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Biological tissues have a complex electrical impedance, which is a function of
frequency, because tissues contain components that have both resistive and charge
storage (capacitive) properties. Bio-impedance is used in a wide variety of researches
and in clinical practice due to its non-destructive and non-invasive nature. It is used to
characterize the state of a tissue or organs and get diagnostic images. Examples
include skin impedance measurements used to assess skin hydration, body
composition analysis, malnutrition states, monitoring of long bone fracture healing,
respiratory and cardiac monitoring, evaluation of neuromuscular disease, detection of
various cancers.
3.1 Principles of Impedance
The earliest impedance measurements are credited to Georg Simon Ohm (17881854). For his initial measurements he used a voltaic cell, probably having copper and
zinc plates, whose voltage varied badly under load. As a result he arrived at an
erroneous logarithmic relationship between the current measured and the length of
wire, which he published in 1851.14 In order to get a more constant voltage Ohm
repeated his measurements using a copper-bismuth thermocouple for a source. His
detector was a torsion galvanometer (invented by Coulomb), a galvanometer whose
deflection was offset by the torque of thin wire whose rotation was calibrated. He
determined "that the force of the current is as the sum of all the tensions, and
inversely as the entire length of the current". Using modern notation this becomes I =
E/R or E =I*R. This is now known as Ohm's Law.15, 16
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Figure 1: Schematic representation of Ohm’s law.
The term impedance was coined by Oliver Heaviside in July 1886.16 Arthur
Kennelly was the first to represent impedance with complex numbers in 1893.
Impedance is defined as the frequency domain ratio of the voltage to the current. In
other words, it is voltage–current ratio for a single complex exponential at a particular
frequency ω.16 In general, impedance will be a complex number, but this complex
number has the same units as resistance, for which the SI unit is the ohm. The symbol
for impedance is usually and it may be represented by writing its magnitude and phase
in the form
It includes both resistance (R) and reactance (X) and according
to Encyclopedia Britannica Online “The resistance component (real part of
impedance) arises from collisions of the current-carrying charged particles with the
internal structure of the conductor. The reactance component (imaginary part of
impedance) is an additional opposition to the movement of electric charge that arises
from the changing magnetic and electric fields in circuits carrying alternating
current.” Resistance and Reactance are not individually significant; together they
determine the magnitude and phase of the impedance, through the following relations,
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Figure 2: Graphical representation of the complex impedance plane.
The impedance of the device can thus be calculated by applying a sinusoidal voltage
to the device in series with a resistor, and measuring the voltage across the resistor
and across the device. Performing this measurement by sweeping the frequencies of
the applied signal provides the impedance phase and magnitude.
Resistance helps to calculate the amount of water in the body. Low resistance,
indicating high conductivity, is due to large amounts of water in the body. Resistance
in the body is proportional to the amount of lean body mass since water is contained
solely within lean body mass.
Reactance is a measure of the cells' ability to store energy. Energy is stored in the cell
membrane therefore this reading gives an indication of the amount of intact cell
membranes in the body. Since intact cellular membranes are contained primarily
within body cell mass, the reactance of the body is proportional to the amount of body
cell mass. The reactance helps to calculate the proportion of the body that is
metabolically active.
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Capacitance is a property that opposes a change in voltage or electric potential across
an object and acts to store energy. A capacitor consists of two conductors; each
oppositely charged and separated by a dielectric material.
Permittivity is a property of the dielectric material and reflects the ability of charges
in the material to move in response to an electric field. Capacitance is a function of
the permittivity and the physical geometry of the object.
Phase Angle: Phase angle is an indicator of cellular health and integrity. Research on
humans has shown that the relationship between phase angle and cellular health is
increasing and nearly linear. A low phase angle is consistent with an inability of cells
to store energy and an indication of breakdown in the selective permeability of
cellular membranes. Cell membranes have high lipid content; therefore this reading
gives an indication of your cell lipid status. A high phase angle is consistent with
large quantities of intact, healthy cell membranes and body cell mass.
Phase angles for adults range from 3 to 10 degrees, with normal values of 6 to 8
degrees. A phase angle of 5 degrees or lower can indicate a serious energy
deficiency. A phase angle higher than 8 degrees is excellent.
Figure 3: Graphical derivation of the phase angle; its relationship with resistance
(R), reactance (Xc), impedance (Z) and the frequency of the applied current.
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Interest and experimentation in the electrical properties of tissues began in the late
1800s. The first electrical impedance measurements of biological variables of the
circulation began at New York Post Graduate Hospital in 1939 with Dr. Jan Nyboer,
Dr. Robert Halsey, Dr. Avrom Barnett and an engineer Mr. Samuel Bagno. In 1938
Mr. Bagno was awarded U.S Patent No. 2,111,135 titled "apparatus and method for
determining impedance angles," that discloses an apparatus for measuring the
electrical phase displacing properties or impedance angle of humans, animals, and
vital tissues. Thomasset A,14,15 conducted the original studies using electrical
impedance measurements as an index of total body water (TBW), using two
subcutaneously inserted needles. In 1967, he was awarded U.S Patent No. 3,316,896
titled "apparatus and methods for the measure of the electrical impedance of living
organisms," which discloses a method for simultaneously and associatively
determining the individual impedances of the extracellular contents and the
intracellular contents of a living organism, which consists in measuring the total
impedance of the organism between two selected points at predetermined frequencies.
Hoffer et al.17 and Nyboer et al.18 first introduced the four-surface electrode BIA
technique. A disadvantage of surface electrodes is that a high current (800 mA) and
high voltage must be utilized to decrease the instability of injected current related to
cutaneous impedance (10000 Ohm/cm2). By the 1970s the foundations of BIA were
established, including those that underpinned the relationships between the impedance
and the body water content of the body. A variety of single frequency BIA analyzers
then became commercially available, and by the 1990s, the market included several
multi-frequency analyzers. Later, Singh B et al.19 in 1979 devised an instrument the
“In vivo dielectric spectrometer” for measuring the relative permittivity and dielectric
loss of body tissues and organs in vivo, over the frequency range 0·1 Hz to about 100
kHz. The results of their study showed differences in relative permittivity and
dielectric loss between different types of tissue.
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3.2
Bioimpedance in various malignancies
Neoplasia is the end product of an unregulated proliferation of cells resulting from the
accumulation of sequential epigenetic and genetic alterations (mutations) in a
precursor cell. The resultant "cancer" is a population of cells that continue to mutate
and that secrete self-­‐perpetuating growth factors and angiogenic factors. One of the
main ways to understand cell function is by the investigation of cell electrical
behavior when subjected to an electric field.
From as early as 1926, researchers have been studying the electrical properties of
breast tumors.20 While there have been varying results; the consensus is that electrical
properties of breast tumors do differ from normal healthy tissue. In 1988, Surowiec et
al.21 performed in vitro tests to determine the variability of properties between
samples of breast carcinoma, samples with a combination of carcinoma and healthy
tissue including the perceived boundary of the lesion, and samples of healthy tissue
only. This group concluded that the dielectric constants and the conductivity of
cancerous tissues differed between the sample groups (as measured for frequencies
from 20 kHz to 100 MHz), although considerable variability existed in the measured
data.
Morimoto T et al.22 in 1990 developed a new impedance analytical system for
studying breast tumors in 54 patients in vivo. Measurements were performed over a
frequency range of 0-200 kHz by the three-electrode method. The biological tissues
were regarded electrically as an equivalent consisting of three parameters namely,
extracellular resistance (Re), intracellular resistance (Ri), and electrical capacitance of
the cell membrane (Cm). It was found that Re and Ri of breast cancers were
significantly higher than those of benign tumors, and that Cm of breast cancers was
significantly lower than that of benign tumors.
Thus, they concluded that the
measurement of the electrical bioimpedance of breast tumors may have value in the
differential diagnosis of breast lesions.
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Figure 4: Extracellular resistance (Re), intracellular resistance (Ri),
and electrical capacitance of the cell membrane (Cm).
Again, in 1993 Morimoto T et al.23 studied electrical bioimpedance of a variety of
tumors and calculated the Re, Ri and Cm in tissues. In this study, the electrical
impedances of various tumors were measured in vivo in 54 patients with breast
disease (31 breast cancers, 13 fibroadenomas, and 10 fibrocystic diseases) and 57
patients with pulmonary disease (44 lung cancers, 5 metastatic pulmonary tumors, 4
pulmonary tuberculoses, and 4 organized pneumonias). Re, Ri, Cm exhibited
significant differences among various tissues and tumors suggesting possible
applications in tumor diagnosis.
Joines WT et al.24 in 1994 measured the electrical conductivity and relative
permittivity of malignant and normal human tissues at frequencies from 50 to 900
MHz. The measurements were made between 23 and 25 degrees C using a network
analyzer connected to a flat-ended coaxial probe that was pressed against the freshly
excised tissue samples. The malignant tissues were of the following normal tissue
origin: bladder, colon, kidney, liver, lung, lymph nodes, mammary gland, spleen, and
testes. The normal tissues included: colon, kidney, liver, lung, mammary gland, and
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muscle. Normal tissue samples of bladder, lymph, spleen, and testes were not
available. In general, at all frequencies tested, both conductivity and relative
permittivity were greater in malignant tissue than in normal tissue of the same type.
Jossinet J et al.25 in 1996 studied the impedance of six groups of breast tissue over a
frequency range of 488 Hz to 1 MHz. The impedance spectra were obtained for 120
samples obtained from 64 patients, with the sample groups divided into three types of
normal breast tissue, two types of benign tissue, and carcinoma. All three articles
present studies using the same data. The first study by Jossinet investigates the
variability of the impedance data within each group by assessing the standard
deviation and the reduced standard error. In the second study by Jossinet, the complex
impedance was plotted versus frequency in a desire to compute the Cole–Cole
parameters. The study involved calculating parameters to try to distinguish the
carcinoma samples from the rest of the samples. Although many of the impedance
spectra do not approximate circular arcs, there are obvious differences observed in the
shape and location of the various impedance loci on the reactance–resistance plane.
The second study suggests that there is a greater difference in characteristics of
cancerous tissues at frequencies over 125 kHz.26
Jossinet and Schmitt,27 attempted, in the third study, to define a new set of eight
parameters by which cancerous tissue can be differentiated from other tissues. They
conclude that no one parameter at a single frequency is sufficient to define the tissue
but that several parameters spanning a range of frequencies are appropriate.
Then Emtestam L et al.28 in 1998, carried out a pilot study for the pre-operative
assessment of nodular basal cell carcinoma (BCC) by measuring electrical impedance.
Four physically distinct indices named magnitude index (MIX), phase index, real part
index and imaginary part index (IMIX) were used and BCC tissue revealed
statistically significant changes of the impedance indices MIX and IMIX.
Chauveau N et al.29 in 1999 have also investigated the need to calculate bioimpedance parameters over a variety of frequency values. For ex vivo samples of
normal and pathological tissues, measurements were obtained for frequencies that
ranged from 10 KHz to 10 MHz. From these measurements and a model that includes
a constant phase element, the extracellular resistance, the intracellular resistance, and
the membrane capacitance were calculated from the measurements and a model that
includes a constant phase element. From these calculated values, three indices are
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defined for classifying tissue pathology. Based on the data presented it is suggested
that these parameters, and the indexes based on them, would allow cancerous tissues
to be differentiated from normal tissues and those with fibrocystic changes.
Thereafter, in 1999, TransScan TS2000 (TransScan Medical, Ltd., Sweden), an
impedance-imaging device for breast cancer detection, was approved for use by the
American Food and Drug Administration as an adjunct to mammography for the
evaluation of equivocal breast lesions. This device maps the capacitance and
conductance of the tissue, in two separate images, in a range of frequencies that are
selectable for any of the available 26 frequencies. The images are obtained via a
square sensor array that is pressed against the breast. The reference electrode was
placed in the patient’s hand.
Lee BR et al.30 in 1999 used bio-impedance for cancer localization in the intact
prostate. Frequencies ranging from 100kHz-4MHz were used to obtain 594
bioimpedance measurements from the six ex-vivo prostate glands. These
measurements were then correlated with histology to determine the presence or
absence of prostate cancer. Prostate cancer was found to have higher impedance, of
932+/-170 ohms, compared to areas of no cancer within the same prostate, 751+/-151
ohms, P < 0.0001, at 2 MHz. This phenomenon was observed across all frequencies
tested. They thus concluded that this technology may improve identification and
localization of cancer within the prostate and also can potentially guide needle
placement during prostate biopsy and thus improve sampling of tumors.
Later on Brown BH et al.31 in 2000 used a pencil probe (diameter 5 mm) to measure
electrical impedance spectra from eight points on the cervix in 124 women with
abnormal cervical smears. Variables that should be sensitive to the expected tissue
changes were calculated and compared with the colposcopic results. Good separation
in the measurements made on normal squamous tissues from those made on
precancerous tissues was achieved.
In 2002, Malich A et al.32 studied electrical impedance scanning (EIS) of lymph nodes
and suggested its potential value as an imaging adjunct in the differentiation of
sonographically equivocal lesions. Best accuracy was achieved at chest/abdominal
wall and inguinal locations whereas the examination of inframandibular and paraaortal lymph nodes was limited.
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Following this, Glickman YA et al.33 in 2003 conducted an animal study which
showed that electrical impedance measurements reflect morphological changes related
to growth of cancerous skin lesions. EIS can be considered as an objective and noninvasive tool for differentiation between benign and malignant skin lesions.
Later, Beetner DG et al.34 in 2003 differentiated among basal cell carcinoma, benign
lesions, and normal skin using electric impedance and concluded that electrical
impedance could be used to provide rapid and noninvasive differentiation of basal cell
carcinoma (BCC) from similar looking benign lesions. Electrical impedance was
measured in vivo from 1 kHz to 1 MHz on 24 human subjects over BCC (19 lesions),
over benign tumors (11 lesions), and over normal skin (all 24 patients). Lesions
ranged from 2-15 mm in diameter. Indexes based on the magnitude (MIX), phase
(PIX), real part (RIX) and imaginary part (IMIX) of impedance were calculated for
each measurement. Significant differences were found between measurements over
BCC, benign lesions and normal skin for indexes MIX, PIX, and IMIX.
Afterwards, Tyna A Hope et al.35 in 2003 presented a paper focusing on electrical
impedance scanning and suggested several benefits of the same including comfort to
the patient, the relatively low cost, and studies suggested that it may be effective in
detecting disease in mammographically dense breasts.
Subsequently Ohmine Y et al.36 in 2004 suggested that local tissues can be diagnosed
differentially and electrically by percutaneous measurement of local bioimpedance
and subsequent estimation of the electrical conductivity of each tissue were calculated
and these were compared with the colposcopic results.
Aberg P et al.37 in 2004 used electrical bioimpedance to assess skin cancers and other
cutaneous lesions with an aim to distinguish skin cancer from benign nevi using
multifrequency impedance spectra. Electrical impedance spectra of about 100 skin
cancers and 511 benign nevi were measured. Impedance of reference skin was
measured ipsilaterally to the lesions. The impedance relation between lesion and
reference skin was used to distinguish the cancers from the nevi. It was found that it is
possible to separate malignant melanoma from benign nevi with 75% specificity at
100% sensitivity, and to distinguish nonmelanoma skin cancer from benign nevi with
87% specificity at 100% sensitivity.
Aberg P et al.38 in 2004 studied skin cancer and other benign lesions like malignant
melanoma, basal cell carcinoma, actinic keratosis, pigmented nevi by electrical
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impedance with a depth selective spectrometer. Impedance spectra of 99 benign nevi,
28 basal cell carcinomas (BCC), and 13 malignant melanomas (MM) were measured
using the two electrode systems. The best separation between nevi and BCC was
achieved using the regular non-invasive probe with a 96% sensitivity and 86%
specificity, whereas the best separation between nevi and MM was achieved using
the microinvasive electrodes (surface furnished with tiny spikes that penetrate stratum
corneum) with a 92% sensitivity and 80% specificity.
Abdul S et al.39 in 2005 carried out a clinical study on the use of impedance
spectroscopy in the detection of cervical intraepithelial neoplasia. 176 women referred
for colposcopy with an abnormal smear (borderline, mild, moderate or severe
dyskaryosis) participated in this study. A 4-electrode impedance probe, 5.5 mm in
diameter, with 2 electrodes injecting a current of 20 AA and the other 2 electrodes
measuring the impedance spectrum, at 8 frequencies was shown to be a promising
cervical screening tool. Cervical impedance spectroscopy has similar sensitivity and
specificity to currently used screening tests for the detection of cervical neoplasia and
also has the potential advantage of providing instant results. Good separation of
cervical epithelial neoplasia from squamous epithelium using this probe was
achieved, but separation from immature metaplastic tissue was less defined.
Gupta D et al.40 in 2008 evaluated a case series of 259 histologically confirmed breast
cancer patients and demonstrated that BIA-derived phase angle is an independent
prognostic indicator in patients with breast cancer. Nutritional interventions targeted
at improving phase angle could potentially lead to an improved survival in patients
with breast cancer.
Halter RJ et al.41 in 2008 conducetd a study to differentiate normal and malignant
prostate tissue using bioimpedance spectra. A probe was designed to measure
impedance spectra over the range of 10 kHz to 1 MHz. Impedance spectra of five ex
vivo prostates were measured in the operating room immediately following radical
prostatectomy. They concluded that conductivity and permittivity are both higher in
normal prostate tissues than they are in malignant tissue making them suitable
parameters for tissue differentiation.
Balasubramani L et al.42 in 2009 evaluated the efficacy of an electrical impedance
probe (Epitheliometer) in the diagnosis of high grade cervical intraepithelial neoplasia
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(CIN) in women referred with cervical smear abnormalities and to assess the effect of
acetic acid (AA) and tissue boundaries on the measurements.
3.3 Bioimpedance in OSCC
Only two in-vivo studies have been carried out till date in OSCC. Ching CT et al.14 in
2010 conducted a preliminary study of the use of bio-impedance in the screening of
squamous tongue cancer by using a disposable probe with 4 silver electrodes
measuring the electrical properties of cancerous tongue tissue (CTT) and surrounding
normal tongue tissue (NTT) in five squamous cell carcinoma patients at six different
frequencies, which were 20 Hz, 50 kHz, 1.3 MHz, 2.5 MHz, 3.7 MHz and 5 MHz.
Four measurement parameters of impedance, phase angle, real part of impedance, and
imaginary part of impedance of tongue were assessed to see if significant difference
in values obtained in CTT and NTT existed. A significant difference (P < 0.05 for the
four measurement parameters) was found at 50 kHz between CTT and surrounding
NTT. Extension of this study was done by Sun TP et al.43 in 2010 in which twelve
tongue cancer patients and twelve healthy subjects participated.
Arias LR et al.44 in 2010 used the electric cell-substrate impedance sensing (ECIS)
system to study the cellular activities of OSCC cells in a real-time and label-free
manner. Various cellular activities, including cell adhesion, spreading, proliferation,
and drug-induced apoptosis and inhibition of apoptosis, were monitored. A linear
relationship was found between the impedance-based cell index and the cell number
in the range of 3500 to 35,000 cells/well. Anti-cancer drug-cisplatin-induced OSCC
cell apoptosis at the minimal concentration of 5 µM after 20 h of treatment and
followed a linear dose-dependent manner in the concentration range from 10 µM to
25 µM. The inhibition of cisplatin-induced apoptosis by the carcinogen, nicotine, at
concentrations from 0.1 µM to 10 µM was monitored. The most significant inhibitory
effect of nicotine on cisplatin-induced apoptosis was observed at concentrations of
0.5–1 µM. The results obtained with impedance method correlated well with
microscopic imaging analysis of cellular morphology and cell viability analysis. This
study demonstrated that the impedance-based method can provide real-time
information about the cellular activity of viable cells and detect drug-induced cellular
activities
much
earlier
than
commonly
used
cell-based
image
analysis.
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This impedance-based method has the potential to provide a useful analytical
approach for cancer research.
Yang L et al.45 in 2011 used electrical impedance-based measurements to distinguish
oral cancer cells and non-cancer oral epithelial cells based on their cellular activities
on the microelectrodes in a real-time and label-free manner. CAL 27 and Het-1A cell
lines were used as the models of oral cancer cells and non-cancer oral epithelial cells,
respectively. Various cellular activities, including cell adhesion, spreading, and
proliferation were monitored. We found that both the kinetics of cell spreading and
the static impedance-based cell index were feasible to distinguish the two cell types.
At each given cell number, CAL 27 cell spreading produced a smaller cell index
change rate that was 60–70% of those of Het-1A cells. When cells were fully spread,
CAL 27 cells generated a cell index more than four times greater than that of Het-1A
cells. Since cell spreading and attachment occurs in the first few hours when they
were cultured on the microelectrodes, this impedance-based method could be a rapid
label-free and non-invasive approach to distinguish oral cancer cells from non-cancer
oral epithelial cells. Cell viability analysis was performed along with the impedancebased analysis. Confocal microscopic imaging analysis showed the difference in cell
morphology and the thickness of cell monolayers between the two cell types.
It has been observed that only two studies have been carried out on OSCC of
tongue.14, 43 No study has been carried out so far on OSCC of the other regions of the
oral cavity. Moreover, no study has been carried out in India on electrical impedance
in OSCC. It is an eminent fact that the molecular changes underlying OSCC in
Eastern countries (India, S.E. Asia) vary widely from those seen in the western world
(U.K., U.S.A., Australia).46 Studies have shown that, p53 mutations are common in
tumours from the West (47%) but are infrequent in the East (7%). Futhermore,
tumours from India and South East Asia are characterised by the involvement of ras
oncogenes, including mutation, loss of heterozygosity (H-ras) and amplification (Kand N-ras), events which are uncommon in the West. Therefore the aim of this study
is to verify whether similar results are obtained in the electrical properties of
cancerous oral tissue and normal oral tissue in the Indian population and thus
establish a new approach, namely bioimpedance, for reliable, low-cost, noninvasive,
and real-time screening or detection of OSCC.
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3.4
Bioimpedance in OPMDs
Though a few studies have been carried out on measurement of bioimpedance in
premalignant lesions of cervix and intraepithelial cervical carcinoma,39,42,47 there is no
study reported in the literature on bioimpedance measurement in any of the OPMD.
So, future research work should be focused on studies on measurement of
bioimpedance in OPMDs to reduce incidence of OSCC transformation.
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