Calibrating a Digital Camera for Use as a Scanner

Abstract

Analyte test system for quantifying the presence of an analyte in a specimen by immunochromatography An analyte testing system for quantifying the presence of an analyte in a specimen by immunochromatography comprises a camera test card depicting a test cassette (10) with immunochromatography, and a portable processor device (16) comprising a digital camera (16a), a light source (16b), and a processor (16c), the software and hardware (16c) configured to estimate the camera and object pose and measure light in the immunochromatographic region of interest. The system enables automatic calibration of the camera and certifies the scanner for use in point-of-care diagnostics.

Description

[Technical field]

[0001] The present invention relates to systems, methods and software for analyzing and determining the presence and content of analytes in test samples subjected to chromatography, particularly lateral flow immunochromatography, through the use of visible light. [Background technology]

[0002] Lateral flow immunochromatographic tests are widely used in analysis and diagnostics (see WO / 2019 / 215199 and references cited therein). The result of a lateral flow test is usually determined by the readout of one or more visible colored lines in the display zone. The intensity of the detectable line is due to the immobilization of the labeled analyte and is proportional to the amount of analyte present in the test sample. However, the optical intensity of the line cannot be easily determined because it depends on many factors, such as the width of the line, as well as the porosity, thickness, and type of the separation material, the pH of the running buffer, temperature, the aging of the sample, the light conditions, and the visual acuity. For this reason, special scanners have been developed to read the light intensity of the test reaction line under pre-controlled light conditions. On the other hand, personal digital devices and smartphones may be used for this purpose, which would bring additional advantages to point-of-care diagnostics and telemedicine. However, the multifaceted variability in the image quality of the cameras and the light source (flashlight, smartphone torch) limits their application. Although the common assay strip format provides rapid results, is easy to operate and cost effective, expensive calibration scanners and housing devices are still used to determine the optical intensity of the response line (see CN1455242A, WO2005 / 066624A1, EP 2 927 688 A1, EP 2 835 643 A1, EP 1 605 249 B1, US200211345).

[0003] US 9,350,956 B2 describes the use of a mobile phone to obtain drug test results instantly. WO2010 / 118124 describes a system for analyzing skin and hair samples, where image data is transmitted to a remote server for analysis. US 8,935,007 B2, EP 2 839 264 B1, EP 2 839 280 B1 disclose systems for point-of-care diagnostics based on a lateral flow chromatography test cassette, a smartphone with a digital camera, and software for the interpretation of the digital images. EP 2 646 809 B1 and EP 3 470 825 A1 teach systems in which the camera software is configured to reject a digital image if the degree of error associated with the rotational misalignment or skew of the imaged test device is greater than a predetermined value. US 9,390,237 B1 (Microlab Devices) and EP 2 923 335 B1 teach handheld camera systems that perform computer-implemented perspective transformation of recorded images based on position information relative to the test cassette. US 9,410,827 B2 and US 9,989,952 B2 claim systems in which the capture of a digital image of a test cassette also includes the capture of calibration data printed on the test cassette. The calibration data is encoded in a two-dimensional barcode and provides direct immunochromatographic assay calibration information that can be used for analysis.

[0004] Article 2 of EU Regulation 2017 / 745 defines a "medical device" as any instrument, apparatus, software, reagent or article intended for use in humans for the diagnosis, prevention, monitoring, prediction, prognosis, treatment or mitigation of disease, injury or disability. This also applies to systems and means for the in vitro testing of specimens derived from the human body. This means that each element of a point-of-care testing system requires certification (CE mark) in the European Economic Area (EEA). Therefore, every single consumer device and application used for point-of-care diagnostics needs to be checked, calibrated, recorded and certified. Meanwhile, smartphone cameras and their light sources (flashlights, smartphone torches) quickly fail due to fingertip tapping or general contamination, and mobile phones and their digital camera devices come in many different brands and versions, which makes the risk of digital images and results being tampered with very high. The entire diagnostic system needs to be considered as a joint set of mobile phone, camera device, camera software and calibrated lateral flow immunoassay. Such systems may be used not only in veterinary diagnostics but also in food control, environmental analysis and other technical fields. Flow-through immunochromatographic tests can be manufactured and certified using standard procedures. If digital images of the exact same test are created with a digital camera and then processed and scanned to obtain accurate quantitative results, the camera equipment is different, so the entire system and each component must be calibrated, documented and approved. This is problematic in the current situation. Summary of the Invention [Problem to be solved by the invention]

[0005] Said object is achieved by a computer implemented test system according to claim 1. Another aspect relates to a camera test card for use with the test system according to claim 1. Preferred embodiments are disclosed in the dependent claims. [Means for solving the problem]

[0006] The present application provides a computer-implemented system for determining the concentration of an analyte in a test sample subjected to immunochromatography, the system comprising: (i) a test cassette adapted to receive an immunochromatographic test and configured to display a region of interest (ROI) having one or more visible response lines and a control line (C) indicative of the presence and amount of an analyte (T) in the test sample, and (ii) a portable processor device comprising a digital camera, a light source and a processor configured to process digital images captured by the camera (16a) and display analytical results. The system, according to the present invention, comprises a camera test card showing one or more test cassettes, (a) data identifying the camera test card, (b) one or more fiducial markers enabling to find a correspondence between points on the camera test card and their projected digital images, and (c) one or more regions of interest (ROIs) printed comparable to the visible response lines of the immunochromatographic test, showing one or more response lines for a test (T) and a control (C), wherein (d) the processor is configured to sequentially analyze the stream of transient digital images for the presence and correspondence of several fiducial markers, and if several fiducial markers are found, (e) calculate a new coordinate system to estimate the position, tilt, rotational alignment, skew and distance between the camera test card and the processor device, and if the overall attitude, angle and distance are found within the tolerance range, (f) the processor is configured to look into the transient digital images for the regions of interest and their light measurements.and (g) offsetting the portable processor device so that the areas of maximum light reflection due to light emitted from the light source are located outside the region of interest (ROI) to eliminate interfering light reflections on the transient digital image, thereby adjusting the position of the portable processor device to provide optimized light conditions on the camera text card and within the region of interest (ROI); and (g) the processor is further adapted to adapt an exposure value (EV) of the digital camera to provide optimized light conditions within the region of interest on the transient digital image. (h) the processor is configured to scan and analyze a region of interest (ROI) on the temporary digital image to determine the optical intensities of the test (T) and control (C) response lines and record the ratio (C / T) of those response lines, (i) the processor is configured to take a digital image of another region of interest (ROI) to determine a second intensity ratio (C / T) of another different response line for the test (T) and control (C), and if both ratios are within the respective tolerance ranges, set the camera parameters to meet a predetermined range of optical intensities for a given different response line (T), thereby calibrating the digital camera and light source based on an optimally positioned portable processor device relative to the camera test card, and if the range of optical intensities of the given response lines is within the tolerance range, (k) the processor is configured to authenticate the camera device, light source and software suitable for a medical device for scanning and evaluating immunochromatographic tests for point-of-care diagnostics and telemedicine.

[0007] In some embodiments, the camera test card preferably shows a region of interest (ROI) in which the response line for a test (T) has a minimum value of light intensity, another region of interest (ROI) in which the response line for the test (T) has a high value of light intensity, and a response line for a control (C) in each ROI of the same light intensity.

[0008] In some embodiments, the system is based on an immunochromatographic test configured to display, for any test sample, a uniform response line of control (C) having a predefined optical intensity.

[0009] In some preferred embodiments, the system includes a processor configured to transfer the analysis data to a physician for remote medical care. The processor may also be configured to transfer credentials of the digital camera, light source, and software to a central device for storage and / or periodic updates. Most preferably, the processor is configured to receive instructions to perform the update of the credentials, if performed by the central device or the physician.

[0010] In some embodiments, the processor may be configured to determine the intensity ratio (T / C) of the response lines (T, C) from a number of stored transient images and select the median ratio (T / C) for quantitative analysis of the optical intensity of the response lines. This allows the system to be protected from any errors. It is also mentioned that the processor is configured to correct the region of interest (ROI) for any degree of error associated with a reference or marker present on the test cassette containing the immunochromatographic test. In some embodiments, the reference or marker may include a machine-readable representation of the data or a link to a data source that provides access to the characteristic data of the lateral flow immunochromatographic test.

[0011] In some other embodiments, the processor may be configured to use external data regarding the location of the region of interest having a visible response line (T, C).

[0012] The immunochromatographic test at the core of the system may be for determining the presence, concentration or amount of allergens in a sample: fecal calprotectin, serum calprotectin, vitamin D in blood or serum, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, albumin, fecal occult blood, gluten immunogenic peptides, bladder cancer markers, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Helicobacter pylori, influenza viruses A and B, troponin I, ringworm, ferritin, D-dimer, CRP, Group A hemolytic streptococcus, Group B hemolytic streptococcus, genetically modified organisms, cereals and products, chickpeas and products, peanuts and products, hazelnuts and products, macadamia and products, mustard and products, soybeans and products, sesame and products, walnuts and products, pistachios and products, lupins and products, celery and products, fish and products, shellfish and products.

[0013] Another aspect of the disclosure is software for use with a test system described as a mobile phone with a digital camera, a light source, and a processor, the software supporting taking digital images, reading machine-readable representations of data, exchanging data and image data with a remote server, and displaying information, data, and test results on a display. The disclosed software may sequentially process a plurality of transient digital images, analyzing each digital image for the presence of a predefined reference image for digital camera pose estimation, and if found and acceptable, the software may analyze each transient digital image for a region of interest consisting of a response line, and if found, the software may examine each region of interest for a measure of reflected light, as well as areas of maximum brightness, absolute brightness, brightness gradient, and if areas of dark pixels are detected, are set to be acceptable. The software may be configured to save image data of the transient digital images for further analysis of the region of interest.

[0014] In some embodiments, the software may be configured to determine the optical intensity of the response lines (T, C) within the region of interest and obtain calibration data and other deposited data for determining a quantitative result of a test sample subjected to an immunochromatographic test. Effect of the Invention

[0015] The disclosure provided is advantageous because it allows the incorporation of mobile phones in systems for point-of-care diagnostics and telemedicine, for example when used as scanners for immunochromatographic testing. The disclosure provides a smartphone-based system that can be adapted to multiple different tests, analytes and test samples and matrices. The immunochromatographic test system no longer requires visual evaluation of the response line or special equipment (holders, tripods, lamps, distance means, etc.) and can be performed anywhere, anytime with any test sample. The position and distance of the mobile phone camera and the light measurements (brightness and brightness gradient) are automatically adjusted, allowing non-experts to obtain more accurate quantitative results.

[0016] The present invention will be described with respect to its advantages, preferred embodiments and examples, but is not limited thereto, the scope of the invention being set forth in the following claims. [Brief description of the drawings]

[0017] [Figure 1A] Top view of an immunochromatographic test cassette showing the data matrix, barcode, sample application pad (S), and regions of interest with control and analyte reaction lines (C-control, T-analyte). Figure 1A: Negative result for the analyte of interest. [Figure 1B] Top view of an immunochromatographic test cassette showing the data matrix, barcode, sample application pad (S), and regions of interest with control and analyte reaction lines (C-control, T-analyte). Figure 1B: Positive result for the analyte of interest. [Diagram 2] This is a transparent image of an immunochromatography test cassette containing test results being photographed with a mobile phone (a smartphone with a camera and a flashlight). [Figure 3-1] 11 is a flowchart showing a procedure for calibration and authentication of a camera device using a light source (flashlight, camera torch). [Figure 3-2] 11 is a flowchart showing a procedure for calibration and authentication of a camera device using a light source (flashlight, camera torch). [Figure 4A] Shown is a copy of the camera test card showing the ArUco markers and a display of two test cassettes showing the regions of interest (viewports). The left test cassette has a region of interest showing a very faint response line (not visible in the printed copy) for the test analyte (T) and a standardized response line for the control (C), while the right test cassette has a predefined positive response line (T) and a standardized response line for the control (C), with both response lines for the control (left, right) having the same predefined optical intensity. [Figure 4B] Shows a photo of a printed camera test card taken under real light conditions using the mobile phone's automatic exposure values ​​(time, shutter). The photo is taken using the camera's flashlight / torch. On the printed camera test card are printed various ArUco markers and two test cassettes with different regions of interest (viewports). The left region of interest shows a faint positive reaction line for the test (T) and a standard reaction line for the control (C), the right region of interest shows a fully positive reaction line for the test (T) and a reaction line for the control (C) with identical optical intensity in both regions of interest. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] This system offers the advantage that the projected transient digital image of the camera test card can be analyzed, data that cannot be corrected eliminated, and compared to a predefined image of known primary data. Thus, the camera test card allows a multidimensional calibration of the system consisting of the camera system, the light or torch system, the exposure system, and the scanner software of the projected image. For this, the transient image data is analyzed, adjusted, and calibrated on the camera test card, and this step can be performed on any smartphone or tablet. Furthermore, the transient digital image is preferentially calibrated only for the region of interest, for example, the region containing the visible response line and the signal of the analyte (analyte), so that the image data is reduced by a factor of 50 to 100 compared to the conventional case. In these systems, a "clean image" (good alignment, contrast, color) of the test cassette is first selected and corrected, but in doing so, it is inevitable to record image data that cannot be corrected before evaluating the optical intensity of the visible response line. These correspond only to the content of the analyte in the test sample. By prioritizing the transient image in the region of interest and the image data that cannot be corrected, a reduction in the data is possible, since the region of interest is only a small part of the entire digital image. As a result, multiple transient images can be processed even on smartphones with limited built-in memory and processing power. Moreover, of the 2 billion cameras manufactured annually for the smartphone and tablet market, more than half are equipped with autofocus. One or more lenses in the camera are moved in and out using microelectromechanical actuators, and an algorithm calculates the image sharpness for that lens position. Therefore, various built-in autofocus algorithms may work against applications that have post-processing algorithms for perspective transformation and sharpness and contrast correction. The systems and methods described herein reduce the amount of transient image data, thus allowing for the immediate evaluation of light measurements in the region of interest. The light measurements in the region of interest are primary optical data that cannot be subsequently corrected by algorithms, since they are directly related to the amount of analyte captured in the response line.When a sequence of transient images of the region of interest is analyzed, images with poor reflected light measurements are discarded, thus lowering the risk of unstable decisions. In other words, the method analyzes the video stream of the region of interest and only those images for light measurements that do not show exceptional characteristics. This is only possible by taking advantage of the camera's characteristics, but also by reducing the data of the analyzed transient images.

[0019] This step also requires estimating the exact pose of the held camera device based on the projected digital image. This can be achieved by placing several ArUco markers or fiducial markers on the camera test card. This is also the first step for the optimization of the required illumination and exposure system, since a focused single light source on a mobile phone can cause interference reflections on the object and cause non-homogeneous illumination in the region of interest of the response line. Therefore, the system processor additionally searches for areas of maximum brightness (hereinafter also called hot spots) on the projected image, and if one of the hot spots is near or inside the region of interest, moves the rear image of the mobile camera that constitutes the frame targeting the actual test cassette so that the region of maximum brightness is finally well outside the region of interest, thereby achieving uniform illumination of the region of interest. The optimal position of the mobile processor device is then recorded with respect to one or more reference images (e.g., QR codes, bar codes, signs or letters) and regions of interest (ROIs) on the test cassette, also described herein as displays or viewports. The region of interest or display includes one or more visible zones, such as bands or lines, indicative of the presence and content of an analyte (T) in a test sample, and a control zone (C). The system is configured in functional combination with a portable processor device, such as a mobile phone, a digital camera, and a light source. The processor device is configured to process digital images captured by the camera and display analytical results. The processor is further configured to sequentially analyze a plurality of transient digital images for the presence of one or more reference images, and if found and correct, perform a pose estimation of the digital camera and the light source, and if within an acceptable range of distances and positions, perform a pose estimation of the digital camera and the light source.The processor analyzes each transient digital image for its region of interest (ROI) and its light measure, and further searches whether a hot spot (area of ​​maximum brightness on the projected digital image) is near or inside the region of interest, and in this case is configured to move the eye image in the frame of the test cassette and / or the region of interest so that the actual hot spot is located significantly outside the region of interest in order to obtain a uniform illumination of the region of interest, and further measures the distance between the eye image and the region of interest. The exposure value of the region of interest on the projected digital image is then gradually lowered until the illumination of the region of interest gives the best result. The best result is obtained with a scan in which the ratio of the light intensities of the test (T) and control (C) response lines on the projected transient digital image matches the predefined focus ratio of the light intensities of the test (T) and control (C) on the camera test card. This procedure is repeated for both simulated regions of interest on the camera test card, one with the faintest response line for the test (T) and the other with the predefined regular optical intensity for the test (T). As a result, the intensity ratio (T. faint / C control ) represents the optical bottom of the measurement range, and the intensity ratio (T regular / C control ) provides a two-point calibration of the measurement range of the scanning. If the system does not detect the optical bottom, the camera or illumination is not suitable for scanning the immunochromatographic test of the present invention. The system fails and is not certified for use in point-of-care diagnostics. If the intensity ratio measured with the camera test card is not within the acceptable range, the system cannot be calibrated and will fail. The camera test card can be used to calibrate the actual immunochromatographic test, since it allows the control response line to be uniform for any test sample. In other words, the control line is used to check whether the immunochromatographic test has been performed correctly, and the test response line (T faint and T regular ) is used as an inherent measure of light intensity.

[0020] When taking digital images, the camera lens and the internal light source (flash) must be within an acceptable distance from the test device. The camera must be located neither too close nor too far from the object. If the distance is small, the flash may overexpose the object and the wet areas on the test strip may become very reflective, which may prevent the analysis of the area of ​​interest. If the camera lens and the flash are too far from the object, the area of ​​interest will not be sufficiently illuminated and, most importantly, will be too small to be analyzed. A predefined acceptable distance and pose of the camera are necessary to obtain a properly illuminated object and also to obtain an area of ​​interest of the appropriate size for determining the light intensity of the test and control lines (T, C). If the distance and pose estimation is satisfactory, the area of ​​interest can be easily identified by the distance to the reference markings on the test device. The analysis of the transient digital image for the measurement of hot spots and their light is as described above and can then be limited to only the area of ​​interest.

[0021] The processor is configured to search for hot spots and determine measures of light reflected from the region of interest in terms of absolute luminance, luminance gradient, areas of shadows and dark pixels, and combinations thereof. It is functionally important that the processor is configured to retrieve and store a plurality of accepted transient image data of the region of interest, so that the light intensities for the tests and controls (T, C) can be determined from a plurality or set of accepted image data. The latter greatly improves the reliability and quality of the recorded image data. In a preferred embodiment, the stored digital images are further corrected for any degree of error related to any rotational misalignment or skew, prior to the determination of the optical intensities (T, C) for the tests and controls. This can be done, for example, based on the response lines, since they must be parallel.

[0022] Analyte lateral flow tests in preferred embodiments use a predefined amount of control to achieve a defined reaction line for the control (C) independent of the presence and content of analyte in the test sample. The control can be, for example, IgG or chicken IgY, which does not recognize and react with the analyte but binds at the control line. The intensity of the control line therefore does not change and is independent of the amount of analyte in the test sample. On the other hand, variability occurs when the sample contains a relatively large amount of analyte. As the analyte is bound by the immunoreactive partner, less unbound immunoreactive partner is available to be bound at the control zone. This results in a weaker intensity of the control zone. This does not happen when a predefined amount of control is added.

[0023] In a most preferred embodiment, the processor is configured to determine intensity ratios (T / C) of zones (T, C) from the multiple stored images and select a median ratio (T / C) for quantitative determination of an analyte in the test sample.

[0024] If the analyte test system consists of a test device with a reference image with a machine-readable representation of data (QR code or barcode), such code may encode or provide access, for example via the internet or by direct telephone line, to immunochromatographic test characteristic data on a server, which can be used to correlate the determined median intensity ratio (T / C) with the quantitative determination of the analyte in the test sample. In a preferred embodiment, the system is therefore configured to exchange data and image data with a remote server.

[0025] The analyte test system may further include a processor configured to employ data relating to the sharpness and contrast of the displayed reference image or data relating to the position of the displayed reference image to identify the position of the visibility zone (T, C) within the region of interest.

[0026] The analyte test system for evaluating the presence of an analyte by lateral flow chromatography comprises a test cassette adapted to accommodate a lateral flow chromatography strip. The strip displays one or more visible lines or bands or response zones indicative of the presence of an analyte in the test samples and controls after use. The test cassette can display one or more reference images, such as bar codes or QR codes, and / or indicia or text. The smartphone must comprise a digital camera, a flash, and a processor. The processor is configured to process the captured image data of the test cassette, the one or more reference images, and the visible lines, zones, or bands for analytes present in the samples and controls. The processor is configured to first analyze image data for one or more reference images to evaluate the distance between the digital camera and the reference images, and if the distance is within a predetermined range, then the processor analyzes the image data for signals of regions of interest and / or visible controls, and if found, for characteristics of reflected light from the chromatography strip (region of interest). The processor is configured to reject any image data if any value assessment relating to the characteristic of the light reflected from the chromatography strip is outside a predetermined range, whereby only captured image data is retrieved and stored for quantitative analysis of analytes that have been pre-tested to be good and valid with respect to the measurement of the light reflected from the chromatography strip. The chromatography strip corresponds to a region of interest.

[0027] The processor may be configured to analyze the characteristics of the reflected light in terms of luminance gradient, absolute luminance, shadow, sharpness, absorbance, transmittance, contrast and combinations thereof. A plurality of sets of valid image data may be acquired and stored by the processor device and processed to select one set of image data for evaluating and quantifying the signal for the analyte. More precisely, the mobile processor device may be configured to supplement and retrieve a non-uniform number of sets of accepted image data, preferably 1-13 sets, more preferably 3-11 sets, most preferably 5-9 sets, so that one set can be selected according to a median value. That region of interest may then be used to evaluate and quantify the presence of the analyte in the test sample. Of course, the processor may analyze each accepted transient image for optical intensity in the visible zone and obtain median values ​​for the T-zone and C-zone.

[0028] In other embodiments, the reference image may be one or more selected from a printed shape, a logo, a barcode, a QR code, visibility lines and dots, the borders of the cassette, shapes and designs on the housing, a chromatography strip, control lines, shapes and designs on the casing. The reference image may be calibration information, the batch number and / or expiry date of the test, or a data matrix (one or more barcodes or QR codes) required to access a website containing all technical data and calibration information regarding the lateral flow test. The smartphone may be capable of sending and receiving data to a remote processing device or server.

[0029] In one embodiment, the processor may be configured to determine an error value range for a characteristic of light received from an area of ​​interest on the test cassette, the error value range being determined by comparing the captured image data to a predetermined characteristic of light reflected from the test cassette.

[0030] In another embodiment, the processor may be configured to add one or more pixel values ​​within the acquired image area (region of interest) to identify the location of the control and test sample signals. The processor may be configured to perform a peak search within the acquired image area (region of interest), the processor being adapted to quantify the intensity of the signals for the control and test samples by calculating the peak height or peak area and determine the concentration of the analyte in the test sample. The mobile processor device may be configured to employ different proportions of contrasting colors or items printed on the casing or test cassette or both to compare the image data captured by the mobile processing device. Most preferred is to convert the RGB data to grayscale for further analysis and quantification of the optical intensity of the visible zone (T, C).

[0031] In another embodiment, the analyte testing system may be adapted to measure allergens in fecal calprotectin, serum calprotectin, vitamin D in blood or serum, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, albumin, fecal occult blood, gluten immunogenic peptides, bladder cancer markers, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium africanum, Helicobacter pylori, influenza viruses A and B, troponin I, ringworm, ferritin, D-dimer, CRP, Group A hemolytic streptococcus, Group B hemolytic streptococcus, genetically modified organisms, cereals and products, chickpeas and products, peanuts and products, hazelnuts and products, macadamia and products, mustard and products, soybeans and products, sesame and products, walnuts and products, pistachios and products, lupins and products, celery and products, fish and products, shellfish and products.

[0032] Another aspect of the present disclosure relates to software for use with a mobile processor device, including a digital camera, a light source, and a processor, for assessing and quantifying the presence and content of an analyte in a test sample by lateral flow chromatography, the software may include means for imaging a test cassette containing a lateral flow chromatography strip and adapted to display one or more visible signals for the presence of an analyte in the test sample and, for control, the test cassette also displays one or more reference images, the software being configured to process the images captured by the camera to obtain image data of the test cassette. The software acquires the one or more reference images and visual signals for the analytes and the controls present in the sample, the software is configured to first analyze the image data for the one or more reference images to evaluate the distance between the digital camera and the reference image, and if found, the software is configured to analyze the image data for the signal of the visible control, and if found, the software is configured to analyze the image data for the characteristics of the light reflected from the chromatography strip, and the software is configured to reject any image if the evaluation of any value related to the characteristics of the light reflected from the chromatography strip is outside of a predetermined range, so that only captured image data is taken for quantitative analysis of the presence of analytes, which image data has been previously tested as good and valid for the characteristics of the light reflected from the chromatography strip. The software may be configured to analyze the characteristics of the reflected light for luminance gradient, absolute luminance, shadow, sharpness, absorbance, contrast, and combinations thereof.

[0033] A preferred embodiment relates to software for use in a mobile phone with a digital camera, a light source and a processor, the software supporting taking digital images, reading machine readable representations of data, exchanging data and image data with a remote server and presenting information, data and test results on a display, the software is configured to sequentially process a number of transient digital images, analyze each digital image for the presence of a reference image having machine readable data, determine the distance between the reference image and a digital camera (16a), if found and acceptable, the software analyzes each transient digital image for a region of interest, if found, examines each region of interest for measures of reflected light, if absolute luminance, brightness gradient, areas of dark pixels are found acceptable, the software is configured to store image data of the transient digital image for further analysis of the region of interest.

[0034] The software is preferably configured to determine the location and optical intensity of the visibility zones (T, C) within the region of interest, and may be further configured to obtain calibration data and other deposited data for determining quantitative results for test samples subjected to lateral flow chromatography.

[0035] The evaluation of the visual signal for the accurate quantification of the analyte requires first the determination of the distance between the lateral flow chromatography test and the camera (if performed with a portable optical device) and then the determination of the light conditions of the imaged chromatographic and / or test cassette. The image itself can be corrected beforehand, if necessary, for example with regard to any rotational deviation or tilt, image stabilization, light temperature and any color tone data. Even the histogram of the image can be post-processed. In terms of the quantitative evaluation of the zones, histograms and gradation data are not very useful if there are shadows in the area of ​​interest, which cannot be automatically removed. Therefore, before a rational evaluation and quantitative evaluation of the zones for tests and controls, the primary measurement of the light in the area of ​​interest (on the chromatographic strip) must be uniform and good. This can be achieved by pre-establishing the application conditions based on the measurement of the light reflection in the area of ​​interest, for example in an area with a colored band or zone.

[0036] When the software determines that the (transient) image has sufficient brightness and the distance is within acceptable limits, the transient image is acquired and stored for post-generation and zone quantitative evaluation. Batch or lot specific calibration data can be used to convert the accepted image data into concentration data of the analyte in the test sample. The evaluation results can be stored and / or appropriately presented on the screen of the handheld camera device. The results may be presented on the display or together with an interpretation. If it is a smartphone, the determined concentration of the analyte in the sample can be transmitted to the supervising physician. This may be mandatory for certain diagnostic analytes and tests. The system can be advantageously used for telemedicine and for the analysis of stool samples. The system relies on and is adapted to the typical hardware of a smartphone, so no external hardware is required other than for the lateral flow test.

[0037] The present disclosure relates to a system for constructing a lateral flow test for use with a personal portable camera terminal (smartphone). The system was originally designed to allow patients and physicians to objectively evaluate the results of lateral flow chromatography performed on biological samples. A representative biological sample is stool, which is difficult to collect. Collection of a stool sample can be a crucial step in determining the diagnosis and appropriate treatment for suspected digestive diseases such as infectious diarrhea. Stool samples may also be required for non-microbiological tests such as fecal occult blood tests (FIT) and FOBT, which are methods for early detection of colon cancer, and for properly monitoring the treatment of inflammatory digestive diseases such as celiac disease. However, collection of stool samples is difficult, and stool samples are generally or rarely collected in a doctor's office. Patient feedback has shown that this process is difficult, even though early fecal occult blood testing has been shown to reduce mortality. Compliance is generally poor (rarely more than 60%), and reasons cited for patient non-compliance include lack of support for returning collected specimens and embarrassment. Patients are not likely to want to drive across town or overland to provide fresh or frozen stool specimens. Remote analysis at the patient's fingertips may therefore be a solution to overcome these barriers. While the testing system of the present invention can be readily adapted to many other specimens, such as food testing, veterinary, agricultural, horticultural, environmental testing, drug testing, etc., this disclosure focuses on stool testing as a representative example that is considered non-limiting.

[0038] The analysis of the visual results is carried out by means of the camera built into the mobile phone or mobile terminal, which analyzes the color information or gray values ​​of the lateral flow test, processes them by algorithms and gives them as numerical values, color codes or letters. In this way, patients or consumers can perform the quantitative lateral flow test themselves and obtain the appropriate information for diagnosis and recommendations. In the case of important clinical parameters and analytes, the system can be adapted and used for automatic certification and authorization, so that the test and analysis are under the supervision of a doctor. In this way, for some analytes, simply the concentration of the analyte present in the sample is indicated, while for others the result shown to the user requires a visit to a doctor's office or hospital. In this way, the smartphone may be necessary for the proper assessment of a disease, and the concentration of allergens or pollution may be given as the absolute value of the content of the analyte in the sample.

[0039] The present disclosure further relates to the longitudinal monitoring of health disorders, particularly chronic gastrointestinal disorders, or inflammatory conditions, and the effectiveness of treatments or medications that may change over time. Results can be displayed visually or numerically as needed, and simultaneously transmitted to a supervising physician for remote medical care, thus saving patients and physicians significant time by avoiding unnecessary consultations, and reducing the embarrassment associated with collecting and analyzing stool samples.

[0040] [Definition] In this context, the term "test cassette" refers to any type of housing or envelope as typically used for lateral flow chromatography strips. The test cassette may have multiple openings to allow application of a liquid with a test sample, and a viewing port for an area to display the results after separation. The term "test cassette" may also refer to the unit that constitutes a lateral flow chromatography strip or a lateral flow immunoassay.

[0041] The term "processor" refers to electronic circuitry that executes the instructions of a computer program by performing arithmetic, logic, control, and input / output (I / O) operations specified by the instructions.

[0042] The term "reference image" refers to codes containing visually recognizable forms, shapes and / or data, such as, but not limited to, printed corporate logos, captions, machine readable fonts, bar codes, QR codes, color codes, batch designations, and geometric elements such as distance lines and dots, borders, outer edges, inner edges, shape designs, test and control lines, etc.

[0043] The term "visible signal" refers to a visually discernible signal, color, shape, geometric structure, line, dot, or reaction zone that appears upon conducting a test reaction on a lateral flow chromatography strip.

[0044] The term "control" refers to a visually recognizable signal, color, shape, geometric structure, line, dot, or zone that confirms that the test has been performed correctly. The present disclosure refers to a control zone on a lateral flow test. Conventional lateral flow tests use the binding of a non-reactive analyte-specific antibody as a control. More precisely, it is a zone with an immobilized antibody that recognizes a species or class-specific antibody, for example, an immobilized goat antibody that binds to a monoclonal mouse anti-analyte antibody.

[0045] A preferred embodiment of the present disclosure contemplates providing a defined amount of non-analyte specific antibody in the application pad of the lateral flow test to obtain a control zone of predetermined intensity, i.e. an intensity that is independent of the amount of analyte present in the test sample. As a result, the term "control" not only refers to conventional controls but also includes "internal standards" of standard intensities in the visual zone (C) for external and internal calibration.

[0046] The term "brightness" refers to the visual perception attribute of a light source appearing to shine or reflect light. In other words, brightness, in this disclosure, refers to the luminance flux, or more precisely, the luminous flux perceived when looking at a target. The target is, in this application, the area of ​​interest of the lateral flow chromatography, hereafter referred to as the viewport or visual area of ​​interest. The term brightness, as used herein, consists of the color appearance of the test and control zones and the generally "white" chromatographic material on the membrane. As is well known, the light reflection or brightness of the target can cause different perceptions of brightness in different situations. This is why the visual interpretation of the lateral flow chromatography zones is not easy. It may be interpreted depending on whether the target is still wet or dry, depending on the ambient light conditions and the shadows in the photo. Among the various color appearance models, we preferably program the software using the RGB color space, where brightness is the arithmetic mean of the red, green, and blue color coordinates, but some components make the reflected light appear brighter than others. Most camera software uses the RGB color space, but there are also representations that are closer to human vision. However, the latter is not relevant for determining the intensity of the signal zone. It is good to use absolute values ​​of the lightness or darkness of the signal zone, but relative values ​​or ratios of the test and control zones are used. To determine the brightness of the viewport or area of ​​interest, a further important parameter is the gradient of brightness, since the "absolute brightness" or luminance (reflected light) must be homogenous over the length and width of the area of ​​interest. Brightness is determined by comparing the brightness of various pixel areas in different areas of the viewport or area of ​​interest. In addition, the viewport is examined for shadows and untypical dark areas. As a result, the examination of the brightness of the viewport, its brightness gradient, and the search for untypical dark areas (photographic shadows) are performed on the transient image before the image data is retrieved and stored for further processing and examination of the visual zone of the lateral flow test.

[0047] The term hotspot refers to the area of ​​maximum brightness on the object and on the projected digital image scanned over it. If the area of ​​maximum brightness overlaps with the area of ​​interest, it should be considered as a direct reflection of light from the light source of the camera or processor device (flashlight or cell phone torch).

[0048] A shadow is a dark area where light from a source is blocked by an opaque object. It occupies the volume behind an illuminated object.

[0049] Sharpness (also called acutance) describes the visual perception related to edge contrast in an image. Sharpness or acutance is related to the amplitude of the brightness derivative with respect to space.

[0050] As used herein, the term "absorption" refers to the physical process of absorbing light. Absorptivity is the common logarithm of the ratio of incident radiant power to transmitted radiant power through a material. It measures the attenuation of the transmitted radiant power.

[0051] The transmittance of a material surface is its effectiveness at transmitting radiant energy.

[0052] In vision, contrast refers to the difference in appearance between two or more areas viewed simultaneously or successively.

[0053] The term "transient image" refers to the transient data in the volatile memory of the processor for evaluating whether the transient image satisfies a first order condition of light reflectance or brightness that allows correlation of the image data with the analyte concentration in the test sample. Prior to retrieval and storage of the image data, the temporary image is cropped and the viewport is cropped. The cropping of the relevant data will be done using reference images (data matrix (QR code), barcode, marker lines, etc.) as they are in a fixed or known spatial relationship to the region of interest or viewport. The data matrix or barcode may also provide access to a server for the exchange of data, especially calibration data.

[0054] The terms "retrieval", "acquisition", "recording" and "storage" of image data refer to the processing of images taken from unexpired test cassettes whose calibration data is available on the server after verification in terms of light scales (brightness, brightness scale, shadows).

[0055] The test system consists of software for a mobile device or smartphone with a camera and a cooperative lateral flow test adapted and designed to meet legal, functional and practical requirements. Since the smartphone is the user's personal smartphone, the method must be compatible with a large number of processor and camera device types, generations and series. Therefore, each element of the test kit and the application software must be tested on the user's personal smartphone or camera to avoid harm to the user. The personal smartphone or camera must first be checked, which can be done for the system and method immediately with a camera test card as shown in Figures 4A and B, on which the areas of interest with defined visibility zones (T, C) are printed as well as the permanent reference markings of the test device. The camera and processor must be able to identify and analyze the areas of interest as well as the print intensity of the visibility zones, and only if this is performed correctly will the application software identify the digital camera as approved and acceptable.

[0056] The software and / or smartphone may then be configured to capture an image from the area of ​​interest of the lateral flow test. For this purpose, the lateral flow strip may be placed in a cassette or envelope with a specific opening for the defined application of the test sample and the readout of the result in the area of ​​interest. The cassette, housing or envelope is a preferred embodiment for reasons of protection against fingerprints etc., but is not an essential feature of the system. The software checks the transient image with regard to the distance between the object (lateral flow test) and the camera and the measurement of the light in the area of ​​interest, and with regard to the subsequent quantification of the analyte, before the image is actually taken, stored and subjected to further analysis and quantification of the coloration or optical intensity of the zone. The main parameters for quantifying the intensity of the coloration in the test and control zones are the distance and the light conditions of the area of ​​interest in the lateral flow test. These are relevant for determining the intensity of the visual signal (line, band, zone). If the lateral flow test complies with many standardizations, the intensity measured in that area is related to the amount of the analyte in question. They are lot and production dependent and therefore need to be carefully determined in advance. It is essential that the reproducibility of the signal multiplied by the reproducibility of the amount of sample applied to the test strip is lower than the tolerance for the quantification of the analyte. If these conditions are met and an internal reference is provided by the control zone, the intensities of the test and control zones can be accurately determined even with a "simple" smartphone camera and the ratio of their intensities is related to the amount or concentration of the analyte in the sample. The intensities of several zones can be used to extend the measurement range. From the manufacturer's point of view, it is easier to have several test zones than to control the breathing of the zones. The machine reading of the reference image, more precisely a barcode or a data matrix (QR code), not only evaluates the distance and the general light conditions but also captures data such as lot number, expiry date, calibration, which can be used to further process and analyze the image. If the visible zone required for a correct reading of the signal is sufficiently illuminated, accurate results can be obtained.The distance between the handheld camera (processor device) and the object (test cassette or area of ​​interest) must also be within a predefined required range in order to mechanically evaluate the light reception on the image.

[0057] The present invention provides a system and software that provides guidance to users without technical talent or expertise, upgrading a mobile phone into an analytical and diagnostic tool that anyone can use. The software can not only perform a quantitative analysis of the bands or zones of the lateral flow test, but also provide instructions on the correct way to perform the lateral flow test and the pre-analytical preparation of a particular biological sample. With the disclosed system, analytical tests can be easily performed at the point of care and the results can be almost instantly imaged, quantified, and / or transmitted to a remote server. The results can be communicated from the server to a general practitioner or medical center, which can also provide or verify the necessary certificates for remote medical care. The remote server can perform advanced image processing analysis or provide diagnostic recommendations. The server can also transfer, return or store the analyzed images and reports locally or in the cloud for quality control, statistics, etc. reasons, and can also store conditions, parameters or attributes related to the tested sample (e.g. time, date, person, GPS data, personal data, other details, etc.).

[0058] As shown in Figure 1, the test cassette (10) has an opening for a region of interest or viewport (12) with visible response lines for control (C) and test (T) (Figure 1A,B). There may be multiple test lines (T) transverse to the flow direction to extend the measurement range of analytes. The top surface of the test cassette (10) further displays two types of reference images (14): a data matrix or QR code (top) and a barcode (bottom right). A spot (S) on the application pad may be applied with a defined amount of test sample for chromatographic separation.

[0059] As shown in FIG. 2, the test system comprises a test cassette (10) and a portable processor device (16), for example a mobile phone (16) with a digital camera (16a) and a screen or display (22) for its user. The mobile phone (16) comprises a light source (16b) and a processor (16c). The mobile phone (16) corrects a transient image including the region of interest or viewport (12) and the reference image (14). The transient image can be viewed by the user on the "eyepiece" display (22) of the smartphone (22) and, if the distance and light scale of the region of interest are correct, the image will be acquired and stored by the mobile phone. In addition, the mobile phone (16) reads data contained in the reference code that provides data for identifying lot numbers, expiration dates, etc., and data for accessing a server that holds the calibration data for retrieval and analysis of the stored image data. The data for accessing the server may be provided in machine-readable form by the reference image (14). The reference images - a QR code and a barcode - are printed adjacent to the test cassette (10) on the casing (18).

[0060] In general, an analyte test system for assessing and quantifying the presence of an analyte in a sample by lateral flow chromatography consists of a test cassette adapted to accommodate a lateral flow chromatography strip and display one or more visible zones for an analyte (T) as well as a control (C) and an internal standard. The test cassette can also display one or more reference images. The system further comprises a mobile terminal comprising a digital camera, a light source and a processor, the processor (16c) being configured to process the transient images captured by the camera (16a) to inspect the transient image data of the test cassette and the one or more reference images. The processor is configured to first analyze the image data for one or more reference images to evaluate the distance between the digital camera and the reference image, and if within a predefined range, the processor analyzes the image data for light measures (brightness, light gradient, luminance, shadow) in the region of interest or viewport. In other words, the processor is configured to first analyze the image for properties of the light reflected from the chromatography strip before performing further analysis. The processor is configured to reject or ignore all images if any value assessment related to a characteristic of the light reflected from the chromatography strip is outside a predetermined range, and only image data that is pre-tested as good and valid data for the measurement of the light reflected from the chromatography strip will be retrieved for quantitative analysis.

[0061] The software may be configured with an acceptable error or variance range for the characteristics of reflected light (luminance). The primary parameters in this context may be selected from the group consisting of absolute brightness (luminance, reflection), brightness gradient in the region of interest (viewport), darkness or shadows corresponding to photographic shadows in the region of interest. The secondary parameters consist of sharpness, absorbance, transmittance, contrast, and combinations thereof. If the measurements are within the acceptable range, a captured transient image can be acquired and processed to analyze the intensity of the visual zone. The software may be configured to sequentially evaluate the brightness and brightness gradient in the viewport region (12). Preferably, the software is configured to sequentially evaluate the viewport region (region of interest) and also the shadow regions (photographic shadows). The software may further be configured to sequentially evaluate not only brightness, brightness gradient, shadows (dark areas), but also reflections (isolated areas of high luminance) and sharpness.

[0062] Following the retrieval of the stored images, the image deviations can be corrected on the basis of the control (C) or reference image (14) on the test cassette (10). This step is advantageously performed after the evaluation of the primary light parameters. Outside of the prior art, when the intensity of the visual zone needs to be quantified, the terminal does not acquire images that do not meet the predetermined criteria for the light measurement, since it cannot be corrected in advance. A precise and predetermined amount of light is essential for the quantitative analysis of analytes by lateral flow chromatography. The correction of light characteristics after image acquisition is always arbitrary and prone to errors, when the camera software creates a "beautiful picture" according to the standards of photography. Only with the appropriate brightness around and above the visual signals (12) corresponding to the control (C) and the analyte (T), and likewise for the reference image (14) on the test cassette (10), a true evaluation and quantification of the concentration of the analyte in the sample is possible.

[0063] As mentioned above, the reference image (14) may be a machine-readable representation of data, such as one or more bar codes and QR codes. The machine-readable data may consist of calibration information, lot numbers and / or expiration dates, but preferably provides information to access a server so that all this data can be exchanged and retrieved from the cloud. The system software may also be configured to transmit the acquired image data and test data to a remote processing device. In the case of personal information, it may be desirable to provide a certificate or authentication data for transmitting the information encrypted.

[0064] The mobile processing device (16) may be configured to acquire, store and / or process a non-uniform number of images of the viewport and lateral flow tests, preferably 1-13, more preferably 3-11, and most preferably 5-9, so that the median value of the test zone, or more precisely the median T / C, can be used as the final test result. Other than in the prior art, the disclosed system and software do not capture multiple images with different exposure settings and combine them to create an image with a "higher dynamic range". In other words, the disclosed system relies on the evaluation of the actual images, and images with exceptional measures of light and reflection are ultimately ignored by selecting the median value of the multiple images captured and processed.

[0065] The processor 16c of the handheld terminal and processor device 16 may be configured to have a tolerance for the brightness or luminance on the test cassette 10 or viewport. This means that the software and device are configured to only reject an image if the light measurements and the brightness in the relevant area are outside of the tolerance and / or vary vertically and longitudinally. The tolerance can also be determined from a reference image 14 on the test cassette 10, but this is less preferred as it does not take into account the wetting of lateral flow chromatography.

[0066] In other embodiments, the processor (16c) may be configured to perform a peak search to quantify the intensities of the control (C) and analyte (T) zones to locate them and determine their ratio via peak height (preferred) or peak area (integrated signal) or any Gaussian range of the peaks, and then determine the concentration of the analyte in the test sample by correlation with the corresponding calibration value.

[0067] The software can be used in any handheld terminal with a camera and a light source. The only other requirement is that the processor device must allow the examination of a series of transient images (video sequences), which is mainly a software issue and not a hardware issue. The system can theoretically be used for any commercially available lateral flow test, where there is a visual band or signal zone. In practice, lateral flow tests must be standardized most carefully, with a constant thickness of the membrane and the separation material on it, and with regard to the application pad, the conjugation zone and the zone of immobilized receptor. The marker dyes must be selected so that the visual signal is obtained properly and proportionately. For accuracy and safety, each POCT may require a machine-readable data matrix or barcode for lot identification, so that lot-specific calibration data can be used. The image with the area of ​​interest of the POCT or rapid test is finally retrieved and stored by the processor or camera. The image is usually returned from the camera in the form of spatially resolved RGB values. That is, for each pixel, a red, green and blue value is delivered. Calculations can be performed in RGB, HSV, HSL, Lab, CMYK, or other color spaces.

[0068] Normalization of the image and removal of rotation errors can be done later, since it simply requires moving pixels according to an algorithm. This can be done by the cassette pictogram and pre-programmed virtual pictograms. Other characteristic features of the POCT cassette that can be used for alignment are a) print-ons on the cassette, such as i) company logos, ii) captions, iii) machine-readable fonts and batch designations, b) geographical and characteristic borders of the test cassette itself, such as i) outer edges, ii) inner edges, other iii) protrusions of the test cassette, etc. The recorded images can at least be present with sufficient resolution to analyze the visual signal without distortion. Since the visual signals or zones can be found with the fixed geometrical variables of the lateral flow test, the orientation of the camera relative to the lateral flow test can be corrected without affecting the analysis of the visual zones.

[0069] The resolution of the camera is not very important in the presence of the intensity ratio of the response lines (T / C) or the misalignment of rotation and tilt. All the fixed information related to length, distance and size in the lateral flow test is pre-programmed into the software and requires calibration for each lot of the device, so that the image resolution provided can be adapted to each individual case.

[0070] Referring to FIG. 3, a flow chart shows that device calibration begins with a camera test card (device check card) configured to identify so-called hot spots (areas of maximum brightness, vertical reflected light) and optimize signal extraction (signal coverage).

[0071] [Pose Estimation] For this purpose, the camera test card is printed with ArUco markers (augmented reality markers, fiducial markers) to determine the position, tilt, rotation and inclination of the device (smartphone) relative to the two-dimensional plane of the camera test card. This device pose estimation is crucial and is based on finding the correspondence between points on the camera test card and their two-dimensional virtual image projection. To make this difficult step easier, it was simplified by having the camera test card pre-printed with fiducial markers. The main advantage of the binary square fiducial marker is that with one marker, it is possible to obtain sufficient correspondences (its four corners) to obtain the camera pose. The ArUco marker is a composite square marker consisting of a wide black border and an internal binary matrix that determines its identifier (ID). The black border facilitates fast detection in the image, and the binarization allows identification and the application of error detection and correction techniques. The internal binarization makes the fiducial markers particularly robust, since it allows the application of error detection and correction techniques (see S. Garrido-Jurado, R. Munoz-Salinas, FJ Madrid-Cuevas, and MJ Marin-Jimenez. 2014. “Automatic generation and detection of highly reliable fiducial markers under occlusion”. Pattern Recognition (Pattern Recogn. 47, 6 (June 2014), 2280-2292. DOI=10.1016 / j.patcog.2014.01.005). The camera check card with the binary fiducial markers is preferably a printed cardboard that is purposely constructed, manufactured and printed for device calibration and authentication.

[0072] Since the accuracy of the system depends heavily on the pose estimation, we perform two different tests to evaluate the sources of error. Projection errors arise from the system and are reflected in the transformation matrix to the virtual image of the camera. To investigate the projection accuracy, we detect the markers in the camera image and determine their relative positions. The centers of the four Aruco markers (or at least three ArUco markers) form a test card coordinate system that replaces the world coordinate system. In the test marker coordinate system, four equidistant points are defined centered on the origin, which become the projected corner points. These points are transformed to projector image coordinates. The square marker card is distorted with an affine transformation so that it appears within these corner points. Ideally, it is a perfect square. The difference in position and rotation between both marker coordinate systems describes the error due to the projection of the system. This method allows a perfect pose estimation of the smartphone relative to the camera check card.

[0073] [Overexposed areas (hot spots)] Following the pose estimation, a search and detection of so-called camera hotspots is performed. A camera hotspot can be defined as the center point of an area where all adjacent pixels are at or close to maximum brightness (total light reflection). This can occur when an object such as a check card in an instant camera is illuminated by a bright single or concentrated light source such as a flashlight or a torch in a smartphone. The size of the hotspot depends on or takes into account the area of ​​maximum brightness. The program library OpenCV (open source computer vision) can be used to determine an ellipse from the detected contours. The determined rectangle is then calculated, for example, by the formula Square root(w 2 +h 2 ) (where w=width and h=height of the rectangle) is used to calculate the size of the hotspot.

[0074] [Signal specifications] We designed two types of camera checks, which are essentially diagrams that match the attributes of the actual test cassette (especially the regions of interest) in terms of reflectance, bands, and signal intensity. Very low and high signals are illustrated (see Figure 4B).

[0075] [Exposure compensation] In most cases, a low signal can be interpreted as being too low because the pixels have been burned, i.e. information has been lost. Since this information cannot be restored, the trick is to under-correct the automatically determined exposure setting to 1 / 3EV (capture the image while darkening it). This allows you to preserve pixel information in bright areas that are difficult to distinguish. A maximum of three adjustments (-1 / 3EV, -2 / 3EV, -1EV) are made.

[0076] [calibration] It should be possible to validate a device for a given test with a certain degree of similarity in calibration results, i.e., eliminating the need for the user to perform a full calibration of the equipment. [Collected information] - Confirmed hotspot (position, size) - Undercorrected value - Final overlay position - Correction signal value - Final scanned image of camera check

[0077] To initially evaluate the suitability of the transient image, 1. the mobile terminal or processor independently determines whether the test cassette is correctly distanced relative to the camera, then batch-specific values ​​are measured in terms of light scale or brightness and brightness gradient on the transient image. Implementation may include registration of the position and size of the machine-readable batch information and the size and position of the printed company logo. 2. The position of both relative to the camera may also be verified based on an overlay displayed on the screen. If the brightness and overlay match, an automatic recording of a transient image is started for further analysis of the visual zone. 3. A combination of points 2 and 1 is also possible, and the image of point 2 is checked in the manner of point 1, and if not satisfactory, the image is normalized (scaled, rotated, shifted, distorted) until the relevant criteria are met.

[0078] Those skilled in the art will appreciate that the framework around the apertures and markers can be used to determine focus, tilt, position, distance, size, and other parameters as well. The cassette can further be printed with individual test codes in the form of bar codes or two-dimensional quick-read codes (QR) to allow for external calibration (standard curves).

[0079] In a typical embodiment, a lateral flow test for the quantitative determination of an analyte in a test sample comprises, in fluid communication with each other, a sample pad for receiving the sample, a filter pad for filtering the sample and homogenizing the flow-through in the wicking direction, a conjugate pad containing a mobile immunoreactant bound to a label, and a water absorbing pad. The separation membrane comprises a membrane having a porous separation material thereon that defines at least one first test zone consisting of immobilized capture molecules of the analyte and one second test zone consisting of immobilized capture molecules of a labeled immunoreactant, and a water-absorbent wicking pad. It is preferred for the disclosed system that the conjugate pad contains at least two labeled but immunogenic and functionally distinct mobile immunoreactants, one of which binds to the analyte to form a labeled complex and the other is inactive with respect to forming a complex with the analyte. This labeling reaction in the second test zone is independent of the reaction of the other labeled immunoreactant with the analyte and capture of the labeled complex in the first test zone, while an inert labeled immunoreactant is provided in a predetermined amount to the conjugate pad to provide an internal reference (C).

[0080] The format of the lateral flow immunoassay can be selected from antigen sandwich assay, antibody assay, or competitive hapten assay. In a preferred embodiment, the lateral flow assay is incorporated into a cassette or envelope with a defined opening for application of the test sample on the sample pad and photographic imaging of the test zone in the viewport. As will be appreciated by those skilled in the art, the immobilized immunoreactant in the first detection zone may be present in more than one line in the first test zone to provide a more dynamic detection range of the analyte. In the most preferred embodiment, the cassette has printed markers indicating one or more positions of the first test zone, and optionally a marker indicating the spatial arrangement of the second test zone, which constitutes an internal reference (C) for proper evaluation and assessment of the zones. In one embodiment, the protein is introduced to the sample pad using a dipstick format, contacting the protein with one end of the test device.

[0081] In another embodiment, the protein is applied onto the sample pad using an applicator, such as a pipette, syringe, dropper, etc. The amount of fluid applied is preferably between about 1-200 μL, more preferably between about 3-100 μL, and most preferably between about 5-50 μL. The sample liquid may be selected from the group consisting of buffered saline, pharmaceutical compositions, and biological fluids. The biological fluid applied directly onto the sample pad may be selected from the group consisting of blood, plasma, fecal extract, fecal fluid, urine, tears, sweat, saliva, and amniotic fluid. The test sample may be a biological fluid of animal or plant origin. The test liquid may also be a mixture of processed foods and / or animal and plant materials.

[0082] In one embodiment, the conjugate pad of the lateral flow test contains a detectable marker. The detectable marker in the conjugate pad may be capable of binding the analyte applied on the sample pad. The conjugate pad may further ensure uniform migration of the detectable marker and the protein (analyte) onto the test membrane. In another embodiment, the detectable marker consists of, but is not limited to, particles, luminescent labels, calorimetric labels, fluorescent labels, chemical labels, enzymes, radioactive labels, metal colloids, and chemiluminescent labels. Gold colloidal spheres are most preferred, but other metal sols and latex microparticles can also be used. Photostable, color-tunable nanoparticles such as carbon, selenium, or quantum dots have also been used as detectable markers. The detectable marker may also be a secondary protein, such as an enzyme, that catalyzes a detection reaction, e.g., a color reaction.

[0083] As mentioned above, the test membrane consists of at least one test zone and at least one control zone. If necessary to increase the quantifiable detection range, two or more test lines with test zones may be used. Of course, one or more test lines (zones) must be upstream of the control zone.

[0084] In one embodiment, the analyte testing device can display (a) a unique code that is a barcode or a quick read QR code, (b) a framed opening in the cassette for either access to the sample pad and visual control of the test zones on the membrane, (c) markers on the cassette indicating the location of the first and second test zones, and (d) a defined area for performing white balancing. The unique code may, for example, encode the manufacturing lot and the QR code encodes data for calibration and interpretation of the test zones, thereby enabling the handheld camera and processor device to operate standalone without an internet connection, provided everything else can be downloaded in advance.

[0085] Another aspect relates to a method for determining the amount or concentration of an analyte in a test sample using a lateral flow immunoassay, the method comprising: (a) taking a digital image of the lateral flow immunoassay using a camera; (b) analyzing the digital image for the location of the second test zone and determining an actual amount of label found in the second test zone based on the digital image; (c) comparing the actual amount of label found in the second test zone with a target amount of label in the second test zone to determine an offset from the respective target values ​​for one or more color channels of the digital image of the second test zone as determined by the calibration digital image of the second test zone; (e) correcting the actual digital image based on the determined offset and further performing white balancing to adjust for the color temperature of the ambient light and / or the assisting flashlight, and optionally the optical characteristics of the camera and lens used in the taken digital image; (f) analyzing the offset-corrected digital image for the location of the first test zone and making a quantitative determination of the label found in the first test zone; and (g) determining the amount of analyte contained in the test sample by comparison with values ​​of a series of calibration standards determined by a lateral flow immunoassay of the same manufacturing lot. [Explanation of symbols]

[0086] 10 Test Devices 12 Lateral flow testing, viewports and regions of interest 14 Reference Image T test zone, response line for analyte C Control Zone, Response Line for Control S Sample Pad 16 Portable processor device 16a Digital Camera 16b Light source 16c processor 18 Casing 20 Reagents 22 Digital Display

Claims

1. 1. A computer-implemented system for determining a concentration of an analyte in a test sample subjected to immunochromatography, the system comprising: a test cassette (10) adapted to receive an immunochromatographic test and configured to display a region of interest (ROI) having one or more visible response lines (12) indicative of the presence and amount of an analyte (T) in the test sample and a control line (C); a portable processor device (16) comprising a digital camera (16a), a light source (16b) and a processor (16c), the processor (16c) configured to process the digital images captured by the camera (16a) and to present analytical results; one or more test cassettes; (i) data identifying a camera test card; (ii) three or more fiducial markers for finding correspondence between points on the camera test card and the projected digital image; (iii) two or more regions of interest (ROIs) printed comparable to the visible response line (12) of the immunochromatographic test and showing two or more response lines (12) for a test (T) and a control (C); Features a camera test card depicting The processor (16c) sequentially analyzes the stream of transient digital images for the presence and correspondence of at least three fiducial markers (14) and, if found, calculates a new coordinate system for estimating position, tilt, rotational alignment, skew and distance between the camera test card and the portable processor device (16), and if the overall pose, angle and distance are found within tolerance, the processor (16c) is configured to check the transient digital image for areas of interest and their light measurements, as well as areas of maximum light reflection, to see if they are close to or within a region of interest (ROI); and offsetting the portable processor device so that the area of ​​maximum light reflection due to light emitted from the light source is located outside the region of interest (ROI) to exclude interfering light reflections on the transient digital image, thereby adjusting the position of the portable processor device to provide optimized light conditions on the camera test card and within the region of interest (ROI); the processor (16c) is further configured to adapt an exposure value (EV) of the digital camera to provide optimized light conditions within the region of interest on the transient digital image; the processor (16c) is configured to scan and analyze a region of interest (ROI) on the transient digital image to determine the optical intensities of test (T) and control (C) response lines and record the ratio of those response lines (C / T); the processor (16c) takes digital images of another region of interest (ROI) to determine a second intensity ratio (C / T) of the other different response lines for test (T) and control (C), and if both ratios are within the tolerance range, sets the camera parameters to meet the given optical intensity range if the optical intensity range of the given different response lines (T) is within the tolerance range based on the portable processor device (16) optimally positioned relative to the configured camera test card, thereby calibrating the digital camera and the light source; The system, wherein the processor (16) is configured to authenticate a camera device, a light source, and software suitable for medical equipment for scanning and evaluation of immunochromatographic tests for point-of-care diagnostics and telemedicine.

2. 2. The system of claim 1, wherein the camera test card depicts a region of interest (ROI) in which a test (T) response line has a minimum light intensity, another region of interest (ROI) in which a test (T) response line has a high light intensity, and a control (C) response line for each region of interest of equal light intensity.

3. 3. A system according to claim 1 or claim 2, based on an immunochromatographic test configured to display a uniform response line of a control (C) having a predetermined light intensity for any test sample.

4. The system of any one of claims 1 to 3, wherein the processor (16c) is configured to transmit the analysis data to a physician for telemedicine.

5. 5. The system of claim 1, wherein the processor (16c) is configured to transfer authentication of the digital camera, the light source and the software to a central unit for storage and / or periodic updating.

6. The system of claim 4, wherein the processor (16c) is configured to receive instructions to perform a certificate update when triggered by the physician.

7. The system of claim 5, wherein the processor (16c) is configured to receive instructions to perform a certificate update when triggered by the central device.

8. A system described in any one of claims 1 to 7, wherein the processor is configured to determine intensity ratios (T / C) of the response lines (T, C) from a number of stored transient images and to select a median ratio (T / C) for quantitative analysis of the optical intensities of the response lines.

9. A system described in any one of claims 1 to 8, wherein the digital image of the region of interest (ROI) is corrected for any degree of error in relation to a fiducial or marker present on a test cassette containing the immunochromatographic test.

10. A system described in any one of claims 1 to 9, wherein the reference or marker (14) includes a machine-readable representation of or a link to data that encodes or provides access to characteristic data of the lateral flow immunochromatographic test.

11. A system described in any one of claims 1 to 10, wherein the processor (16c) is configured to employ external data regarding the position of an area of ​​interest having the visible response line (T, C).

12. A system as claimed in any one of claims 1 to 11, which measures the presence, concentration or amount of allergens in fecal calprotectin, serum calprotectin, vitamin D in blood or serum, luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone, albumin, stool occult blood, gluten immunogenic peptides, bladder cancer markers, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Helicobacter pylori, influenza viruses A and B, troponin I, ringworm, ferritin, D-dimer, CRP, Group A hemolytic streptococcus, Group B hemolytic streptococcus, genetically modified organisms, cereals and products thereof, chickpeas and products thereof, peanuts and products thereof, hazelnuts and products thereof, macadamia and products thereof, mustard and products thereof, soybeans and products thereof, sesame and products thereof, walnuts and products thereof, pistachios and products thereof, lupins and products thereof, celery and products thereof, fish and products thereof, or shellfish and products thereof.

13. Software for use with the system of any one of claims 1 to 12, in a mobile phone (16) including a digital camera (16a), a light source (16b) and a processor (16c), for assisting in taking digital images, reading machine-readable representations of data, exchanging data and image data with a remote server, and displaying information, data and test results on a display, comprising: The software sequentially processes a number of transient digital images, analysing each digital image for the presence of a predefined reference image (14) for pose estimation of the digital camera (16a), and if found and allowed: the software is configured to analyze each transient digital image for a region of interest comprising the response line, and if found, to analyze the region of interest; The software examines each region of interest for measures of reflected light, and if the absolute luminance, luminance gradient, and area of ​​dark pixels are determined to be acceptable, the software analyzes each region of interest; The software is configured to store image data of the transient digital image for further analysis of the region of interest.

14. The software described in claim 13, configured to determine the light intensity of the response lines (T, C) within a region of interest, and further configured to obtain calibration data and other deposited data for determining quantitative results of subjects subjected to immunochromatographic testing.

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