Method and apparatus for selecting a position for reading an image on a slide medium - Patents.com

Abstract

The present disclosure is directed to methods and devices for identifying target locations on a reaction cell and performing an assay using the target locations. In an exemplary embodiment, a method for performing at least one assay includes acquiring at least one image of a fluid sample located on the reaction cell and generating a series of differential data including a plurality of differential data points based on the at least one image. The method further includes determining an image gradient data point for each of the plurality of differential data points and determining a target location of the fluid sample within the reaction cell based on the image gradient data points. The method further includes performing at least one assay using the target location of the fluid sample within the reaction cell. [Selected figure] Figure 9A

Description

[Technical field]

[0001] The present invention relates generally to methods and apparatus for positioning a target location on a reaction cell, such as a dry slide or other porous / solid medium, so that an assay can be performed using the target location, and more specifically to methods and apparatus for positioning the reading center of a fluid sample as a target location on a solid medium. [Background technology]

[0002] One method of performing the assay is to perform the assay on a solid or porous medium. Discharged However, the problem with this method is that the axially symmetric gradient Discharged The problem is that the image profile may not be flat, radiating from the center of the sampled location. This problem can occur, for example, due to inaccuracies in the weighing, media position, or varying media, resulting in the sample being placed at an unintended location. Discharge If the area for performing the assay is a predetermined location, Assay Another problem is that there may be defects in the reaction image due to optical imperfections (e.g., dust, coating defects, etc.) or unintended flow patterns that can degrade the assay signal. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure is directed to methods and apparatus for determining a target location in a reaction cell, such as a dry slide or other solid medium, for example, by obtaining an average optical intensity in a predetermined area centered on a target location of an assay signal, and performing an assay using the target location. In an exemplary embodiment that can be used with any other embodiment disclosed herein, a method of performing at least one assay includes obtaining at least one image of a fluid sample located on a reaction cell, generating a set of differential data including multiple differential data points based on the at least one image, determining an image gradient data point for each of the multiple differential data points, determining a target location of the fluid sample in the reaction cell based on the image gradient data points, and performing at least one assay using the target location of the fluid sample in the reaction cell.

[0004] In another embodiment, which can be used with any other embodiment disclosed herein, the reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

[0005] In another embodiment, which can be used with any other embodiment disclosed herein, the target location is approximately the center of a uniform region of the fluid sample within the reaction cell.

[0006] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, the method includes performing multiple assays using target locations from multiple images.

[0007] In another embodiment, which can be used with any other embodiment disclosed herein, the derivative data points are first derivatives based on color in the at least one image, in an embodiment, the color in the at least one image represents light intensity.

[0008] In another embodiment, which can be used with any other embodiment disclosed herein, determining the target location of the fluid sample includes at least one of: (i) using a first image gradient data point that has a lower image gradient than a second image gradient data point; or (ii) excluding a second image gradient data point that has a higher image gradient than the first image gradient data point. In an embodiment, this means that a first derivative is taken across the entire image and then all pixels that have a first derivative value higher than a threshold are removed, so that the centers of the remaining pixels can be used to find the target location.

[0009] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, the method includes removing image defects from at least one image.

[0010] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, at least one image is a two-dimensional image.

[0011] In another embodiment, which can be used with any other embodiment disclosed herein, the step of determining the target location of the fluid sample includes detecting or measuring an indicator reaction of an indicator molecule in the reaction cell formed from at least one reagent that binds to the fluid sample.

[0012] In another embodiment, which can be used with any other embodiment disclosed herein, the method includes forming a read area around the target location, and performing at least one assay includes using the read area.

[0013] In a general embodiment that can be used with any other embodiment disclosed herein, a method for performing at least one assay includes obtaining at least one image of a fluid sample located on a reaction cell, creating a differential dataset including a plurality of differential data points based on the at least one image, determining a target location of the fluid sample based on the plurality of differential data points, forming a read area around the target location, and performing at least one assay using the read area.

[0014] In another embodiment, which can be used with any other embodiment disclosed herein, the reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

[0015] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, the reading area exhibits a generally circular shape in at least one image.

[0016] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, the reading area exhibits a generally oval shape in at least one image.

[0017] In another embodiment, which can be used with any other embodiment disclosed herein, the method includes determining an image gradient data point for each of the plurality of differential data points, and determining a target location of the fluid sample based on the image gradient data points.

[0018] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, the method includes performing multiple assays using target locations derived from multiple images.

[0019] In another embodiment, which can be used in conjunction with any other embodiment disclosed herein, at least one image is a two-dimensional image.

[0020] In a general embodiment that can be used with any other embodiment disclosed herein, an apparatus for performing at least one assay comprises a slide receiving position configured to receive at least one reaction cell in which a fluid sample is located, an imaging device positioned and arranged relative to the slide receiving position to obtain at least one image of the fluid sample located in the reaction cell, and a control unit configured to (i) determine a target position within the fluid sample located in the reaction cell by analyzing differential data points from the at least one image, and (ii) perform at least one assay based on the target position.

[0021] In another embodiment, which can be used with any other embodiment disclosed herein, the reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

[0022] In another embodiment, which can be used with any other embodiment disclosed herein, the solid medium comprises a second reaction cell.

[0023] In another embodiment, which can be used with any other embodiment disclosed herein, the target location is approximately the center location of the uniform region of said fluid sample within the reaction cell.

[0024] In another embodiment, which can be used with any other embodiment disclosed herein, the control unit is configured to determine the target location by analyzing image gradient data points for each of the plurality of differential data points.

[0025] In another embodiment, which can be used with any other embodiment disclosed herein, the control unit is configured to determine the target location by at least one of: (i) using a first image gradient data point that has a lower image gradient than a second image gradient data point; or (ii) excluding a second image gradient data point that has a higher image gradient than the first image gradient data point. In an embodiment, this means that a first derivative is taken over the entire image, and then all pixels that have a first derivative value higher than a threshold are removed, so that the center of the remaining pixels may be used to find the target location.

[0026] In another embodiment, which can be used with any other embodiment disclosed herein, the control unit is configured to determine the target position approximately at the geometric center of a first image gradient data point having a lower image gradient than a second image gradient data point.

[0027] In another embodiment, which can be used with any other embodiment disclosed herein, the control unit is configured to perform a plurality of different assays using target locations from a plurality of images.

[0028] In another embodiment, which can be used with any other embodiment disclosed herein, the target location of the fluid sample within the reaction cell corresponds to a location where an indicator reaction occurs such that as target molecules in the fluid sample react with a reagent within the reaction cell, an indicator is developed for indication.

[0029] In another embodiment, which may be used with any other embodiment disclosed herein, any of the structures and features disclosed in association with Figures 1-37 may be combined with any other structure and feature disclosed in association with Figures 1-37.

[0030] Therefore, in light of the present disclosure and the above aspects, it is an advantage of the present disclosure to provide improved methods and apparatus for determining target locations in a dry slide, reaction cuvette, or other solid media reaction cell.

[0031] The advantages discussed herein may be found in one or some, and perhaps not all, of the embodiments disclosed herein. Additional features and advantages are described herein or will be apparent from the following detailed description and drawings. [Brief description of the drawings]

[0032] Embodiments of the present disclosure will now be described in more detail, by way of example only, with reference to the accompanying drawings, in which:

[0033] [Figure 1] 1 shows a cross-sectional view of an exemplary embodiment of a solid medium according to the present disclosure. [Diagram 2] FIG. 2 shows an exploded perspective view of the solid medium of FIG. [Diagram 3] 2 shows a diagram of an exemplary embodiment of an assay device holding the solid medium of FIG. 1 according to the present disclosure. [Figure 4] 1 shows a diagram of an exemplary embodiment of a sample ejection mechanism for ejecting a fluid sample onto a solid medium according to the present disclosure. [Diagram 5] 5 shows a diagram of an exemplary embodiment of a liquid lens formed by a fluid sample on a solid medium by ejection as shown in FIG. 4. [Figure 6] 6A-6C show diagrams of an exemplary embodiment of a top perspective view and a bottom view illustrating the exemplary embodiment of the dispensing described in FIG. 5. [Figure 7A] 1 shows a diagram of an exemplary pattern that may be formed by a fluid sample on a solid medium. [Figure 7B] 1 shows a diagram of an exemplary pattern that may be formed by a fluid sample on a solid medium. [Figure 7C] 1 shows a diagram of an exemplary pattern that may be formed by a fluid sample on a solid medium. [Figure 8]FIG. 1 shows an exemplary embodiment diagram of a method for performing at least one assay according to the present disclosure. [Figure 9A] 1 shows a diagram of an exemplary embodiment of a method for determining a target location in an image of a fluid sample on a solid medium according to the present disclosure. [Figure 9B-1] 1 shows a diagram of an exemplary embodiment of a method for determining a target location in an image of a fluid sample on a solid medium according to the present disclosure. [Figure 9B-2] 1 shows a diagram of an exemplary embodiment of a method for determining a target location in an image of a fluid sample on a solid medium according to the present disclosure. [Figure 10] 1A-1D show diagrams of different exemplary patterns that can be formed by a fluid sample on a stationary medium. [Figure 11] 1 shows an original image and the first spatial derivative that may be formed by a fluid sample on a solid medium. [Figure 12] 1 shows an illustration of different imperfections that can form in an image of a fluid sample on a solid medium. [Figure 13] FIG. 1 shows a diagram illustrating how a speck of dust on a solid medium can cause a spike in the corresponding image. [Figure 14] FIG. 1 illustrates how the method according to the present disclosure improves the accuracy of an assay. [Figure 15] FIG. 1 illustrates how the method according to the present disclosure improves the accuracy of an assay. [Figure 16] FIG. 6 shows diagrams of the solid medium embodiments of FIGS. 1-5 according to exemplary embodiments of the present disclosure. [Figure 17] 4 shows a diagram of an image recorded by the assay system of FIG. 3 including stagnation area samples of respective slides according to an exemplary embodiment of the present disclosure. [Figure 18] 1 illustrates an image recorded by an assay system where dust or another contamination is present in the sample, according to an exemplary embodiment of the present disclosure. [Figure 19] FIG. 19 illustrates the analysis of a sample performed by the image-based assay system of FIG. 18. [Figure 20]1 illustrates an image recorded by an assay system where dust or another contamination is present in the sample, according to an exemplary embodiment of the present disclosure. [Figure 21] FIG. 21 illustrates the analysis of a sample performed by the image-based assay system of FIG. 20. [Figure 22] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 23] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 24] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 25] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 26] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 27] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 28] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 29] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 30] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 31] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 32] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 33] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 34] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 35] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Diagram 36] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. [Figure 37] 22A-22D show diagrams of test result data comparing the analytical performance of the solid media of FIGS. 1-21 with known solid media, according to exemplary embodiments of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present disclosure relates to methods and devices for determining target locations on a reaction cell and performing an assay using the target locations. As described in detail below, the disclosed methods and devices are advantageous, for example, in improving computation time, reducing sensitivity of off-center measurements, avoiding edges / frames and minimizing the effects of non-assay signals, and detecting and removing image defects (e.g., dust, coating defects, wicking, etc.), and reducing bias and outliers. Image defects may also include foreign particles, air bubbles, scratches, etc. Although the present disclosure describes the reaction cell as a dry slide, reaction cuvette, or solid media, those skilled in the art will understand that the present disclosure can be used with other types of reaction cells, such as porous media such as nitrocellulose, semi-porous and solid media such as Ouchterlony plates, liquid reagent devices such as cuvettes, flow cells, or reaction tubes, and / or solid media chips with embedded lateral flow arrays with optical measurement zones.

[0035] 1 and 2 illustrate an exemplary embodiment of a reaction cell that can be used according to the present disclosure. In FIGS. 1 and 2, the reaction cell is a solid medium 10 that can be used in an assay system 20 according to the present disclosure. The solid medium 10 can be, for example, a single or multi-layer thin film element onto which a fluid sample is dispensed. In the illustrated embodiment, the solid medium 10 includes multiple layers, including a first layer 12, which can be an upper slide mounting layer configured to provide an upper base layer with an opening through which a liquid sample is received. The first layer can be configured to spread a fluid sample thereon. The solid medium 10 also includes a second layer 14, which can be a reagent layer that includes a reagent configured to react with the fluid sample for a particular assay. The second layer 14 can also include a support layer that provides support or rigidity to the reagent layer. The solid medium further includes a third layer 16, which can be a filter layer that provides a low wavelength cutoff filter used in optical analysis. The third layer 16 can include a lower slide mounting layer configured to provide a lower base layer with an opening therethrough for optical analysis.

[0036] 3 illustrates an exemplary embodiment of an assay system 20 configured to determine target locations within an image of a fluid sample on a solid medium 10 and perform one or more assays using the target locations. As illustrated, the assay system 20 can include a slide receiving location 22 configured to receive at least one solid medium 10, a sample ejection mechanism 32 configured to eject a sample fluid onto the solid medium 10, an imaging device 24 positioned and arranged corresponding to the solid medium receiving location 22 to obtain at least one image of the fluid sample located on the solid medium 10, a light source 26 (e.g., one or more light emitting diode lights) configured to project light onto the solid medium 10 such that the light may be modulated by the liquid sample ejected onto the solid medium 10, and optionally an optical filter 28 configured to modulate the light from the light source 26 to a particular wavelength specific to the assay being performed.

[0037] In the illustrated embodiment, the slide receiving position 22 is configured to receive and hold the solid medium 10 while a fluid sample is applied to the solid medium 10 and / or while the solid medium 10 is illuminated by the light source 26 and imaged by the imaging device 24. In the illustrated embodiment, the slide receiving position 22 includes at least one bracket that creates a first opening 22a and a second opening 22b. The first opening 22a of the slide receiving position 22 allows the fluid sample to be applied to the solid medium 10 (e.g., through an opening in the first layer 12), while the second opening 22b of the slide receiving position 22 allows the solid medium 10 to be illuminated and imaged (e.g., through an opening in the third layer 16) after the fluid sample has reacted with a reagent. It should further be appreciated that addition of a fluid sample may occur before the solid foreign medium 10 is received by the slide receiving position 22, and that illumination and imaging of the solid medium 10 may occur at a first opening 22a at the top of the slide receiving position 22 opposite a second opening 22b at the bottom of the slide receiving position 22.

[0038] 2 may further include a control unit 30 configured to control one or more components of the assay system 20, determine a target location of a fluid sample on the solid medium 10, and analyze the fluid sample on the solid medium 10 according to the methods described herein. The control unit 30 may include a processor and non-transitory memory that stores instructions for performing the methods described herein, where the processor executes the instructions for controlling one or more components of the assay system 20 to perform one or more assays.

[0039] 4 illustrates a fluid sample being dispensed onto the solid medium 10 by a sample dispensing mechanism 32 according to an exemplary embodiment of the present disclosure. In an embodiment, the fluid sample may be dispensed onto the solid medium 10 via the sample dispensing mechanism 32 manually controlled by a user before the solid medium 10 is inserted into the slide receiving position 22 of the assay system 20. The sample dispensing mechanism 32 may be, for example, a pipette or other fluid dispensing mechanism known in the art. Alternatively, the assay system 20 may include a sample dispensing mechanism 32 as shown in FIG. 3. Here, the sample dispensing mechanism 32 may be controlled by a user or by the control unit 30 to dispense the fluid sample onto (or into) the solid medium 10 before or after the solid medium 10 is inserted into the slide receiving position 22 of the assay system 20.

[0040] Figure 4 shows the time t 1 From 5 1 shows a sample dispensing mechanism 32 dispensing a fluid sample onto the solid medium 10 over a period of time. During this time, stagnation and lateral flow create a spatial gradient for the concentration of the coated material in the solid medium 10. As shown, over a period of time t 1 At time t 2 At time t , the fluid sample continues to be ejected from the sample ejection mechanism 32 onto the solid medium 10, and the formation of a stagnation region 44 begins. 3At time t , the fluid sample continues to be ejected from the sample ejection mechanism 32 onto the solid medium 10, spreading throughout the solid medium 10 and increasing the size of the retention region 44. 4 At time t the sample dispensing mechanism 32 begins to stop dispensing the fluid sample, causing fluid necking between the sample dispensing mechanism 32 and the solid medium 10, thereby concentrating the fluid sample in a stagnation region 44 directly below the sample dispensing mechanism 32. 5 At this point, the sample dispensing mechanism 32 is no longer ejecting fluid sample, leaving behind a stagnation region 44. In the illustrated embodiment, the stagnation region 44 corresponds to a region of least variability. In some embodiments, the stagnation region 44 has a relatively low optical density variability and / or is relatively rich in indicator (e.g., sample dye).

[0041] Figure 5 illustrates a liquid lens 46 formed by the fluid sample on the solid medium 10 due to the stagnation region 44 created by the dispensing process of Figure 4. As shown, the fluid sample forms a circular liquid lens 46 on the solid medium 10. As shown in Figure 5, the circular liquid lens 46 is formed by an axisymmetric fluid flow from the sample dispensing mechanism 32, which results in an axisymmetric distribution on the coated material of the solid medium 10.

[0042] FIG. 6 shows a top perspective view and a bottom view illustrating an example embodiment of the distribution of liquid lenses 46 across the stagnation region 44. The bottom plot of FIG. 6 shows the light intensity in two-dimensional space (i.e., x and y space) across all pixels in the image. The center of the liquid lens 46 has a lower light intensity, while the edge of the liquid lens 46 has a higher light intensity. Graph 70 of FIG. 6 has the same information as graph 72, but is displayed in a three-dimensional format with a z-axis to represent light intensity, and the x- and y-axes represent pixel location. If a pixel (x- and y-coordinates) has a high light intensity, the z-value of graphs 70 and 72 will be large. Light intensity is measured in a value called "AD count" ("ADC"). Bright pixels have a higher AD count value than dark pixels.

[0043] Due to differences in assay kinetics and uniformity of the coated material of the solid medium 10, there are different patterns that may form in images taken of a fluid sample on the solid medium 10. Figures 7A-7C show three exemplary patterns that may be formed. Figure 7A shows a concave pattern. Figure 7B shows a convex pattern, and Figure 7C shows a complex pattern. All three patterns in Figures 7A-7C share the common feature of a central stagnation region 44 and Z-axis symmetric radiation perturbed by local non-uniformity. The present disclosure uses this common feature to locate the center of a uniform region (e.g., relatively uniform light intensity) of a fluid sample on the solid medium as a target location for use in performing one or more assays.

[0044] Figure 8 illustrates a method 100 for performing at least one assay by acquiring images and processing the images as disclosed herein. In an embodiment, the control unit 30 may include a processor and non-transitory memory that stores instructions for performing the steps of the method 100. The processor executes the instructions to cause the components of the assay system 20 to perform the steps of Figure 8. It should be understood by one of ordinary skill in the art that one or more of the steps illustrated in Figure 8 may be omitted, and / or additional steps may be added, and / or the order of certain steps may be rearranged without departing from the spirit and scope of the present disclosure.

[0045] At step 102, the imaging device 24 records at least one image of the fluid sample dispensed onto the solid medium 10. For example, the image can be recorded while the light source 26 is projecting light onto the solid medium 10. The light from the light source 26 is configured to transmit light emitted by target molecules in the sample that have reacted with one or more reagents on the solid medium 10 to provide a visual indication of the reaction (e.g., an indicator reaction). In some examples, the optical filter 28 and / or a filter provided by the solid medium 10 are configured to modulate the light from the light source 28. The imaging device 24 can be, for example, a charge-coupled device ("CCD") camera, which can record a two-dimensional image showing, for example, a generally oval or circular fluid sample on the solid medium 10. In an embodiment, the wavelengths required for a particular assay can be programmed or provided to the control unit 30, which can control the light source 26 and the optical filter 28 such that the correct wavelength of light for the assay being performed is projected onto the solid medium 10 while the image is being recorded by the imaging device 24.

[0046] In step 104, the control unit 30 normalizes the image and determines a target location for the assay. Step 104 is shown and described in more detail in FIG. 9 and corresponding description below. As described in more detail below, step 104 includes one or more steps of generating a set of differential data including a plurality of differential data points based on the image, determining an image gradient data point for each of the plurality of differential data points, determining a target location of the fluid sample on the solid medium based on the image gradient data points, and removing image defects from the image.

[0047] In step 106, the control unit 30 uses the target location determined in step 104, for example, by detecting light intensity or other values ​​at the target location and / or at a designated area around the target location. In an embodiment, the target location can be used as a center point to form a designated area around the target location. For example, the target location can be used to create a read area that is used to detect a signal from light projected onto the solid medium 10. U.S. Provisional Application No. 62 / 693,120, entitled "Dry Slide Assay Using Reduced Read Window," filed July 2, 2018, further describes methods for creating a read area around the target location, which is incorporated by reference and relied upon herein. In other embodiments, the target location can be used, for example, to align the instrument measurement system and / or optics, predict maintenance intervals, and / or minimize spectral errors due to reaction cell interference.

[0048] In step 108, the control unit 30 calculates an average light intensity of the image signal from the light projected onto the solid medium 10, for example, at the target location and / or within a read area created from the target location. In an embodiment, the average light intensity is the sum of the AD count values ​​within the read area divided by the sum of the total area (total number of pixels) of the read area.

[0049] In step 110, the control unit 30 calculates the response using the target position. For endpoint assays (e.g., UREA), the response can be calculated by the signal difference between two different reads of the same slide or solid medium 10. For rate assays (e.g., AST), the response can be calculated from the ratio of the change in signal over time from multiple reads of the same single slide or solid medium 10. Because the position of the slide or solid medium 10 may change slightly over time due to rotation of the incubator, it is desirable to find the target position of each image on the same slide medium 10 over time.

[0050] At step 112, the control unit 30 finishes performing the assay by calculating the concentration of the fluid sample, for example, using a calibration curve relating response to concentration. The calibration curve may be obtained in a calibration process using fluids of known concentrations and the responses obtained corresponding to those concentrations. In the calibration curve, the concentration is a known function of the response. In an embodiment, the control unit 30 may perform multiple different assays using target locations from multiple images.

[0051] 9A and 9B show a method 200 that illustrates step 104 of method 100 in more detail. Specifically, Figures 9A and 9B show algorithms specifically designed to detect target locations (Figure 9A) and to handle image defects (e.g., correct image defects or report errors) (Figure 9B) to improve assay accuracy and reduce assay bias and outliers. As described in more detail below, in detecting target locations (Figure 9A), the first derivative of the image at an assay-specific step size is used to obtain the image gradient. Then, a threshold is applied to remove high gradient regions. Finally, the center of the remaining pixels of the image is used as the target location.

[0052] After finding the target position (e.g., the center of a homogeneous region with the least variation and the greatest signal), the image quality can be checked by checking the distance between the target position and the synchronization position, detecting the number of spikes associated with the target position, and analyzing the axial symmetry and noise level of the target position (FIG. 9B). If an assay-specific threshold is exceeded in one or more of the checked quantities, an error code is issued for the image, indicating that the analyzed fluid sample may be deemed unreliable and that the method 100 should be restarted with a new fluid sample. If no error is found, the average optical intensity of the image at the target position can be calculated as described above. In some embodiments, if the number of spikes is below the threshold, the method 200 is configured to remove the spikes from the image, thereby improving the reproducibility of the assay system. However, if the number of spikes exceeds the threshold, the image is discarded or associated with an error code and / or an alert.

[0053] It should be understood by one of ordinary skill in the art that one or more steps shown in Figures 9A and 9B may be omitted, and / or additional steps may be added, and / or the order of certain steps may be rearranged without departing from the spirit and scope of the present disclosure. As with method 100, it should be understood that control unit 30 may include a processor and non-transitory memory that stores instructions for performing the steps of the method of Figures 9A and 9B. The processor executes the instructions to cause the components of assay system 20 to perform the disclosed steps.

[0054] Step 200 begins with an image recorded by imaging device 22 in step 102 of Fig. 8. In step 200, control unit 30 analyzes the image by associating the image with metadata related to, for example, the assay type, the default center 40 of the fluid sample, the type of pattern detected for the fluid sample (e.g., concave, convex, complex), etc. The parameters stored in the metadata can be pre-programmed and stored in the memory of control unit 30, or can be entered by a user for a particular assay. The default center 40 of the fluid sample can be, for example, the center of the solid medium 10, or the center of the dispense location where the center of the fluid sample is expected to be.

[0055] The type of pattern detected for the fluid sample can be planar or non-planar (e.g., concave, convex, or more complex shapes) depending on, for example, the sensitivity to redistribution of soluble materials in the solid medium 10, the analyte / reagent concentration, and / or the kinetics of the chemistry. FIG. 10 shows different exemplary patterns formed by fluids ejected onto the solid medium 10, while FIG. 11 shows diagrams showing original images and first spatial derivatives for different assays. FIGS. 10 and 11 show that there are smaller values ​​in the stagnation region 44. As shown, the fluid sample can have, for example, stagnation regions 44 with very small gradients (e.g., peak or valley intensity), washout regions with larger gradients, and / or edges with large gradients (from the wet-dry interface or slide frame). U.S. Provisional Application No. 62 / 693,120, filed July 2, 2018, entitled "Dry Slide Assay Using Reduced Read Window," further describes how washout regions can affect images and cause large gradients, which description is incorporated and relied upon herein by reference. According to this method, when an appropriate threshold is applied, all large gradient regions can be removed and retention regions 44 can be identified.

[0056] In step 202, the control unit 30 normalizes the image by converting it to a flat-field image if it is not already in this shape. In an embodiment, normalizing the image includes normalizing the light energy applied to the entire reading area to reduce errors. In an embodiment, the normalization has two steps. The first step includes subtracting the dark read signal from the raw signal. The dark read signal is the digital signal from the CCD sensor with the light source turned off. This calculation minimizes the background digital noise. The second step is to normalize the dark correction signal using the flat-field signal by multiplying the signal by a flat-field function. The flat-field function is obtained by reading a standard surface that is a uniform, flat, white and / or reflective surface. The flat-field function may be a function that produces a value of "1" over the entire surface of the standard surface. Assuming that the measured light intensity distribution is f=f(x,y), the flat-field is F=1 / f.

[0057] In step 204, the control unit 30 determines or accepts assay-specific parameters, such as, for example, image crop size, step size, threshold parameters, etc. The assay-specific parameters can be pre-programmed and stored in the memory of the control unit 30, or can be input by a user for a particular assay. The image crop size can be, for example, the size intended for the image to be cropped in step 206, and can be input or automatically generated by the control unit 30. In an exemplary embodiment, the image crop size can be about 4.5 mm in diameter around the center point (e.g., default center 40). The step size and threshold parameters are similarly input or are pre-defined values ​​used in later steps of the process. Each assay has its own crop size, step size, and threshold (e.g., URIC crop size=3.64 mm, step size 0.28 mm, and threshold=10).

[0058] In step 206, the control unit 30 crops a region 46 of the image. In an example, the control unit 30 crops the region 46 based on an expected location of a default center 40 of the fluid sample on the solid medium 10. In an embodiment, the default center 40 is known from step 200, for example, as the center of the solid medium 10 or as the center of the ejection location where the center of the fluid sample is expected. Thus, the image can be cropped, for example, as a circle (e.g., a circle having a diameter of 4.5 millimeters ("mm")) surrounding the default center 40 based on the image crop size determined in step 204. In another embodiment, the image can be cropped based on the detected light intensity without being limited to a particular size. U.S. Provisional Application No. 62 / 693,120, filed July 2, 2018, entitled "Dry Slide Assay Using Reduced Read Window," further describes how to crop a region of an image to create a read region, the description of which is incorporated and relied upon herein by reference.

[0059] In step 208, the control unit 30 records a default center 40 of the cropped image. In an embodiment, the default center 40 is known from step 102 and the image is cropped based on the default center 40. In another embodiment, the default center 40 does not correspond to the center of the cropped image and may be determined based on the newly cropped image. For example, the image may be cropped based on light intensity in step 206 and then the default center 40 may be recorded as the geometric center of the cropped image.

[0060] In steps 210-214, the control unit 30 creates a set of derivative data including a plurality of derivative data points based on the cropped image. In an embodiment, the derivative data points may be first derivatives based on colors in the image, and each may include an image gradient data point that defines a binary image. In an embodiment, at least one image color represents light intensity. In an embodiment, the first derivative is indicative of image light intensity in space. For example, each pixel in the image has a unique light intensity measured in AD counts (i.e., ADC). The first derivative may be the difference in light intensity between pixels at a predefined distance divided by the distance between the pixels.

[0061] In step 210, the control unit 30 creates a set of derivative data by taking the absolute value of the first derivative (e.g., slope) of the cropped image at the assay-specific step size determined in step 204. The first derivative can be calculated using a central difference method using the assay-specific step size in the cropped image. For example, the set of derivative data can be calculated using the following equation: Slope 1 =max(|du / dx|,|du / dy|) where u is the light intensity (AD count value) at a pixel in the image, and x and y are the coordinate location of that pixel. The slope can be calculated along both the x and y directions, and the maximum absolute value can be defined as the slope. The step size for calculating the derivative is assay specific. As explained in more detail below, the slope 1 (Slope 1 ) is less than the threshold, then the mass value is "1", otherwise the mass value is "0".

[0062] In steps 212 and 214, the control unit 30 determines an image gradient data point for each of a plurality of differential data points of the set of differential data, thereby generating a binary image. 1If the slope is greater than an assay specific threshold, the control unit 30 sets the image gradient data point to zero (0). That is, pixels with higher gradients are set to zero (0) to remove spikes, edges, etc. from the binary image. Next, in step 214, the control unit 30 sets the image gradient data point equal to a value of one if the image gradient data point is greater than a value of zero (0). That is, 1 If exceeds the threshold, then the image gradient data point at each pixel location (e.g., the mass at that location in the binary image) is zero (0), and the slope 1 is below the threshold and is 1. In an embodiment, (ii) a first image gradient data point having a lower image gradient than a second image gradient data point is included or set to 1, and / or (iii) a second image gradient data point having a higher image gradient than the first image gradient data point is excluded or set to zero (0).

[0063] At step 216, the control unit 30 determines a target location of the fluid sample (e.g., elliptical or circular) based on the image gradient data points. For example, the target location may be calculated as the center of gravity of the binary image (e.g., the binary image shown in FIG. 9A). In an embodiment, the center location is calculated using the following formula:

number

[0064] The centroid of the binary image may then be designated as a target location of the fluid sample on the solid medium 10 , which is approximately the geometric center of the homogenous region of the fluid sample on the solid medium 10 .

[0065] In steps 218-252 of FIG. 9B, the control unit 30 is configured to detect and / or remove defects from the image (e.g., due to image imperfections, dust, coating defects, wicking, foreign particles, air bubbles, scratches, etc.) and / or determine an error condition based on the target location (e.g., the center of the fluid sample determined from step 216). The error may also be indicative of a problem with the reflectometer illumination system of the assay system 20, such as dirty projector optics. For example, as illustrated by FIG. 12, there are many different imperfections in the image (e.g., dust, off-center ejection, wicking, foreign particles, air bubbles, scratches, etc.) that can adversely affect the assay.

[0066] In step 218, the control unit 30 compares the target location determined in step 216 with the previously determined default center 40. In an embodiment, if the distance between the default center and the target location is greater than a threshold value, the control unit 30 may report an error in step 220 and terminate the analysis in step 222. This may mean that the analyzed fluid sample is considered unreliable and the method 100 should then be restarted with a new fluid sample.

[0067] In step 224, the control unit 30 determines whether the flatness of the image is less than a threshold value. In an embodiment, the flatness of the image is the maximum of two linear slopes (absolute value and value normalized by ADC average) across the central vertical length and horizontal length of the cropped image. Since the AD count value (ADC) varies over a large range (e.g., 10,000 to 50,0000) depending on the analyte concentration in the sample, the AD count value is normalized by dividing the AD count value by its average value. In this way, the change in slope is related to the flatness rather than the analyte concentration. Then, the average value of the AD count value is calculated, and the AD count value is divided by the average value. The absolute value of the slope is used to determine whether the image is flat, so the slope can be positive or negative. The image can be concave or convex in light intensity distribution, but is axially symmetric about the center (discharge position).

[0068] If the flatness of the image is below the threshold in step 226, the control unit 30 may report an error in step 228 and terminate the analysis in step 122. This may indicate that the analyzed fluid sample is considered unreliable and the method 100 should then be restarted with a new fluid sample.

[0069] In step 230, the control unit 30 creates a first derivative of the cropped image with a determined step size (e.g., a step size of 1). For example, the first derivative data may be calculated using the following formula: Slope 2 =|du / dx|+|du / dy| can be determined using

[0070] In step 232, the controller 30 calculates the slope within the new center region. 2 (Slope 2) and obtain the mean (MEAN) and standard deviation (SD). In step 234, the control unit 30 determines whether the standard deviation / mean is below a threshold. If the standard deviation / mean is below a threshold, the control unit 30 may report an error in step 236 and terminate the analysis in step 238. This may mean that the analyzed fluid sample is considered unreliable and the method 100 should then be restarted with a new fluid sample.

[0071] In step 240, the controller 30 may define two assay-specific constants, SpikeValueMax and n. In an embodiment, the spike maximum value (SpikeValueMax) represents a value determined by assay performance experiments and is provided for each assay, while n represents a positive number and is used to calculate a value to define the spike detection threshold, the spike gradient limit (SpikeGradientLimit). The controller 30 then calculates, for example, SpikeValueMax = n, using the following formula: SpikeGradientLimit=min(SpikeValueMax, MEAN+n*SD) In the above formula, the minimum of SpikeValueMax and MEAN+n*SD is used to define the spike slope limit, which is the threshold used to detect if the slope within the image capture region exceeds the limit.

[0072] In steps 242 and 244, the controller 30 removes large gradient spikes from the cropped image. In an exemplary embodiment, the spikes are removed by defining a spike mask, where the slope of a point is 1 (Slope 1 ) is greater than the SpikeGradientLimit, the spike mask is equal to a value of zero (0) and the slope of a point 1If is less than the spike slope limit, then the spike mask is equal to a value of 1. In another embodiment, spikes may be removed based on being above or below a predetermined threshold.

[0073] In step 246, the control unit 30 determines the total number of spikes detected in steps 240 and 242. If the number of spikes detected and removed from the image is low, the remaining pixels in the image are still used to calculate the signal of that solid medium 10. However, if the number of detected spikes exceeds a predefined limit based on the assay experiment, the control unit 30 may report an error in step 248 and terminate the analysis in step 252. This may mean that the analyzed fluid sample is considered unreliable and the method 100 should be restarted with a new fluid sample.

[0074] In step 250 , the control unit 30 integrates within the cropped image defined by the target locations, removes the detected defects, and makes the resulting image available for use in step 106 of the method 100 .

[0075] 13 shows an example of how a speck of dust on the solid medium 10 can cause a spike in the corresponding image. As shown, removing the spikes can also remove bias to an assay performed using the image, improving the reliability of the assay.

[0076] Figures 14 and 15 show the advantages achieved by the method of the present disclosure. In Figure 14, TPxt means that the assay is TP ("total protein") in multi-test format (two tests per slide). COM (shown as circles) represents the accuracy when reading at the target position found using the method of the present disclosure. PS (shown as squares) represents the accuracy when reading at a pre-specified center (e.g., the expected dispense position) without knowing the actual dispense position. The coefficient of variation (CV%) at each reading diameter is the average of all samples tested.

[0077] FIG. 16 illustrates an embodiment of the solid medium 10 of FIGS. 1-5 according to an exemplary embodiment of the present disclosure. The solid medium 10 of FIG. 16 includes two chemical tips 1602a and 1602b that correspond to the stagnation region 44 discussed above. As discussed above, the stagnation region corresponds to the region of the chemical chip directly below the opening of the fluid metering tip. The stagnation region 44 may also be characterized by little or no radial fluid flow. There is a fluid flow vector located radially outward from the spot center and area that points away from the center of the chemical chip. There is some amount of washout of the chemical chip reagents in this region. The stagnation zone or region 44 does not experience this washout and may have a different optical density when the chemical reaction is completed compared to the region outside of the stagnation region 44. In some instances, the stagnation zone or region 44 does not have a significant level of radial fluid flow, which creates an area of ​​low optical variability and an area of ​​high indicator (dye) presence.

[0078] In some examples, the chemical chip and / or retention area 44 corresponds to a target location of the fluid sample in the reaction cell. The target location or chemical chip corresponds to a location where an indicator reaction occurs such that the indicator is expressed for indication as a target molecule in the fluid sample reacting with a reagent in the reaction cell. In some examples, the chemical chips 1602a and 1602b may each include a reaction cuvette filled with liquid. In these examples, imaging of the reaction cuvette may show air bubbles, debris, and / or scratches in the cuvette walls. Identified defects can be identified and their impact mitigated in relation to the analysis and assay results.

[0079] The use of chemical chips 1602 reduces the area required for the sample and allows multiple samples to be distributed on a slide. Each of the chemical chips 1602 may receive the same fluid from the sample to allow for the same or different analyses using the same solid medium 10. Alternatively, each of the chemical chips 1602 may receive different fluids to allow for the same or different analyses using the same slide 10. Additionally, while FIG. 16 shows two chemical chips 1602, in other examples, the solid medium 10 may include three, four, etc. additional chemical chips. It should be appreciated that including multiple chemical chips on the solid medium 10 improves the operational efficiency of the assay system 20 since multiple analyses can be performed on the same solid medium 10 without moving the solid medium. In contrast, a solid medium with only a single chemical chip would require the processing of two or more separate solid media to perform the same analyses as a single solid medium 10 with multiple chemical chips.

[0080] FIG. 16 also shows examples of chemical solid media combinations. The combinations are triglyceride-cholesterol (TRI-CHOL), total bilirubin-alkaline phosphatase (TBIL-ALKP), alanine aminotransferase-aspartate aminotransferase (ALTV-AST), urea-creatinine (UREA-CREA), glucose-calcium (GLU-C a), and Albumin-Total Protein (ALB-TP). It should be understood that other combinations may be created and implemented on the solid medium 10.

[0081] FIG. 17 shows an image recorded by the assay system of FIG. 3 including a sample placed on the chemical tip 1602 of each solid medium 10 according to an exemplary embodiment of the present disclosure. As described above, ejection of the fluid sample forms a liquid lens 46 on the chemical tip 1602. The exemplary solid medium 10 of FIG. 17 is configured to reduce sample size and increase the operational efficiency of the assay system 20 while maintaining analytical performance compared to known conventional single slide tests. Due to the small sample volume placed in each chemical tip 1602, the size of the area for analytical measurement is reduced. In some examples, the assay system 20 may use digital chemistry techniques, as discussed above, to mitigate possible performance sensitivity. As shown in FIG. 17, digital chemistry uses wavelength specific LEDs to record or capture an image of the solid medium 10 in a digital imaging reflectometer. The assay system 20 uses one or more imaging algorithms to improve chemistry results (as described above) by ensuring analysis of optimal areas of the sample even in the presence of measurement location variations or containments (e.g., dust).

[0082] As shown in Figure 17, the assay system 20 uses an imaging algorithm to determine a center 40, shown as the center 40a-40d of each tip 1602a-1602d. As mentioned above, the center 40 may or may not be the same as the default center, which is the theoretical spot center if the dispensing tip and slide were perfectly aligned. The assay system 20 then analyzes a particular radius around the center 40. This radius is between 1 mm and 8 mm, preferably about 2.25 mm, as shown by the dashed line in the figure.

[0083] FIG. 18 illustrates an image 1800 recorded by the assay system 20 of FIG. 3 where the sample contains dust or another contaminant 1802, according to an exemplary embodiment of the present disclosure. FIG. 19 illustrates an analysis of the sample of FIG. 18 performed by the assay system 20 using the methods disclosed herein. A first graph 1902 illustrates the results of the analysis before the anomaly 1802 is detected and removed. A second graph 1904 illustrates the results of the analysis after the anomaly 1802 is detected and removed. In the illustrated example, an imaging algorithm detects the anomaly and removes it from a processed image 1906 (derived from the image 1800 of FIG. 18). As a result, the anomaly is not included in subsequent analysis of the sample. In some examples, the assay system 20 removes the anomaly by changing the pixel color associated with the anomaly to a color that matches the surrounding pixel colors or to a pixel color that indicates no data for subsequent analysis.

[0084] FIG. 20 shows another image 2000 recorded by the assay system 20 of FIG. 3, where dust or another contaminant is included in the sample, according to an exemplary embodiment of the present disclosure. FIG. 21 shows an analysis of the sample performed by the assay system 20. A first graph 2102 shows the analysis results before an anomaly is detected and removed. A second graph 2104 shows the analysis results after the anomaly is detected and removed. As in the previous example, the imaging algorithm detects the anomaly and removes it from the processed image 2106 (derived from the image 2000 of FIG. 20). As a result, the anomaly is not included in the subsequent analysis of the sample. In addition, the assay system 20 is configured to move the center 40 of the image 2106 to reduce or minimize the number of anomaly-corrected locations included within the analysis field (e.g., a circle around the center).

[0085] 22-37 show test result data comparing the analytical performance of the exemplary solid media 10 of FIGS. 1-21 with known slides or solid media. In the exemplary examples of FIGS. 22 and 23, the solid media 10 were analyzed using the assay system 20, while the known slides or solid media were analyzed using a known conventional assay system. In the examples, six of the exemplary solid media 10 for two serum concentrations were evaluated using quality control materials on the exemplary assay system 20. The total intra-laboratory precision of performance for a single calibration (reported as coefficient of variance (%CV)) was evaluated according to CLSI EP05 guidelines with two runs per day, two replicates per run over 20 days, for a total of 80 replicates. The worst intra-laboratory precision of the exemplary solid media 10 analyzed by the assay system 20 was compared to a corresponding single prior art test slide analyzed using a conventional assay system.

[0086] The %CV corresponds to the ratio of the standard deviation of the sample data to the mean and provides an indication of the precision and reproducibility of the assay, with lower values ​​corresponding to higher precision and reproducibility.

[0087] FIG. 22 is a diagram of table 2200 showing two serum levels for each slide chemistry (UREA, TRIG, GLU, ALB, TBIL, ALTV, CREA, CHOL, CA, TP, ALKP, and AST) and the corresponding %CV for the prior art slides using the conventional assay system and the exemplary solid medium 10 using the exemplary assay system 20. As shown, out of the 24 different tests, the solid medium 10 using the assay system 20 had a lower %CV in 19 of the 24 different tests. FIG. 23 shows a graph 2300 illustrating the difference in %CV for each of the different slide chemistry tests between the known prior art slides and the exemplary solid medium 10 disclosed herein analyzed using the assay system 20. As shown, using the exemplary solid medium 10 with the assay system 20 provided a better %CV in most slide chemistry tests compared to the prior art slides.

[0088] In the exemplary embodiment of Figures 24-37, an external precision test was performed. In this test, prior art test slides were analyzed using a conventional assay system. Test slides with 12 chemistries were analyzed for two serum concentrations. Statistical data outliers were removed from the analysis. A Shapiro-Wilk normality test was performed on the remaining data, and sets showing significant departures from normality were removed. %CV was calculated for each slide serum and reagent lot combination. %CV measurements were averaged within reagent lots and compared to the worse intra-laboratory precision of the exemplary solid media (10) analyzed with the exemplary assay system 20.

[0089] FIG. 24 shows a diagram of table 2400 illustrating two serum levels for each slide chemistry (UREA, TRIG, GLU, ALB, TBIL, ALTV, CREA, CHOL, CA, TP, ALKP, and AST) and the corresponding %CV for the prior art slides using the conventional assay system and the exemplary solid medium 10 using the exemplary assay system 20. As shown, out of the 24 different tests, the solid medium 10 using the assay system 20 had a lower %CV in 20 of the 24 different tests. FIG. 25 shows a graph 2500 illustrating the %CV difference for each of the different slide chemistry tests between the known prior art slides and the exemplary solid medium 10 disclosed herein analyzed using the assay system 20.

[0090] 26-37 show graphs depicting results for each reagent lot number for an exemplary solid medium 10 and known prior art slides. In the graphs, solid medium 10 was provided in three lots and is referred to as "XT···Lot Number." In addition, data relating to the prior art slides is referred to as "ST···Field derived" and "ST Average." Each graph provides the precision %CV for serum (PVI) and (PVII), respectively.

[0091] Graph 2600 of FIG. 26 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for TRIG slide chemistry reagents compared to prior art slides analyzed by a conventional assay system. Graph 2700 of FIG. 27 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for CHOL slide chemistry reagents compared to known prior art slides analyzed by a conventional system, except for lot 3 of PVI serum, which had a slightly higher %CV. Graph 2800 of FIG. 28 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for GLU slide chemistry reagents compared to prior art slides analyzed by a conventional assay system. Graph 2900 of FIG. 29 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for CA slide chemistry reagents compared to prior art slides analyzed by a conventional assay system.

[0092] Further, graph 3000 of FIG. 30 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the TP slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. Graph 3100 of FIG. 31 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the ALB slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. Graph 3200 of FIG. 32 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the ALV slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. Graph 3300 of FIG. 33 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the AST slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system.

[0093] Graph 3400 in FIG. 34 shows that the exemplary solid medium 10 analyzed by assay system 20 had a higher %CV for the UREA slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. However, the difference in %CV between the exemplary system and the conventional system is less than 0.5%. Graph 3500 in FIG. 35 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the CREA slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. Graph 3600 in FIG. 36 shows that the exemplary solid medium 10 analyzed by assay system 20 had about the same or lower %CV for the ALKP slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system. Graph 3700 in FIG. 37 shows that the exemplary solid medium 10 analyzed by assay system 20 had a lower %CV for the TBIL slide chemistry reagent compared to the prior art slides analyzed by the conventional assay system.

[0094] (Conclusion) It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present subject matter and without diminishing its intended advantages. Accordingly, such changes and modifications are intended to be covered by the appended claims.

[0095] Unless otherwise indicated, all numerical values ​​expressing properties such as amounts of ingredients, molecular weights, reaction conditions, and the like used in the specification and claims should be understood in all instances as being modified by the term "about". Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be construed, at the very least, in light of the number of reported significant digits and by applying ordinary rounding approaches. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0096] As used in the context of this disclosure (particularly in the context of the claims below), the terms "a," "an," "the," and similar referents should be construed to include both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Limitations of value ranges herein are merely intended to serve as a shorthand method for individually referring to each separate value falling within a range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all exemplary or exemplary language (e.g., "such as") provided herein is intended merely to further clarify the disclosure and does not pose a limitation on the scope of the claimed disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0097] Use of the term "or" in the claims is used to mean "and / or," unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, however, the present disclosure supports a definition that refers to alternatives only and "and / or."

[0098] Grouping of alternative elements or embodiments of the disclosure disclosed herein should not be construed as limiting. The elements of each group may be referenced and claimed individually or in any combination with other elements of the group or other elements found herein. It is anticipated that one or more elements of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the modified group, and thus fulfills the written description of all Markush groups used in the appended claims.

[0099] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Of course, variations of these preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that such variations will be adopted by those skilled in the art as appropriate, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, all possible combinations of the elements described above in any possible variations are encompassed by the present disclosure unless otherwise indicated herein or clearly contradicted by context.

[0100] Certain embodiments disclosed herein may be further limited in the claims using the terms "consisting of" or "consisting essentially of." When used in a claim, whether added at the time of filing or amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claim to those materials or steps specified and that do not materially affect the basic and novel characteristics. The claimed embodiments of the present disclosure are essentially or explicitly described and enabled herein.

[0101] It is further understood that the embodiments of the present disclosure disclosed herein are illustrative of the principles of the present disclosure. Other modifications that may be employed are within the scope of the present disclosure. Thus, by way of example, but not limitation, alternative configurations of the present disclosure may be utilized in accordance with the teachings herein. Thus, the present disclosure is not limited to that precisely as shown and described.

[0102] [Note] [Appendix 1] obtaining, in the control unit, at least one image of the fluid sample located on the reaction cell; generating, via the control unit, a set of differential data based on the at least one image, the set including a plurality of differential data points; determining, via said control unit, an image gradient data point for each of said plurality of differential data points; determining, via the control unit, a target position of the fluid sample within the reaction cell based on the image gradient data points; and performing at least one assay using the target location of the fluid sample within the reaction cell via the control unit and an associated assay system; 2. A method for performing at least one assay comprising:

[0103] [Appendix 2] The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette. The method according to claim 1,

[0104] [Appendix 3] the target location comprises an approximate center of a uniform region of the fluid sample within the reaction cell; 3. The method according to claim 1 or 2,

[0105] [Appendix 4] performing a plurality of assays using target locations from the plurality of images; 4. The method according to claim 1, 2 or 3.

[0106] [Appendix 5] the derivative data points are first derivatives based on color in the at least one image; 5. The method according to claim 1, 2, 3 or 4.

[0107] [Appendix 6] The step of determining the target location of the fluid sample comprises: (i) using a first image gradient data point having a lower image gradient than a second image gradient data point; or (ii) excluding a second image gradient data point having a higher image gradient than the first image gradient data point; At least one of 6. The method according to claim 1, 2, 3, 4 or 5.

[0108] [Appendix 7] removing image defects from the at least one image via the control unit. 7. The method according to claim 1 or 6,

[0109] [Appendix 8] said at least one image being a two-dimensional image; 8. The method according to claim 1 or 7,

[0110] [Appendix 9] forming, via the control unit, a read area around the target location, and performing at least one assay includes using the read area. 9. The method according to claim 1 or 8,

[0111] [Appendix 10] determining the target location of the fluid sample includes detecting or measuring an indicator reaction of an indicator molecule in the reaction cell formed from at least one reagent that binds to the fluid sample; The method according to claim 1,

[0112] [Appendix 11] obtaining, via the control unit, at least one image of the fluid sample located on the reaction cell; generating, via the control unit, a set of differential data based on the at least one image, the set including a plurality of differential data points; determining, via the control unit, a target location for the fluid sample based on the plurality of differential data points; forming, via the control unit, a reading area around the target location; and performing at least one assay using said read area via said control unit and associated assay system; Including, A method for performing at least one assay.

[0113] [Appendix 12] The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette. 12. The method according to claim 11,

[0114] [Appendix 13] The reading area exhibits a substantially circular shape in at least one image. 13. The method according to claim 11 or 12,

[0115] [Appendix 14] the reading area is displayed in the at least one image as a substantially oval shape; 14. The method according to claim 11, 12 or 13,

[0116] [Appendix 15] determining, via the control unit, an image gradient data point for each of the plurality of differential data points; and determining the target location of the fluid sample based on the image gradient data points. 15. The method according to claim 11, 12, 13 or 14.

[0117] [Appendix 16] performing, via said control unit and said associated assay system, a plurality of assays using target locations derived from the plurality of images. 16. The method according to claim 11 or 15,

[0118] [Appendix 17] The at least one image is a two-dimensional image. 17. The method according to claim 11 or 16,

[0119] [Appendix 18] a slide receiving location configured to receive at least one reaction cell having a fluid sample disposed therein; an imaging device positioned and arranged relative to the slide receiving location to obtain at least one image of the fluid sample located within the reaction cell; and a control unit configured to (i) determine a target location within the fluid sample located in the reaction cell by analyzing differential data points from the at least one image; and (ii) perform at least one assay based on the target location; 16. An apparatus for performing at least one assay comprising:

[0120] [Appendix 19] The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette. 19. The apparatus of claim 18.

[0121] [Appendix 20] The solid medium comprises a second reaction cell; 20. The device of claim 19,

[0122] [Appendix 21] the target location comprises an approximate center location of a uniform region of the fluid sample; 21. The device according to claim 18 or 20.

[0123] [Appendix 22] the control unit is configured to determine the target location by analyzing image gradient data points for each of the plurality of differential data points. 22. The device of claim 18, 19, 20, or 21.

[0124] [Appendix 23] the control unit is configured to determine the target location by at least one of: (i) using a first image gradient data point having a lower image gradient than a second image gradient data point; or (ii) excluding a second image gradient data point having a higher image gradient than the first image gradient data point. 23. The apparatus of claim 22.

[0125] [Appendix 24] the control unit is configured to determine the target location at approximately a geometric center of a first image gradient data point having a lower image gradient than a second image gradient data point. 23. The apparatus of claim 22.

[0126] [Appendix 25] the control unit is configured to perform a plurality of different assays using target locations from the plurality of images. 26. The device according to claim 18 or 25.

[0127] [Appendix 26] the target location of the fluid sample within the reaction cell corresponds to a location within the reaction cell where an indicator reaction occurs such that an indicator is developed for indication as target molecules in the fluid sample react with a reagent within the reaction cell; 26. The device according to claim 18 or 25.

Claims

1. obtaining, in the control unit, an image of the fluid sample located on the reaction cell; determining, via the control unit, a default center corresponding to at least one of a center of the reaction cell and a center of a dispensing position of the fluid sample onto the reaction cell; generating, via the control unit, a set of differential data based on the image, the set including a plurality of differential data points; determining, via the control unit, an image gradient data point for each of the plurality of differential data points to generate a binary image; comparing each of the plurality of differential data points to a threshold; assigning a value of "0" to the image gradient data points corresponding to the derivative data points having values ​​large compared to a threshold to remove unwanted image artifacts; and assigning a value of "1" to the image gradient data points that are to be retained; Steps by determining, via said control unit, a centroid of said binary image using said image gradient data points; designating, via the control unit, the determined centroid as a target position for the fluid sample on the reaction cell; comparing, via the control unit, the target position with the default center; and via said control unit and an associated assay system, performing at least one assay using said target position of said fluid sample on said reaction cell if the distance between said default center and said target position is less than a threshold value; A method of performing at least one assay comprising:

2. The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

2. The method of claim 1 .

3. the target location includes a center of a uniform area of ​​the fluid sample on the reaction cell; 2. The method of claim 1 .

4. performing a plurality of assays using target locations from the plurality of images; 2. The method of claim 1 .

5. the plurality of derivative data points are first derivatives based on color in the image; 2. The method of claim 1 .

6. The step of determining a centroid of the binary image comprises: (i) using a first image gradient data point having a lower image gradient than a second image gradient data point; or (ii) excluding second image gradient data points having a higher image gradient than the first image gradient data points; At least one of 2. The method of claim 1 .

7. removing image defects from the image via the control unit.

2. The method of claim 1 .

8. The image is a two-dimensional image.

2. The method of claim 1 .

9. forming, via the control unit, a read area around the target location, and performing the at least one assay includes using the read area.

2. The method of claim 1 .

10. obtaining an image of the fluid sample includes detecting or measuring an indicator reaction of an indicator molecule on the reaction cell formed from at least one reagent that binds with the fluid sample; 2. The method of claim 1 .

11. obtaining, via the control unit, an image of the fluid sample located on the reaction cell; determining, via the control unit, a default center corresponding to at least one of a center of the reaction cell and a center of a dispensing position of the fluid sample onto the reaction cell; generating, via the control unit, a set of differential data based on the image, the set including a plurality of differential data points; determining, via the control unit, an image gradient data point for each of the plurality of differential data points to generate a binary image; comparing each of the plurality of differential data points to a threshold; assigning a value of "0" to the image gradient data points corresponding to the derivative data points having values ​​large compared to a threshold to remove unwanted image artifacts; and assigning a value of "1" to the image gradient data points that are to be retained; Steps by determining, via said control unit, a centroid of said binary image using said image gradient data points; designating, via the control unit, the determined centroid as a target location for the fluid sample; forming, via the control unit, a reading area around the target location; comparing, via the control unit, the target position with the default center; and via said control unit and associated assay system, performing at least one assay using said read area if the distance between said default center and said target location is less than a threshold value; Including, A method for performing at least one assay.

12. The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

12. The method of claim 11 .

13. The reading area represents a circular shape in the image.

12. The method of claim 11 .

14. The reading area is displayed as an oval in the image.

12. The method of claim 11 .

15. performing, via said control unit and said associated assay system, a plurality of assays using target locations derived from the plurality of images.

12. The method of claim 11 .

16. The image is a two-dimensional image.

12. The method of claim 11 .

17. a slide receiving location configured to receive at least one reaction cell having a fluid sample disposed therein; an imaging device positioned and arranged relative to the slide receiving location to obtain an image of the fluid sample located on the reaction cell; determining a default center corresponding to at least one of a center of the reaction cell and a center of a dispensing position of the fluid sample onto the reaction cell; generating a set of differential data based on the image, the set including a plurality of differential data points; determining an image gradient data point for each of the plurality of differential data points to generate a binary image; comparing each of the plurality of differential data points to a threshold; assigning a value of "0" to the image gradient data points corresponding to the derivative data points having values ​​large compared to a threshold to remove unwanted image artifacts; and assigning a value of "1" to the image gradient data points that are to be retained; generating the binary image by determining a centroid of said binary image using said image gradient data points; designating the determined centroid as a target location within the fluid sample located above the reaction cell; Comparing the target location to the default center; and performing at least one assay based on the target location if the distance between the default center and the target location is less than a threshold value; A control unit configured as described above.

16. An apparatus for performing at least one assay comprising:

18. The reaction cell comprises a solid medium, a dry slide, or a reaction cuvette.

20. The apparatus of claim 17 .

19. The solid medium comprises a second reaction cell.

20. The apparatus of claim 18.

20. the target location comprises a center location of a uniform region of the fluid sample; 20. The apparatus of claim 17 .

21. (i) using a first image gradient data point having a lower image gradient than a second image gradient data point, or (ii) using a second image gradient data point having a higher image gradient than the first image gradient data point. and excluding gradient data points.

20. The apparatus of claim 17 .

22. the control unit is configured to determine the target location at a geometric center of a first image gradient data point having a lower image gradient than a second image gradient data point.

20. The apparatus of claim 17 .

23. the control unit is configured to perform a plurality of different assays using target locations from the plurality of images.

20. The apparatus of claim 17 .

24. the target location of the fluid sample on the reaction cell corresponds to a location on the reaction cell where an indicator reaction occurs such that as target molecules in the fluid sample react with a reagent, an indicator is developed for indication; 20. The apparatus of claim 17 .

25. and reporting, via the control unit, an error indicating that a new fluid sample should be dispensed onto a new reaction cell if the distance between the default center and the target location is equal to or greater than a threshold value.

2. The method of claim 1 .

Images