WO2025042317A1

WO2025042317A1 - Methods and apparatus for measuring electrical impedance and assessing biological conditions of tissue samples

Info

Publication number: WO2025042317A1
Application number: PCT/SE2024/050669
Authority: WO
Inventors: Nima ASKARY LORD; Rasmus Larsson; Andreas Berndtsson; Ali AKDIS; Yasutaka MITAMURA; Jeremy BOST
Original assignee: Scibase AB
Current assignee: Scibase AB
Priority date: 2023-08-18
Filing date: 2024-07-04
Publication date: 2025-02-27
Anticipated expiration: 2026-02-18

Abstract

The present invention generally relates to the field of evaluation of biological conditions of tissue and tissue samples. In particular, the present invention relates to methods and apparatus for evaluating ex vivo tissue samples in for example medical pathology, and in particular to methods and apparatus that characterizes ex vivo tissue samples using precise measurements of electrical impedance of the tissue.

Description

METHODS AND APPARATUS FOR MEASURING ELECTRICAL IMPEDANCE AND ASSESSING BIOLOGICAL CONDITIONS OF TISSUE SAMPLES

Technical field

Background art

Epithelial tissues consist of layers of specialized cells closely bound together with a primary function to form a physical and chemical barrier between the body and the external environment. The epithelial barrier protects the internal tissues from environmental stresses, by minimizing water loss and preventing the entry of pathogens, pollutants, toxins and allergens through skin or mucosa. Recent genome-wide association studies have shown that the role of barrier function of the epithelium is essential in several allergic diseases. Barrier defects have been reported in atopic dermatitis, asthma, chronic rhino sinusitis, allergic rhinitis, eosinophilic esophagitis and colitis. This defect is a starting point of chronic inflammation and allergen sensitization and allows tissue-damaging factors to enter the deeper tissue and thus activate immune and inflammatory responses.

Skin has two barrier structures: the stratum corneum and tight junctions (TJ). The stratum corneum is the outermost layer of the epidermis, consisting of terminally differentiated keratinocytes, called corneocytes, which form a densely packed and extensively cross-linked lipid-protein matrix. The proteins filaggrin, loricrin and involucrin have a pivotal role in skin barrier function by interacting with keratin intermediate filaments. The most important component of the epithelial barrier is represented by the TJ that seal paracellular spaces at the very apical side in the mucosa and at the level of stratum granulosum in the skin between neighboring epithelial cells. TJ are responsible for the epithelial permeability, by controlling the paracellular flux of ions and bigger molecules, and physically separate the two different compartments. TJ are necessary for appropriate epithelial cell differentiation and function, with strong involvement in signal transduction and epithelial proliferation and differentiation. They form large complexes in the cell membrane consisting of three major types of transmembrane proteins: the claudin family, the tight junction associated MARVEL (MAL and related proteins for vesicle trafficking and membrane link) protein family, single-span proteins such as the immunoglobulin-like proteins junction adhesion molecules (JAM) and coxsackie and adenovirus receptor (CAR). Intracellularly, the transmembrane proteins bind to several scaffold proteins such as the zonula oc- cludens family (ZO) and are consequently connected to the actin cytoskeleton.

Historically, the epithelial barrier has been possible to assess in vitro, by measuring trans-epithelial electrical resistance (TEER), which represents the opposition of the epithelium to the passage of a steady electrical current. For this purpose, epithelial cells are cultured to an air liquid interface (ALI) in transwell plates. The confluence of the cellular integrity determines a sharp increase in TEER, indicating a low ion flux and a tight epithelial barrier; while the disruption of junctional complexes results in reduction of TEER. In addition, TEER measurements show good negative correlation with fluoresceinated dextran passage as demonstrated in ALI cultures of different tissues.

In vivo there are few noninvasive methods to assess epithelial barrier function. One of these is the quantification the transepidermal water loss (TEWL) in the skin across the stratum corneum. Although TEWL increases in proportion to the level of damage, it is also affected by environmental factors such as humidity, temperature, season and moisture content of the skin. Other used noninvasive methods include the stratum corneum hydration, colorimetry, skin surface pH and sebometry. They provide information on different characteristics and / or condition of the skin, but they don’t directly measure the barrier function.

The impairment of the epithelial barrier function is associated with various skin and mucosal allergic, metabolic and autoimmune disorders.^1-5 The prevalence of these diseases has been markedly increasing in the Western world since the 1960s and continues to increase in developing countries.^6-8 The epithelial barrier hypothesis has been recently proposed to explain the reasons for such an increase.⁶’⁹¹⁰ Like many other diseases, allergic diseases result from complex gene-environment interactions. Changes in genetic factors are unlikely to be the underlying cause of the prevalence increase since such a rise occurred relatively rapidly. Instead, increasing evidence points to environmental factors playing a key role.^711-13 Indeed, epidemiological studies have shown that exposure to multiple environmental factors, such as air pollutants, tobacco smoke, fragrances and preservatives, can contribute to the development and exacerbation of asthma and other allergies.^712-15

Hence, as understood there is would be of a great value to find improved and more accurate tools that can be used to analyze and assess biological conditions such as epithelial skin barrier function in a test environment on ex-vivo skins samples.

Summary of the invention

An object of the present invention is to provide medical apparatus and methods for analyzing and assessing biological conditions on ex vivo tissue samples.

This and other objects of the present invention are achieved by a device and method as claimed in the independent claims. Further embodiments are defined in the dependent claims.

The present invention is based on a deeper understanding of dielectric properties of various tissues, which now makes it possible to establish a method to assess the biological conditions, such as for example epithelial barrier function on ex vivo skin samples. The inventive system and method can, for example, be used in test environment and diagnostic instrument for skin inflammatory disorders with a barrier defect. The electrical impedance (El) spectroscopy is a relatively new technique that previously has been used as means to characterize skin tumors. Electrical signal are transmitted through the skin at several depths and frequencies and the impedance response is measured, influenced by certain properties of tissue integrity. Generally, when there is an alteration in tissue structure and cellular composition, there is an imprinting in the electrical impedance spectrum related to the type of the tissue alteration. In some diseases, such as melanoma, the measurements of tissue El have been used for diagnosis, assessment of disease progression and evaluation of therapy. The inventors have now found that El spectroscopy technique can be used on ex vivo skin samples to investigate and assess effects on skin samples from, for example, chemical substances such as detergents. Epithelial skin barrier function or skin barrier integrity can be evaluated/quantified. An impaired skin barrier is a pre-cursor to many disorders such as atopic dermatitis. Further, the efficiency of various treatments can be assessed. Quantifying degree of sensitivity for allergens and toxic /irritant substances, both on skin and oral cavity is further applications of the present invention.

According to an aspect of the present invention, there is provided a test kit for ex vivo tissue sample analysis comprising: a medical device holder configured to hold a medical device in a fixed position in relation an ex vivo tissue sample, wherein an electrode array of said medical device abuts said tissue sample with a predetermined pressure, a medical device further comprising: an impedance measuring unit connected to said electrode array and configured to pass electrical current into the tissue sample via electrodes of said electrode array to obtain tissue impedance data of a tissue region of the tissue sample, said tissue impedance data comprising a plurality of impedance values measured in the tissue region; and an electronic computer communicating with the impedance measuring unit to control activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum; and for each measurement in said measurement loop, control a first electrode in an electrode pair of said electrode array to inject a current into or to apply a voltage to the tissue sample and a second electrode of said pair to measure the resulting current or the resulting voltage from the tissue sample.

According to another aspect of the present invention, there is provided a method for ex vivo tissue sample analysis comprising the steps of: arranging a medical device in a medical device holder in a fixed position in relation an ex vivo tissue sample, wherein an electrode array of said medical device abuts said tissue sample with a predetermined pressure, said medical device further comprising: an impedance measuring unit connected to said electrode array and configured to pass electrical current into the tissue sample via electrodes of said electrode array to obtain tissue impedance data of a tissue region of the tissue sample, said tissue impedance data comprising a plurality of impedance values measured in the tissue region; and instructing an electronic computer communicating with the impedance measuring unit to control activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum; and for each measurement in said measurement loop, control a first electrode in an electrode pair of said electrode array to inject a current into or to apply a voltage to the tissue sample and a second electrode of said pair to measure the resulting current or the resulting voltage from the tissue sample.

In embodiments of the present invention, the medical device includes a probe for measuring electrical impedance of tissue of a subject. The probe comprises a plurality of electrodes, the electrodes being adapted to be placed in di- rect contact with the skin sample, and being connectable to an impedance measuring circuit adapted to apply a voltage and to measure a resulting current to determine an impedance signal. In preferred embodiments, the probe further comprises a switching circuit for selectively activate electrode pairs by connecting at least two of electrodes with the impedance measuring circuit and disconnecting the remaining electrodes from the impedance circuit, wherein the voltage is applied at the two electrodes and the resulting current is measured between the at least two electrodes. The switching circuit is adapted to receive control signals instructing the switching circuit to activate electrode pairs in accordance with a predetermined activation scheme, the predetermined activation scheme including to activate adjacent electrodes in a successive manner to gradually scan tissue of the subject at a first tissue depth so as to obtain a sequence of impedance signals from a selected tissue depth.

According to embodiments, the probe is provided with electrodes that have an elongated rectangular shape and are arranged at the probe in parallel rows. However, there are a number of alternative designs. For example, the electrodes may be arranged as concentric rings, or as squares. The electrodes may be arranged with micro-needles wherein each electrode comprises at least one spike. The spikes are laterally spaced apart from each other and having a length being sufficient to penetrate at least into and/or through the stratum corneum. In an alternative embodiment, the electrodes are non-invasive and each electrode has a substantially flat surface adapted to be placed against the tissue of the subject. It is also possible to combine electrodes provided with micro-needles with non-invasive electrodes.

WO 01/52731 discloses example medical electrodes for sensing electric bio-potentials created within the body of a living subject. The electrode comprises a number of micro-needles adapted to penetrate the skin. The micro-needles are long enough to reach the stratum corneum and penetrate at least into the stratum corneum and are electrically conductive on their surface and connected to each other to form an array. In EP 1437 091, an apparatus for diagnosis of biological conditions using impedance measurements of organic and biological material is disclosed. The apparatus comprises a probe including a plurality of electrodes, where each electrode is provided with a number of micro-needles each having a length being sufficient to penetrate at least into stratum corneum. The micro-needles according to EP 1437 091 are also “nail-like”, i.e. they have stem having a substantially circular cross-section with a constant or a gradually decreasing diameter and a tip-portion with a substantially spherical or needle- shaped tip.

In embodiments of the present invention, the probe may have a spherical shape, i.e. the surface of the probe provided with electrodes is spherically shaped.

As the skilled person realizes, steps of the methods according to the present invention, as well as preferred embodiments thereof, are suitable to realize as computer program or as a computer readable medium.

Generally, all terms used in the claims and description are to be interpreted according to their ordinary meaning in the technical field, unless explicitly defined otherwise herein. All references to “a/an/the [element, device, component, unit, means, step, etc.]” are to be interpreted openly as referring to at least one instance of the element, device, component, unit, means, step, etc., unless explicitly stated otherwise. The steps of any method disclosed herein do not have to be performed in the exact order disclosed unless explicitly defined otherwise herein.

Further objects and advantages of the present invention will be discussed below by means of exemplifying embodiments.

Brief description of the drawings

Exemplifying embodiments of the invention will be described below with reference to the accompanying drawings, in which:

Fig. 1 is a schematic block diagram of one embodiment of a medical device which can be used in a medical system according to the present invention; Fig. 2 is a schematic block diagram of a test equipment according to the present invention;

Fig. 3 is a schematic block diagram of a test equipment according to the present invention;

Fig. 4 illustrates test results achieved with the test equipment and method according to the present invention;

Fig. 5 is a schematic block diagram of the method according to the present invention.

Description of exemplifying embodiments

The following is a description of exemplifying embodiments in accordance with the present invention. This description is not to be taken in limiting sense, but is made merely for the purposes of describing the general principles of the invention. Even though particular types of probes including micro-invasive as well as non-invasive will be described, the invention is also applicable to other types of probes.

Thus, preferred embodiments of the present invention will now be described for the purpose of exemplification with reference to the accompanying drawings, wherein like numerals indicate the same elements throughout the views. It should be understood that the present invention encompasses other exemplary embodiments that comprise combinations of features as described in the following. Additionally, other exemplary embodiments of the present invention are defined in the appended claims.

Referring first to Fig. 1, a general description of a medical device which can be used in a medical system according to the present invention will be given. The system 10 comprises a medical device 5 including an impedance measuring circuit or unit 2 adapted to obtain impedance data of a tissue region or sample, for example, ex vivo tissue samples.

The tissue impedance measurement for obtaining the impedance data of the target tissue region may be performed by means of a probe 8 integrated in the medical device 5 or a probe being external to the medical device 5 and connected to the medical device 5. Irrespective of being external or integrated, the probe may comprise a plurality of electrodes or an array of electrodes 14 adapted to be placed in contact with the tissue to be analysed, typically skin or skin samples. The tissue impedance may be measured by applying an AC voltage over a pair of electrodes and measure the resulting current passing the same pair of electrodes. In embodiments, 2-point measurements are used by applying voltage and measure current over one pair of electrodes. The remaining electrodes may be grounded or free floating. In embodiments of the present invention, the probe 8 comprises, for example, seven or five electrodes, e.g. shaped as rectangular electrode bars. The electrodes are adapted to be placed in direct contact with the skin sample.

For example, adjacent electrodes can be separated with a distance of about 0.3 mm and having a length of about 5 mm, which has shown to be a practical and useful configuration for detections of diseased conditions such as malignant melanoma, both with regard to spatial resolution in a lateral dimension and in a depth dimension. A skin area of about 5x5 mm or about 25 mm² is thus covered by the probe and at high frequencies, above about 100 kHz, the deepest tissue layer being reached is about 2.5 mm which has been proven to be a clinical relevant depth. However, as the skilled person realizes, the probe may include more or less than five electrodes, for example 3 or 7 electrodes. Further, other electrode dimensions and other spacing between adjacent electrodes are conceivable, for example, electrodes having a width of about 4 mm and a length of about 8 mm.

In particular embodiments of the present invention, EIS measurements can be made using Nevisense® (SciBase, Sweden), a device established for skin cancer detection and skin barrier research. EIS is the measure of a material’s opposition to the flow of alternating currents at various frequencies. Specifically, tissue EIS values can reflect the pathophysiological status of the tissue. Normal and abnormal tissues differ with regards to cell size, shape, orientation, compactness, water content and structure of cell membranes. The system measures the electrical impedance at 35 different frequencies distributed between 1 kHz and 2.5 MHz at four depths in 10 permutations, resulting in 700 data points per measurement. The applied voltage and current are limited to 150 mV and 75 pA, respectively, and its safety has been proven in humans. The broad frequency range allows the collection of information on both intra- and extracellular tissue properties. Nevisense® is equipped with a handpiece and a disposable 5 mm by 5 mm electrode.

In general, by selecting adjacent pairs of electrodes, the topmost layer of a skin sample can be scanned in steps, and by selecting pairs that are spaced further apart, i.e. electrode pairs with one or more intermediate electrodes, the resulting current path allows for measurement at deeper skin layers. In this exemplifying embodiment of the probe according to the present invention, there are ten possible ways of selecting electrode pairs. The possibility to measure inter alia the topmost skin layer in small (determined inter alia by the spacing between adjacent electrodes and the frequency of the applied current) consecutive partitions is important since it allows for detection of small anomalies in the skin and tissue. Each electrode of the probe may be set in four different states including inject (the electrode is set to inject measurement current into the tissue), measure (the resulting current from the tissue is measured via the electrode), ground (the electrode is grounded to prevent leakage of superficial current when measurements are performed using other electrodes) and floating (the electrode is disconnected).

The medical device 5 may be communicatively connected to an electronic computer 4, which however may be integrated in the medical device. The electronic computer 4 may include storage units 3 for storing, for example, obtained impedance data performed on the patient. The electronic computer 4 may also include a processing circuit 7 adapted to process obtained impedance data to reduce the number of variables by removing insignificant variables by performing linear or non-linear projections of the impedance data to lower subspaces. In preferred embodiments of the present invention, principal component analysis (PCA) is used. An alternative approach is to use parallel factor analysis (PARAFAC). Further, classification rules determined by means of, for example, linear discriminant analysis (LDA) or soft independent modelling of class analogy (SIMCA) may be used to improve the evaluation.

The electronic computer 4 may further be configured to control, for example, switching cycles/sequences of the electrodes 14 in accordance with a predetermined activation procedure or scheme. This predetermined activation scheme may include an activation of a particular electrode pair or selected electrode pairs to perform a number of subsequent measurements in accordance with a predetermined measurement loop, or an activation of adjacent electrode in a successive manner to gradually scan tissue of the subject at a first tissue depth, which scanned tissue depends to a large extent on spacing between activated electrode pairs so as to obtain a matrix of impedance signals from different tissue depths.

Moreover, the electronic computer 4 may communicate with display means for displaying, for example, a biological condition status.

According to embodiments of the present invention, each electrode is provided with micro-needles, thereby forming a micro-needled surface. As has been discussed above, the probe, in preferred embodiments, may include five rectangular areas or bars. In this configuration, each bar contains an array of, for example, 57 (19 x 3) micro-needles. Each bar is about 0.3 mm wide and 5 mm long. The distance between adjacent bars is about 0.2-0.5 mm. The active part of the probe is thus about 5 x 5 mm. Each micro-needle has a length of approximately 100 micrometer, as measured from its base, and a thickness of at least 20 micrometer. The electrode bars and micro-needles can be made of plastic material in a moulding process. The material could be made intrinsically conductive or covered with a conductive layer such as gold. In an alternative embodiment, the electrode bars and micro-needles are made of silicon and covered with gold having a thickness of at least 1 micrometer. However, other materials comprising a conductive surface with similar dimensions would work, but it should be selected to be biocompatible. In, for example, the patent applications EP 1959828, EP 1600104, and EP 1437091 by the same applicant, different probe concepts having such micro-needles are described.

In another embodiment, the electrode bars are non-invasive and substantially flat. In, for example, US 5,353,802 by the same applicant, a probe concept including non-invasive electrodes has been described.

In other embodiments of the present invention, the probe is spherically shaped, i.e. the surface including the electrodes that is pressed against the skin or tissue during a measurement has a spherical shape. This also means that the electrodes may be at least partly spherically shaped.

For example, each spike may have a length of 0.01 to 1 mm. The spikes may be arranged on electrodes, in turn arranged on the probe, where each electrode may comprise from at least two spikes to about 100-200 spikes in certain applications, and any number in between. In the patent US 9,636,035 by the same applicant, examples of preferred embodiments of spike designs are described. By such configurations of spikes an increased versatility and increased adaptability in terms of capacity requirements can be achieved, in addition to possibly alleviating the problem of non-linear effects of Stratum Corneum.

The electronic computer 4 may be configured to pre-process the impedance data, for example, reduction of noise content and/or reduction of the dimensionality. The noise reduction may include reduction of noise in the impedance magnitude and / or phase angle spectra. The noise reduction may for example be made with the use of a Savitsky-Golay smoothing filter. Further, the preprocessing may comprise detection and correction of spikes or other artefacts, enabling removal of spikes or artefacts in the impedance spectrum, i.e. magnitude and/or phase angle spectra. Spikes may for example be detected with a me- dian filter with an adequate window size. Data points of the filtered data that differ too much from raw data may be considered to be a spike or other artefact and may be corrected by e.g. linear interpolation.

The electronic computer 4 may further comprise a pre-filter enabling rejection of measurement that do not fulfill one or a few specific criteria, such as cut-offs. The pre-filter may be applied on impedance data that has been corrected /adjusted e.g. by pre-processing as discussed above. For example, the magnitude values and / or phase angle values may all be required to fall within a specified magnitude range of a specified phase range, respectively, in order for a measurement not to be rejected. If the measurement is on a human/animal skin, the criteria, such as the ranges, may be set such as non-physiological measurements are rejected. Also, a specific criteria may be set for a certain value relating to a specific frequency.

Moreover, the electronic computer 4 may further include a classifier to assess whether quality of measured impedance data is good. This procedure may be combined with pre-processing and / or pre-filtering to further improve quality of the data. Examples of such classification include assessment of the variation, e.g. the variance or standard deviation, of magnitude and / or phase angle in different permutations at one or a plurality of frequencies. Another examples are the absolute value of magnitude and/or phase angle may be studied, for example the median value or average value, or skewness of magnitude or phase angle.

The medical device 5 may further include a communication unit 12 capable of transmitting/ receiving data to/from the electronic computer 4, if externally arranged and/or other external units 15, such as a laptop computer, a handheld computer/device, a database, a cloud-based arrangement, etc., directly with the unit or network itself or via a wireless network 16. In this way, the device 10 may be supplied with, for example, clinical data for use in the evaluation. Moreover, data obtained with the medical device 5 such as impedance data from measurements can also be downloaded to external devices 15 via the communication unit With reference now to Fig. 2, an embodiment of the test kit, or analysis system, in accordance with the present invention will be described. In preferred embodiments, a medical device as shown in Fig. 1 is used in the analysis system or test kit 20, for example, Nevisense® (SciBase, Sweden), a device established for skin cancer detection and skin barrier research. Further, ex-vivo human skin samples, such as NativeSkin® provided by GenoSkin Inc. (Toulouse, France, www.genoskin.com) can be used in the present method and test kit.

In embodiments of the present invention, the medical device 5, or probe 8 is arranged in a medical device holder 21 such that the electrodes 14 are put in contact with the surface of the skin sample 22. A pressure indicator or pressure sensor 24 may be connected to the probe 8, or integrated into the probe 8, to measure an applied pressure from the probe 8 on the skin sample 22. In preferred embodiments, the pressure P (see Fig. 3) should be low, or around 0 - 0.005 N, or around 0.001 - 0.01 N. The probe 8, and the electrodes 14, is preferably arranged in the medical device holder 21 such that an angle 32 between the surface of the skin sample 22 and an axis perpendicular to a surface of the electrodes 14 is around 90 degrees, or between 88 - 92 degrees. During measurements, the medical device 5, and the probe 8, is preferably instructed to execute a measurement loop including measurements between a second electrode 14b and a fourth electrode 14d, and the second electrode 14b and the third electrode 14c, and the third electrode 14c and the fourth electrode 14d, as illustrated in Fig. 3. This measurement loop is preferable repeated for 30 - 60 times, and the first and last measurement are discarded. However, as understood, other sequences of measurements can be applied, for example using all electrodes 14a - 14e, or only second and fourth electrode, or alternating between second and third and third and fourth. Preferable, the measurement loop is initiated after a predetermined period of time from that the electrodes are put into contact with the skin sample 22, for example, 15 - 20 seconds, or 10 - 30 seconds. As understood by a person skilled in the art, there are a number of other conceivable measurement loops. The above mentioned session or loop is just exemplary. For example, if a probe with more or less number of electrodes, another measurement session or loop will be used.

According to a preferred embodiment of the method 50 according to the present inventions, a ex-vivo skin sample 22 is placed in the test equipment 20, in step 51. At step 52, which however, is optional, the skin sample 22 is padded with a solution such as saline. At step 53, the probe 8, or medical device 5, is arranged in the medical device holder 21 as discussed above with reference to Fig. 2 and 3. At step 54, the medical device 5 is instructed to initiate a measurement session or loop, for example, by clicking ’’start” on a graphical user interface 7, and the measurement is interrupted at step 55 after a predetermined period of time, a predetermined number of measurements, or manually by clicking ’’stop” on the graphical user interface 7.

In Fig. 4, test results achieved with the inventive test kit, or analysis system, and method in accordance with the present invention are shown. The impairment of the epithelial barrier function is associated with various skin allergic and inflammatory disorders. Electrical impedance spectroscopy (EIS) is a noninvasive tool to detect skin barrier function in vivo. Ex-vivo experiments with human skin (NativeSkin®) is a model that exhibits normal skin barrier function and contains almost all cell types. The aim of this study was to investigate how laundry detergent cause skin barrier dysfunction and as can be seen in Fig. 4, the results using the inventive system and method are strong. This is discussed more thoroughly under the section ’’Test results” below.

It is to be understood that in the context of the present invention and in relation to electrical components electrically connected to each other, the term connected is not limited to mean directly connected, but also encompasses functional connections having intermediate components. For example, on one hand, if an output of a first component is connected to an input of a second component, this comprises a direct connection. On the other hand, if an electrical conductor directly supplies a signal from the output of the first component substantially unchanged to the input of the second component, alternatively via one or more additional components, the first and second components are also connected. However, the connection is functional in the sense that a gradual or sudden change in the signal from the output of the first component results in a corresponding or modified change in the signal that is input to the second component.

Although exemplary embodiments of the present invention has been shown and described, it will be apparent to those having ordinary skill in the art that a number of changes, modifications, or alterations to the inventions as described herein may be made. Thus, it is to be understood that the above description of the invention and the accompanying drawings is to be regarded as a nonlimiting example thereof and that the scope of protection is defined by the appended patent claims.

Test results

The impairment of the epithelial barrier function is associated with various skin and mucosal allergic, metabolic and autoimmune disorders.^1-5 The prevalence of these diseases has been markedly increasing in the Western world since the 1960s and continues to increase in developing countries.^6-8 The epithelial barrier hypothesis has been recently proposed to explain the reasons for such an increase.⁶’⁹¹⁰ Like many other diseases, allergic diseases result from complex geneenvironment interactions. Changes in genetic factors are unlikely to be the underlying cause of the prevalence increase since such a rise occurred relatively rapidly. Instead, increasing evidence points to environmental factors playing a key role.^711-13 Indeed, epidemiological studies have shown that exposure to multiple environmental factors, such as air pollutants, tobacco smoke, fragrances and preservatives, can contribute to the development and exacerbation of asthma and other allergies.^712-15 A growing number of studies indicated an epidemiological association between professional and domestic cleaning agents and the increased prevalence of asthma, rhinitis and allergic contact dermatitis.^16-29 Certain cleaning products were also associated with the risk of exacerbating asthma symptoms.³⁰³¹ Therefore, as detergents are present daily in our lives, we urgently need to improve our understanding of how exposure to even low doses of cleaning products may contribute to allergic disorders. Detergents can also be present as residues on surfaces and remain on clothing after rinsing during laundry, thus getting in contact with the skin and the respiratory mucosa.³² Interestingly, the post-rinse doses have been shown to affect the barriers of epithelial tissues from asthmatic, chronic obstructive pulmonary disease and healthy subjects.³²

One of the suggested mechanisms through which detergents might induce allergen sensitization is through a disrupted epithelial barrier. In air-liquid interface cultures of human bronchial epithelial cells, laundry detergents were shown to reduce the barrier function affecting the expression of tight junctions (TJs).³² These intercellular junctions consist of transmembrane and cytoplasmic proteins, also known as scaffold proteins. They represent one of the major contributions to barrier function, sealing paracellular spaces between neighboring epithelial cells at the very apical side in the mucosa and at the level of stratum granulosum in the skin.¹’³³³⁴ In the epidermis, in addition to TJs, the stratum corneum contributes to forming a physically stronger barrier than mucosal membranes due to the expression of cornified envelope proteins, such as the filament-forming filaggrin (fig), the structural protein loricrin (lor) and its interacting partner involucrin (ivl), and the profilaggrin-like protein hornerin.³⁵³⁶

The present study aims to evaluate the in vivo effects of household laundry detergents on skin barrier integrity, even used at low concentration. We treated mouse skin with different dilutions of two household laundry detergents, and we assessed the epidermal barrier function using electrical impedance spectroscopy (EIS), previously described by our group as a valid method to detect skin barrier function in vivo.³⁷ In addition, we determined transepidermal water loss (TEWL), a second method previously validated for skin barrier integrity for comparison.³⁸ Even when diluted 10’000 times, the epicutaneous application of household laundry detergents caused a reduction in skin barrier after only four hours, together with significant changes in skin barrier-related and proinflammatory molecules in skin transcriptome and proteome, most of which correlated with the skin barrier EIS and TEWL measurements.

Epicutaneous treatments on Ex-vivo human skin

Ex-uwo human skin samples (NativeSkin®) were purchased (GenoSkin Inc., Toulouse, France, www.genoskin.com). Adult human skin explants were obtained from three healthy female donors (32, 37 and 57 years old) undergoing plastic surgery and bio-stabilized for up to 7 days. All samples were provided as 15 mm diameter round natural human skin biopsies in propriatory custom built plastic inserts.

Detergent B was diluted at the concentrations of 1:200, 1:1’000, 1:5’000 and 1:25’000 (v/v) and sodium dodecyl sulfate (SDS) was diluted at the concentrations of 5 mg/mL, 1 mg/mL, 0.2 mg/mL, and 0.04 mg/mL (w/v). Ex-uwo human skin samples were treated with 60 pl of diluted household laundry detergent B and SDS. Detergent, SDS and PBS were applied on the surface of ex-uwo human skin for 6 hours. The surface of NativeSkin samples were washed out with PBS three times after the stimulation.

EIS measurements

EIS measurements were made using Nevisense® (SciBase, Sweden), a device established for skin cancer detection and skin barrier research. EIS is the measure of a material’s opposition to the flow of alternating currents at various frequencies. Specifically, tissue EIS values can reflect the pathophysiological status of the tissue. Normal and abnormal tissues differ with regards to cell size, shape, orientation, compactness, water content and structure of cell membranes. The system measures the electrical impedance at 35 different frequencies distributed between 1 kHz and 2.5 MHz at four depths in 10 permutations, resulting in 700 data points per measurement. The applied voltage and current are limited to 150 mV and 75 pA, respectively, and its safety was proven in humans.³⁹ The broad frequency range allows the collection of information on both intra- and extracellular tissue properties. Nevisense is equipped with a handpiece and a disposable 5 mm by 5 mm electrode. A single Nevisense impedance measurement takes less than a minute in total and consists of first moistening the skin with a physiological saline solution, then applying the electrode to the area to be examined for an 8 second EIS measurement.

For ex-uwo human skin, the surface of NativeSkin samples was moistened with PBS and wiped off with sterilized cotton gauze before each measurement. The electrical impedance was measured at 35 different frequencies distributed between 1 kHz and 2.5 MHz at two depths in three permutations after the optimization. EIS measurements were performed at 0, 6, 12, and 24 hours after the treatment. For each time point, measurements were made in triplicates.

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4. Soyka MB, Wawrzyniak P, Eiwegger T, et al. Defective epithelial barrier in chronic rhinosinusitis: the regulation of tight junctions by IFN-y and IL-4. J Allergy Clin Immunol. 2012;130(5):1087-1096.el010.

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Claims

1. A medical system for ex vivo tissue sample analysis comprising: a medical device holder configured to hold a medical device in a fixed position in relation an ex vivo tissue sample, wherein an electrode array of said medical device abuts said tissue sample with a predetermined pressure; a medical device further comprising: an impedance measuring unit connected to said electrode array and configured to pass electrical current into the tissue sample via electrodes of said electrode array to obtain tissue impedance data of a tissue region of the tissue sample, said tissue impedance data comprising a plurality of impedance values measured in the tissue region; and an electronic computer communicating with the impedance measuring unit to control activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum; and for each measurement in said measurement loop, control a first electrode in an electrode pair of said electrode array to inject a current into or to apply a voltage to the tissue sample and a second electrode of said pair to measure the resulting current or the resulting voltage from the tissue sample.

2. The system according to claim 1, wherein said electronic computer is operating according to a stored program to apply an evaluation procedure for analyzing biological conditions of the tissue sample on the basis of the measured impedance data for the tissue region and to evaluate the obtained data set of impedance values to provide an outcome indicating a biological condition status.

3. The system according to claim 1 or 2, wherein said medical device includes a probe for measuring electrical impedance of tissue of a subject, said device comprising a plurality of electrodes, the electrodes being adapted to be placed in direct contact with the skin sample, and being connectable to an impedance measuring circuit adapted to apply a voltage and to measure a resulting current to determine an impedance signal.

4. The system according to claim 3, wherein said probe comprises a switching circuit for selectively activate electrode pairs by connecting at least two of electrodes with the impedance measuring circuit and disconnecting the remaining electrodes from the impedance circuit, wherein the voltage is applied at the two electrodes and the resulting current is measured between the at least two electrodes.

5. The system according to claim 4, wherein said switching circuit is adapted to receive control signals from said electronic computer instructing the switching circuit to activate electrode pairs in accordance with a predetermined activation scheme, the predetermined activation scheme including to activate adjacent electrodes in a successive manner to gradually scan tissue of the subject at a first tissue depth so as to obtain a sequence of impedance signals from a selected tissue depth.

6. A test kit for ex vivo tissue sample analysis comprising: a medical device holder configured to hold a medical device in a fixed position in relation an ex vivo tissue sample, wherein an electrode array of said medical device abuts said tissue sample with a predetermined pressure, a medical device further comprising: an impedance measuring unit connected to said electrode array and configured to pass electrical current into the tissue sample via electrodes of said electrode array to obtain tissue impedance data of a tissue region of the tissue sample, said tissue impedance data comprising a plurality of impedance values measured in the tissue region; and an electronic computer communicating with the impedance measuring unit to control activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum; and for each measurement in said measurement loop, control a first electrode in an electrode pair of said electrode array to inject a current into or to apply a voltage to the tissue sample and a second electrode of said pair to measure the resulting current or the resulting voltage from the tissue sample.

7. A method for ex vivo tissue sample analysis comprising the steps of: arranging a medical device in a medical device holder in a fixed position in relation an ex vivo tissue sample, wherein an electrode array of said medical device abuts said tissue sample with a predetermined pressure, measuring an impedance by passing an electrical current into the tissue sample via electrodes of said electrode array to obtain tissue impedance data of a tissue region of the tissue sample, said tissue impedance data comprising a plurality of impedance values measured in the tissue region; and applying a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum.

8. The method according to claim 7, further comprising: instructing an electronic computer to control activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement loop including performing a number of subsequent measurements at predetermined frequencies in a predetermined frequency spectrum; and for each measurement in said measurement loop, control a first electrode in an electrode pair of said electrode array to inject a current into or to apply a voltage to the tissue sample and a second electrode of said pair to measure the resulting current or the resulting voltage from the tissue sample.