Cell analysis in bodily fluids, in particular blood

Abstract

Devices and methods for analyzing leukocytes in an analyte, such as a liquid sample, are disclosed.

Description

[0001] Cross-reference to related applications

[0002] This application is a divisional application of the Chinese national phase application with application number 201980067048.5 and filing date 2019-08-16. Like the parent application, this application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 764,887 filed on August 16, 2018 and U.S. Provisional Patent Application No. 62 / 719,201 filed on August 17, 2018, and is hereby incorporated by reference in its entirety by virtue of the contents of the above provisional patent applications. The entire disclosure of any publication or patent document mentioned herein is incorporated by reference in its entirety. Technical Field

[0003] The present invention relates particularly to devices and methods for performing biological and chemical assays, such as, but not limited to, assays related to leukocyte analysis. Background Art

[0004] In biological and chemical analysis (eg, diagnostic testing), there is often a need to quickly and easily measure and / or detect an analyte in a sample or a portion of a sample. The present invention provides devices and methods for achieving these goals. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Those skilled in the art will appreciate that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present invention in any way. In the drawings, some of the drawings are not drawn to scale. In the drawings that provide experimental data points, the lines connecting the data points are used only to guide the observation of the data and have no other purpose.

[0006] Figure 1 An embodiment of a QMAX (Q: quantification; M: amplification; A: reagent addition; X: acceleration) device, also known as a compression-regulated open flow (CROF) device, is shown, comprising a first plate and a second plate. Panel (A) shows a perspective view of the plates in an open configuration when separated; panel (B) shows a perspective view and a cross-sectional view of a sample being deposited on the first plate in the open configuration; and panel (C) shows a perspective view and a cross-sectional view of the QMAX device in a closed configuration.

[0007] Figure 2 The graph shows the comparison of white blood cell (WBC) counting accuracy, field of view (FoV), and QMAX gap (thickness of the sample layer). Figure (A) shows a graph of WBC counting accuracy versus QMAX gap size, with an effective FoV of 4mm. 2 , 16mm 2 , 36mm 2 , 64mm 2 and 100mm 2Figure (B) shows a plot of WBC count accuracy FoV with QMAX gap size of 2 pm, 3 pm, 5 pm, 6.2 pm, 10 pm, and 30 pm.

[0008] Figure 3 Figure (A) shows a plot of WBC missing count percentage versus QMAX gap size (thickness of sample layer) of 2 pm, 5 pm, 10 pm, and 30 pm. Figure (B) shows a plot of QMAX transmittance at 500 nm wavelength (which is close to the fluorescence of WBC) versus QMAX transmittance at 500 nm wavelength (which is close to the fluorescence of WBC) versus QMAX gap size.

[0009] Figure 4 Figure shows theoretical calculation of self- overlap rate of WBC cells versus QMAX gap.

[0010] Figure 5 Figure shows a schematic exploded view of an optical adapter device for attaching a QMAX device to a mobile communication device.

[0011] Figure 6 Figure shows a schematic cross-sectional view of details of a system with a QMAX device that can be used to test a sample in a fluorescence illumination mode, and in particular shows an optical adapter.

[0012] Figure 7 Figure shows (a) a photo of a QMAX device and (b) a photo of a QMAX device and an adapter on a smart phone.

[0013] Figure 8 Figure shows (a) a bright field image of HgB in the device at a wavelength of about 520 nm, (b) a bright field image of WBC in the device at an excitation of about 490 nm and emission beyond 500 nm, (c) a bright field image of RBC in the device, (d) a bright field image of WBC and PLT in the device at an excitation of about 490 nm and emission beyond 500 nm, where the whole blood is internally photographed by an apple phone based optical system.

[0014] Figure 9 Figure shows exemplary HgB, WBC, RBC, PLT analysis results of a whole blood sample using a QMAX device, and compared to a commercial blood cell counter such as Horiba Pentra 60C. The results show that the device and method has better accuracy compared to the commercial machine.

[0015] The following detailed description of example embodiments, taken in conjunction with the accompanying drawings, illustrates some embodiments of the present application. The specific embodiments described herein are illustrative of certain embodiments of the present application but are not meant to be limiting as to the scope of the application. The section headings and any subtitles used herein are for organizational purposes only and are not meant to be used to construe the limitations of the subject matter described herein under those headings and / or subtitles.

[0016] Any publication mentioned herein is referred to for its disclosure prior to the filing date of this application. This citation of a publication is not to be construed as an admission that the present application is not entitled to antedate this publication by virtue of prior application. Further, the dates of publication provided can be different from the actual publication dates which can need to be independently confirmed.

[0017] Among other things, the present application provides devices, systems and methods for performing biological and chemical assays using QMAX cards.

[0018] The example embodiments disclosed herein can be combined with biological / chemical devices and assays including, but not limited to, those disclosed, described and / or referenced in the application PCT / US2016 / 046437, which is hereby incorporated by reference in its entirety.

[0019] The embodiments in these applications incorporated herein can be considered to be combined with each other or as a single application, rather than as discrete and independent documents.

[0020] Further, the example embodiments disclosed herein are applicable to embodiments including, but not limited to, biological / chemical assays, QMAX cards and systems, QMAX with hinges, notches, grooved edges and sliders, assays and devices with uniform sample thickness, smart phone detection systems, cloud computing designs, various detection methods, labels, capture agents and detection agents, analytes, diseases, applications and samples; various embodiments are disclosed, described and / or referenced in the above-mentioned applications, all of which are hereby incorporated by reference in their entirety.

[0021] Examples of QMAX devices with hinges (QMAX cards)

[0022] Figure 1A general QMAX (Q: quantification; M: magnification; A: addition of reagents; X: acceleration; also known as a Compressed Regulated Open Flow (CROF) device) is shown. The general QMAX device comprises a first plate 10 and a second plate 2. In particular, Figure (A) shows a perspective view of the first plate 10 and the second plate 20, where the first plate has spacers. However, it should be noted that the spacers can also be fixed on the second plate 20 (not shown) or on both the first plate 10 and the second plate 20 (not shown). Figure (B) shows a perspective view and a cross-sectional view of depositing a sample 90 on the first plate 10 in an open configuration. However, it should be noted that the sample 90 can also be deposited on the second plate 20 (not shown) or on both the first plate 10 and the second plate 20 (not shown). Figure (C) shows (i) the use of the first plate 10 and the second plate 20 to distribute the sample 90 (the sample flows between the inner surfaces of the plates) and reduce the sample thickness, and (ii) the use of the spacers and the plates to regulate the sample thickness in the closed configuration of the QMAX device. The inner surface of each plate has one or more binding sites and / or storage sites (not shown).

[0023] In some embodiments, the spacers 40 have a predetermined uniform height and a predetermined uniform spacer spacing. In the closed configuration, as shown in Figure (C) of the Figure 1 The spacing between the plates and thus the thickness of the sample 90 is regulated by the spacers 40. In some embodiments, the uniform thickness of the sample 90 is substantially similar to the uniform height of the spacers 40. It should be noted that although the spacers 40 are shown fixed on one of the plates, in some embodiments, the spacers are not fixed. For example, in certain embodiments, the spacers are mixed with the sample such that when the sample is compressed into a thin layer, the spacers, as rigid beads or particles of uniform size, regulate the thickness of the sample layer. Figure 1

[0024] QMAX assay

[0025] In biological and chemical assays (i.e., tests), devices and / or methods that simplify assay operations or accelerate assay speed are generally of great value.

[0026] In QMAX (Q: quantification; M, magnification; A, addition of reagents; X: acceleration; also known as a Compressed Regulated Open Flow (CROF) assay platform), the QMAX card uses two plates to shape the sample into a thin layer (e.g., by compression) (as shown in Figure 1 In certain embodiments, plate manipulation requires multiple changes in the relative positions of the two plates (referred to as: plate configurations) by human hands or other external forces. It is necessary to design the QMAX card to make manual operation easy and fast.

[0027] ​One of the plate configurations in a QMAX assay is an open configuration in which the two plates are completely or partially separated (the spacing between the plates is not controlled by spacers) and sample can be deposited. Another configuration is a closed configuration in which at least a portion of the sample deposited in the open configuration is compressed by the two plates into a layer of very uniform thickness, the uniform thickness of the layer being defined by the inner surfaces of the plates and adjusted by the plates and spacers.

[0028] In a QMAX assay operation, the operator needs to first bring the two plates into an open configuration ready for sample deposition, then deposit the sample on one or both plates, and finally close the plates into a closed position. In certain embodiments, the two plates of a QMAX card are initially on top of each other and need to be separated to enter the open configuration for sample deposition. This separation is difficult to do by hand when one of the plates is a thin plastic film (175 pm thick PMA). The present invention aims to provide devices and methods that make the operation of certain assays, such as QMAX card assays, easy and fast.

[0029] In some embodiments, the QMAX device contains a hinge that connects the two or more plates, enabling the plates to open and close in a manner similar to a book.

[0030] In certain embodiments, the hinge is configured such that the hinge can self-retain the angle between the plates after adjustment.

[0031] In certain embodiments, the hinge is configured such that the material of the hinge can retain the QMAX card in a closed configuration, such that the entire QMAX card can be slid into and out of a card slot without causing accidental separation of the two plates.

[0032] Another aspect of the present invention is to provide an opening mechanism, such as but not limited to a notch on the edge of the plate or a strip attached to the plate, such that the user can more easily manipulate the positioning of the plates, such as but not limited to separating the plates by hand.

[0033] Another aspect of the present invention is to provide a hinge that can control the rotation of more than two plates.

[0034] The term "compression open flow (COF)" refers to a method of changing the shape of a flowable sample deposited on a plate by (i) placing another plate on top of at least a portion of the sample, and (ii) then compressing the sample between the two plates by pushing the two plates towards each other; wherein the compression reduces the thickness of at least a portion of the sample and causes the sample to flow into the open space between the two plates. The term "compression regulated open flow" or "CROF" (or "self-calibrating compression open flow" or "SCOF" or "SCCOF") (also referred to as QMAX) refers to a specific type of COF, wherein the final thickness of the portion or the entire sample after compression is "regulated" by spacers, wherein spacers are placed between the two plates. Here, the CROF device can be used interchangeably with the QMAX device.

[0035] Unless otherwise stated, the terms "spacer" or "stopper" refer to a mechanical object that sets a limit to the minimum spacing between two plates when placed between the two plates, which limit can be reached when the two plates are compressed together. That is, during compression, the spacer will stop the relative movement of the two plates to prevent the plate spacing from becoming smaller than a pre-set (i.e. predetermined) value.

[0036] The terms "spacer has a predetermined height" and "spacer has a predetermined spacer spacing" mean that the values of the spacer height and the spacer spacing, respectively, are known prior to the QMAX process. If the values of the spacer height and the spacer spacing are not known prior to the QMAX process, the values of the spacer height and the spacer spacing are not predetermined. An example of a non-predetermined spacer spacing is the case where beads are sprayed on the plates as spacers, wherein the beads land at random positions on the plates. Another example of a non-predetermined spacer spacing is that the spacers move during the QMAX process.

[0037] In the QMAX process, the term "spacer is fixed to its respective plate" means that the spacer is connected to a location of the plate and remains connected to that location during the QMAX process (i.e. the position of the spacer on the respective plate does not change). An example of "spacer is fixed to its respective plate" is that the spacer is integrally made of a piece of material of the plate, and the position of the spacer relative to the plate surface does not change during the QMAX process. An example of "spacer is not fixed to its respective plate" is that the spacer is adhered to the plate by an adhesive, but during the QMAX process, the adhesive is not able to keep the spacer in its original position on the plate surface during the QMAX process, and the spacer moves away from its original position on the plate surface.

[0038] In the QMAX process, the term "open configuration" of two plates refers to a configuration in which the two plates are either partially or completely separated, and the spacing between the plates is not regulated by spacers

[0039] The term "closed configuration" of the two plates in a QMAX process refers to a configuration in which the plates face each other, the spacer and the relevant volume of the sample are between the plates, the relevant spacing between the plates and thus the thickness of the relevant volume of the sample are regulated by the plates and the spacer, wherein the relevant volume is at least a portion of the entire volume of the sample.

[0040] In a QMAX process, the term "the thickness of the sample is regulated by the plates and the spacer" refers to the fact that, for given conditions of the plates, the sample, the spacer and the plate compression method, the thickness of at least one port of the sample in the closed configuration of the plates can be predetermined according to the properties of the spacer and the plates.

[0041] In a QMAX device, the term "inner surface" or "sample surface" of a plate refers to the surface of the plate that contacts the sample, while the other surface of the plate (that does not contact the sample) is referred to as "outer surface".

[0042] Unless specifically stated, the term "height" or "thickness" of an object in a QMAX process refers to the dimension of the object in a direction perpendicular to the surface of the plate. For example, the spacer height is the dimension of the spacer in a direction perpendicular to the surface of the plate, and spacer height and spacer thickness mean the same thing.

[0043] Unless specifically stated, the term "area" of an object in a QMAX process refers to the area of the object that is parallel to the surface of the plate. For example, the spacer area is the area of the spacer that is parallel to the surface of the plate.

[0044] The term QMAX device refers to a device that performs a QMAX (e.g., CROF) process on a sample, with or without a hinge connecting the two plates.

[0045] The terms "QMAX device with a hinge" and "QMAX card" are interchangeable.

[0046] The terms "angle self-maintain", "angle self-maintaining" or "rotation angle self-maintaining" refer to the property of a hinge that substantially maintains the angle between the two plates after an external force that moves the plates from an initial angle is removed from the plates.

[0047] QMAX devices and assays for cell counting

[0048] QMAX devices can be used to analyze fluid samples, such as, but not limited to, biological fluid samples. In some embodiments, QMAX devices are used to analyze blood samples. For example, in certain embodiments, QMAX devices are used to measure the quantity of certain analytes, such as the count of red blood cells (RBCs), white blood cells (WBCs), and / or subtypes of certain blood cells. In certain embodiments, QMAX devices can be used for WBC counts. In certain embodiments, staining reagents can be used to label cells and structures, such as, but not limited to, RBCs, WBCs (including WBC subtypes), and platelets.

[0049] As shown in FIGS. 1A and IB, the QMAX device can be used to analyze a sample in an open configuration, as shown in FIG. 1A, or in a closed configuration, as shown in FIG. IB. In the open configuration, the sample is deposited on one or both plates, and the plates are not yet pressed together. In the closed configuration, the plates are pressed together, and the sample is compressed into a very uniform thickness layer that is trapped between the plates and defined by the interior surfaces of the plates. Figure 1 As shown, various parameters of the QMAX device can vary based on the particular test. For example, in some embodiments, the spacer height is less than 0.2 pm, 0.5 pm, 0.8 pm, 1 pm, 1.2 pm, 1.5 pm, 1.8 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, 16 pm, 17 pm, 18 pm, 19 pm, 20 pm, 25 pm, 30 pm, 35 pm, 40 pm, 45 pm, 50 pm, 60 pm, 70 pm, 75 pm, 80 pm, 90 pm, 100 pm, 125 pm, 150 pm, 175 pm, 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 600 pm, 700 pm, 800 pm, 900 pm, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, or a range between any two of the recited values. In the closed configuration, the uniform thickness of the sample layer is substantially the same as the gap between the QMAX plates, which is substantially the same as the spacer height. Thus, a description of the spacer height also applies to the thickness of the sample layer and the QMAX gap, and vice versa.

[0050] In some embodiments of a QMAX assay, a sample is deposited on one or both plates in an open configuration; the plates are then pressed into a closed configuration such that at least a portion of the sample is compressed into a very uniform thickness layer that is trapped on the plates and defined by the interior surfaces of the plates. In some embodiments, an analyte in the sample is measured. In certain embodiments, the analyte is a type of cell that can be counted. For example, in certain embodiments, the sample is a blood sample and the analyte is a red blood cell; in certain embodiments, the sample is a blood sample and the analyte is a white blood cell; in certain embodiments, the sample is a blood sample and the analyte is a white blood cell subtype (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes).

[0051] In some embodiments, an image of the sample layer can be captured using a camera when the QMAX device is in the closed configuration. In certain embodiments, the camera can have a field of view (FoV) defined as the area in which the camera can capture an image of the sample. In certain embodiments, the camera is part of a device, such as but not limited to a mobile device. In certain embodiments, the mobile device is a smartphone, a tablet computer, or a laptop computer. In some embodiments, the mobile device is a mobile communication device, such as a smartphone. In certain embodiments, the camera has one lens; in certain embodiments, the camera has two lenses aligned parallel to each other.

[0052] In some embodiments, different spacer heights (and thus different sample thicknesses and QMAX gaps) can affect the accuracy of the count of certain cells, such as but not limited to white blood cells and subtypes of white blood cells. For example, for counting white blood cells (WBCs), the spacer height and the FoV can affect the accuracy and consistency of the count results. With an acceptable level of consistency, the direct count results can be adjusted to reflect the true number of cells, providing a basis for diagnosis and health guidance. In certain embodiments, one factor to consider is the consistency of the “miss rate,” which is the deviation of the results of the method being tested from the true number, which is typically established with a well-defined and well-accepted method. It should also be noted that the methods disclosed herein can be applied not only to WBC counting, but also to other assays.

[0053] The devices and methods of the present invention can be used to (1) count white blood cells, (b) count white blood cell subtypes (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes), and (3) differentiate white blood cells, wherein the device further comprises a spacer that regulates the spacing between the sample contact areas when the plate is in the closed configuration.

[0054] In some embodiments, the average thickness of the uniform thickness layer is in the range of 5.0 pm to 8.5 pm.

[0055] In some embodiments, the average thickness of the uniform thickness layer is in the range of 7.5 pm to 10.5 pm.

[0056] In some embodiments, the average thickness of the uniform thickness layer is in the range of 9.5 pm to 12.5 pm.

[0057] In some embodiments, the average thickness of the uniform thickness layer is in the range of 9.5 pm to 12.5 pm.

[0058] In some embodiments, the average thickness of the uniform thickness layer is in the range of 11.5 pm to 13.5 pm.

[0059] In some embodiments, the average thickness of the uniform thickness layer is in the range of 12.5 pm to 14.5 pm.

[0060] In some embodiments, the average thickness of the uniform thickness layer is in the range of 13.5 pm to 16 pm.

[0061] In some embodiments, the spacer height is in the range of 5.0 pm to 8.5 pm.

[0062] In some embodiments, the spacer height is in the range of 7.5 pm to 10.5 pm.

[0063] In some embodiments, the spacer height is in the range of 9.5 pm to 12.5 pm.

[0064] In some embodiments, the spacer height is in the range of 9.5 pm to 12.5 pm.

[0065] In some embodiments, the spacer height is in the range of 11.5 pm to 13.5 pm.

[0066] In some embodiments, the spacer height is in the range of 12.5 pm to 14.5 pm.

[0067] In some embodiments, the spacer height is in the range of 13.5 pm to 16 pm.

[0068] In some embodiments, the field of view for counting and differentiating WBCs is 0.1 mm 2 , 10 mm 2 , 50 mm 2 , 100 mm 2 , or a range between any two of the values.

[0069] In some embodiments, when the gap size of the QMAX device is about 10 pm, the FoV is greater than 36 mm 2 , thereby the WBC counting and differentiation accuracy is less than 5%.

[0070] In some embodiments, when the gap size of the device is 10 pm, the FoV is greater than 16 mm 2 , thereby the WBC counting and differentiation accuracy is less than 10%.

[0071] In some embodiments, when the gap size of the device is 10 pm, the FoV is greater than 2 mm 2 , thereby the WBC counting and differentiation accuracy is less than 20%.

[0072] In some embodiments, the field of view is in the range of 0.1 mm 2 to 10 mm 2within the range of 10 mm to 50 mm, the preferred gap size of the device is within the range of 5 pm to 30 pm, 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 10%.

[0073] In some embodiments, the field of view is within the range of 0.1 mm 2 to 10 mm 2 , the preferred gap size of the device is within the range of 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 20%.

[0074] In some embodiments, the field of view is within the range of 10 mm 2 to 50 mm 2 , the preferred gap size of the device is within the range of 5 pm to 30 pm, 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 10%.

[0075] In some embodiments, the field of view is within the range of 10 mm 2 to 50 mm 2 , the preferred gap size of the device is within the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 20%.

[0076] In some embodiments, the field of view is within the range of 50 mm 2 to 100 mm 2 , the preferred gap size of the device is within the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, 30 pm to 50 pm, whereby the counting and differentiation accuracy is less than 10%.

[0077] In some embodiments, the height of the spacer is preferably within the range of 2 pm to 5 pm, whereby the WBC missing count is less than 15%.

[0078] In some embodiments, the height of the spacer is preferably within the range of 2 pm to 5 pm, 5 pm to 10 pm, whereby the WBC missing count is less than 30%.

[0079] In some embodiments, the height of the spacer is preferably within the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, whereby the WBC missing count is less than 60%.

[0080] In some embodiments, the distance of the sample to the lens is within the range of 2 mm to 5 mm.

[0081] In some embodiments, the distance of the sample to the lens is within the range of 4 mm to 7 mm.

[0082] In some embodiments, the distance of the sample to the lens is within the range of 6 mm to 9 mm.

[0083] In some embodiments, the sample-to-lens distance is in the range of 8 mm to 11 mm.

[0084] In some embodiments, the sample-to-lens distance is in the range of 10 mm to 13 mm.

[0085] In some embodiments, the sample-to-lens distance is in the range of 12 mm to 15 mm.

[0086] Examples of QMAX devices for white blood cell counts

[0087] Figure 2 A comparison of white blood cell (WBC) count accuracy, field of view (FoV), QMAX gap (thickness of sample layer) is illustrated. Undiluted blood is deposited on one or both plates of a QMAX device in an open configuration; the plates are pressed into a closed configuration such that at least part of the sample is compressed into a uniform thickness layer; an image of the compressed sample is captured using a camera in a smartphone; WBC count is performed by image analysis.

[0088] Figure 2 Figure (A) of shows a plot of WBC count accuracy versus QMAX gap size with effective FoV of 4 mm 2 , 16 mm 2 , 36 mm 2 , 64 mm 2 , and 100 mm 2 ; Figure (B) shows a plot of WBC count accuracy FoV with QMAX gap size of 2 pm, 3 pm, 5 pm, 6.2 pm, 10 pm, and 30 pm. Results are also summarized in Table 1.

[0089] Table 1 - WBC count accuracy, field of view, QMAX gap comparison In this set of experiments, the first plate of the QMAX device was 1 mm thick PMMA with printed acridine orange dye, the second plate was an X-plate with spacers made on 175 pm thick PMMA with post size of 30 x 40 pm, spacer distance of 80 pm. 1 pL of fresh blood without any anticoagulant was used in the test and deposited on the first plate. Count accuracy is defined as the standard deviation of the number of counts for all fields on the card with a specific FoV. This count accuracy represents the case when fields with FoV in the sample layer are randomly picked for measurement, which represents how the accuracy of the average number of all fields. In general, WBC count is more accurate with larger field of view and larger QMAX gap. In essence, the count accuracy here reflects the consistency of the method with a specific gap size and field of view.

[0090] Table 2 shows the relationship between WBC missing count and correction factor versus QMAX gap. In this context, missing count rate is defined as the percentage difference between the inverse calculated WBC concentration (from count number, count area, packing factor, gap size) and the true WBC concentration of the sample (measured by a calibrated commercial blood machine).

[0091] Correction factor = 1 / (1 - missing count rate).

[0092] Table 2 - WBC missing count, correction factor versus QMAX gap

[0093] QMAX gap size (pm) WBC missing count WBC correction factor 2 0％ 1 3 0％ 1 5 10％ 1.1 10 25％ 1.3 30 50％ 2.0

[0094] As shown in Table 2, the missing count rate increases with gap size (and thus spacer height and sample thickness). In addition, further experiments show that differentiated WBC (granulocytes, lymphocytes, monocytes) counts have similar missing count rates as WBC total counts. In addition, WBC missing count rate is not affected by field of view.

[0095] Figure 3 Figure (A) of Example 1 shows a plot of WBC missing count percentage versus QMAX gap size (thickness of sample layer; height of spacer) of 2 pm, 5 pm, 10 pm, and 30 pm. Figure 3 Figure (B) of Example 1 shows a plot of QMAX transmittance versus QMAX gap size at 500 nm wavelength (which is close to the fluorescence of WBC).

[0096] As shown in Example 1, Figure 3 Figures (A) and (B), more WBCs are missing counted as the gap size is larger (thicker blood film). One of the reasons is that the fluorescence of WBC is darkened and blocked by RBCs with thicker blood film, as shown in (b) transmittance versus gap size. Thus, the larger the QMAX gap, the more WBCs are missing counted. However, as shown in Figures (A) and (B) of Example 1, Figure 2 the count precision, which reflects the consistency of counts at a particular gap size and field of view, is higher at larger gap size and larger field of view, respectively. Thus, in some embodiments, certain gap size (and thus spacer height) and / or field of view size can be selected to achieve an acceptable level of consistency, and / or to prevent high level of missing counts.

[0097] With the correction factor based on missing count rate, the count result can be adjusted to provide more accurate and consistent numbers for medical and health purposes. In some embodiments, the final number is equal to the count result multiplied by the correction factor. In certain embodiments, the correction factor can be obtained / calculated from Table 2 and / or Figure 3 Example 2.

[0098] Figure 4The calculation of the self-overlap rate of WBC cells versus QMAX gap is shown. The results are also shown in Table 3. Generally, more WBCs overlap when the gap size is large, especially greater than 30 pm.

[0099] Table 3 - Comparison of QMAX gap size, WBC distance, overlap rate

[0100]

[0101]

[0102] Exemplary embodiments with 8 to 12 pm gap

[0103] Experiments (see, e.g., Figures 2-4 ) show that for the measurement of WBCs in undiluted blood samples, a spacer height of 5 to 15 pm provides more accurate results than a spacer height of 2 to 3 pm in a given field of view provided by a camera (e.g., a camera in a mobile phone). In some embodiments, a QMAX device for WBC measurement has a spacer height of 5 to 15 pm. In certain embodiments, a QMAX device has a spacer height of 10 pm, while the same sample thickness uniformity can be achieved. In some embodiments, such a column height is advantageous for imaging and counting white blood cells in undiluted blood.

[0104] Exemplary embodiments of optical adapters

[0105] In some embodiments, a QMAX device (e.g., in the form of a QMAX card) with a sample can be inserted into an adapter, which can be attached to a device containing a camera and / or an illumination source. In certain embodiments, the device is a mobile communication device, such as but not limited to a smart phone.

[0106] Figure 5 A schematic exploded view of an optical adapter device for attaching a QMAX device to a mobile communication device and for measuring an analyte in a sample is shown. Here, the optical adapter device 18 is in a system 19 containing a mobile communication device (smart phone) 1.

[0107] The adapter 18 contains a holder shell 2 that fits over the upper portion of the smart phone 1; an optical box 3 attached to the shell 2, including a socket slot 4, an optical chamber 3C, a plurality of tracks 6b and 6t that allow the rod 8 to slide in, and a rubber door 16 that is inserted into a plurality of grooves 4s to cover the socket slot 4. An optical insert 7 is mounted on top of the optical chamber 3C, with an exit aperture 7L and an entry aperture 7C to the light source 1L and camera 1C (see Figure 6) is aligned. The lens 11 is mounted in the access aperture 7C of the optical insert 7 and is configured so that the sample in the sample slide 5 inserted into the receptacle slot 4 is within the working distance of the camera 1C (see Figure 6 ). The lens 11 is used to magnify the image of the sample captured by the camera 1C (see below). Figure 6 ). A long pass optical filter 12 is mounted on top of the lens 11 in the entrance aperture 7C. A pair of right angle mirrors 13 and 14 are mounted on the bottom of the optical chamber 3C and are configured so that the mirrors 13 and 14 are aligned with the light source 1L and the camera 1C, respectively (see Figure 6 ). Below Figure 6 The operation of mirrors 13 and 14 as brightfield illumination optics in apparatus 18 is described in .

[0108] The rod 8 comprises two horizontal rods: the upper horizontal rod comprises a bandpass optical filter 15 mounted in a slot 8a, and the lower horizontal rod comprises a light absorber 9 mounted on a horizontal plane 8b and a reflector 10 mounted on an inclined plane 8c. Figure 6 1 and 10 are described in conjunction with the operation of the optical filter 15, the light absorber 9, and the reflector 10 as the fluorescence illumination optics in the device 18. The upper horizontal rod of the rod 8 slides along the track 6t in the box 3, and the lower horizontal rods 8b and 8c slide along the track 6b in the box 3. The rod 8 stops at two different positions in the box 3 to switch between brightfield illumination optics and fluorescence illumination optics. The rod 8 is fully inserted into the box 3 to switch the device 18 to operate with the fluorescence illumination optics. A ball plunger 17 is mounted on the side wall of the track 6t to stop the rod 8 at a predetermined position when the rod 8 is pulled outward from the box 3 to switch the device 18 to operate with the brightfield illumination optics.

[0109] Figure 6 A schematic cross-sectional view of a system for testing a sample in fluorescence illumination mode is shown, and in particular details of the optical adapter are shown. Figure 5 The function of the elements described. Figure 5The light source 1L emits a light beam BF1 away from the smartphone 1. The optical filter 15 allows the passage of the light beam BF1 having a specific wavelength range that matches the excitation wavelength of the fluorescent sample in the sample slide 5. A portion of the light beam BF1 is incident on the edge of the transparent sample slide 5 and is coupled to a waveguide light beam BF3 that travels in the sample slide 5 and is incident on the sample region underneath the lens 11. A portion of the light beam BF1 is incident on the mirror 10. The tilted mirror 10 deflects the beam BF1 to a beam BF2 and back-illuminates the sample region in the sample slide 5 directly underneath the lens 11 at a large tilt angle. The remaining portion of the light beam BF1 having a large angle of divergence, i.e., a light beam BF4, is incident on the absorber 9 and is absorbed such that the reflected light of the light beam BF4 does not enter the camera 1C at a small angle of incidence. The light from the sample region underneath the lens 11 passes through the lens 11 and is filtered by the long-pass filter 12 such that only the light within the specified wavelength range emitted by the fluorescent sample in the sample slide 5 enters the camera 1C to form an image. The smartphone 1 captures and processes the image to obtain some properties of the sample. The rubber door 16 is inserted into the device 18 to cover the sample slide 5, preventing ambient light from entering the device 18 to affect the test.

[0110] In some embodiments, Figure 5 and 6 The adapter described in the Summary can be used to measure a blood sample, such as an undiluted whole blood sample. In certain embodiments, the analyte can be WBC, which requires the rod 8 to be inserted for optimal reading. In some embodiments, the adapter comprises:

[0111] (a) an attachment member configured to attach the adapter to a device comprising a light source and a camera;

[0112] (b) a card slot configured to receive a sample card containing a liquid sample compressed into a uniform thickness layer, wherein when the sample card is inserted into the card slot, the sample is located in the field of view of the camera and the light source;

[0113] (c) an optical filter configured to filter light from the light source to form a first light beam having a specific wavelength range, wherein a portion of the first light beam is incident on the edge of the sample card and travels in the sample card to illuminate the sample;

[0114] (d) a mirror configured to deflect a portion of the first light beam to form a second light beam that back-illuminates the sample at a tilt angle;

[0115] (e) an absorber configured to absorb a remaining portion of the first light beam having an angle of divergence.

[0116] In some embodiments, a method of measuring an analyte in a liquid sample, such as but not limited to WBCs, can comprise:

[0117] (a) obtaining a liquid sample;

[0118] (b) compressing at least a portion of the sample into a uniform thickness layer using a sample card,

[0119] (c) inserting the sample card into an adapter device configured to attach to a device comprising a light source and a camera;

[0120] (d) illuminating the sample with light from the light source, wherein

[0121] i. the light is filtered by an optical filter of the adapter device to form a first light beam having a specific wavelength range, a portion of the first light beam illuminating an edge of the sample card and traveling in the sample card to illuminate the sample;

[0122] ii. a portion of the first light beam is deflected by a mirror of the adapter device to form a second light beam that retro-illuminates the sample at an oblique angle; and

[0123] iii. a remaining portion of the first light beam having a divergence angle is absorbed by an absorber of the adapter device.

[0124] In some embodiments, the method further comprises:

[0125] (a) capturing an image of the sample in the uniform thickness layer using the camera;

[0126] (b) analyzing the image to enumerate the analyte in the image; and

[0127] (c) calculating a concentration of the analyte in the sample based on the uniform thickness, a field of view of the camera, a number of the analyte, and a predetermined correction factor;

[0128] wherein the field of view is a range of a field in which the camera captures the image;

[0129] wherein the correction factor is determined by an error count ratio, the error count ratio depending on the field of view, the uniform thickness, and a property of the analyte.

[0130] Exemplary embodiments for WBC measurement

[0131] For device or method embodiments of the present invention, the device can further comprise a plurality of reagent layers on one or both plates, the plurality of reagent layers comprising an anti-adhesion reagent, a cell lysis reagent, a cell staining reagent, a release time control material, and any combination thereof.

[0132] In some embodiments, each reagent layer coated on the plate has a thickness of 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, or a range between any two of the values.

[0133] In some embodiments, the anti-adhesion agent comprises ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate, K2EDTA, or K3EDTA, or any combination thereof.

[0134] In some embodiments, the cell stain comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-Grünwald stain, Leishman's stain ("polychrome" methylene blue (i.e., demethylated to azure) and eosin), Erythrosin B stain (Erythrosin B), and other fluorescent dyes including but not limited to acridine orange dye, 3,3-dihexyloxacarbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, brilliant sulfofluor yellow and diaminostilbene disulfonic acid derivatives, Erythrosin B or trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4,6-diamidino-2-phenylindole, dihydrochloride), or any combination thereof.

[0135] In some embodiments, the cell lysis agent comprises ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, or other acids and bases, or any combination thereof.

[0136] In some embodiments, the release time control material comprises albumin, carbomer, carboxymethylcellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol, or any combination thereof.

[0137] In some embodiments of the method embodiments of the present application, the RBC, platelets, or both in the sample are lysed prior to detecting and / or measuring the WBC.

[0138] In some embodiments of the method embodiments of the present application, the WBC, platelets, or both are lysed in the sample prior to detecting the RBC.

[0139] In some embodiments of the method embodiments of the present application, the RBC, WBC, or both are lysed in the sample prior to detecting the PLT.

[0140] Other examples of the present application

[0141] Other embodiments in accordance with the presently disclosed subject matter are described in the following enumerated paragraphs.

[0142] Correction factor and field of view

[0143] A1. A method for analyzing an analyte in a liquid sample, comprising:

[0144] (a) obtaining a liquid sample;

[0145] (b) compressing at least a portion of the sample into a uniform thickness layer,

[0146] (c) capturing an image of the sample in the uniform thickness layer with a camera, wherein the image shows the analyte; and

[0147] (d) analyzing the image to enumerate the analyte in the image,

[0148] (e) calculating a concentration of the analyte in the sample based on the uniform thickness, a field of view of the camera, the enumeration of the analyte, and a predetermined correction factor;

[0149] wherein the field of view is a range of a field in which the camera captures the image;

[0150] wherein the correction factor is determined by an error count ratio, which depends on the field of view, the uniform thickness, and properties of the analyte.

[0151] Illumination of WBCs

[0152] B1. An adapter device for analyzing an analyte in a liquid sample, comprising:

[0153] (a) an attachment member configured to attach the adapter to an apparatus comprising a light source and a camera;

[0154] (b) a card slot configured to receive a sample card containing a liquid sample compressed into a uniform thickness layer, wherein when the sample card is inserted into the card slot, the sample is located in a field of view of the camera and the light source;

[0155] (c) an optical filter configured to filter light from the light source to form a first light beam having a specific range of wavelengths, wherein a portion of the first light beam illuminates an edge of the sample card and travels in the sample card to illuminate the sample;

[0156] (d) a mirror configured to deflect the portion of the first light beam to form a second light beam that retro-illuminates the sample at an oblique angle;

[0157] (e) an absorber configured to absorb a remaining portion of the first light beam having a divergence angle.

[0158] B2. A method for analyzing an analyte in a liquid sample, comprising:

[0159] (a) obtaining a liquid sample;

[0160] (b) compressing at least a portion of the sample into a uniform thickness layer with a sample card,

[0161] (c) inserting the sample card into an adapter device, the adapter device configured to attach to a device containing a light source and a camera;

[0162] (d) illuminating the sample with light from the light source, wherein

[0163] i. the light is filtered by an optical filter of the adapter device to form a first light beam having a specific range of wavelengths, a portion of the first light beam illuminating an edge of the sample card and traveling in the sample card to illuminate the sample;

[0164] ii. a portion of the first light beam is deflected by a mirror of the adapter device to form a second light beam that back-illuminates the sample at an oblique angle; and

[0165] iii. a remaining portion of the first light beam having a divergence angle is absorbed by an absorber of the adapter device.

[0166] B3. The kit of embodiment 35A, further comprising:

[0167] (a) capturing an image of the sample in the uniform thickness layer with the camera;

[0168] (b) analyzing the image to enumerate the analytes in the image; and

[0169] (c) calculating a concentration of the analytes in the sample based on the uniform thickness, a field of view of the camera, a number of the analytes, and a predetermined correction factor;

[0170] wherein the field of view is a range of a field in which the camera captures the image;

[0171] wherein the correction factor is determined by an error count ratio, the error count ratio depending on the field of view, the uniform thickness, and properties of the analytes.

[0172] Additional features:

[0173] C1. The device or method of any of the above embodiments, wherein the liquid sample is a blood sample.

[0174] C2. The device or method of any of the above embodiments, wherein the analyte is a white blood cell (WBC).

[0175] C3. The device or method of any of the above embodiments, wherein the analyte is a WBC subtype.

[0176] C4. The device or method of any of the above embodiments, wherein the analyte is a neutrophil, an eosinophil, a basophil, a lymphocyte, or a monocyte.

[0177] C5. The device or method of any of the preceding embodiments, wherein the analyte is fluorescently labeled.

[0178] C6. The device or method of any of the preceding embodiments, wherein the uniform thickness is in the range of 5 to 30 pm.

[0179] C7. The device or method of any of the preceding embodiments, wherein the uniform thickness is in the range of 8 to 12 pm.

[0180] C8. The device or method of any of the preceding embodiments, wherein the uniform thickness is about 10 pm.

[0181] C9. The device or method of any of the preceding embodiments, wherein the field of view (FOV) is equal to or greater than 4 mm 2 .

[0182] C10. The device or method of any of the preceding embodiments, wherein the field of view (FOV) is equal to or greater than 16 mm 2 .

[0183] C11. The device or method of any of the preceding embodiments, wherein the field of view (FOV) is equal to or greater than 36 mm 2 .

[0184] C12. The device or method of any of the preceding embodiments, wherein the field of view (FOV) is equal to or greater than 64 mm 2 .

[0185] C13. The device or method of any of the preceding embodiments, wherein the field of view (FOV) is equal to or greater than 100 mm 2 .

[0186] C14. The device or method of any of the preceding embodiments, wherein the correction factor is 1 when the sample thickness is 2 mm, 1 when the sample thickness is 3 mm, 1.1 when the sample thickness is 5 mm, 1.3 when the sample thickness is 10 mm, and 2.0 when the sample thickness is 30 mm.

[0187] C15. The device or method of any of the preceding embodiments, wherein the analyte is fluorescently labeled, and the wavelength range of the first light beam matches the excitation wavelength of the fluorescence of the labeled analyte.

[0188] C15. The device or method of any of the preceding embodiments, wherein the adapter device further comprises a housing member.

[0189] C16. The device or method of any of the preceding embodiments, wherein the adapter device further comprises a stem that can be inserted into or withdrawn from the housing member.

[0190] C17. The device or method of any of the preceding embodiments, wherein the mirror and the absorber are mounted on the stem.

[0191] C18. The device or method of any of the preceding embodiments, wherein the adapter device comprises a card slot having a safety opening that allows insertion of a sample card and prevents ambient light from entering the card slot.

[0192] WBC analysis device

[0193] AA1. A device for analyzing white blood cells in a blood sample, comprising:

[0194] a first plate, a second plate, and spacers, wherein:

[0195] i. the plates are movable relative to each other into different configurations;

[0196] ii. one or both plates are flexible;

[0197] iii. each of the plates comprises an inner surface having a sample contact area for contacting a blood sample;

[0198] iv. one or both of the plates comprises spacers permanently fixed on the sample contact area of the respective plate;

[0199] v. the spacers have:

[0200] (a) a predetermined substantially uniform height having a value selected in the range of 2 pm to 30 pm,

[0201] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0202] (c) a ratio of the width to the height equal to or greater than 1;

[0203] (d) a predetermined fixed, non-randomized spacer pitch in the range of 10 pm to 200 pm (micrometers);

[0204] (e) a fill factor equal to 1% or more, wherein the fill factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0205] (f) a product of the fill factor and the Young's modulus is 2 MPa or more.

[0206] One of the configurations is an open configuration, in which the two plates are partially or completely separated, the spacing between the plates is not regulated by spacers, and a sample is deposited on one or both of the plates; and

[0207] Another of the configurations is a closed configuration, which is configured after the sample is deposited in the open configuration; and in which at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, in which the uniform thickness of the layer is limited by the sample contact area of the two plates and is regulated by the plates and the spacers.

[0208] AA2. A device for analyzing white blood cells in a blood sample, comprising:

[0209] a first plate, a second plate, spacers, and an adapter, wherein:

[0210] i. the plates are movable relative to each other into different configurations;

[0211] ii. one or both plates are flexible;

[0212] Each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0213] iv. one or both of the plates comprises spacers permanently fixed on the sample contact area of the respective plate;

[0214] v. the spacers have:

[0215] (a) a predetermined substantially uniform height having a value selected in the range of 2 pm to 30 pm,

[0216] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0217] (c) a ratio of the width to the height equal to or greater than 1;

[0218] (d) a predetermined fixed, non-random spacer pitch in the range of 10 pm to 200 pm;

[0219] (e) a fill factor equal to 1% or more, where the fill factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0220] (f) a product of the fill factor and the Young's modulus is 2 MPa or more.

[0221] vi. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) a plate in a closed configuration to slide into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing that is configured to cause at least a portion of the sample contact area to be imaged by the camera;

[0222] wherein one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0223] the other of the configurations is a closed configuration configured after the sample is deposited following the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact areas of the two plates and regulated by the plates and the spacers.

[0224] AA3. A device for analyzing white blood cells in a blood sample, comprising:

[0225] a first plate, a second plate, spacers, and an adapter, wherein:

[0226] i. the plates are movable relative to each other into different configurations;

[0227] ii. one or both plates are flexible;

[0228] iii. each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0229] iv. one or both of the plates comprises spacers permanently fixed on the sample contact area of the respective plate;

[0230] v. the spacers have:

[0231] (a) a predetermined substantially uniform height having a value selected in the range of 10 pm to 50 pm,

[0232] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0233] (c) a ratio of the width to the height equal to or greater than 1;

[0234] (d) a predetermined fixed, non-random spacer spacing in the range of 10 pm to 200 pm;

[0235] (e) a packing factor equal to 3% or greater, where the packing factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0236] (f) the product of the packing factor and the Young's modulus is 2 MPa or greater.

[0237] vi. one or both of the plates contains a reagent coated on the sample contact area of the respective plate;

[0238] vii. the reagent has at least one of: (a) a component that stains WBCs; (b) a component that evenly distributes RBCs; (c) a component that lyses RBCs;

[0239] viii. the adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in the closed configuration to slide into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing that is configured to cause at least a portion of the sample contact area to be imaged by the camera;

[0240] where one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0241] where the other of the configurations is a closed configuration configured after the sample is deposited following the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, where the uniform thickness of the layer is limited by the sample contact areas of the two plates and regulated by the plates and the spacers.

[0242] AA4. A method for analyzing white blood cells in a blood sample, comprising:

[0243] (a) obtaining a blood sample;

[0244] (b) obtaining the device of AA1 or AA2 or AA3;

[0245] (c) depositing the blood sample on one or both of the plates when the plates are configured in the open configuration,

[0246] (d) forcing the two plates to form the closed configuration after (c); and

[0247] (e) capturing an image of the sample in the layer of uniform thickness when the plates are in the closed configuration; and

[0248] (f) analyzing the images to determine the concentration of white blood cells in the sample.

[0249] AA5. A method for white blood cell and sub-type (including neutrophils, eosinophils, basophils, lymphocytes and monocytes) counting using a single device, comprising:

[0250] (a) obtaining a blood sample;

[0251] (b) obtaining a device according to any of the above embodiments, wherein the spacer height is 5 pm to 40 pm,

[0252] (c) depositing the blood sample in one or both of the plates when the plates are configured in an open configuration,

[0253] (d) forcing the two plates to form a closed configuration after (c);

[0254] (e) capturing images of the sample in a layer of uniform thickness when the plates are in a closed configuration; and

[0255] (f) analyzing the images to determine the respective numbers of white blood cells, neutrophils, lymphocytes, monocytes, eosinophils and basophils by counting the number of cells in the images and analyzing the fluorescent color (meaning emission wavelength range) and shape of each white blood cell.

[0256] BB1. A device or method according to any of the above embodiments, wherein the blood sample is undiluted.

[0257] BB2. A device or method according to any of the above embodiments, wherein the staining and shape of white blood cells provide fluorescent color, structure and size differentiation of white blood cells and their sub-types, and white blood cell differentiation.

[0258] A device or method according to any of the above embodiments, wherein fluorescent color (meaning emission wavelength range) is used for WBC counting and differentiation;

[0259] A device or method according to any of the above embodiments, wherein size is used for WBC counting and differentiation;

[0260] A device or method according to any of the above embodiments, wherein structure of WBC is used for WBC counting and differentiation;

[0261] A device or method according to any of the above embodiments, wherein both color (meaning emission wavelength range) and structure of WBC are used for WBC counting and differentiation;

[0262] A device or method according to any of the above embodiments, wherein color (meaning emission wavelength range) of white blood cells is differentiated by red, green, blue channels of the image.

[0263] The apparatus or method according to any of the preceding embodiments, wherein the color (meaning the emission wavelength range) of the white blood cells is differentiated by a filter at different wavelengths in front of the camera.

[0264] The apparatus or method according to any of the preceding embodiments, wherein the color (meaning the emission wavelength range) of the white blood cells is differentiated by a filter at different wavelengths in front of the light source

[0265] The apparatus or method according to any of the preceding embodiments, wherein the color (meaning the emission wavelength range) of the white blood cells is from WBC staining by a chemical such as acridine orange dye.

[0266] The apparatus or method according to any of the preceding embodiments, wherein the color (meaning the emission wavelength range) of the white blood cells is from WBC staining by different chemicals.

[0267] The apparatus or method according to any of the preceding embodiments, wherein the white blood cells are stained by propidium iodide (PI), fluorescein isothiocyanate (FITC), and basic orange (BOM); PI is a red nucleic acid dye that stains DNA and shows red fluorescence; FITC stains cytoplasmic proteins and shows green fluorescence; BO21 is a green nucleic acid dye that stains DNA and shows green fluorescence; the combination of these three fluorescent dyes can differentiate different types of white blood cells, such as lymphocytes, monocytes, neutrophils, and eosinophils.

[0268] The apparatus or method according to any of the preceding embodiments, wherein the shape of the white blood cells is analyzed by machine learning.

[0269] The apparatus or method according to any of the preceding embodiments, wherein the color (meaning the emission wavelength range) and shape of the white blood cells are analyzed by machine learning.

[0270] BB3. The apparatus or method according to any of the preceding embodiments, wherein the apparatus further comprises a multi-reagent layer on one or both plates, including an anti-adhesion, cell lysis, cell staining, release time control material layer, or a combination thereof.

[0271] CC1. The apparatus or method according to any of the preceding embodiments, wherein the column height is in the range of 5 pm to 15 pm.

[0272] CC2. The apparatus or method according to any of the preceding embodiments, wherein the column height is in the range of 8 pm to 12 pm.

[0273] CC3. The apparatus or method according to any of the preceding embodiments, wherein the column height is about 10 pm.

[0274] CC3. The device or method of any of the preceding embodiments, wherein the column height is about 30 pm.

[0275] CC4. The device or method of any of the preceding embodiments, wherein the device is configured to count white blood cells.

[0276] CC5. The device or method of any of the preceding embodiments, wherein the device is configured to count white blood cell subtypes (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes).

[0277] CC6. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 7.5 pm to 10.5 pm.

[0278] CC7. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 9.5 pm to 12.5 pm.

[0279] CC8. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 11.5 pm to 13.5 pm.

[0280] CC9. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 12.5 pm to 14.5 pm.

[0281] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 13.5 pm to 15 pm.

[0282] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 15 pm to 18 pm.

[0283] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 18 pm to 25 pm.

[0284] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 25 pm to 30 pm.

[0285] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 30 pm to 35 pm.

[0286] CC10. The device or method of any of the preceding embodiments, wherein the spacer height is in a range of 35 pm to 40 pm.

[0287] CC10. The device or method of any of the above embodiments, wherein the spacer height is in the range of 40 pm to 50 pm.

[0288] CC11. The device or method of any of the above embodiments, wherein the field of view for counting and differentiating WBCs is 0.1 mm 2 , 10 mm 2 , 50 mm 2 , 100 mm 2 , or a range between any two of the values.

[0289] CC12. The device or method of any of the above embodiments, wherein the FoV is greater than 36 mm 2 when the gap size of the device is 10 pm, whereby the WBC counting and differentiation accuracy is less than 5%.

[0290] CC13. The device or method of any of the above embodiments, wherein the FoV is greater than 16 mm 2 when the gap size of the device is 10 pm, whereby the WBC counting and differentiation accuracy is less than 10%.

[0291] CC14. The device or method of any of the above embodiments, wherein the FoV is greater than 2 mm 2 when the gap size of the device is 10 pm, whereby the WBC counting and differentiation accuracy is less than 20%.

[0292] CC15. The device or method of any of the above embodiments, wherein the field of view is 0.1 mm 2 to 10 mm 2 , the preferred gap size of the device is in the range of 10 pm to 30 pm, 30 pm to 50 pm, whereby the counting and differentiation accuracy is less than 10%.

[0293] CC16. The device or method of any of the above embodiments, wherein the field of view is 0.1 mm 2 to 10 mm 2 , the preferred gap size of the device is in the range of 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 20%.

[0294] CC17. The device or method of any of the above embodiments, wherein the field of view is 10 mm 2 to 50 mm 2 , the preferred gap size of the device is in the range of 5 pm to 30 pm, 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 10%.

[0295] CC18. The device or method of any of the above embodiments, wherein the field of view is 10 mm 2 to 50 mm 2 and the preferred gap size of the device is in the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, whereby the counting and differentiation accuracy is less than 20%.

[0296] CC19. The device or method of any of the above embodiments, wherein the field of view is 50 mm 2 to 100 mm 2 and the preferred gap size of the device is in the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, 30 pm to 50 pm, whereby the counting and differentiation accuracy is less than 10%.

[0297] CC20. The device or method of any of the above embodiments, wherein the height of the spacer is in the range of 2 pm to 5 pm, whereby the WBC missing count is less than 15%.

[0298] CC21. The device or method of any of the above embodiments, wherein the height of the spacer is in the range of 2 pm to 5 pm, 5 pm to 10 pm, whereby the WBC missing count is less than 30%.

[0299] CC22. The device or method of any of the above embodiments, wherein the height of the spacer is preferably in the range of 2 pm to 5 pm, 5 pm to 10 pm, 10 pm to 30 pm, whereby the WBC missing count is less than 60%.

[0300] CC23. The device or method of any of the above embodiments, wherein the distance of the sample to the phone lens is in the range of 2 mm to 5 mm.

[0301] CC24. The device or method of any of the above embodiments, wherein the distance of the sample to the phone lens is in the range of 4 mm to 7 mm.

[0302] CC25. The device or method of any of the above embodiments, wherein the distance of the sample to the phone lens is in the range of 6 mm to 9 mm.

[0303] CC26. The device or method of any of the above embodiments, wherein the distance of the sample to the phone lens is in the range of 8 mm to 11 mm.

[0304] CC27. The device or method of any of the above embodiments, wherein the distance of the sample to the phone lens is in the range of 10 mm to 13 mm.

[0305] CC28. The device or method of any of the previous embodiments, wherein the sample-to-telephone lens distance is in the range of 12 mm to 15 mm.

[0306] Other examples of blood cell counts

[0307] A device for analyzing white blood cells in a blood sample, comprising:

[0308] a first plate, a second plate, spacers, and an adapter, wherein:

[0309] i. the plates are movable relative to each other into different configurations;

[0310] ii. one or both plates are flexible;

[0311] iii. each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0312] iv. one or both of the plates comprises spacers permanently affixed on the sample contact area of the respective plate;

[0313] v. the spacers have:

[0314] (a) a predetermined substantially uniform height having a value selected in the range of 10 pm to 50 pm,

[0315] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0316] (c) a ratio of the width to the height equal to or greater than 1 ;

[0317] (d) a predetermined fixed, non-randomized spacer pitch in the range of 10 pm to 200 pm;

[0318] (e) a packing factor equal to 3% or more, where the packing factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0319] (f) a product of the packing factor and the Young’s modulus is 2 MPa or more.

[0320] vi. one or both of the plates comprises a reagent coated on the sample contact area of the respective plate;

[0321] vii. the reagent has at least one of: (a) a component that stains WBCs; (b) a component that evenly distributes RBCs; (c) a component that lyses RBCs; (d) a component that dilutes blood;

[0322] viii. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) a plate in a closed configuration to slide into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing configured to cause at least a portion of the sample contact area to be imaged by the camera;

[0323] wherein one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0324] wherein the other of the configurations is a closed configuration configured after the sample is deposited following the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact areas of the two plates and regulated by the plates and the spacers.

[0325] A device for analyzing hemoglobin in a blood sample, comprising:

[0326] a first plate, a second plate, spacers, and an adapter, wherein:

[0327] i. the plates are movable relative to each other into different configurations;

[0328] ii. one or both plates are flexible;

[0329] iii. each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0330] iv. one or both of the plates comprises spacers permanently fixed on the sample contact area of the respective plate;

[0331] v. the spacers have:

[0332] (a) a predetermined substantially uniform height having a value selected in the range of 10 μm to 50 μm,

[0333] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0334] (c) a ratio of the width to the height equal to or greater than 1;

[0335] (d) a predetermined fixed, non-random spacer spacing in the range of 10 μm to 200 μm;

[0336] (e) a packing factor equal to 3% or greater, where the packing factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0337] (f) the product of the packing factor and the Young's modulus is 2 MPa or greater.

[0338] vi. one or both of the plates comprises a reagent coated on the sample contact area of the respective plate;

[0339] vii. the reagent has at least one of: (a) a component that causes the RBCs to distribute uniformly; (b) a component that causes the RBCs to lyse; (c) a component that causes the blood to dilute;

[0340] viii. the adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to attach to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in the closed configuration to slide into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from the outer surface of the camera;

[0341] where one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0342] where the other of the configurations is a closed configuration, the closed configuration is configured after the sample is deposited following the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, where the uniform thickness of the layer is limited by the sample contact areas of the two plates and regulated by the plates and the spacers.

[0343] A device for analyzing red blood cells in a blood sample, comprising:

[0344] a first plate, a second plate, spacers, and an adapter, where:

[0345] i. the plates are movable relative to each other into different configurations;

[0346] ii. one or both plates are flexible;

[0347] iii. each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0348] iv. one or both of the plates comprises a spacer permanently affixed on the sample contact area of the respective plate;

[0349] v. the spacers have:

[0350] (a) a predetermined substantially uniform height having a value selected in the range of 1.5 pm to 8 pm,

[0351] (b) a shape of the column having a substantially uniform cross-section and a flat top surface;

[0352] (c) a ratio of width to said height equal to or greater than 1 ;

[0353] (d) a predetermined fixed, non-random spacer pitch in the range of 10 pm to 200 pm;

[0354] (e) a fill factor equal to 3% or greater, where fill factor is the ratio of spacer contact area (on the plate) to total plate area; and

[0355] (f) a product of said fill factor and said Young’s modulus is 2 MPa or greater.

[0356] vi. one or both of the plates comprises a reagent coated on a sample contact area of the respective plate;

[0357] vii. the reagent has at least one of: (a) a component that causes uniform distribution of RBCs; (b) a component that reduces aggregation of RBCs; (c) a component that stains RBCs; (d) a component that dilutes blood;

[0358] viii. the adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plates to be slid into the slot in a closed configuration, and (2) when the plates are in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing configured to cause at least a portion of the sample contact area to be imaged by the camera;

[0359] where one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0360] where the other of the configurations is a closed configuration configured after the sample is deposited following the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is substantially stagnant relative to the plates, where the uniform thickness of the layer is limited by the sample contact areas of the two plates and regulated by the plates and the spacers.

[0361] An apparatus for analyzing platelets in a blood sample, comprising:

[0362] a first plate, a second plate, spacers, and an adapter, where:

[0363] i. the plates are movable relative to each other into different configurations;

[0364] ii. one or both plates are flexible;

[0365] iii. each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample.

[0366] iv. one or both of the plates comprises spacers permanently fixed on the sample contact area of the respective plate;

[0367] v. the spacers have:

[0368] (a) a predetermined substantially uniform height having a value selected in the range of 1.5 pm to 30 pm,

[0369] (b) a shape of a column having a substantially uniform cross-section and a flat top surface;

[0370] (c) a ratio of width to said height equal to or greater than 1;

[0371] (d) a predetermined fixed, non-random, spacer pitch in the range of 10 pm to 200 pm;

[0372] (e) a packing factor equal to 3% or more, where the packing factor is the ratio of the spacer contact area (on the plate) to the total plate area; and

[0373] (f) a product of the packing factor and the Young's modulus is 2 MPa or more.

[0374] vi. one or both of the plates comprises a reagent coated on the sample contact area of the respective plate;

[0375] vii. the reagent has at least one of: (a) a component that homogenizes the PLT distribution; (b) a component that reduces PLT aggregation; (c) a component that stains the PLT; (d) a component that dilutes the blood;

[0376] viii. the adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plates in the closed configuration to be slid into the slot, and (2) when the plates are in the slot, at least a portion of the sample area is less than 2 cm from the outer surface of the camera;

[0377] wherein one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not regulated by the spacers, and a sample is deposited on one or both of the plates; and

[0378] One of the configurations is the open configuration, which is configured after the sample is deposited in the open configuration; and in the open configuration, at least a portion of the sample is compressed by the two plates into a layer of substantially uniform thickness and is substantially stagnant relative to the plates, where the uniform thickness of the layer is limited by the sample contact area of the two plates and is adjusted by the plates and spacers.

[0379] Spacers

[0380] According to the present invention, the spacing between the two plates and thus the sample thickness is controlled by using spacers.

[0381] Spacer height. In some embodiments, all spacers have the same predetermined height. In some embodiments, the spacers have different predetermined heights. In some embodiments, the spacers can be divided into groups or regions, where each group or region has its own spacer height. In certain embodiments, the predetermined height of the spacers is the average height of the spacers. In some embodiments, the spacers have approximately the same height. In some embodiments, a percentage number of the spacers have the same height.

[0382] The height of the spacers is selected by the desired adjusted spacing between the plates and / or the adjusted final sample thickness and residual sample thickness. The spacer height (predetermined spacer height), the spacing between the plates and / or the sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 pm or less, 2 pm or less, 3 pm or less, 5 pm or less, 10 pm or less, 20 pm or less, 30 pm or less, 50 pm or less, 100 pm or less, 150 pm or less, 200 pm or less, 300 pm or less, 500 pm or less, 800 pm or less, 1 mm or less, 2 mm or less, 4 mm or less, or in a range between any two of the recited values.

[0383] The spacer height, the spacing between the plates and / or the sample thickness is in one preferred embodiment 1 nm to 100 nm, in another preferred embodiment 100 nm to 500 nm, in a separate preferred embodiment 500 nm to 1000 nm, in another preferred embodiment 1 pm (i.e. 1000 nm) to 2 pm, in a separate preferred embodiment 2 pm to 3 pm, in another preferred embodiment 3 pm to 5 pm, in a separate preferred embodiment 5 pm to 10 pm, and in another preferred embodiment 10 pm to 50 pm, in a separate preferred embodiment 50 pm to 100 pm.

[0384] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 1.5 pm and 2.5 pm.

[0385] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 2.5 pm and 4 pm.

[0386] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 4 pm and 6 pm.

[0387] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 6 pm and 10 pm.

[0388] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 10 pm and 15 pm.

[0389] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 15 pm and 25 pm.

[0390] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 25 pm and 35 pm.

[0391] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 35 pm and 50 pm.

[0392] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 50 pm and 100 pm.

[0393] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 100 pm and 150 pm.

[0394] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is between 150 pm and 200 pm.

[0395] The spacer height is related to and limited by the incident light source power density when testing whole blood samples.

[0396] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is less than 2 pm, less than 5 pm, less than 10 pm, at an incident light source power of 0.1 W / cm 2 to 5 W / cm 2 .

[0397] In a preferred embodiment, the spacer height, the spacing between the plates, and / or the sample thickness is less than 2 pm, less than 5 pm, less than 10 pm, at an incident light source power of 0.1 W / cm 2 to 5 W / cm2 In the case of the spacer height, the spacing between the plates and / or the sample thickness, in a preferred embodiment, is less than 10 μm, less than 20 μm, less than 30 μm.

[0398] In a preferred embodiment, when the incident light source power is 0.1 W / cm 2 to 5W / cm 2 In the case of the spacer height, the spacing between the plates and / or the sample thickness, in a preferred embodiment, is less than 30 μm, less than 40 μm, less than 50 μm.

[0399] In a preferred embodiment, when the incident light source power is 5W / cm 2 Up to 50W / cm 2 In the case of the spacer height, the spacing between the plates and / or the sample thickness, in a preferred embodiment, is less than 10 μm, less than 20 μm, less than 30 μm.

[0400] In a preferred embodiment, when the incident light source power is 5W / cm 2 Up to 50W / cm 2 In the case of the spacer height, the spacing between the plates and / or the sample thickness, in a preferred embodiment, is less than 30 μm, less than 40 μm, less than 50 μm.

[0401] In a preferred embodiment, when the incident light source power is 5W / cm 2 Up to 50W / cm 2 In a preferred embodiment, the spacer height, the spacing between the plates and / or the sample thickness is less than 50 μm, less than 100 μm, less than 150 μm, less than 200 μm.

[0402] In a preferred embodiment, when the incident light source power is 50W / cm 2 to 500W / cm 2 In a preferred embodiment, the spacer height, the spacing between the plates and / or the sample thickness is less than 50 μm, less than 100 μm, less than 150 μm, less than 200 μm.

[0403] In some embodiments, the spacer height is precisely controlled. The relative accuracy of the spacers (i.e., the ratio of the deviation to the desired spacer height) is 0.001% or less, 0.01% or less, 0.1% or less, 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or a range between any two of these values.

[0404] In some embodiments, the spacer height, the spacing between the plates, and / or the sample thickness is: (i) equal to or slightly greater than the smallest dimension of the analyte, or (ii) equal to or slightly greater than the largest dimension of the analyte. By "slightly greater" is meant about 1% to 5% greater, and any value in between the two values.

[0405] In some embodiments, the spacer height, the spacing between the plates, and / or the sample thickness is: (i) equal to or slightly greater than the smallest dimension of the analyte, or (ii) equal to or slightly greater than the largest dimension of the analyte. By "slightly greater" is meant about 1% to 5% greater, and any value in between the two values.

[0406] For example, red blood cells have a disc shape with a smallest dimension of 2 pm (disc thickness) and a largest dimension of 11 pm (disc diameter). In embodiments of the present application, the spacer is selected so that the inner surface spacing of the plates in the relevant area is, in one embodiment, 2 pm (equal to the smallest dimension), in another embodiment, 2.2 pm, or in another embodiment, 3 pm (50% greater than the smallest dimension), or in another embodiment, 5 pm, but less than the largest dimension of the red blood cell. Such embodiments have certain advantages in blood cell counting. In one embodiment, for red blood cell counting, an undiluted whole blood sample is confined in the spacing by having the inner surface spacing be 2 pm or 6 pm and any value in between the two values; on average, each red blood cell (RBC) does not overlap with others, allowing for accurate counting of red blood cells visually.

[0407] For example, white blood cells have a dimension of 5 pm to 20 pm. In embodiments of the present application, the spacer is selected so that the inner surface spacing of the plates in the relevant area is, in one embodiment, 5 pm (equal to the smallest dimension), in another embodiment, 10 pm, or in another embodiment, 30 pm (50% greater than the smallest dimension), or in another embodiment, 5 pm, but less than the largest dimension of the red blood cell. Such embodiments have certain advantages in blood cell counting. In one embodiment, for white blood cell counting, an undiluted whole blood sample is confined in the spacing by having the inner surface spacing be 5 pm or 30 pm and any value in between the two values, allowing for accurate counting of red blood cells visually.

[0408] In some embodiments, the spacer height, the spacing between the plates, and / or the sample thickness is: (i) equal to or slightly less than the smallest dimension of the analyte, or (ii) equal to or slightly less than the largest dimension of the analyte. By "slightly less" is meant about 1% to 5% less, and any value in between the two values.

[0409] In some embodiments, the spacer height, the spacing between the plates, and / or the sample thickness is: (i) equal to or slightly less than the smallest dimension of the analyte, or (ii) equal to or slightly less than the largest dimension of the analyte. By "slightly less" is meant about 1% to 5% less, and any value in between the two values.

[0410] In the present disclosure, in some embodiments, the plates and spacers are used not only to adjust the thickness of the sample, but also to adjust the orientation and / or surface density of the analyte / entity in the sample when the plates are in the closed configuration. A thinner thickness of the sample results in fewer analyte / entity per surface area (i.e., a smaller surface concentration) when the plates are in the closed configuration.

[0411] Spacer lateral dimension. For open spacers, the lateral dimension can be characterized by its lateral dimension (sometimes referred to as width) in both orthogonal directions x and y. The lateral dimension of the spacer is the same or different in each direction. In some embodiments, the lateral dimension in each direction (x or y) is 1 nm or less, 3 nm or less, 5 nm or less, 7 nm or less, 10 nm or less, 20 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 pm or less, 2 pm or less, 3 pm or less, 5 pm or less, 10 pm or less, 20 pm or less, 30 pm or less, 50 pm or less, 100 pm or less, 150 pm or less, 200 pm or less, 300 pm or less, or 500 pm or less, or in a range between any of these two values.

[0412] In some embodiments, the lateral dimension of the spacer is 5 pm to 10 pm.

[0413] In some embodiments, the lateral dimension of the spacer is 10 pm to 15 pm.

[0414] In some embodiments, the lateral dimension of the spacer is 15 pm to 20 pm.

[0415] In some embodiments, the lateral dimension of the spacer is 20 pm to 25 pm.

[0416] In some embodiments, the lateral dimension of the spacer is 25 pm to 30 pm.

[0417] In some embodiments, the lateral dimension of the spacer is 30 pm to 40 pm.

[0418] In some embodiments, the lateral dimension of the spacer is 40 pm to 50 pm.

[0419] In some embodiments, the lateral dimension of the spacer is 50 pm to 70 pm.

[0420] In some embodiments, the lateral dimension of the spacer is 70 pm to 90 pm.

[0421] In some embodiments, the lateral dimension of the spacers is 90 pm to 120 pm.

[0422] In some embodiments, the lateral dimension of the spacers is between 20 times and 40 times the center wavelength of the incident light.

[0423] In some embodiments, the lateral dimension of the spacers is between 40 times and 80 times the center wavelength of the incident light.

[0424] In some embodiments, the lateral dimension of the spacers is between 80 times and 120 times the center wavelength of the incident light.

[0425] In some embodiments, the lateral dimension of the spacers is between 120 times and 80 times the center wavelength of the incident light.

[0426] In some embodiments, the ratio of the lateral dimensions in the x and y directions is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or a range between any two of the values. In some embodiments, different ratios are used to adjust the sample flow direction; the greater the ratio, the more flow is along one direction (the larger dimension direction).

[0427] In some embodiments, the different lateral dimensions of the spacers in the x and y directions are used as (a) using the spacers as scale markers to indicate the orientation of the plate, (b) using the spacers to create more sample flow in the preferred direction, or both.

[0428] In preferred embodiments, the period, width, and height of the spacers are substantially the same. In some embodiments, all of the spacers have the same shape and dimensions. In some embodiments, the spacers have different lateral dimensions.

[0429] For enclosed spacers, in some embodiments, the inner lateral shape and dimensions are selected based on the total volume of the sample to be enclosed by the enclosed spacers, where the volume dimensions have been described in this disclosure; and in certain embodiments, the outer shape and dimensions are selected based on the strength required to support the pressure of the liquid against the spacers and the compression pressure of the pressing plate.

[0430] In certain embodiments, the aspect ratio of the height to the average lateral dimension of the column spacers is 100,000, 10,000, 1,000, 100, 10, 1, 0.1, 0.01, 0.001, 0.0001, 0,00001, or a range between any two of the values.

[0431] Spacer spacing. The spacer can be a single spacer or a plurality of spacers on the plate or in the sample-related area. In some embodiments, the spacers on the plate are configured and / or arranged in an array, and the array is fixed-spaced, a non-fixed-spaced array, or fixed-spaced at some locations on the plate and non-fixed-spaced at other locations.

[0432] In some embodiments, the spaced array of spacers is arranged in a lattice of squares, rectangles, triangles, hexagons, polygons, or any combination thereof, where combination means that different locations of the plate have different spacer lattices.

[0433] In some embodiments, the spacer spacing of the spacer array is fixedly spaced in at least one direction of the array (ie, uniform spacer spacing). In some embodiments, the spacer spacing is configured to improve uniformity between plate spacing in the closed configuration.

[0434] In some embodiments, the distance between adjacent spacers (i.e., the spacer pitch) is 1 μm or less, 5 μm or less, 7 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 40 μm or less, 50 μm or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less, 200 μm or less, 300 μm or less, 400 μm or less, or in a range between any two of said values.

[0435] In certain embodiments, the spacer pitch is 400 μm or less, 500 μm or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm or less, 10 mm or less, or any range therebetween. In certain embodiments, the spacer pitch is 10 mm or less, 20 mm or less, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or any range therebetween.

[0436] The distance between adjacent spacers (i.e., the spacer spacing) is selected so that for given properties of the plate and sample, in the closed configuration of the plate, in some embodiments, the sample thickness between two adjacent spacers varies by at most 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or within any range between the stated values; or in certain embodiments, by at most 80%, 100%, 200%, 400%, or within a range between any two of the stated values.

[0437] Obviously, in order to maintain a given sample thickness variation between two adjacent spacers, a closer spacer spacing is required when using a more flexible plate.

[0438] In preferred embodiments, the spacer pitch is between 20 pm and 50 pm.

[0439] In preferred embodiments, the spacer pitch is between 50 pm and 80 pm.

[0440] In preferred embodiments, the spacer pitch is between 80 pm and 100 pm.

[0441] In preferred embodiments, the spacer pitch is between 100 pm and 150 pm.

[0442] In preferred embodiments, the spacer pitch is between 150 pm and 200 pm.

[0443] In preferred embodiments, the spacer pitch is between 200 pm and 250 pm.

[0444] In preferred embodiments, the spacer pitch is between 250 pm and 300 pm.

[0445] In preferred embodiments, the spacer pitch is between 300 pm and 400 pm.

[0446] In preferred embodiments, the spacer pitch is between 400 pm and 500 pm.

[0447] In preferred embodiments, the spacers are a fixed-spaced square array, wherein the spacers are posts having a height of 2 pm to 6 pm, an average lateral dimension of 10 pm to 40 pm, and a spacer pitch of 1 pm to 100 pm.

[0448] In preferred embodiments, the spacers are a fixed-spaced square array, wherein the spacers are posts having a height of 2 pm to 6 pm, an average lateral dimension of 10 pm to 50 pm, and a spacer pitch of 100 pm to 250 pm.

[0449] In preferred embodiments, the spacers are a fixed-spaced square array, wherein the spacers are posts having a height of 10 pm to 50 pm, an average lateral dimension of 20 pm to 50 pm, and a spacer pitch of 1 pm to 100 pm.

[0450] In preferred embodiments, the spacers are a fixed-spaced square array, wherein the spacers are posts having a height of 10 pm to 50 pm, an average lateral dimension of 20 pm to 50 pm, and a spacer pitch of 100 pm to 250 pm.

[0451] The spacing of the array of spacers is, in one preferred embodiment, 1 nm to 100 nm, in another preferred embodiment, 100 nm to 500 nm, in a separate preferred embodiment, 500 nm to 1000 nm, in another preferred embodiment, 1 μm (i.e., 1000 nm) to 2 μm, in a separate preferred embodiment, 2 μm to 3 μm, in another preferred embodiment, 3 μm to 5 μm, in a separate preferred embodiment, 5 μm to 10 μm, in another preferred embodiment, 10 μm to 50 μm, in a separate preferred embodiment, 50 μm to 100 μm, in a separate preferred embodiment, 100 μm to 175 μm, in a separate preferred embodiment, 175 μm to 300 μm.

[0452] Spacer density. The spacers are arranged on the respective plate with a surface density of greater than 1 per μm 2 greater than 1 per 10 μm 2 greater than 1 per 100 μm 2 greater than 1 per 500 μm 2 greater than 1 per 1000 μm 2 greater than 1 per 5000 μm 2 greater than 1 per 0.01 mm 2 greater than 1 per 0.1 mm 2 greater than 1 per 1 mm 2 greater than 1 per 5 mm 2 greater than 1 per 10 mm 2 greater than 1 per 100 mm 2 greater than 1 per 1000 mm 2 greater than 1 per 10000 mm 2 greater than 1, or in a range between any two of the foregoing values. In some embodiments, the spacers have a density of at least 1 / mm 2 at least 10 / mm 2 at least 50 / mm 2 at least 100 / mm 2 at least 1,000 / mm 2 or at least 10,000 / mm 2 .

[0453] Spacer area fill factor is defined as the ratio of spacer area to total area or the ratio of spacer spacing to width. In some embodiments, the fill factor is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, or in a range between any two of the foregoing values. In certain embodiments, the fill factor is at least 2.3%.

[0454] In a device comprising two plates and spacers, the fourth power of the spacer pitch (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5 x 10 6 μm 3 / GPa or less.

[0455] In a device comprising two plates and spacers, the fourth power of the spacer pitch (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5 x 10 5 μm 3 / GPa or less.

[0456] In a device comprising two plates and spacers, the spacers have a columnar shape, a substantially planar top surface, a predetermined substantially uniform height, and a predetermined constant spacer pitch that is at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one).

[0457] In a device comprising two plates and spacers, the spacers have a columnar shape, a substantially planar top surface, a predetermined substantially uniform height, and a predetermined constant spacer pitch that is at least about 2 times larger than the size of the analyte, wherein the Young's modulus of the spacer multiplied by the fill factor of the spacer is equal to or greater than 2 MPa, wherein the fill factor is the ratio of the spacer contact area to the total plate area, and wherein for each spacer, the ratio of the lateral dimension of the spacer to its height is at least 1 (one), wherein the fourth power of the spacer pitch (ISD) divided by the thickness (h) and Young's modulus (E) of the flexible plate (ISD 4 / (hE)) is 5 x 10 6 μm 3 / GPa or less.

[0458] In a device comprising two plates and spacers, the ratio of the spacer pitch of the spacer to the average width of the spacer is 2 or greater, and the fill factor of the spacer multiplied by the Young's modulus of the spacer is 2 MPa or greater.

[0459] Area, width, and length of the card

[0460] The device, kit, system, or method according to any of the above embodiments, wherein the area of either plate depends on the specific application.

[0461] The device, kit, system, or method of any of the above embodiments, wherein the area of at least one of the plates is 1 mm 2 (squared millimeters) or less, 10 mm 2 (squared millimeters) or less, 25 mm 2 (squared millimeters) or less, 50 mm 2 (squared millimeters) or less, 75 mm 2 (squared millimeters) or less, 1 cm 2 (squared centimeters) or less, 2 cm 2 (squared centimeters) or less, 3 cm 2 (squared centimeters) or less, 4 cm 2 (squared centimeters) or less, 5 cm 2 (squared centimeters) or less, 10 cm 2 (squared centimeters) or less, 100 cm 2 (squared centimeters) or less, 500 cm 2 (squared centimeters) or less, 1000 cm 2 (squared centimeters) or less, 5000 cm 2 (squared centimeters) or less, 10000 cm 2 (squared centimeters) or less, or a range between any of the two values.

[0462] The device, kit, system, or method of any of the above embodiments, wherein the area of at least one plate of the QMAX card is in a range of 500 to 1000 mm 2 .

[0463] The device, kit, system, or method of any of the above embodiments, wherein one plate has an area of about 600 mm 2 and another plate has an area of about 750 mm 2 .

[0464] The device, kit, system, or method of any of the above embodiments, wherein at least one plate of the QMAX card is 1 mm or less, 5 mm or less, 10 mm or less, 15 mm or less, 20 mm or less, 25 mm or less, 30 mm or less, 35 mm or less, 40 mm or less, 45 mm or less, 50 mm or less, 100 mm or less, 200 mm or less, 500 mm or less, 1000 mm or less, 5000 mm or less, or a range between any of the two values.

[0465] The device, kit, system, or method of any of the above embodiments, wherein the width of at least one plate of the QMAX card is in a range of 20 mm to 30 mm.

[0466] The device, kit, system, or method of any of the above embodiments, wherein one plate has a width of about 22 mm and another plate has a width of about 24 mm.

[0467] The device, kit, system or method according to any of the preceding embodiments, wherein the at least one plate of the QMAX card is 1 mm or less, 5 mm or less, 10 mm or less, 15 mm or less, 20 mm or less, 25 mm or less, 30 mm or less, 35 mm or less, 40 mm or less, 45 mm or less, 50 mm or less, 100 mm or less, 200 mm or less, 500 mm or less, 1000 mm or less, 5000 mm or less, or in a range between any two of the values.

[0468] The device, kit, system or method according to any of the preceding embodiments, wherein the at least one plate of the QMAX card has a length in the range of 20 to 40 mm.

[0469] The device, kit, system or method according to any of the preceding embodiments, wherein one plate has a length of about 27 mm and the other plate has a length of about 32 mm.

[0470] The device, kit, system or method according to any of the preceding embodiments, wherein one plate has a length of about 27 mm and this plate has a width of about 22 mm.

[0471] The device, kit, system or method according to any of the preceding embodiments, wherein one plate has a length of about 32 mm and this plate has a width of about 24 mm.

[0472] The device, kit, system or method according to any of the preceding embodiments, wherein one plate has a length of about 27 mm and the other plate has a length of about 32 mm, and one plate has a width of about 22 mm and the other plate has a width of about 24 mm.

[0473] Shape of the card

[0474] The device, kit, system or method according to any of the preceding embodiments, wherein the two plates have a shape that is circular, elliptical, rectangular, triangular, polygonal, annular, or any superposition of these shapes.

[0475] The device, kit, system or method according to any of the preceding embodiments, wherein the two (or more) plates of the QMAX card can have the same size and / or shape, or different sizes and / or shapes.

[0476] The device, kit, system or method according to any of the preceding embodiments, wherein at least one of the two (or more) plates of the QMAX card has rounded corners for user safety considerations, wherein the diameter of the rounded corners is 100 pm or less, 200 pm or less, 500 pm or less, 1 mm or less, 2 mm or less, 5 mm or less, 10 mm or less, 50 mm or less, or in a range between any of these two values.

[0477] The device, kit, system or method according to any of the preceding embodiments, wherein the plates can have any shape, preferably a shape that allows for open flow and adjustment of sample thickness of the compression of the sample.

[0478] The device, kit, system or method according to any of the preceding embodiments, wherein the special shape of the plates is advantageous.

[0479] Thickness of the card

[0480] The device, kit, system or method according to any of the preceding embodiments, wherein the thickness, width and / or length of the two (or more) plates of the QMAX card can be the same or different.

[0481] The device, kit, system or method according to any of the preceding embodiments, wherein the average thickness of at least one of the plates is 2 nm or less, 10 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 1000 nm or less, 2 pm (micrometer) or less, 5 pm or less, 10 pm or less, 20 pm or less, 50 pm or less, 100 pm or less, 150 pm or less, 200 pm or less, 300 pm or less, 500 pm or less, 800 pm or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, 5 mm or less, 10 mm or less, 20 mm or less, 50 mm or less, 100 mm or less, 500 mm or less, or in a range between any of these two values.

[0482] The device, kit, system or method according to any of the preceding embodiments, wherein the thickness of at least one of the plates is in the range of 0.5 mm to 1.5 mm.

[0483] The device, kit, system or method according to any of the preceding embodiments, wherein the thickness of at least one of the plates is about 1 mm.

[0484] The device, kit, system or method according to any of the preceding embodiments, wherein the thickness of at least one of the plates is in the range of 0.15 mm to 0.2 mm.

[0485] The device, kit, system or method of any of the above embodiments, wherein the thickness of at least one of the plates is about 0.175 mm.

[0486] The device, kit, system or method of any of the above embodiments, wherein the thickness of at least one of the plates is in the range of 0.01 mm to 0.15 mm.

[0487] The device, kit, system or method of any of the above embodiments, wherein the thickness of at least one of the plates is about 0.025 mm.

[0488] The device, kit, system or method of any of the above embodiments, wherein the thickness of at least one of the plates is about 0.05 mm.

[0489] The device, kit, system or method of any of the above embodiments, wherein the thickness of at least one of the plates is about 0.1 mm.

[0490] The device, kit, system or method of any of the above embodiments, wherein the thickness of any of the plates is non-uniform across the plate.

[0491] The device, kit, system or method of any of the above embodiments, wherein different plate thicknesses at different locations can be used to control plate bending, folding, sample thickness adjustment, etc.

[0492] Notch

[0493] The device, kit, system or method of any of the above embodiments, wherein one or more notches are on one or more sides of one of the plates for easy peeling off of another plate and separating the two plates.

[0494] The device, kit, system or method of any of the above embodiments, wherein the shape of the notch is circular, elliptical, rectangular, triangular, polygonal, annular, or any superposition of these shapes.

[0495] The device, kit, system or method of any of the above embodiments, wherein the size of the notch is 1 mm 2 (square millimeter) or less, 10 mm 2 or less, 25 mm 2 or less, 50 mm 2 or less, 75 mm 2 or less or in a range between any of the two values.

[0496] The device, kit, system or method of any of the above embodiments, wherein the area of each notch on the QMAX card is in the range of 10 to 30 mm 2 .

[0497] The device, kit, system or method according to any of the preceding embodiments, wherein the notch is semicircular with a diameter of 3 to 6 mm.

[0498] The device, kit, system or method according to any of the preceding embodiments, wherein the notch has a width of 3 mm and a length of 6 mm.

[0499] The device, kit, system or method according to any of the preceding embodiments, wherein the notch is located on the short width side of the thicker plate.

[0500] The device, kit, system or method according to any of the preceding embodiments, wherein two notches are located on the two long width sides of the thicker plate.

[0501] Hinge

[0502] The device, kit, system or method according to any of the preceding embodiments, wherein the size of the hinge is variable and can be adjusted according to the size of the plate and the specific needs of the device application.

[0503] The device, kit, system or method according to any of the preceding embodiments, wherein the shape of the hinge is circular, elliptical, rectangular, triangular, polygonal, annular or any superposition of these shapes.

[0504] The device, kit, system or method according to any of the preceding embodiments, wherein the length of the hinge link is less than 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 100 mm, 200 mm or 500 mm, or in a range between any of these two values.

[0505] The device, kit, system or method according to any of the preceding embodiments, wherein the length of the hinge link is about 20 mm.

[0506] The device, kit, system or method according to any of the preceding embodiments, wherein the width of the hinge link is less than 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 10 mm, 15 mm, 20 mm, 25 mm, 30 mm, 40 mm, 50 mm, 100 mm, 200 mm or 500 mm, or in a range between any of these two values.

[0507] The device, kit, system or method according to any of the preceding embodiments, wherein the width of the hinge link is about 6 mm.

[0508] The device, kit, system, or method of any of the above embodiments, wherein the length of the hinge link is about 20 mm and the width of the hinge link is about 6 mm.

[0509] The device, kit, system, or method of any of the above embodiments, wherein the ratio of the length of the hinge link to the length of the plate edge to which the hinge link is aligned is less than 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or a range between any of these two values.

[0510] The device, kit, system, or method of any of the above embodiments, wherein the ratio of the length of the hinge link to the length of the plate edge to which the hinge link 36 is aligned is 1, indicating that the hinge link completely covers the hinged edge.

[0511] The device, kit, system, or method of any of the above embodiments, wherein the total area of the hinge is less than 1 mm 2 , 5 mm 2 , 10 mm 2 , 20 mm 2 , 30 mm 2 , 40 mm 2 , 50 mm 2 , 100 mm 2 , 200 mm 2 , 500 mm 2 , or a range between any of these two values.

[0512] The device, kit, system, or method of any of the above embodiments, wherein the width of the hinge link is about 120 mm 2 .

[0513] The device, kit, system, or method of any of the above embodiments, wherein the ratio of the total size of the hinge to the total size of one of the plates is less than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, or a range between any of these two values.

[0514] The device, kit, system, or method of any of the above embodiments, wherein the ratio of the total size of the hinge to the total size of one of the plates is about 0.16 to 0.20.

[0515] The device, kit, system, or method of any of the above embodiments, wherein different layers of the hinge have the same or different thicknesses.

[0516] The device, kit, system or method of any of the above embodiments, wherein any of the layers of the hinge has a thickness of 0.1 pm, 1 pm, 2 pm, 3 pm, 5 pm, 10 pm, 20 pm, 30 pm, 50 pm, 100 pm, 200 pm, 300 pm, 500 pm, 1 mm, 2 mm, and a range between any of the two values.

[0517] The device, kit, system or method of any of the above embodiments, wherein any of the layers of the hinge has a thickness in the range of 25 pm to 50 pm.

[0518] The device, kit, system or method of any of the above embodiments, wherein any of the layers of the hinge has a thickness in the range of 50 pm to 75 pm.

[0519] The device, kit, system or method of any of the above embodiments, wherein the hinge has a thickness of about 68 pm.

[0520] The device, kit, system or method of any of the above embodiments, wherein the length of the hinge linker is about 20 mm, the width of the hinge linker is about 6 mm, and the thickness of the hinge linker is about 68 pm.

[0521] The socket slot

[0522] The device, kit, system or method of any of the above embodiments, wherein the receiving area of the socket slot or the lateral area covered by the sliding track has an area greater than or equal to the area of the QMAX device.

[0523] The device, kit, system or method of any of the above embodiments, wherein the shape of the receiving area of the socket slot is circular, elliptical, rectangular, triangular, polygonal, annular, or any superposition of these shapes.

[0524] The device, kit, system or method of any of the above embodiments, wherein the average gap size of the sliding track is greater than the average thickness of the device by 100 nm, 500 nm, 1 pm, 2 pm, 5 pm, 10 pm, 50 pm, 100 pm, 300 pm, 500 pm, 1 mm, 2 mm, 5 mm, 1 cm, or a range between any of the two values.

[0525] The device, kit, system or method of any of the above embodiments, wherein the average gap size of the slot is greater than the average thickness of the device by 50 pm to 300 pm.

[0526] The device, kit, system or method of any of the above embodiments, wherein the receiving area of the socket slot is greater than the area of the device by 1 mm2 (mm2) or less, 10 mm 2 (mm2) or less, 25 mm 2 (mm2) or less, 50 mm 2 (mm2) or less, 75 mm 2 (mm2) or less, 1 cm 2 (mm2) or less, 2 cm 2 (mm2) or less, 3 cm 2 (mm2) or less, 4 cm 2 (mm2) or less, 5 cm 2 (mm2) or less, 10 cm 2 (mm2) or less, 100 cm 2 (mm2) or less, or within a range between any two of the values.

[0527] The device, kit, system or method according to any of the preceding embodiments, wherein the shape of one of the plates or both of the plates is the same as the shape of the socket slot.

[0528] The device, kit, system or method according to any of the preceding embodiments, wherein the socket slot has the shape of a box with one open surface, with a length of 31 mm, a width of 27 mm and a height of 2.5 mm.

[0529] The device, kit, system or method according to any of the preceding embodiments, wherein the QMAX device, when fully engaged, is at most only partially located within the socket slot, and the shape of the portion of one of the plates or both of the plates is the same as the shape of the socket slot.

[0530] Reagent

[0531] The device, kit, system or method according to any of the preceding embodiments, wherein a dye for staining WBCs is applied to the first plate or the second plate or both.

[0532] The device, kit, system or method according to any of the preceding embodiments, wherein a dye for staining WBCs and PLTs is applied to the first plate or the second plate or both.

[0533] The device, kit, system or method according to any of the preceding embodiments, wherein a dye for staining PLTs is applied to the first plate or the second plate or both.

[0534] The device, kit, system or method according to any of the preceding embodiments, wherein the reagent is applied in an array by droplet printing.

[0535] The device, kit, system or method according to any of the preceding embodiments, wherein the reagent is applied by spray.

[0536] The device, kit, system, or method of any of the above embodiments, wherein the reagent is applied by contact printing.

[0537] The device, kit, system, or method of any of the above embodiments, wherein the reagent is applied by contact printing.

[0538] The device, kit, system, or method of any of the above embodiments, wherein a dye that stains RBCs is applied to the first plate or the second plate or both.

[0539] The device, kit, system, or method of any of the above embodiments, wherein a surfactant used to separate and round RBCs is applied to the first plate or the second plate or both.

[0540] The device, kit, system, or method of any of the above embodiments, wherein a chemical that lyses RBCs is applied to the first plate or the second plate or both.

[0541] The device, kit, system, or method of any of the above embodiments, wherein acridine orange is applied to the first plate or the second plate or both.

[0542] The device, kit, system, or method of any of the above embodiments, wherein Zwittergent is applied to the first plate or the second plate or both.

[0543] The device, kit, system, or method of any of the above embodiments, wherein methylene blue and Zwittergent are applied to the first plate or the second plate or both.

[0544] The device, kit, system, or method of any of the above embodiments, wherein acridine orange and Zwittergent are applied to the first plate or the second plate or both.

[0545] The device, kit, system, or method of any of the above embodiments, wherein YOYO dye and Zwittergent are applied to the first plate or the second plate or both.

[0546] The device or method of any of the above embodiments, wherein the device further comprises a plurality of reagent layers on one or both plates, the plurality of reagent layers comprising an anti-adhesion, a cell lysis, a cell staining, a release time control material layer, and combinations thereof.

[0547] wherein each layer applied on the plate has a thickness of 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, or a range between any of the two values.

[0548] wherein the anti-adhesion agent comprises ethylenediaminetetraacetic acid (EDTA), disodium ethylenediaminetetraacetate, K2EDTA, K3EDTA, and the like;

[0549] wherein the cell stain comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's stain ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), Erythrosin B stain (Erythrosin B), and other fluorescent dyes including, but not limited to, acridine orange dye, 3,3-dihexyloxy carbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, brilliant sulfofluor yellow and diaminostilbene disulfonic acid derivatives, Erythrosin B or trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4,6-diamidino-2-phenylindole, dihydrochloride), YOYO;

[0550] wherein the cell stain comprises Wright's stain (eosin, methylene blue), Giemsa stain (eosin, methylene blue, and azure B), May-grünwald stain, Leishman's stain ("polychromatic" methylene blue (i.e., demethylated to azure) and eosin), Erythrosin B stain (Erythrosin B), and other fluorescent dyes including, but not limited to, acridine orange dye, 3,3-dihexyloxy carbocyanine (DiOC6), propidium iodide (PI), fluorescein isothiocyanate (FITC) and basic orange 21 (BO21) dyes, ethidium bromide, brilliant sulfofluor yellow and diaminostilbene disulfonic acid derivatives, Erythrosin B or trypan blue, Hoechst 33342, trihydrochloride, Trihydrate, DAPI (4,6-diamidino-2-phenylindole, dihydrochloride), YOYO, acid fuchsin, hematoxylin, Hoechst stains including Hoechst 33258 and Hoechst 33342, methyl green, methylene blue, nile blue, nile red, osmium tetroxide, rhodamine, safranin, meosic-AAPV-AMC, CFSE, BCECF / AM, silver nitrate, neutral red, pyronine Y, calcein-AM, dihydroethidium, dimethylthiazolyl cyanine FF, rhodamine 123, 4-methylumbelliferone palmitate, fast blue B salt, fluorescein, CH dipotassium salt, DAPI dilactate, propidium iodide;

[0551] wherein the cell lysis agent comprises ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, other acids and bases, and the like;

[0552] wherein the release time control material comprises albumin, carbomer, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol, and the like.

[0553] In some embodiments, a chemical with a certain concentration is coated on the plate and dissolved into the blood to achieve uniform distribution of red blood cells in the device.

[0554] In some embodiments, a chemical with a certain concentration is coated on the plate and dissolved into the blood to lyse red blood cells in the device,

[0555] Wherein the coating can be on the first plate or the second plate or both.

[0556] In certain embodiments, the chemical coated in the device includes but is not limited to surfactants, Zwittergent, ASB-14, ASB-16, CHAPS, cationic surfactant N-N-[tris(hydroxymethyl)methyl]-N-alkyl-N,N-dimethylammonium chloride (lla), llb, llc, lld, CTAC, Tween 20, Tween 40, Tween 60, Tween 80, sodium lauryl sulfate (SLS), ammonium lauryl sulfate, CTAB, sodium lauryl ether sulfate (SLES), sodium myreth sulfate, docusate, perfluorooctyl sulfonate, alkyl-aryl ether phosphates, alkyl ether phosphates, CTAB, cetylpyridinium chloride (CPC), benzalkonium chloride (BAC), benzethonium chloride (BZT), dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), cocamidopropyl hydroxysultaine, cocamidopropyl betaine, narrow-range ethoxylates, octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, nonxynols, Triton X-100, polyethoxylated tallow amine, cocamide monoethanolamine, cocamide diethanolamine, poloxamer, glycerol monostearate, glycerol monolaurate, sorbitan laurate, sorbitan monostearate, sorbitan tristearate, alkyl polyglucose, lauryl glucoside, octyl glucoside, lauryl dimethylamine oxide, dimethyl sulfoxide, phosphine oxide.

[0557] In some embodiments, the agent coated in the device that causes lysis of red blood cells includes but is not limited to Pluronic F-127, Cremophor, Pluronic F-68, Myrj 52, Brij 35, sodium oleate, sodium dodecyl sulfate, Tween 20, Tween 40, Tween 60, Tween 80, SLS, CTAB, CTAC, tamoxifen, saponins, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, ABS-14, ABS-16, antimalarial drugs (quinine compounds), arsenic, dapsone, metals (chromium / chromate, platinum salts, nickel compounds, copper, lead, cisplatin), nitrite, nitrofurantoin, penicillin, fentanyl (marlofen), rho immunoglobulin, ribavirin, sulfonamides, sulfones.

[0558] In some embodiments, the anticoagulant coated in the device includes, but is not limited to, EDTA, such as disodium ethylenediaminetetraacetate (K2EDTA), trisodium ethylenediaminetetraacetate (K3EDTA), coumarin (vitamin K antagonist), warfarin (Coumadin), umbelliferone, benzofurantoin, atromentin, phenindione, heparin, fondaparinux, and idraparinux, dabigatran, rivaroxaban, apixaban, edoxaban, betrixaban, NOAC, hirudin, recombinant hirudin, bivalirudin, agratroban, dabigatran, batroxobin, ecarin, vitamin E, sodium citrate, citrate dextrose, oxalate such as fluoroate oxalate, deltaparin, desirudin, enoxaparin.

[0559] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm2 2 , 5 ng / mm2 2 , 8 ng / mm2 2 , 12 ng / mm2 2 , 15 ng / mm2 2 , 25 ng / mm2 2 , 35 ng / mm2 2 , 50 ng / mm2 2 , 80 ng / mm2 2 , or a range between any two of these values.

[0560] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 100 ng / mm2 2 , 120 ng / mm2 2 , 150 ng / mm2 2 , 180 ng / mm2 2 , 200 ng / mm2 2 , 300 ng / mm2 2 , 400 ng / mm2 2 , 500 ng / mm2 2 , 800 ng / mm2 2 , 1000 ng / mm2 2 , or a range between any two of these values.

[0561] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm

[0562] In certain embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of these values.

[0563] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 3 ng / mm 2 , 5 ng / mm 2 , 8 ng / mm 2 , 12 ng / mm 2 , 15 ng / mm 2 , 25 ng / mm 2 , 35 ng / mm 2 , 50 ng / mm 2 , 80 ng / mm 2 , 100 ng / mm 2 , or in a range between any two of these values.

[0564] In some embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred area concentration of 100 ng / mm 2 , 120 ng / mm 2 , 150 ng / mm 2 , 180 ng / mm 2 , 200 ng / mm 2 , 300 ng / mm 2 , 400 ng / mm 2 , 500 ng / mm 2 , 800 ng / mm 2 , 1000 ng / mm 2 , or in a range between any two of these values.

[0565] In some embodiments, to achieve uniform distribution of red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in blood of 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 1.0 mg / mL, 2 mg / mL, or in a range between any two of said values.

[0566] In certain embodiments, to lyse red blood cells in the device, Zwittergent is coated on the plate at a preferred final concentration in blood of 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 25 mg / mL, 50 mg / mL, or in a range between any two of said values.

[0567] The device, kit, system, or method according to any of the above embodiments, wherein acridine orange is coated on the plate at an area concentration of 0.5 ng / mm 2 , 1 ng / mm 2 , 2 ng / mm 2 , 3 ng / mm 2 , 5 ng / mm 2 , 8 ng / mm 2 , 10 ng / mm 2 , 15 ng / mm 2 , 20 ng / mm 2 , 30 ng / mm 2 , or in a range between any two of said values.

[0568] The device, kit, system, or method according to any of the above embodiments, wherein acridine orange is coated on the plate at an area concentration of 3 to 10 ng / mm 2 and Zwittergent is coated on the plate at an area concentration of 3 to 10 ng / mm 2 .

[0569] The device, kit, system, or method according to any of the above embodiments, wherein acridine orange is coated on the plate at an area concentration of 5 to 20 ng / mm 2 and Zwittergent is coated on the plate at an area concentration of 10 to 30 ng / mm 2 .

[0570] Materials

[0571] The device, kit, system or method of any of the above embodiments, wherein the material of the first plate and the second plate is poly(methyl methacrylate), polystyrene, polycarbonate, polyethylene terephthalate, polyamide, polyester, polyethylene, polypropylene, polyurethane, polyvinyl chloride, acrylonitrile butadiene styrene, polyepoxide, polytetrafluoroethylene, phenolic, furan, silicone, polylactic acid, polyimide, or the like.

[0572] The device, kit, system or method of any of the above embodiments, wherein the surface of the first plate and the second plate is silicon oxide or silicon nitride.

[0573] The device, kit, system or method of any of the above embodiments, wherein the surface of the first plate and the second plate is treated to be hydrophilic.

[0574] The device, kit, system or method of any of the above embodiments, wherein the surface of the first plate and the second plate is treated to not bind cells.

[0575] Transfer tube

[0576] The device, kit, system or method of any of the above embodiments, wherein blood is transferred from the finger directly onto the QMAX card.

[0577] The device, kit, system or method of any of the above embodiments, wherein blood is transferred from the body surface to the QMAX card using a transfer tube having a volume of 2 μΐ, 3 μΐ, 5 μΐ, 8 μΐ, 10 μΐ, 15 μΐ, or a range between any two of the values.

[0578] Landing marker

[0579] The device, kit, system or method of any of the above embodiments, wherein the landing marker of the blood droplet is on the outer surface of the first plate or the second plate.

[0580] The device, kit, system or method of any of the above embodiments, wherein the landing marker of the blood droplet is outside the field of view of the image.

[0581] The device, kit, system or method of any of the above embodiments, wherein the landing marker of the blood droplet is close to the center of the card.

[0582] The device, kit, system or method of any of the above embodiments, wherein the landing marker of the blood droplet is a small dot or a small cross.

[0583] Fill marker

[0584] The device, kit, system, or method of any of the above embodiments, wherein the fill marker for informing the user of the minimum volume and footprint area of the sample in the device is on an outer surface of the first plate or the second plate.

[0585] The device, kit, system, or method of any of the above embodiments, wherein the fill marker for informing the user of the minimum volume and footprint area of the sample in the device is outside the field of view of the image.

[0586] The device, kit, system, or method of any of the above embodiments, wherein the fill marker for informing the user of the minimum volume and footprint area of the sample in the device is near the center of the card.

[0587] The device, kit, system, or method of any of the above embodiments, wherein the fill marker for informing the user of the minimum volume and footprint area of the sample in the device is circular or rectangular.

[0588] Example QMAX device measures complete blood count and compares to commercial machine

[0589] Figure 7 An example device and method for measuring complete blood count (CBC) using a QMAX device is shown. The device measures all CBC parameters without dilution. Preliminary tests show that the device measures accurately compared to a commercial machine.

[0590] Figure 7 A photograph of (a) a QMAX device and (b) a QMAX device and adapter on a smartphone is shown.

[0591] The device was fabricated using PMMA material. The device can be made of polystyrene, PMMA, PC, COC, COP, or other plastic materials.

[0592] The plate 1 used in this example has a thickness of 950 pm to 1050 pm. The plate 1 has a preferred thickness range of 200 pm to 1500 pm.

[0593] The plate 2 used in this example has a thickness of 170 pm to 180 pm. The plate 2 has a preferred thickness range of 50 pm to 250 pm.

[0594] One device that measures RBC and PLT in an experiment has a post height of 5 pm, a post-to-post distance of 90 pm, and a post size of 20 pm. The post can have a post height of 2 pm to 6 pm, a post-to-post distance of 50 pm to 200 pm, and a post size of 5 pm to 40 pm.

[0595] One device that measures HgB and WBC in an experiment has a post height of 30 pm, a post-to-post distance of 80 pm, and a post size of 30 pm. The post can have a post height of 20 pm to 50 pm, a post-to-post distance of 50 pm to 200 pm, and a post size of 10 pm to 50 pm.

[0596] Acridine orange dye for staining WBC and PLT and Zwittergent for partitioning RBC are coated on the plate 1.

[0597] Acridine orange is coated on the plate at an area concentration of 10-80 ng / mm 2 Zwittergent is coated on the plate at an area concentration of 20-130 ng / mm 2 .

[0598] In some other examples, a staining reagent is coated on one or both of the plates. A cell separation reagent is coated on one or both of the plates. The cell lysis reagent is coated on one or both of the plates.

[0599] When measuring and analyzing a whole blood sample using such a device, the following steps are included:

[0600] (a) obtaining a whole blood sample (can refer to a fresh blood draw or K2EDTA venous whole blood) and the device;

[0601] (b) depositing the sample on one or both of the plates when the plates are configured in an open configuration,

[0602] (c) after (b), forcing the two plates to form a closed configuration; and

[0603] (d) while the plates are in the closed configuration, causing light to shine on the device and capturing an image of the sample in the device; and

[0604] (e) analyzing the image to analyze the whole blood count in the device.

[0605] Figure 8 Brightfield images of (a) HgB in the device at a wavelength of about 520 nm, (b) WBC in the device at an excitation of about 490 nm and an emission beyond 500 nm, (c) RBC in the device, (d) WBC and PLT in the device at an excitation of about 490 nm and an emission beyond 500 nm, where the whole blood interior is photographed by an optical system based on an apple phone are shown.

[0606] The red blood cells in (a) with a post height of 30 pm are multilayered, which is beneficial for the determination of HgB. The red blood cells in (c) with a post height of 5 pm become a single layer and are countable in the magnified image.

[0607] Leukocytes and platelets were stained with AO dye and the fluorescent image was a bright spot. Leukocytes in the 5 pm and 30 pm spacing device became a monolayer and were countable in the magnified image. Platelets in the 5 pm spacing device were monolayered and countable in the magnified image.

[0608] Full blood samples (venous blood in K2EDTA tubes) from 50-100 patients were measured using the QMAX device and compared to a commercial hemocytometer such as the Horiba Pentra 60C. 9uL of full blood was dropped onto plate 2 and squeezed through plate 1. The card was then read by a smartphone-based optical system as shown in Figure 8

[0609] Figure 9 Exemplary HgB, WBC, RBC, PLT analysis results of full blood samples using the QMAX device are shown and compared to a commercial hemocytometer such as the Horiba Pentra 60C. The results show that the device and method have good accuracy compared to the commercial machine.

[0610] In detail, compared to the Horiba Pentra 60C, the HgB readings of the commercial machine have R2=98.5% in the measurement range of 7 g / dL to 20 g / dL, the WBC readings have R2=99.3% in the measurement range of 0.4x10 3 / μL to 0.4x10 3 / μL, the RBC readings have R2=98.2% in the measurement range of 21x10 6 / μL to 5.2x10 6 / μL, and the PLT readings have R2=93% in the measurement range of 21x10 3 / μL to 581x10 3 / μL.

[0611] Example 2 QMAX device measures WBC and WBC differentiation

[0612] Figure 10 One exemplary result of measuring WBC and WBC differentiation using the QMAX device is shown. The device measures all WBC and 3 subtypes (granulocytes, monocytes, lymphocytes) without dilution. Preliminary tests show that the device measures accurately compared to the commercial machine.

[0613] Figure 10 ​Fluorographs of WBCs in one QMAX device are shown (a) taken by a smartphone optical system, and (b) statistical count summary of WBCs in the device, plotting WBC counts, green channel intensity per WBC, red channel intensity.

[0614] The device is the same as the WBC device in Example 1.

[0615] Acridine Orange dye, which is used to stain WBCs, is coated on the plate 1. When AO binds to DNA, AO intercalates DNA as a monomer, producing intense green fluorescence under blue excitation. When it binds to RNA and proteins, it forms electrostatic complexes in the form of polymers, producing red fluorescence under blue excitation. Since the DNA / RNA ratio of the 3 subtypes (granulocytes, monocytes, lymphocytes) is different, differentiation of WBCs can be achieved by analyzing the green and red fluorescence ratio of each WBC.

[0616] Acridine Orange is coated on the plate at an area concentration of 10-80 ng / mm 2 and Zwittergent is coated on the plate at an area concentration of 20-130 ng / mm 2 .

[0617] When measuring and analyzing a whole blood sample using such a device, the following steps are included:

[0618] (a) obtaining a whole blood sample (can be a finger prick or K2EDTA venous whole blood) and the device;

[0619] (b) depositing the sample in one or both of the plates when the plates are configured in an open configuration,

[0620] (c) after (b), forcing the two plates to form a closed configuration; and

[0621] (d) when the plates are in a closed configuration, causing light to be irradiated on the device and capturing an image of the sample in the device; and

[0622] (e) analyzing the image to analyze the whole blood count in the device.

[0623] The wavelength of the excitation illumination is 450-480 nm, and the emission is long pass with a cutoff wavelength around 520 nm, and the green (550 nm) and red (650 nm) fluorescence of each WBC can be observed from the camera.

[0624] According to the results, the white blood cells are stained with AO dye, which are colored dots on the fluorescence image, with clear three colors (green, yellow, and red), as Figure 10(a) shown, which corresponds to lymphocytes (more DNA), monocytes (balanced DNA, RNA), and granulocytes (more RNA). The color of each WBC is analyzed using machine learning and software and is separated into 3 clusters as Figure 10 (b) shown.

[0625] iMOST HgB + WBC + WBC differentiation QMAX card example

[0626] In a preferred embodiment, the spacing between the plates and / or the sample thickness is about 30 pm.

[0627] In a preferred embodiment, the spacing between the plates and / or the sample thickness is between 20 pm and 40 pm.

[0628] The spacers are rectangular in shape with rounded corners.

[0629] The lateral dimension of the spacers is about 30 pm x 40 pm.

[0630] The lateral dimension of the spacers is 10 pm to 40 pm.

[0631] The diameter of the rounded corners of the spacers is 10 pm.

[0632] The spacers are in a rectangular lattice array.

[0633] The spacer-to-spacer distance of the spacers is about 80 pm.

[0634] The spacer-to-spacer distance of the spacers is 70 pm to 150 pm.

[0635] The length of one plate of the Q-card is 27 mm and the width of the plate is 22 mm.

[0636] The length of one plate of the Q-card is 32 mm and the width is 24 mm.

[0637] The area of one plate is about 600 mm 2 and the area of the other plate is about 750 mm 2 .

[0638] The thickness of one plate of the Q-card is about 175 pm.

[0639] The thickness of one plate of the Q-card is about 1 mm.

[0640] The notch area on the QMAX card is in the range of 10 to 30 mm 2 .

[0641] The notch is semicircular with a diameter of 3-6 mm.

[0642] The notch has a width of 3 mm and a length of 6 mm.

[0643] The width of the hinge joint is about 6 mm.

[0644] The length of the hinge joint is about 20 mm.

[0645] The hinge has a thickness of about 70 pm.

[0646] The reagents are applied into an array by droplet printing.

[0647] The reagents are applied by spraying.

[0648] Acridine orange or other staining reagent is applied to the first plate or the second plate or both.

[0649] Zwittergent or other detergent is applied to the first plate or the second plate or both.

[0650] Acridine orange is applied to the plate at an area concentration of 10-60 ng / mm 2 Zwittergent is applied to the plate at an area concentration of 20-130 ng / mm 2 .

[0651] The material of the first plate and the second plate is poly(methyl methacrylate).

[0652] The landing mark for the blood droplet is on the outer surface of the first plate or the second plate.

[0653] The landing mark for the blood droplet is a small dot or a small cross.

[0654] The landing mark for the blood droplet is outside the field of view of the image.

[0655] The landing mark for the blood droplet is close to the center of the card.

[0656] At least one of the plates is transparent.

[0657] iMOST RBC+PLT QMAX card example

[0658] Same as 1 above, except that:

[0659] In a preferred embodiment, the spacing between the plates and / or the sample thickness is about 5 pm.

[0660] In a preferred embodiment, the spacing between the plates and / or the sample thickness is between 2 pm and 7 pm.

[0661] The lateral dimension of the spacer is about 30 pm to 40 pm.

[0662] The lateral dimension of the spacer is 5 pm to 40 pm.

[0663] Acridine orange or other staining reagent is applied to the first plate or the second plate or both.

[0664] Zwittergent or other detergent is coated onto the first plate or the second plate or both.

[0665] Acridine orange is coated on the plate at an area concentration of 10-60 ng / mm 2 Zwittergent is coated on the plate at an area concentration of 20-130 ng / mm 2 .

Claims

1. A method for analyzing an analyte in a liquid sample, comprising: (a) obtaining the liquid sample; (b) compressing at least a portion of the sample into a layer of uniform thickness, (c) capturing an image of the sample in the uniform thickness layer with a camera, wherein the image shows the analyte; and (d) analyzing the image to enumerate analytes in the image, (e) calculating the concentration of the analyte in the sample based on the uniform thickness, the field of view of the camera, the analyte enumeration, and a predetermined correction factor; wherein the field of view is the extent of the field over which the camera captures the image; wherein the correction factor is determined by a false count ratio that depends on the field of view, the uniform thickness, and the nature of the analyte.

2. A device for analyzing leukocytes in a blood sample, comprising: A first plate, a second plate, and a spacer, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both plates are flexible; iii. Each of the plates comprises an inner surface having a sample contact area for contacting a blood sample; iv. One or both of said plates comprises said spacer permanently affixed to said sample contact area of ​​the respective plate. v. The spacer has: (a) a predetermined substantially uniform height having a value selected in the range of 2 μm to 50 μm, (b) a pillar shape having a substantially uniform cross-section and a flat top surface; (c) the ratio of width to said height is equal to or greater than 1; (d) a predetermined fixed, non-random spacer spacing in the range of 10 μm to 200 μm (micrometers); (e) a fill factor equal to 1% or greater, wherein the fill factor is the ratio of the spacer contact area (on the panel) to the total panel area; as well as (f) The product of the filling factor and the Young's modulus is 2 MPa or more. wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; and wherein another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact area of ​​the two plates and adjusted by the plates and the spacers.

3. A device for analyzing leukocytes in a blood sample, comprising: A first plate, a second plate, a spacer, and an adapter, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both plates are flexible; iii. Each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample. iv. One or both of said plates comprises said spacer permanently affixed to said sample contact area of ​​the respective plate. v. The spacer has: (a) a predetermined substantially uniform height having a value selected in the range of 2 μm to 50 μm, (b) a pillar shape having a substantially uniform cross-section and a flat top surface; (c) the ratio of width to said height is equal to or greater than 1; (e) a predetermined fixed, non-random spacer spacing within the range of 10 μm to 200 μm; (e) a fill factor equal to 1% or greater, wherein the fill factor is the ratio of the spacer contact area (on the panel) to the total panel area; and (f) The product of the filling factor and the Young's modulus is 2 MPa or more. vi. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in a closed configuration to be slid into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing that is configured to image at least a portion of the sample contact area by the camera; wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; and wherein another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact area of ​​the two plates and adjusted by the plates and the spacers.

4. A device for analyzing leukocytes in a blood sample, comprising: A first plate, a second plate, a spacer, and an adapter, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both plates are flexible; iii. Each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample. iv. One or both of said plates comprises said spacer permanently affixed to said sample contact area of ​​the respective plate. v. The spacer has: (a) a predetermined substantially uniform height having a value selected within the range of 10 μm to 50 μm, (b) a pillar shape having a substantially uniform cross-section and a flat top surface; (c) the ratio of width to said height is equal to or greater than 1; (f) a predetermined fixed, non-random spacer spacing within the range of 10 μm to 200 μm; (e) a fill factor equal to 3% or greater, wherein the fill factor is the ratio of the spacer contact area (on the panel) to the total panel area; and (f) The product of the filling factor and the Young's modulus is 2 MPa or more. Vi. One or both of said plates comprises a reagent coated on said sample contact area of ​​said respective plate; vii. The reagent has at least one of the following: (a) a component for staining WBC; (b) a component for uniformly distributing the RBC; (c) a component for lysing the RBC; viii. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in a closed configuration to be slid into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing that is configured to image at least a portion of the sample contact area by the camera; wherein one of the configurations is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by a spacer, and the sample is deposited on one or both of the plates; and Another of the configurations is a closed configuration, which is configured after the sample is deposited in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by two plates into a layer of very uniform thickness and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact area of ​​the two plates and adjusted by the plates and spacers.

5. A method for analyzing leukocytes in a blood sample, comprising: (a) obtaining a blood sample; (b) obtaining a device according to any preceding claim; in (c) depositing the blood sample on one or both of the plates when the plates are configured in the open configuration, (d) after (c), forcing the two plates into a closed configuration; (e) capturing an image of the sample in the uniform thickness layer when the plate is in the closed configuration; as well as (g) analyzing the image to determine the concentration of leukocytes in the sample.

6. A method for counting leukocytes and subtypes (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes) using a single device, comprising: (a) obtaining a blood sample; (b) obtaining a device according to any preceding claim, wherein the spacer height is 5 μm to 40 μm, (c) depositing the blood sample on one or both of the plates when the plates are configured in an open configuration, (d) after (c), forcing the two plates into a closed configuration; (e) capturing an image of the sample in the layer of uniform thickness when the plate is in the closed configuration; and (f) analyzing the image to determine the corresponding number of leukocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils by counting the number of cells in the image and analyzing the fluorescent color (meaning the emission wavelength range) and shape of each leukocyte.

7. An adapter device for analyzing an analyte in a liquid sample, comprising: (a) an attachment member configured to attach the adapter to a device comprising a light source and a camera; (b) a card slot configured to receive a sample card containing a liquid sample compressed into a layer of uniform thickness, wherein when the sample card is inserted into the card slot, the sample is located in the field of view of the camera and the light source; (c) an optical filter configured to filter the light from the light source to form a first light beam having a specific wavelength range, wherein a portion of the first light beam illuminates an edge of the sample card and travels in the sample card to illuminate the sample; (d) a reflective mirror configured to deflect a portion of the first light beam to form a second light beam that illuminates the sample in reverse at an oblique angle; (e) an absorber configured to absorb a remaining portion of the first light beam having a divergent angle.

8. A method for analyzing an analyte in a liquid sample, comprising: (a) obtaining the liquid sample; (b) compressing at least a portion of the sample into a layer of uniform thickness using a sample card, (c) inserting the sample card into an adapter device configured to be attached to an apparatus comprising a light source and a camera; (d) irradiating the sample with light from the light source, wherein i. The light is filtered by the optical filter of the adapter device to form a first light beam having a specific wavelength range, a portion of which illuminates the edge of the sample card and travels in the sample card to illuminate the sample; ii. a portion of the first beam is deflected by the reflector of the adapter device to form a second beam that illuminates the sample in reverse at an oblique angle; as well as iii. The remaining portion of the first light beam having a divergent angle is absorbed by the absorber of the adapter device.

9. A device for analyzing leukocytes in a blood sample, comprising: A first plate, a second plate, a spacer, and an adapter, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both plates are flexible; iii. Each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample. iv. One or both of said plates comprises said spacer permanently affixed to said sample contact area of ​​the respective plate. v. The spacer has: (a) a predetermined substantially uniform height having a value selected within the range of 5 μm to 150 μm, (b) a pillar shape having a substantially uniform cross-section and a flat top surface; (c) the ratio of width to said height is equal to or greater than 1; (h) a predetermined fixed, non-random spacer spacing within the range of 10 μm to 200 μm; (e) a fill factor equal to 3% or greater, wherein the fill factor is the ratio of the spacer contact area (on the panel) to the total panel area; and (f) The product of the filling factor and the Young's modulus is 2 MPa or more. Vi. One or both of said plates comprises a reagent coated on said sample contact area of ​​said respective plate; vii. The reagent has at least one of the following: (a) a component for staining WBC; (b) a component for uniformly distributing the RBC; (c) a component for lysing the RBC; (d) a component that dilutes the blood; viii. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in a closed configuration to be slid into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, and (d) an optical system in the housing that is configured to image at least a portion of the sample contact area by the camera; wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; and wherein another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact area of ​​the two plates and adjusted by the plates and the spacers.

10. A device for analyzing hemoglobin in a blood sample, comprising: A first plate, a second plate, a spacer, and an adapter, wherein: i. The plates are movable relative to each other into different configurations; ii. One or both plates are flexible; iii. Each of the plates comprises an inner surface having a sample contact area for contacting a fluid sample. iv. One or both of said plates comprises said spacer permanently affixed to said sample contact area of ​​the respective plate. v. The spacer has: (a) a predetermined substantially uniform height having a value selected within the range of 10 μm to 150 μm, (b) a pillar shape having a substantially uniform cross-section and a flat top surface; (c) the ratio of width to said height is equal to or greater than 1; (d) a predetermined fixed, non-random spacer spacing within the range of 10 μm to 200 μm; (e) a fill factor equal to 3% or greater, wherein the fill factor is the ratio of the spacer contact area (on the panel) to the total panel area; and (f) The product of the filling factor and the Young's modulus is 2 MPa or more. vi. One or both of the plates contain reagents coated on the sample contact areas of the respective plates; vii. The reagent has at least one of the following: (a) a component that uniformly distributes the RBC; (b) a component that lyses the RBC; (c) a component that dilutes the blood; viii. The adapter comprises: (a) a housing, (b) an attachment member on the housing that allows the adapter to be attached to a mobile phone having a camera, (c) a slot in the housing that allows (1) the plate in a closed configuration to be slid into the slot, and (2) when the plate is in the slot, at least a portion of the sample area is less than 2 cm from an outer surface of the camera, wherein one of the configurations is an open configuration in which the two plates are partially or fully separated, the spacing between the plates is not adjusted by the spacer, and the sample is deposited on one or both of the plates; and wherein another of the configurations is a closed configuration configured after the sample is deposited in the open configuration; and in the closed configuration, at least a portion of the sample is compressed by the two plates into a layer of very uniform thickness and is essentially stagnant relative to the plates, wherein the uniform thickness of the layer is limited by the sample contact area of ​​the two plates and adjusted by the plates and the spacers.

Images