Method and apparatus for measuring electrical impedance and assessing biological condition of tissue sample

Abstract

The present invention relates generally to the field of biological condition assessment of tissues and tissue samples. In particular, the present invention relates to methods and devices for assessing ex vivo tissue samples, for example in medical pathology, and in particular to methods and devices for characterizing ex vivo tissue samples using precise electrical impedance measurements of tissue.

Description

Technical Field

[0001] This invention generally relates to the field of assessing the biological condition of tissues and tissue samples. Specifically, the invention relates to methods and apparatus for assessing ex vivo tissue samples, for example, in medical pathology, and particularly to methods and apparatus for characterizing ex vivo tissue samples using precise electrical impedance measurements of tissues. Background Technology

[0002] Epithelial tissue is composed of tightly packed, multilayered specialized cells, whose primary function is to form a physical and chemical barrier between the body and the external environment. The epithelial barrier protects internal tissues from environmental stress by minimizing water loss and preventing the invasion of pathogens, pollutants, toxins, and allergens through the skin or mucous membranes. Recent genome-wide association studies have demonstrated the crucial role of epithelial barrier function in various allergic diseases. Barrier defects have been reported in atopic dermatitis, asthma, chronic sinusitis, allergic rhinitis, eosinophilic esophagitis, and colitis. This defect is the starting point for chronic inflammation and allergen sensitization, allowing tissue-damaging factors to penetrate deeper tissues, thereby activating immune and inflammatory responses.

[0003] The skin has two barrier structures: the stratum corneum and tight junctions (TJs). The stratum corneum, the outermost layer of the epidermis, is composed of terminally differentiated keratinocytes (called corneocytes), which form a densely packed and extensively cross-linked lipid-protein matrix. Filamentin, nautiloidin, and inner nautiloidin play key roles in skin barrier function by interacting with intermediate keratin filaments. The most important component of the epithelial barrier is represented by the TJ, which closes the bypass gaps between adjacent epithelial cells on the apical side of the mucosa and at the level of the granular layer in the skin. The TJ is responsible for epithelial permeability by controlling the bypass flux of ions and larger molecules and physically separating two distinct compartments. The TJ is essential for proper epithelial cell differentiation and function and is deeply involved in signal transduction as well as epithelial proliferation and differentiation. They form large complexes within the cell membrane, containing three main types of transmembrane proteins: the claudin family, the tight junction-associated MARVEL family (MAL and related proteins used for vesicle transport and membrane junctions), and single-pass transmembrane proteins (such as the junction adhesion molecule (JAM) and the Coxsackievirus and Adenovirus Receptor (CAR)). Intracellularly, transmembrane proteins bind to various scaffold proteins (such as the zonal occludens (ZO) family) and subsequently link to the actin cytoskeleton.

[0004] Historically, the epithelial barrier has been assessed in vitro by measuring transepithelial electrical resistance (TEER), which represents the resistance of the epithelium to the passage of steady currents. For this purpose, epithelial cells were cultured in Transwell plates to the air-liquid interface (ALI). A sharp increase in TEER was determined by the degree of confluence of cells with high cell integrity, indicating low ion flux and a tight epithelial barrier; conversely, disruption of the junctional complex led to a decrease in TEER. Furthermore, TEER measurements showed a strong negative correlation with the permeability of fluorescein-labeled dextran, which has been confirmed in ALI cultures of different tissues.

[0005] There are few non-invasive methods available for assessing epithelial barrier function in vivo. One of these is quantifying trans-epidermal water loss (TEWL) across the stratum corneum. Although TEWL increases proportionally with the degree of damage, it is also affected by environmental factors such as humidity, temperature, season, and skin hydration. Other commonly used non-invasive methods include stratum corneum hydration, colorimetric methods, skin surface pH, and sebum assays. These provide information about different skin characteristics and / or conditions but do not directly measure barrier function.

[0006] Impaired epithelial barrier function is associated with a variety of skin and mucous membrane allergies, metabolic disorders, and autoimmune diseases. 1-5 Since the 1960s, the prevalence of these diseases has risen significantly in the Western world and continues to increase in developing countries. 6-8 The epithelial barrier hypothesis has recently been proposed to explain the cause of this type of growth. 6,9,10 Like many other diseases, allergic diseases stem from complex gene-environment interactions. Changes in genetic factors are unlikely to be the root cause of the increased prevalence, as such increases occur relatively rapidly. Instead, mounting evidence suggests that environmental factors play a crucial role. 7,11-13 In fact, epidemiological studies have shown that exposure to a variety of environmental factors (such as air pollutants, tobacco smoke, fragrances and preservatives) can contribute to the development and progression of asthma and other allergies. 7,12-15

[0007] Therefore, as is understood, developing improved and more accurate tools to analyze and assess biological conditions (such as epithelial-skin barrier function) of ex vivo skin samples in testing settings will be of great value. Summary of the Invention

[0008] One object of the present invention is to provide a medical device and method for analyzing and evaluating the biological condition of ex vivo tissue samples.

[0009] This and other objectives of the invention are achieved by the apparatus and method claimed in the independent claims. Further embodiments are defined in the dependent claims.

[0010] This invention is based on a deep understanding of the dielectric properties of various tissues, which makes it possible to develop methods for assessing biological conditions, such as epithelial barrier function in ex vivo skin samples. The systems and methods of this invention can be used, for example, in testing environments and diagnostic instruments for inflammatory skin conditions with barrier defects. Electrical impedance (EI) spectroscopy is a relatively new technique that has previously been used to characterize skin tumors. Electrical signals are transmitted through the skin at multiple depths and frequencies, and the impedance response is measured, influenced by specific characteristics of tissue integrity. Typically, when tissue structure and cellular composition change, an imprint related to the type of tissue alteration appears in the EI spectrum. In certain diseases, such as melanoma, tissue EI measurements have been used for diagnosis, disease progression assessment, and therapy evaluation. The inventors have now discovered that EI spectroscopy can be used on ex vivo skin samples to study and evaluate the effects of chemicals, such as detergents, on skin samples. Epithelial-skin barrier function, or skin barrier integrity, can be assessed / quantified. A damaged skin barrier is a precursor to many conditions, such as atopic dermatitis. Furthermore, the effectiveness of various treatments can be evaluated. The quantification of sensitivity to allergens and toxic / irritating substances in the skin and mouth is a further application of this invention.

[0011] According to one aspect of the invention, a test kit for analyzing ex vivo tissue samples is provided, comprising: a medical device holder configured to hold a medical device in a fixed position relative to the ex vivo tissue sample, wherein an electrode array of the medical device abuts the tissue sample at a predetermined pressure; the medical device further comprising: an impedance measurement unit connected to the electrode array and configured to transmit current to the tissue sample via electrodes of the electrode array to obtain tissue impedance data of a tissue region of the tissue sample, the tissue impedance data including a plurality of impedance values ​​measured in the tissue region; and an electronic computer communicating with the impedance measurement unit to control the activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement cycle, including performing multiple subsequent measurements at a plurality of predetermined frequencies in a predetermined spectrum; and for each measurement in the measurement cycle, controlling a first electrode of an electrode pair of the electrode array to inject current into the tissue sample or apply a voltage to the tissue sample, and controlling a second electrode of the electrode pair to measure a result current or a result voltage from the tissue sample.

[0012] According to another aspect of the present invention, a method for analyzing ex vivo tissue samples is provided, comprising the following steps: A medical device is positioned in a fixed location relative to an ex vivo tissue sample in a medical device holder, wherein the electrode array of the medical device abuts against the tissue sample at a predetermined pressure. The medical device further includes: an impedance measurement unit connected to the electrode array and configured to transmit current to the tissue sample via the electrodes of the electrode array to obtain tissue impedance data of a tissue region of the tissue sample, the tissue impedance data including multiple impedance values ​​measured in the tissue region; and an electronic computer communicating with the impedance measurement unit to control the activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement cycle, including performing multiple subsequent measurements at multiple predetermined frequencies in a predetermined spectrum; and for each measurement in the measurement cycle, controlling a first electrode in an electrode pair of the electrode array to inject current into the tissue sample or apply a voltage to the tissue sample, and controlling a second electrode in the electrode pair to measure a resulting current or a resulting voltage from the tissue sample.

[0013] In embodiments of the invention, the medical device includes a probe for measuring the electrical impedance of a subject's tissue. The probe includes a plurality of electrodes adapted to directly contact a skin sample and connectable to an impedance measurement circuit adapted to apply a voltage and measure a resulting current to determine an impedance signal. In a preferred embodiment, the probe further includes a switching circuit for selectively activating an electrode pair by connecting at least two electrodes to the impedance measurement circuit and disconnecting the remaining electrodes from the impedance circuit, wherein a voltage is applied to the two electrodes and a resulting current is measured between the at least two electrodes. The switching circuit is adapted to receive a control signal that instructs the switching circuit to activate the electrode pair according to a predetermined activation scheme, the predetermined activation scheme including a sequence of sequentially activating adjacent electrodes to progressively scan the subject's tissue at a first tissue depth, thereby obtaining an impedance signal from a selected tissue depth.

[0014] According to one embodiment, the probe is provided with electrodes having an elongated rectangular shape, which are arranged in parallel rows on the probe. However, several alternative designs exist. For example, the electrodes may be arranged in concentric rings or in a square configuration. The electrodes may be provided with microneedles, wherein each electrode includes at least one spike. The spikes are spaced apart from each other laterally and have a length sufficient to penetrate at least to and / or through the stratum corneum. In alternative embodiments, the electrodes are non-invasive, and each electrode has a generally flat surface adapted to be placed against the subject's tissue. Electrodes with microneedles can also be combined with non-invasive electrodes.

[0015] WO 01 / 52731 discloses an example medical electrode for sensing electrophysiological potentials generated within a living subject. The electrode comprises multiple microneedles adapted to penetrate the skin. The microneedles are long enough to reach and penetrate at least into the stratum corneum, have conductive surfaces, and are interconnected to form an array. EP 1 437 091 discloses a device for diagnosing biological conditions using impedance measurements of organic and biological materials. This device includes a probe comprising multiple electrodes, each electrode having multiple microneedles, each microneedle having a length sufficient to penetrate at least into the stratum corneum. The microneedles according to EP 1 437 091 are also “nail-shaped,” meaning they have a stem with a generally circular cross-section and a constant or gradually decreasing diameter, and a tip portion with a generally spherical or needle-shaped tip.

[0016] In embodiments of the present invention, the probe may have a spherical shape, that is, the probe surface with electrodes is spherical.

[0017] As will be understood by those skilled in the art, the steps of the method according to the invention and its preferred embodiments are suitable for implementation as a computer program or a computer-readable medium.

[0018] Generally, all terms used in the claims and specification should be interpreted according to their ordinary meaning in the technical field, unless otherwise expressly defined herein. All references to “a / an / the [element, device, component, unit, apparatus, step, etc.]” ​​should be openly interpreted as referring to at least one instance of that element, device, component, unit, apparatus, step, etc., unless expressly stated otherwise. The steps of any method disclosed herein need not be performed in the exact order disclosed, unless expressly defined otherwise.

[0019] Further objects and advantages of the present invention will be discussed below through exemplary embodiments.

[0020] Brief description of the attached figures

[0021] Exemplary embodiments of the present invention will be described below with reference to the accompanying drawings, wherein: Figure 1 This is a schematic block diagram of an embodiment of a medical device that can be used in a medical system according to the present invention; Figure 2 This is a schematic block diagram of the testing equipment according to the present invention; Figure 3 This is a schematic block diagram of the testing equipment according to the present invention; Figure 4 The test results obtained using the test equipment and method according to the present invention are shown; Figure 5 This is a schematic block diagram of the method according to the present invention. Detailed Implementation

[0022] The following describes exemplary embodiments of the invention. This description should not be construed as limiting, but is for the purpose of illustrating the general principles of the invention. Although specific types of probes, including microinvasive and non-invasive probes, will be described, the invention is also applicable to other types of probes.

[0023] Therefore, preferred embodiments of the invention will be described below for illustrative purposes with reference to the accompanying drawings, wherein the same reference numerals denote the same elements in the various views. It should be understood that the invention covers other exemplary embodiments constituted by combinations of the features described below. Furthermore, other exemplary embodiments of the invention are defined in the appended claims.

[0024] First refer to Figure 1 A general description of a medical device that can be used in a medical system according to the invention will be given. System 10 includes a medical device 5 that includes an impedance measurement circuit or unit 2 adapted to acquire impedance data of a tissue region or sample (e.g., an ex vivo tissue sample).

[0025] Tissue impedance measurements for acquiring impedance data of a target tissue region can be performed using a probe 8 integrated into a medical device 5 or a probe located externally to and connected to the medical device 5. Whether external or integrated, the probe may include multiple electrodes or an array of electrodes 14 adapted to contact the tissue to be analyzed (typically skin or a skin sample). Tissue impedance can be measured by applying an alternating current to a pair of electrodes and measuring the resulting current flowing through the same pair of electrodes. In an embodiment, a two-point measurement method is used, i.e., applying a voltage to a pair of electrodes and measuring the current. The remaining electrodes may be grounded or left to float freely. In embodiments of the invention, the probe 8 may include, for example, seven or five electrodes, e.g., in the shape of a rectangular electrode strip. The electrodes are adapted to directly contact the skin sample.

[0026] For example, the spacing between adjacent electrodes can be approximately 0.3 mm and the length approximately 5 mm. This configuration has proven practical and effective for detecting disease conditions such as malignant melanoma in terms of spatial resolution in both the lateral and depth dimensions. Therefore, the skin area covered by the probe is approximately 5 × 5 mm, or approximately 25 square millimeters, and the deepest tissue layer that can be reached at frequencies above approximately 100 kHz is approximately 2.5 mm, a depth that has proven clinically relevant. However, as those skilled in the art will understand, the probe may include more or fewer than five electrodes, such as three or seven electrodes. Furthermore, other electrode sizes and other spacings between adjacent electrodes are conceivable, such as electrodes with a width of approximately 4 mm and a length of approximately 8 mm.

[0027] In a specific embodiment of the invention, EIS measurements can be performed using the Nevisense® (SciBase, Sweden), a device established for skin cancer detection and skin barrier research. EIS is a measurement of the impedance of a material to the flow of alternating current at different frequencies. Specifically, tissue EIS values ​​reflect the pathophysiological state of the tissue. Normal and abnormal tissues differ in cell size, shape, orientation, density, water content, and cell membrane structure. This system measures electrical impedance at 35 different frequencies between 1 kHz and 2.5 MHz, at four depths, in 10 different arrangements, generating 700 data points per measurement. The applied voltage and current are limited to 150 mV and 75 μA, respectively, and its safety has been validated in humans. The wide frequency range allows for the collection of information on intracellular and extracellular tissue characteristics. The Nevisense® is equipped with a handle and disposable 5 mm × 5 mm electrodes.

[0028] Typically, by selecting adjacent electrode pairs, the outermost layer of a skin sample can be scanned in a stepwise manner; by selecting electrode pairs with greater spacing (i.e., electrode pairs with one or more intermediate electrodes), the resulting current path allows for measurements in deeper layers of skin. In this exemplary embodiment of the probe of the present invention, there are ten possible electrode pair selection methods. The ability to measure the outermost layer of skin in small, continuous partitions (determined at least in part by the spacing between adjacent electrodes and the frequency of the applied current) is crucial, as it allows for the detection of minute abnormalities in the skin and tissue. Each electrode of the probe can be configured in four different states, including injection (the electrode is configured to inject a measurement current into the tissue), measurement (measuring the resulting current from the tissue via the electrode), grounding (the electrode is grounded to prevent leakage of surface current when measurements are performed using other electrodes), and floating (the electrode is disconnected).

[0029] Medical device 5 may communicate with computer 4, but the computer may also be integrated into the medical device. Computer 4 may include storage unit 3 for storing, for example, acquired impedance data performed on a patient. Computer 4 may also include processing circuitry 7 adapted to process the acquired impedance data by linearly or nonlinearly projecting the impedance data to a lower subspace to remove insignificant variables, thereby reducing the number of variables. In a preferred embodiment of the invention, principal component analysis (PCA) is used. An alternative method is parallel factor analysis (PARAFAC). Furthermore, the evaluation can be improved by classification rules determined, for example, by linear discriminant analysis (LDA) or soft independent modelling of class analogy (SIMCA).

[0030] The computer 4 can also be configured to control the switching cycle / sequence of the electrodes 14 according to a predetermined activation program or scheme. The predetermined activation scheme may include activating specific electrode pairs or selected electrode pairs to perform multiple subsequent measurements according to a predetermined measurement cycle, or activating adjacent electrodes in a sequential manner to progressively scan the subject's tissue at a first tissue depth, the scanned tissue depending largely on the spacing between the activated electrode pairs, thereby acquiring impedance signal matrices from different tissue depths.

[0031] In addition, the electronic computer 4 can communicate with a display device to display, for example, biological conditions.

[0032] According to embodiments of the invention, each electrode is provided with microneedles, thereby forming a microneedle surface. As discussed above, the probe may comprise five rectangular regions or strips in a preferred embodiment. In this configuration, each strip contains, for example, an array of 57 (19 × 3) microneedles. Each strip is approximately 0.3 mm wide and approximately 5 mm long. The distance between adjacent strips is approximately 0.2 mm to 0.5 mm. Thus, the active portion of the probe is approximately 5 × 5 mm. The length of each microneedle (measured from its base) is approximately 100 micrometers, and its thickness is at least 20 micrometers. The electrode strips and microneedles can be made from a plastic material by a molding process. This material may be made intrinsically conductive or coated with a conductive layer (e.g., gold). In an alternative embodiment, the electrode strips and microneedles are made of silicon and coated with gold with a thickness of at least 1 micrometer. However, other materials with conductive surfaces and similar dimensions may also be suitable, but should be chosen to be biocompatible. Different probe concepts with such microneedles are described, for example, in the same applicant's patent applications EP 1959828, EP 1600104, and EP 1437091.

[0033] In another embodiment, the electrode strip is non-invasive and generally flat. For example, a probe concept including non-invasive electrodes is described in US 5,353,802, filed by the same applicant.

[0034] In other embodiments of the invention, the probe is spherical, meaning that the surface containing the electrode, which is pressed against the skin or tissue during measurement, has a spherical shape. This also means that the electrode may be at least partially spherical.

[0035] For example, the length of each spike can be from 0.01 mm to 1 mm. The spikes can be arranged on electrodes, which in turn are arranged on probes, wherein each electrode in some applications may include any number of spikes, from at least two to about 100 to 200. Preferred embodiments of the spike design are described in the same applicant's patent US 9,636,035. Such a spike configuration enables enhanced versatility and adaptability in terms of capacity requirements, and may also mitigate the problem of nonlinear effects of the stratum corneum.

[0036] The computer 4 can be configured to preprocess impedance data, such as reducing noise content and / or reducing dimensionality. Noise reduction may include reducing noise in the impedance amplitude and / or phase angle spectrum. For example, noise reduction can be achieved by using a Savitsky-Golay smoothing filter. Furthermore, preprocessing may include the detection and correction of spikes or other artifacts, thereby removing spikes or artifacts from the impedance spectrum (i.e., the amplitude and / or phase angle spectrum). For example, spikes can be detected using a median filter with an appropriate window size. Data points in the filtered data that differ excessively from the original data can be considered spikes or other artifacts and can be corrected, for example, by linear interpolation.

[0037] The electronic computer 4 may also include a pre-filter capable of rejecting measurements that do not meet one or a few specific criteria (e.g., cutoff values). The pre-filter can be applied to impedance data that has been corrected / adjusted, for example, through preprocessing as discussed above. For example, to prevent a measurement from being rejected, the amplitude and / or phase angle values ​​may both fall within a specified amplitude range or a specified phase range, respectively. If the object of measurement is human / animal skin, the criteria (e.g., range) can be set to reject non-physiological measurements. Furthermore, specific criteria can be set for specific values ​​associated with a specific frequency.

[0038] Furthermore, the electronic computer 4 may include a classifier to assess whether the quality of the measured impedance data is good. This process can be combined with preprocessing and / or pre-filtering to further improve data quality. Examples of such classification include evaluating the variation (e.g., variance or standard deviation) of amplitude and / or phase angle in different permutations at one or more frequencies. Another example is studying the absolute values ​​of amplitude and / or phase angle, such as the median or mean, or the skewness of amplitude or phase angle.

[0039] The medical device 5 may also include a communication unit 12, which is capable of transmitting / receiving data directly to / from the computer 4 (if externally located) and / or other external units 15 (e.g., laptop computers, handheld computers / devices, databases, cloud-based configurations, etc.) or via a wireless network 16. In this way, the device 10 can be provided with, for example, clinical data for evaluation. Furthermore, data acquired using the medical device 5 (e.g., impedance data from measurements) can also be downloaded to the external device 15 via the communication unit 12.

[0040] Now refer to Figure 2 This section will describe embodiments of the test kit or analysis system according to the present invention. In a preferred embodiment, the analysis system or test kit 20 uses, for example... Figure 1 The medical devices shown, such as Nevisense® (SciBase, Sweden), have been established for skin cancer detection and skin barrier research. Additionally, ex vivo human skin samples (such as NativeSkin® provided by GenoSkin Inc. (Toulouse, France, www.genoskin.com)) can be used in this method and test kit.

[0041] In an embodiment of the invention, the medical device 5 or probe 8 is arranged in a medical device holder 21 such that the electrode 14 is in contact with the surface of the skin sample 22. A pressure indicator or pressure sensor 24 may be connected to or integrated into the probe 8 to measure the pressure applied by the probe 8 to the skin sample 22. In a preferred embodiment, the pressure P (see...) Figure 3 The value should be low, or approximately 0 to 0.005 N, or approximately 0.001 N to 0.01 N. The probe 8 and electrode 14 are preferably arranged in the medical device holder 21 such that the angle 32 between the surface of the skin sample 22 and the axis perpendicular to the surface of the electrode 14 is approximately 90 degrees, or between 88 and 92 degrees. During measurement, the medical device 5 and probe 8 are preferably instructed to perform measurement cycles including measurements between the second electrode 14b and the fourth electrode 14d, between the second electrode 14b and the third electrode 14c, and between the third electrode 14c and the fourth electrode 14d, such as... Figure 3As shown. This measurement cycle is preferably repeated 30 to 60 times, with the first and last measurements discarded. However, as understood, other measurement sequences can be applied, such as using all electrodes 14a to 14e, or only the second and fourth electrodes, or alternating between the second and third electrodes and the third and fourth electrodes. Preferably, the measurement cycle is initiated after a predetermined time period (e.g., 15 to 20 seconds, or 10 to 30 seconds) following contact between the electrodes and the skin sample 22. As understood by those skilled in the art, a variety of other conceivable measurement cycles exist. The above session or cycle is merely an example. For example, if the probe has more or fewer electrodes, another measurement session or cycle would be used.

[0042] According to a preferred embodiment of method 50 of the present invention, in step 51, the ex vivo skin sample 22 is placed in the testing device 20. In step 52 (optional), the skin sample 22 is moistened with a solution (e.g., physiological saline). In step 53, the probe 8 or medical device 5 is positioned as described above. Figure 2 and 3 The discussed method is arranged in the medical device holder 21. In step 54, the medical device 5 is instructed to start a measurement session or cycle, for example by clicking "start" on the graphical user interface 7, and in step 55, the measurement is interrupted after a predetermined time period, a predetermined number of measurements, or by manually clicking "stop" on the graphical user interface 7.

[0043] exist Figure 4 The results obtained using the test kit or analytical system and method of this invention are shown. Impairment of epithelial barrier function is associated with various skin allergies and inflammatory conditions. Electrical impedance spectroscopy (EIS) is a non-invasive tool for detecting skin barrier function in vivo. In vitro experiments using human skin (NativeSkin®) are a model that demonstrates normal skin barrier function and includes almost all cell types. The aim of this study is to investigate how laundry detergents cause skin barrier dysfunction, and how... Figure 4 As shown, the results obtained using the system and method of this invention are significant. This will be discussed in more detail in the "Test Results" section below.

[0044] It should be understood that, in the context of this invention, the term "connection" with respect to electrically connected electrical components is not limited to the meaning of a direct connection, but also encompasses a functional connection with intermediate components. For example, on the one hand, if the output of a first component is connected to the input of a second component, this includes a direct connection. On the other hand, if an electrical conductor directly provides a signal from the output of the first component to the input of the second component (or via one or more additional components) with substantially unchanged signal, the first and second components are also considered connected. However, this connection functionally means that a gradual or sudden change in the signal output by the first component will cause a corresponding or modified change in the signal input to the second component.

[0045] Although exemplary embodiments of the invention have been shown and described, it will be apparent to those skilled in the art that various changes, modifications, or alterations can be made to the invention described herein. Therefore, it should be understood that the foregoing description and drawings of the invention should be considered non-limiting examples, and the scope of protection is defined by the appended claims.

[0046] Test Results

[0047] Impaired epithelial barrier function is associated with a variety of skin and mucous membrane allergies, metabolic disorders, and autoimmune diseases. 1-5 Since the 1960s, the prevalence of these diseases has risen significantly in the Western world and continues to increase in developing countries. 6-8 The epithelial barrier hypothesis has recently been proposed to explain the cause of this type of growth. 6,9,10 Like many other diseases, allergic diseases stem from complex gene-environment interactions. Changes in genetic factors are unlikely to be the root cause of the increased prevalence, as such increases occur relatively rapidly. Instead, mounting evidence suggests that environmental factors play a crucial role. 7,11-13 In fact, epidemiological studies have shown that exposure to a variety of environmental factors (such as air pollutants, tobacco smoke, fragrances and preservatives) can contribute to the development and progression of asthma and other allergies. 7,12-15 A growing body of research suggests an epidemiological link between occupational and household cleaning products and an increased prevalence of asthma, rhinitis, and allergic contact dermatitis. 16-29 Some cleaning products are also associated with an increased risk of worsening asthma symptoms. 30,31 Therefore, given the ubiquity of detergents in our daily lives, there is an urgent need to deepen our understanding of how even low-dose exposure to cleaning products can contribute to allergic reactions. Detergents can also remain on surfaces and on clothing after washing and rinsing, thus coming into contact with the skin and respiratory mucous membranes. 32 Interestingly, the post-rinse dose has been shown to affect the barrier function of epithelial tissue in asthmatic, chronic obstructive pulmonary disease, and healthy subjects. 32

[0048] One suggested mechanism by which detergents may induce allergen sensitization is through disruption of the epithelial barrier. In air-fluid interface cultures of human airway epithelial cells, laundry detergents have been shown to reduce barrier function and affect tight junction (TJ) expression. 32 These intercellular connections are composed of transmembrane proteins and cytoplasmic proteins (also known as scaffold proteins). They represent one of the major contributors to barrier function, closing the bypass spaces between adjacent epithelial cells at the apical side of the mucosa and at the granular layer level in the skin. 1,33,34 In the epidermis, in addition to TJ, the stratum corneum also forms a stronger physical barrier than the mucous membrane by expressing keratinizing capsule proteins such as filament-forming filaggrin (flg), structural protein loricrin (lor), its interacting partner involucrin (ivl), and hornerin. 35,36

[0049] This study aimed to evaluate the in vivo effects of household laundry detergents, even at low concentrations, on skin barrier integrity. We treated mouse skin with different dilutions of two household laundry detergents and assessed epidermal barrier function using electroimpedance spectroscopy (EIS), a method our group has previously described as effective for in vivo assessment of skin barrier function. 37 In addition, we measured transepidermal water loss (TEWL), a method previously validated for skin barrier integrity assessment for comparison. 38 Even when diluted 10,000 times, topical application of household laundry detergent to the skin resulted in a decrease in the skin barrier within just four hours, accompanied by significant changes in skin barrier-related molecules and pro-inflammatory molecules in the skin transcriptome and proteome, most of which were related to the results of skin barrier EIS and TEWL measurements.

[0050] In vitro Topical treatment of human skin

[0051] In vitro Human skin samples (NativeSkin®) were purchased from GenoSkin Inc. (Toulouse, France, www.genoskin.com). Adult cadaveric skin explants were obtained from three healthy female donors who underwent plastic surgery (aged 32, 37, and 57) and were biostably preserved for up to 7 days. All samples were provided in the form of 15 mm diameter circular natural human skin biopsies, housed in proprietary custom-made plastic inserts.

[0052] Detergent B was diluted at concentrations of 1:200, 1:1000, 1:5000, and 1:25000 (v / v volume ratio), and sodium dodecyl sulfate (SDS) was diluted at concentrations of 5 mg / mL, 1 mg / mL, 0.2 mg / mL, and 0.04 mg / mL (weight / volume). In vitro Human skin samples were treated with 60 μL of diluted household laundry detergent B and SDS. The detergent, SDS, and PBS were applied to the isolated human skin surface for 6 hours. NativeSkin sample surfaces were washed three times with PBS after stimulation.

[0053] EIS measurement

[0054] EIS measurements were performed using Nevisense® (SciBase, Sweden), a device established for skin cancer detection and skin barrier research. EIS is a measurement of the impedance of a material to the flow of alternating current at different frequencies. Specifically, tissue EIS values ​​reflect the pathophysiological state of the tissue. Normal and abnormal tissues differ in cell size, shape, orientation, density, water content, and cell membrane structure. This system measures electrical impedance at 35 different frequencies between 1 kHz and 2.5 MHz, at four depths, in 10 different arrangements, generating 700 data points per measurement. The applied voltage and current are limited to 150 mV and 75 μA, respectively, and its safety has been validated in humans. 39 A wide frequency range allows for the collection of information on intracellular and extracellular tissue properties. Nevisense is equipped with a handpiece and disposable 5mm x 5mm electrodes. A single Nevisense impedance measurement takes less than one minute, including first moistening the skin with saline solution, then applying the electrodes to the area to be examined for an 8-second EIS measurement.

[0055] for In vitro Human skin samples were moistened with PBS and dried with sterile gauze before each measurement. Optimized, electrical impedance tomography (EIS) was measured at 35 different frequencies between 1 kHz and 2.5 MHz at two depths in three different arrangements. EIS measurements were performed at 0, 6, 12, and 24 hours post-treatment. Three replicate measurements were performed at each time point.

[0056] References

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Claims

1. A medical system for analyzing ex vivo tissue samples, comprising: A medical device holder configured to hold a medical device in a fixed position relative to an ex vivo tissue sample, wherein the electrode array of the medical device abuts the tissue sample at a predetermined pressure; Medical equipment, which also includes: An impedance measurement unit, connected to the electrode array and configured to transmit current to the tissue sample via the electrodes of the electrode array to obtain tissue impedance data of a tissue region of the tissue sample, the tissue impedance data including multiple impedance values ​​measured in the tissue region; and An electronic computer, which communicates with the impedance measurement unit to control the activation of the electrode array, operates according to a stored program to: The application measurement cycle includes performing multiple subsequent measurements at multiple predetermined frequencies within a predetermined spectrum; and For each measurement in the measurement cycle, the first electrode in the electrode pair of the electrode array is controlled to inject current into the tissue sample or apply voltage to the tissue sample, and the second electrode in the electrode pair is controlled to measure the resulting current or resulting voltage from the tissue sample.

2. The system of claim 1, wherein the computer operates according to a stored program to apply an evaluation process, the evaluation process analyzing the biological condition of the tissue sample based on measured impedance data of the tissue region and evaluating the obtained impedance value dataset to provide results indicative of the biological condition status.

3. The system of claim 1 or 2, wherein the medical device includes a probe for measuring the electrical impedance of a subject's tissue, the device including a plurality of electrodes adapted to directly contact a skin sample and connectable to an impedance measurement circuit adapted to apply a voltage and measure a resulting current to determine an impedance signal.

4. The system of claim 3, wherein the probe includes a switching circuit for selectively activating an electrode pair by connecting at least two electrodes to an impedance measurement circuit and disconnecting the remaining electrodes from the impedance circuit, wherein a voltage is applied to the two electrodes and a resultant current is measured between the at least two electrodes.

5. The system of claim 4, wherein the switching circuit is adapted to receive a control signal from the computer, the control signal instructing the switching circuit to activate an electrode pair according to a predetermined activation scheme, the predetermined activation scheme comprising activating adjacent electrodes sequentially to progressively scan subject tissue at a first tissue depth, thereby obtaining an impedance signal from a selected tissue depth.

6. A test kit for analyzing ex vivo tissue samples, comprising: A medical device holder configured to hold a medical device in a fixed position relative to an ex vivo tissue sample, wherein the electrode array of the medical device abuts the tissue sample at a predetermined pressure; The medical device further includes: an impedance measurement unit connected to the electrode array and configured to transmit current to the tissue sample via electrodes of the electrode array to obtain tissue impedance data of a tissue region of the tissue sample, the tissue impedance data including a plurality of impedance values ​​measured in the tissue region; And an electronic computer that communicates with the impedance measurement unit to control the activation of the electrode array, the electronic computer operating according to a stored program to: apply a measurement cycle, including performing multiple subsequent measurements at multiple predetermined frequencies in a predetermined spectrum; and for each measurement in the measurement cycle, controlling a first electrode in the electrode pair of the electrode array to inject current into the tissue sample or apply a voltage to the tissue sample, and controlling a second electrode in the electrode pair to measure the resulting current or resulting voltage from the tissue sample.

7. A method for analyzing isolated tissue samples, comprising the following steps: A medical device is positioned in a medical device holder relative to an ex vivo tissue sample, wherein the electrode array of the medical device abuts against the tissue sample at a predetermined pressure. Impedance is measured by transmitting current into the tissue sample via the electrodes of the electrode array to obtain tissue impedance data of a tissue region of the tissue sample, the tissue impedance data including multiple impedance values ​​measured in the tissue region; as well as The application measurement cycle includes performing multiple subsequent measurements at multiple predetermined frequencies within a predetermined spectrum.

8. The method according to claim 7, further comprising: The computer is instructed to control the activation of the electrode array, which operates according to a stored program to: apply a measurement cycle, including performing multiple subsequent measurements at multiple predetermined frequencies in a predetermined spectrum; and for each measurement in the measurement cycle, controlling the first electrode in the electrode pair of the electrode array to inject current into the tissue sample or apply voltage to the tissue sample, and controlling the second electrode in the electrode pair to measure the resulting current or resulting voltage from the tissue sample.

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