numerical grading system , which depends upon the differentiation of tumor cells. A Grade I lesion is highly differentiated while grade IV is poorly differentiated. Using standard procedure, patients with their number were grouped as follows:
Description of the equipment
In TDR, a step-like pulse produced by a pulse generator propagates through the coaxial line and is detected from the sample section placed at the end of the line . The reflected pulse also propagates through the same line. The difference between the reflected and incident pulses recorded in the time domain contains the signature of the sample. Details of the set-up in our experiments are as follows:
The pulse generator
The pulse generator is the main unit of the setup. The unit generates a pulse with a rise time in the range of picosecond and having a peak voltage of at least 1 V on 50 impedance transmission line. The PCI-3125 system  was used as handheld pulse generator. The unit was very compact having 130 gm weight and size of (153 X 76 X 3) mm.
Before using the pulse generator, all parameters used for this purpose need to be optimized. All parameters were optimized individually by noise corresponding to different parameters set in an experiment. For this, experiments were performed on a healthy subject. In each experiment, noise is estimated by the standard deviation in base line of the reflected pulse. These parameters and their optimized values are summarized as follows:
Microstrip mouth sensor
The mouth sensor was designed on a micro strip line. The sensor could be removed from the connector for cleaning purposes. On one side of the microstrip line, a conducting copper line of width about 1 mm was deposited. The width of the copper strip was chosen such that the characteristic impedance was 50 ohms. The length of the strip line was about 10 cm with the other end rounded up so that it could be placed easily in the mouth. Eight cm of the probe was covered with teflon tape, and 2 cm was kept open. While doing a measurement, care was taken to keep this open area in the mouth in such a way that it touched the area of interest in the subject's mouth. The other side of the microstrip line was used as a ground.
An IBM laptop computer was used as dedicated system for the TDR system. The pulse generator was plugged into the system along with the dedicated software. The complete setup with all components is shown in Figure 1. A typical operating window is shown in Figure 2.
Figure 1 : Time Domain Reflectometer along with sampling probe and dedicated computer.
Figure 2 : A typical operating Window.
The data analysis
It was found that there are certain limitations in performing the experiments in vivo . The probe placed in the subject's mouth can only be placed for a limited time. The probe cannot be held steady because the subject cannot hold his mouth steady for a long time. The number of averages in vitro (n) should be the largest possible n for the best signal to noise ratio, but in vivo n must be practical. Two warm up pulses along with four averages required about four minutes. This time was found to be practical for the experiments.
Due to this limitation, the noise in recorded signals becomes significant. When the time domain data are transformed to frequency domain, one gets noisy frequency domain spectra. It was not possible to determine reliable values of dielectric parameters from the spectra. Due this limitation, it was decided not to transform time domain data to frequency domain, and instead to determine dielectric parameters. The features were directly extracted from time domain signals using the procedure as follows:
Figure 3 : Incident and Reflected pulses (a) probe in air (red) (b) probe placed in mouth(blue) (Sampling rate :2400 MHz).
Figure 4 : Reflected pulses only (a) probe in the air (red) (b) probe in the mouth (blue).
Figure 5: Selected part of reflected pulses with baseline shifted to zero.
A typical example of p-features extracted for different
groups is given in Figure 6. Codes for LDA were written using
Matlab. As discussed earlier, the measurements have been
classified in three categories and five groups as follows:
Group g1: Subjects with no tobacco using habits
Group g2: Subjects with tobacco using habits
Group g3-g5: Subjects with known cases of cancer. As mentioned in the previous section, these are further classified in three categories, as grade I (g3) , II (g4) and III (g5).
Figure 6 : One example of p-feature vector.
The clusters corresponding to group 1 with other groups are shown in Figures 7a-7d. In these figures, the center of each cluster is represented by a black circle and rectangular boundaries represent the most likely region for the cluster around the center. Figure 8 gives all clusters corresponding to each group together. From these figures, one may conclude as follows:
Figure 7 : Clusters corresponding to (a) groups g1 and g2 , (b) groups g1 and g3, (c) groups g1 and g4 (d) groups g1 and g5.
Figure 8 : Clusters corresponding all groups g1 to g5.
From the above discussion, we may summarize as follows:
It was found that the handheld time domain reflectometry can easily be used in vivo . The frequency domain spectra were noisier than the data in the time domain. The noise can be reduced by increasing the number of pulses averaged, which will, in turn, increase acquisition time. The subject will not remain comfortable if the signal integration time is too long. The optimum time was found to be about four minutes, corresponding to two warm-up pulses and four averages.
Another difficulty in the presently- used TDR versus the conventional TDR was finding an appropriate thickness of the sample. As in conventional TDR, SubMiniature Version A (SMA) lines are used to determine the effective length of the sample. Here we have used a microstrip line, an unconventional transmission line. The surface of the strip line remains in contact with the sample. It is very difficult to estimate the effective sample length, and there is a need to compensate for surface effect, as well.
Due to the difficulties mentioned above, it was decided not to adopt the conventional method of material characterization based on values of dielectric parameters and conductivity. A new set of feature vectors were extracted from reflected pulses acquired in the time domain. The data suggested that it was possible to diagnose cancerous cells with the feature vectors by using the LDA technique. However, the method needs to be cross validated and detection limits determined using more cases to enhance confidence in the results.
The authors declare that they have no competing interests.
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The financial support from TePP, TIFAC, New Delhi, India is thankfully acknowledged. We are also thankful to the ethics committee at BJMC, Pune for permission to conduct the clinical study on patients in the hospital.
Editor: JIAN-XIN YU, The University of Texas Southwestern Medical
Center at Dallas, USA.
EIC: Robert A. Lodder University of Kentucky, USA.
Received: 11-Oct-2013 Revised: 22-Nov-2013
Re-revised: 28-Dec-2013 Accepted: 30-Dec-2013
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