CELL ANALYSIS IN BODY FLUIDS, ESPECIALLY BLOOD

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] 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.FIELD

[0002] Among other things, the present invention is related to methods of performing biological and chemical assays, such as but not limited to assay related to analysis of white blood cells.BACKGROUND

[0003] In biological and chemical assays (e.g. diagnostic testing), it is often necessary to measure and / or detect analytes of a sample or a part of the sample, quickly and simply. The current invention provides methods for achieving these goals Patent applications US2016 / 0356999 A1, US2017 / 293133 A1 and US2018 / 0202903 A1 disclose methods and devices known in the art.BRIEF DESCRIPTION OF THE DRAWINGS

[0004] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. In some Figures, the drawings are in scale. In the figures that present experimental data points, the lines that connect the data points are for guiding a viewing of the data only and have no other means. Fig. 1 shows an embodiment of a QMAX (Q: quantification; M: magnifying; A: adding reagents; X: acceleration; also known as compressed regulated open flow (CROF)) device, which comprises a first plate and a second plate. Panel (A) shows the perspective view of the plates in an open configuration when the plates are separated apart; panel (B) shows the perspective view and a sectional view of depositing a sample on the first plate at the open configuration; panel (C) the perspective view and a sectional view of the QMAX device in a closed configuration. Fig. 2 illustrates white blood cell (WBC) counting accuracy vs. field of view (FoV) vs. QMAX gap (thickness of sample layer). Panel (A) shows plots of WBC counting accuracy vs. QMAX gap size with effective FoV of 4 mm 2< , 16 mm 2< , 36 mm 2< , 64 mm 2< , and 100 mm 2< ; panel (B) shows plots of WBC counting accuracy FoV with QMAX gap size of 2 um, 3 um, 5 um, 6.2 um, 10 um and 30 um. Fig. 3. Panel (A) illustrates plots of WBC miss count percentage vs. QMAX gap size (thickness of sample layer) of 2um, 5um, 10um and 30um. Panel (B) illustrates plots of QMAX transmittance at 500nm wavelength (which is close to fluorescence of WBCs) vs. QMAX gap size. Fig. 4 shows the theoretical calculation of self-overlap rate of WBC cell vs. QMAX gap. Fig. 5 shows a schematic exploded view of an optical adaptor device for attaching the QMAX device to a mobile communication device. Fig. 6 shows a schematic sectional view with details of a system that can be used to test a sample in fluorescent illumination mode, and particularly of the optical adapter. Fig. 7 shows the (a) the photo of one QMAX device and (b) the photo of QMAX device and adapter on a smartphone. Fig. 8 shows (a) the bright field image of HgB in device at wavelength around 520 nm, (b) fluoresce field image of WBC in device at excitation around 490 nm and emission over 500 nm, (c) the bright field image of RBC in device, (d) fluoresce field image of WBC and PLT in device at excitation around 490 nm and emission over 500 nm, with whole blood inside taken by iphone based optical system. Fig. 9 shows the example HgB, WBC, RBC, PLT analyze results of whole blood samples using QMAX device and compared with commercial hemocytometer as Horiba Pentra 60C. The results show good accuracy of the device and method compared with commercial machine. DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0005] The following detailed description illustrates some embodiments of the invention by way of example and not by way of limitation. The section headings and any subtitles used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. The contents under a section heading and / or subtitle are not limited to the section heading and / or subtitle, but apply to the entire description of the present invention.

[0006] The present invention provides methods of performing biological and chemical assays using a QMAX card.

[0007] The exemplary embodiments herein disclosed can be combined with the bio / chemical devices and assays including, but not limited to, the devices and assays as disclosed, described, and / or referred to in the following publication: WO 2017 / 027643 A1.

[0008] Moreover, the exemplary embodiments disclosed herein are applicable to embodiments including but not limited to: bio / chemical assays, QMAX cards and systems, QMAX with hinges, notches, recessed edges and sliders, assays and devices with uniform sample thickness, smartphone detection systems, cloud computing designs, various detection methods, labels, capture agents and detection agents, analytes, diseases, applications, and samples; the various embodiments are disclosed, described, and / or referred to in the aforementioned applications.Examples of QMAX Device with Hinges (QMAX Card)

[0009] Fig. 1 shows an embodiment of a generic QMAX (Q: quantification; M: magnifying; A: adding reagents; X: acceleration; also known as compressed regulated open flow (CROF)) device. The generic QMAX device comprises a first plate 10 and a second plate 2. In particular, panel (A) shows the perspective view of a first plate 10 and a second plate 20 wherein the first plate has spacers. It should be noted, however, that the spacers can also be fixed on the second plate 20 (not shown) or on both first plate 10 and second plate 20 (not shown). Panel (B) shows the perspective view and a sectional view of depositing a sample 90 on the first plate 10 at an open configuration. It should be noted, however, that the sample 90 also 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). Panel (C) illustrates (i) using the first plate 10 and second plate 20 to spread the sample 90 (the sample flow between the inner surfaces of the plates) and reduce the sample thickness, and (ii) using the spacers and the plate to regulate the sample thickness at the closed configuration of the QMAX device. The inner surfaces of each plate have one or a plurality of binding sites and or storage sites (not shown).

[0010] In some embodiments, the spacers 40 have a predetermined uniform height and a predetermined uniform inter-spacer distance. In the closed configuration, as shown in panel (C) of Fig. 1, the spacing between the plates and the 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 Fig. 1 shows the spacers 40 to be 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 so that when the sample is compressed into a thin layer, the spacers, which is rigid beads or particles that have a uniform size, regulate the thickness of the sample layer.QMAX Assay

[0011] In biological and chemical assaying (i.e. testing), a device and / or a method that simplifies assaying operation or accelerates assaying speed is often of great value.

[0012] In the QMAX (Q: quantification; M: magnifying; A: adding reagents; X: acceleration; also known as compressed regulated open flow (CROF)) assay platform, a QMAX card uses two plates to manipulate the shape of a sample into a thin layer (e.g. by compressing) (as illustrated in Fig. 1). In certain embodiments, the plate manipulation needs to change the relative position (termed: plate configuration) of the two plates several times by human hands or other external forces. There is a need to design the QMAX card to make the hand operation easy and fast.

[0013] In QMAX assays, one of the plate configurations is an open configuration, wherein the two plates are completely or partially separated (the spacing between the plates is not controlled by spacers) and a sample can be deposited. Another configuration is a closed configuration, wherein at least part of the sample deposited in the open configuration is compressed by the two plates into a layer of highly uniform thickness, the uniform thickness of the layer is confined by the inner surfaces of the plates and is regulated by the plates and the spacers.

[0014] In a QMAX assay operation, an operator needs to first make the two plates to be in an open configuration ready for sample deposition, then deposit a sample on one or both of the plates, and finally close the plates into a close position. In certain embodiments, the two plates of a QMAX card are initially on top of each other and need to be separated to get into an open configuration for sample deposition. When one of the plate is a thin plastic film (175 um thick PMA), such separation can be difficult to perform by hand. The present invention intends to provide the devices and methods that make the operation of certain assays, such as the QMAX card assay, easy and fast.

[0015] In some embodiments, the QMAX device comprises a hinge that connects the two or more plates, so that the plates can open and close in a similar fashion as a book.

[0016] In certain embodiments, the hinge is configured so that the hinge can self-maintain the angle between the plates after adjustment.

[0017] In certain embodiments, the hinge is configured so that the material of the hinge, which maintain the QMAX card in the closed configuration, such that the entire QMAX card can be slide in and slide out a card slot without causing accidental separation of the two plates.

[0018] Another aspect of the present invention is to provide opening mechanisms such as but not limited to notches on plate edges or strips attached to the plates, making is easier for a user to manipulate the positioning of the plates, such as but not limited to separating the plates of by hand.

[0019] Another aspect of the present invention is to provide a hinge that can control the rotation of more than two plates.

[0020] The term "compressed open flow (COF)" refers to a method that changes the shape of a flowable sample deposited on a plate by (i) placing other plate on top of at least a part 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 a thickness of at least a part of the sample and makes the sample flow into open spaces between the plates. The term "compressed regulated open flow" or "CROF" (or "self-calibrated compressed open flow" or "SCOF" or "SCCOF") (also known as QMAX) refers to a particular type of COF, wherein the final thickness of a part or entire sample after the compression is "regulated" by spacers, wherein the spacers are placed between the two plates. Here the CROF device is used interchangeably with the QMAX device.

[0021] The term "spacers" or "stoppers" refers to, unless stated otherwise, the mechanical objects that set, when being placed between two plates, a limit on the minimum spacing between the two plates that can be reached when compressing the two plates together. Namely, in the compressing, the spacers will stop the relative movement of the two plates to prevent the plate spacing becoming less than a preset (i.e. predetermined) value.

[0022] The term "a spacer has a predetermined height" and "spacers have a predetermined inter-spacer distance" means, respectively, that the value of the spacer height and the inter spacer distance is known prior to a QMAX process. It is not predetermined, if the value of the spacer height and the inter-spacer distance is not known prior to a QMAX process. For example, in the case that beads are sprayed on a plate as spacers, where beads are landed at random locations of the plate, the inter-spacer distance is not predetermined. Another example of not predetermined inter spacer distance is that the spacers moves during a QMAX processes.

[0023] The term "a spacer is fixed on its respective plate" in a QMAX process means that the spacer is attached to a location of a plate and the attachment to that location is maintained during a QMAX (i.e. the location of the spacer on respective plate does not change) process. An example of "a spacer is fixed with its respective plate" is that a spacer is monolithically made of one piece of material of the plate, and the location of the spacer relative to the plate surface does not change during the QMAX process. An example of "a spacer is not fixed with its respective plate" is that a spacer is glued to a plate by an adhesive, but during a use of the plate, during the QMAX process, the adhesive cannot hold the spacer at its original location on the plate surface and the spacer moves away from its original location on the plate surface.

[0024] The term "open configuration" of the two plates in a QMAX process means a configuration in which the two plates are either partially or completely separated apart and the spacing between the plates is not regulated by the spacers

[0025] The term "closed configuration" of the two plates in a QMAX process means a configuration in which the plates are facing each other, the spacers and a 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, is regulated by the plates and the spacers, wherein the relevant volume is at least a portion of an entire volume of the sample.

[0026] The term "a sample thickness is regulated by the plate and the spacers" in a QMAX process means that for a give condition of the plates, the sample, the spacer, and the plate compressing method, the thickness of at least a port of the sample at the closed configuration of the plates can be predetermined from the properties of the spacers and the plate.

[0027] The term "inner surface" or "sample surface" of a plate in a QMAX device refers to the surface of the plate that touches the sample, while the other surface (that does not touch the sample) of the plate is termed "outer surface".

[0028] The term "height" or "thickness" of an object in a QMAX process refers to, unless specifically stated, the dimension of the object that is in the direction normal to a surface of the plate. For example, spacer height is the dimension of the spacer in the direction normal to a surface of the plate, and the spacer height and the spacer thickness means the same thing.

[0029] The term "area" of an object in a QMAX process refers to, unless specifically stated, the area of the object that is parallel to a surface of the plate. For example, spacer area is the area of the spacer that is parallel to a surface of the plate.

[0030] The term of QMAX device refers the device that perform a QMAX (e.g. CROF) process on a sample, and have or not have a hinge that connect the two plates.

[0031] The term "QMAX device with a hinge and "QMAX card" are interchangeable.

[0032] The term "angle self-maintain", "angle self-maintaining", or "rotation angle self-maintaining" refers to the property of the hinge, which substantially maintains an angle between the two plates, after an external force that moves the plates from an initial angle into the angle is removed from the plates.QMAX Device and Assay for Cell Counting

[0033] The QMAX device can be used to analyze fluid samples, such as but not limited to biological fluid samples. In some embodiments, the QMAX device is used to analyze a blood sample. For example, in certain embodiments, the QMAX device is used to measure the amount of certain analytes, e.g. counting of red blood cells (RBC), white blood cells (WBC), and / or subtypes of certain blood cells. In certain embodiments, the QMAX device can be used for the counting of WBC. In certain embodiments, staining reagents can be used to label the cells and structures, such as but not be limited to RBC, WBC (including WBC subtypes), and platelets.

[0034] As shown in Fig. 1, various parameters of the QMAX device can vary based on specific tests. For example, in some embodiment, the spacer height is less than 0.2 um, 0.5 um, 0.8 um, 1 um, 1.2 um, 1.5 um, 1.8 um, 2 um, 3 um, 4 um, 5 um, 6 um, 7 um, 8 um, 9 um, 10 um, 11 um, 12 um, 13 um, 14 um, 15 um, 16 um, 17 um, 18 um, 19 um, 20 um, 25 um, 30 um, 35 um, 40 um, 45 um, 50 um, 60 um, 70 um, 75 um, 80 um, 90 um, 100 um, 125 um, 150 um, 175 um, 200 um, 250 um, 300 um, 350 um, 400 um, 450 um, 500 um, 600 um, 700 um, 800 um, 900 um, 1 mm, 2 mm, 3 mm, 4 mm, 5mm, 10 mm, or in a range between any of the two 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. Therefore, the descriptions to the spacer height also apply to the thickness of the sample layer and the QMAX gap, and vice versa.

[0035] In some embodiments of the QMAX assay, the sample is deposited to one or both of the plates in the open configuration; then the plates are pressed into a closed configuration so that at least part of the sample compressed into a layer of highly uniform thickness, which is stagnant to the plates and confined by the inner surfaces of the plates. In some embodiments, an analyte in the sample is measured. In certain embodiments, the analyte is a type of cells that can be counted. For example, in certain embodiments the sample is a blood sample and the analyte is red blood cells; in certain embodiments the sample is a blood sample and the analyte is white blood cells; in certain embodiments the sample is a blood sample and the analyte is white blood cell sub-types (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes).

[0036] In some embodiments, when the QMAX device is in the closed configuration, a camera can be used to capture images of the sample layer. In certain embodiments, the camera can have a field of view (FoV), which is defined as the area of sample of which the image can be captured by the camera. 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 smart phone, a tablet computer, or a laptop computer. In some embodiments, the mobile device is a mobile communication device such as a smart phone. In certain embodiments, the camera has one lens; in certain embodiments, the camera has two lenses that are aligned parallel to each other.

[0037] In some embodiments, different spacer height (hence different sample thickness and QMAX gap) can affect the accuracy of the counting of certain cells, such as but not limited to white blood cells and sub-types of white blood cells. For example, for counting white blood cells (WBC), spacer height and FoV can affect the accuracy and consistency of the counting results. With an acceptable level of consistency, the direct counting results can be adjusted to reflect the real number of cells, providing basis for diagnostics and health guidance. In certain embodiments, one factor that needs to be considered is the consistency of "miss count" rate, which is the deviation of the results with a method being tested from the real number, which is usually established with a well-defined and well-accepted method. It should also be noted that the method herein disclosed can be applied to not only WBC counting, but also other assays.

[0038] The device and method of the current invention can be used to (1) count the white blood cells, (b) count the white blood cells sub-types (including neutrophils, eosinophils, basophils, lymphocytes, and monocytes), and (3) differentiate white blood cells, wherein the device further comprises spacers that regulate the spacing between the sample contact areas when the plates are in a closed configuration.

[0039] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 5.0 um to 8.5 um.

[0040] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 7.5 um to 10.5 um.

[0041] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 9.5 um to 12.5 um.

[0042] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 9.5 um to 12.5 um.

[0043] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 11.5 um to 13.5 um.

[0044] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 12.5 um to 14.5 um.

[0045] In some embodiments, the average thickness of the layer of uniform thickness is in the range of 13.5 um to 16 um.

[0046] In some embodiments, the spacer height is in the range of 5.0 um to 8.5 um.

[0047] In some embodiments, the spacer height is in the range of 7.5 um to 10.5 um.

[0048] In some embodiments, the spacer height is in the range of 9.5 um to 12.5 um.

[0049] In some embodiments, the spacer height is in the range of 9.5 um to 12.5 um.

[0050] In some embodiments, the spacer height is in the range of 11.5 um to 13.5 um.

[0051] In some embodiments, the spacer height is in the range of 12.5 um to 14.5 um.

[0052] In some embodiments, the spacer height is in the range of 13.5 um to 16 um.

[0053] 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.

[0054] In some embodiments, when the gap size of the QMAX device is about 10 um, the FoV is larger than 36 mm 2< , thereby the WBC counting and differentiation accuracy is less than 5%.

[0055] In some embodiments, when the gap size of device is 10 um, the FoV is larger than 16 mm 2< , thereby the WBC counting and differentiation accuracy is less than 10%.

[0056] In some embodiments, when the gap size of device is 10 um, the FoV is larger than 2 mm 2< , thereby the WBC counting and differentiation accuracy is less than 20%.

[0057] In some embodiments, the field of view is in the range of 0.1 mm 2< to 10 mm 2< , the preferred gap size of device is in the range of 10 um to 30 um, 30 um to 50 um, thereby the counting and differentiation accuracy is less than 10%.

[0058] In some embodiments, the field of view is in the range of 0.1 mm 2< to 10 mm 2< , the preferred gap size of device is in the range of 10 um to 30 um, thereby the counting and differentiation accuracy is less than 20%.

[0059] In some embodiments, the field of view is in the range of 10 mm 2< to 50 mm 2< , the preferred gap size of device is in the range of 5 um to 10 um, 10 um to 30 um, thereby the counting and differentiation accuracy is less than 10%.

[0060] In some embodiments, the field of view is in the range of 10 mm 2< to 50 mm 2< , the preferred gap size of device is in the range of 2um to 5 um, 5 um to 10 um, 10 um to 30 um, thereby the counting and differentiation accuracy is less than 20%.

[0061] In some embodiments, the field of view is in the range of 50 mm 2< to 100 mm 2< , preferred gap size of device is in the range of 2 um to 5 um, 5 um to 10 um, 10 um to 30 um, 30 um to 50 um thereby the counting and differentiation accuracy is less than 10%.

[0062] In some embodiments, the spacer has a height of preferred range of 2 um to 5um, thereby the WBCs missing counting is less than 15%.

[0063] In some embodiments, the spacer has a height of preferred range of 2 um to 5um, 5 um to 10 um, thereby the WBCs missing counting is less than 30%.

[0064] In some embodiments, the spacer has a height of preferred range of 2 um to 5um, 5 um to 10 um, 10um to 30um thereby the WBCs missing counting is less than 60%.

[0065] In some embodiments, the sample to camera lens distance is in the range of 2 mm to 5 mm.

[0066] In some embodiments, the sample to camera lens distance is in the range of 4 mm to 7 mm.

[0067] In some embodiments, the sample to camera lens distance is in the range of 6 mm to 9 mm.

[0068] In some embodiments, the sample to camera lens distance is in the range of 8 mm to 11 mm.

[0069] In some embodiments, the sample to camera lens distance is in the range of 10 mm to 13 mm.

[0070] In some embodiments, the sample to camera lens distance is in the range of 12 mm to 15 mm.Examples of QMAX Device for Counting White Blood Cells

[0071] Fig. 2 illustrates white blood cell (WBC) counting accuracy vs. field of view (FoV) vs. QMAX gap (thickness of sample layer). Undiluted blood was deposited on one or both of the plates of the QMAX device in the open configuration; the plates were pressed into a closed configuration so that at least part of the sample was compressed into a layer of uniform thickness; a camera in a smart phone was used to capture images of the compressed sample; the number of WBC was counted by analyzing the images.

[0072] Panel (A) of Fig. 2 shows plots of WBC counting accuracy vs. QMAX gap size with effective FoV of 4 mm 2< , 16 mm 2< , 36 mm 2< , 64 mm 2< , and 100 mm 2< ; panel (B) shows plots of WBC counting accuracy FoV with QMAX gap size of 2 um, 3 um, 5 um, 6.2 um, 10 um and 30 um. The results are also summarized in Table 1. Table 1 - WBC counting accuracy vs. Field of View vs. QMAX gapField of View (mm 2< )QMAX gap size (um)2356.21030461%57%39%24%15%12%1633%27%15%12%8%8%3620%13%8%7%4%4%647%7%6%6%3%2%1006%6%3%5%2%3%

[0073] In this set of experiments, the first plate of the QMAX device is 1 mm thick PMMA with printed acridine orange dye, and the second plate is X-Plate with spacers having 30 x 40 um pillar size, 80 um inter spacing distance, made on 175 um thick PMMA. 1uL fresh blood without any anticoagulant was used in the test and deposited on the first plate. Counting accuracy is defined as the counting number's standard deviation for all the fields on card with a specific FoV. This counting accuracy represents the case when a field with FoV in the sample layer is randomly picked for measure, how accurate it represents the average number of all the fields. Generally, WBC counting is more accurate with larger field of view and larger QMAX gap. In essence, counting accuracy here reflects the consistency of the method with specific gap size and field of view.

[0074] Table 2 shows the relationship between WBC miss counting and correction factor vs. QMAX gap. Herein, miss counting rate is defined as the percentage difference between the back- calculated WBC concentration (from counting number, counting area, filling factor, gap size) and sample's real WBC concentrations (measured by calibrated commercial hematology machine). Correction factor = 1 / 1 − Missing Counting Rate . Table 2 - WBC miss counting & correction factor vs. QMAX gapQMAX gap size (um)WBC miss countingWBC correction factor20%130%1510 %1.11025 %1.33050 %2.0

[0075] As shown in Table 2, the miss counting rate increases with the gap size (thus spacer height and sample thickness). Furthermore, additional experiments show that differentiated WBC (Granulocytes, Lymphocyte, Monocyte) counting has similar miss counting rate with WBC total counting. In addition, WBC miss counting rate is not influenced by field of view.

[0076] Panel (A) of Fig. 3 illustrates plots of WBC miss count percentage vs. QMAX gap size (thickness of sample layer; spacer height) of 2um, 5um, 10um and 30um. Panel (B) of Fig. 3 illustrates plots of QMAX transmittance at 500nm wavelength (which is close to fluorescence of WBCs) vs. QMAX gap size.

[0077] As shown in Fig. 3, panels (A) and (B), more WBCs are miss counted with larger gap size (thicker blood film). One of the reason is fluorescence from WBC is dimmed and blocked by the RBCs with thicker blood film as shown in the (b) transmittance vs. gap size. Therefore, it means larger QMAX gap, more WBC are miss-counted. However, as shown in Fig. 2, panels (A) and (B), the counting accuracy, which reflects the consistency of the counting at certain gap sizes and field of view, is higher with a larger gap size and a larger field of view, respectively. Therefore, in some embodiments, certain gap sizes (thus spacer heights) and / or field of view size can be chosen to obtain an acceptable level of consistency, and / or prevent high level of miss count.

[0078] With the correction factor, which is based on the miss counting rate, the counting result can be adjusted to provide a more accurate and consistent number for medical and health purposes. In some embodiments, the final number equals the counting results multiplies the correction factor. In certain embodiments, the correction factor can be obtained / calculated from Table 2 and / or Fig. 3.

[0079] Fig. 4 shows the calculation of self-overlap rate of WBC cell vs. QMAX gap. The results are also shown in Table 3. In general, more WBCs are overlapped when the gap size is larger, especially larger than 30 um. Table 3 - QMAX gap size vs. WBC distance vs. Overlap rateCROF gap (um) Cell 2D Distance (um) Overlap Rate 23200%101400%30801%50602%70505%100459%3002566% Exemplary embodiments with a gap of 8 to 12 um

[0080] The experiments (see e.g. Figs. 2-4) show that for the measurement of WBC in undiluted blood sample, with a given field of view provide by a camera (e.g. camera in a mobile phone), a spacer height of 5 to 15 um provides more accurate results than spacer height of 2 um to 3 um. In some embodiments, a QMAX device for WBC measurement has spacer height of 5 to 15 um. In certain embodiments, the QMAX device has a spacer height of 10 um, while a same of a similar sample thickness uniformity can be achieved. In some embodiments, such pillar heights have advantage for imaging and counting the white blood cells in an undiluted blood.Exemplary embodiments of optical adapter

[0081] In some embodiments, the QMAX device (e.g. in the form of a QMAX card) with sample can be inserted into an adaptor, which can be attached to a device that comprises 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.

[0082] Fig. 5 shows a schematic exploded view of an optical adaptor device for attaching the QMAX device to a mobile communication device and for measurement of an analyte in the sample. Here the optical adaptor device 18 is in system 19, which comprises the mobile communication device (smart phone) 1.

[0083] adaptor 18 comprises a holder case 2 fitting over the upper part of smartphone 1; an optical box 3 attached to case 2 including a receptacle slot 4, an optics chamber 3C, track 6b and 6t allowing lever 8 to slide in, and a rubber door 16 inserted into trench 4s to cover receptacle slot 4. An optics insert 7 is fitted into the top of optics chamber 3C with an exit aperture 7L and an entrance aperture 7C in it aligning with light source 1L and camera 1C (referring to Fig. 6) in smartphone 1. A lens 11 is mounted in entrance aperture 7C in optics insert 7 and configured so that the sample in sample slide 5 inserted into receptacle slot 4 is located within the working distance of the camera 1C (referring to Fig. 6). Lens 11 serves to magnify the images of the sample captured by camera 1C (referring to Fig. 6). A long-pass optical filter 12 is mounted on top of lens 11 in entrance aperture 7C. A pair of right angle mirrors 13 and 14 are mounted on the bottom of optics chamber 3C and configured so that mirror 13 and mirror 14 are aligned with light source 1L and camera 1C (referring to Fig. 6) respectively. Mirror 13 and mirror 14 whose operation as bright-field illumination optics in device 18 is described below in Fig. 6.

[0084] Lever 8 comprises two level bars: the upper-level bar comprises a band-pass optical filter 15 mounted in slot 8a, and the lower-level bar comprises a light absorber 9 mounted on the horizontal plane 8b and a reflective mirror 10 mounted on the tilted plane 8c. The optical filter 15, light absorber 9 and mirror 10 whose operation as fluorescent illumination optics in device 18 is described in Fig. 6. The upper-level bar of lever 8 slides along track 6t in box 3 and lower-level bar 8b and 8c slides along track 6b in box 3. Lever 8 stops at two different positions in box 3 to switch between bright-field illumination optics and fluorescent illumination optics. Lever 8 is fully inserted into box 3 to switch device 18 to work with fluorescent illumination optics. Ball plunger 17 is mounted on the sidewall of track 6t to stop lever 8 at a pre-defined position when lever 8 being pulled outward from box 3 to switch device 18 to work with bright-field illumination optics.

[0085] Fig. 6 shows a schematic sectional view with details of a system that can be used to test a sample in fluorescent illumination mode, and particularly of the optical adaptor. This Fig. illustrates the functionality of the elements that were described above with reference to FIG. 5. Lever 8 (shown in FIG. 5) is fully inserted into device 18 so that light absorber 9 and tilted mirror 10 are under the view of camera 1C and light source 1L, and block the light path between light source 1L and the pair of mirrors of 13 and 14. And band-pass optical filter 15 is right under the light source 1L. Light source 1L emits light beam BF1 away from smartphone 1. Optical filter 15 allows beam BF1 with specific wavelength range which matches the excitation wavelength of the fluorescent sample in sample slide 5 to go through. Part of beam BF1 illuminates on the edge of transparent sample slide 5 and couples to waveguide beam BF3 travelling in sample slide 5 and illuminates the sample area under the lens 11. Part of beam BF1 illuminates on mirror 10. Tilted mirror 10 deflects beam BF1 to beam BF2 and back-illuminates the sample area in sample slide 5 right under lens 11 in large oblique angle. The remaining part of beam BF1 with large divergence angle (i.e., beam BF4) illuminates on absorber 9 and get absorbed so that no reflected light of beam BF4 gets into the camera 1C in small incidence angle. The light coming from the sample area under the lens 11 goes through the lens 11 and is filtered by long-pass filter 12 so that only light in a specify wavelength range that is emitted by the fluorescent sample in sample slide 5 gets into camera 1C to form an image. Smartphone 1 captures and processes the image to get some property of the sample. Rubber door 16 is inserted into device 18 to cover sample slide 5 to prevent ambient light getting into device 18 to affect the test.

[0086] The adapter as described in Fig. 5 and 6 can be used to measure a blood sample, e.g. undiluted whole blood sample. In certain embodiments, the analyte can be WBC, which requires the lever 8 to be inserted for optimal reading.

[0087] The adapter comprises: (a) an attachment member configured to attach the adapter to an apparatus that comprises a light source and a camera; (b) a card slot configured to accommodate a sample card, which contains a liquid sample that is compressed into a layer of uniform thickness, wherein when the sample card inserted into the card slot, the sample is positioned under the view of the camera and the light source; (c) an optical filter configured to filter light from the light source to form a first beam with a specific wavelength range, wherein a part of the first beam illuminates on the edge of the sample card and travels in the sample card to illuminate the sample; (d) a mirror configured to deflect part of the first beam to form a second beam that back-illuminates the sample in an oblique angle; (e) an absorber configured to absorb a remaining part of the first beam that has a divergence angle.

[0088] In some embodiments, the method to measure an analyte, such as but not limited to WBC, in a liquid sample, can comprises: (a) obtaining the liquid sample; (b) compressing at least part of the sample into a layer of uniform thickness with a sample card, (c) inserting the sample card into an adaptor device, which is configured to be attached to an apparatus that comprises a light source and a camera; (d) illuminating the sample with light from the light source, wherein i. the light is filtered by an optical filter of the adapter device to form a first beam with a specific wavelength range, part of the first beam illuminating on the edge of the sample card and travels in the sample card to illuminate the sample; ii. part of the first beam is deflected by a mirror of the adapter device to form a second beam that back-illuminates the sample in an oblique angle; and iii. a remaining part of the first beam that has a divergence angle is absorbed by an absorber of the adapter device.

[0089] In some embodiment, the method can further comprise: (a) capturing images of the sample in the layer of uniform thickness with the camera; (b) analyzing the images to enumerate the analyte in the images; and (c) calculating the concentration of the analyte in the sample based on the uniform thickness, a field of view of the camera, analyte number, and a predetermined correction factor; wherein the field of view is the extent of the field in which the camera captures the images; wherein the correction factor is determined by a miscount ratio, which is dependent on the field of view, the uniform thickness, and properties of the analyte. Exemplary embodiments for WBC measurement

[0090] For the device or method embodiments of the current invention, the device can further comprise, on one or both plates, multi reagent layers including anti-conglutination reagents, cell lysing reagents, cell staining reagents, release time control material, and any combinations thereof.

[0091] In some embodiments, each reagent layer coated on the plates has a thickness of 10nm, 100nm, 200nm, 500nm, 1um or in a range between any two of the values.

[0092] In some embodiments, the anti-conglutination agent comprises ethylenediaminetetraacetic acid (EDTA), EDTA disodium, K2EDTA, or K3EDTA, or any combinations thereof.

[0093] In some embodiments, the cell stain agent comprises Wright's stain (Eosin, methylene blue), Giemsa stain (Eosin, methylene blue, and Azure B), May-Grünwald stain, Leishman's stain ("Polychromed" methylene blue (i.e. demethylated into various azures) and eosin), Erythrosine B stain (Erythrosin B), and other fluorescence stain including but not limit to Acridine orange dye, 3,3-dihexyloxacarbocyanine (DiOC6), Propidium Iodide (PI), Fluorescein Isothiocyanate (FITC) and Basic Orange 21 (BO21) dye, Ethidium Bromide, Brilliant Sulfaflavine and a Stilbene Disulfonic Acid derivative, Erythrosine B or trypan blue, Hoechst 33342, Trihydrochloride, Trihydrate, or DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride), or any combinations thereof.

[0094] In some embodiments, the cell lysing agent comprises ammonium chloride, sodium bicarbonate, ethylenediaminetetraacetic acid (EDTA), acetic acid, citric acid, or other acid and base, or any combinations thereof.

[0095] In some embodiments, the release time control material comprises albumin, carbomers, carboxymethyl cellulose, carrageenan, chitosan, dextrin, polyethylene glycol, polyvinylpyrrolidone, or polyvinyl alcohol, or any combinations thereof.

[0096] In some embodiments of the method embodiments of the current invention, the RBCs, platelets, or both are lysed in the sample before the detection and / or measurement of WBCs.

[0097] In some embodiments of the method embodiments of the current invention, the WBCs, platelets, or both are lysed in sample before the detection of RBCs.

[0098] In some embodiments of the method embodiments of the current invention, the RBCs, WBCs, or both are lysed in sample before the detection of PLTs.Example: QMAX device measure complete blood count and compare with commercial machine

[0099] One example device and method using QMAX device to measure complete blood count (CBC) is shown in Fig. 7. The device is able to measure all the CBC parameters without dilution. The preliminary test shows the results using such device is accurate compared with commercial machine.

[0100] Fig. 7 shows the (a) the photo of one QMAX device and (b) the photo of QMAX device and adapter on a smartphone.

[0101] The device was fabricated with the materials of PMMA. The device can be fabricated with the materials of polystyrene, PMMA, PC, COC, COP, or another plastic.

[0102] The plate 1 used in the example has a thickness of 950 um to 1050 um. The plate 1 have a preferred thickness range of 200 um to 1500 um.

[0103] The plate 2 used in the example has a thickness of 170 um to 180 um. The plate 2 have a preferred thickness range of 50 um to 250 um.

[0104] One device to measure RBC and PLT in the experiment has a pillar height 5 um, inter pillar distance of 90 um, and a pillar size 20 um. The pillar can have a pillar height from 2 um to 6 um with a inter pillar distance of 50 um to 200 um and a pillar size 5 um to 40 um.

[0105] One device to measure HgB and WBC in the experiment has a pillar height 30 um, inter pillar distance of 80 um, and a pillar size 30 um. The pillar can have a pillar height from 20 um to 50 um with a inter pillar distance of 50 um to 200 um and a pillar size 10 um to 50 um.

[0106] The acridine orange dye for staining WBC and PLT, and the Zwittergent for distribute the RBC is coated on the plate 1.

[0107] The acridine orange is coated on the plate with an area concentration of 10 to 80 ng / mm 2< and Zwittergent is coated on the plate with an area concentration of 20 to 130 ng / mm 2< .

[0108] In some other examples, the staining reagent is coated on one of the plate or both plates. The cell separation reagent is coated on one of the plate or both plates. The cell lysing reagent is coated on one of the plate or both plates.

[0109] When measuring and analyzing whole blood sample using such device, comprising following steps: (a) obtaining a whole blood sample (can be finger prick fresh blood or K 2 EDTA venous whole blood) and a device; (b) depositing the sample on one or both of the plates when the plates are configured in the open configuration, (c) after (b), forcing the two plates into a closed configuration; and (d) illuminating the light on the device and capturing images of sample in the device while the plates are the closed configuration; and (e) analyzing the images to analyze complete blood count in the device.

[0110] Fig. 8 shows (a) the bright field image of HgB in device at wavelength around 520 nm, (b) fluoresce field image of WBC in device at excitation around 490 nm and emission over 500 nm, (c) the bright field image of RBC in device, (d) fluoresce field image of WBC and PLT in device at excitation around 490 nm and emission over 500 nm, with whole blood inside taken by iPhone based optical system.

[0111] The red blood cell in (a) with pillar height 30 um become multilayers, thus good for HgB measurement. The red blood cell in (c) with a pillar height 5 um become a monolayer and countable in the zoom-in image.

[0112] The white blood cell and platelet is stained with AO dye and is bright dots in the fluorescence image. The white blood cell in both 5 um and 30 um spacing device become a monolayer and countable in the zoom-in image. The platelet in 5 um spacing device is a monolayer and countable in the zoom-in image.

[0113] Whole blood samples (venous in K2EDTA tube) from 50 to 100 patients are measured by QMAX device and compared with commercial hemocytometer as Horiba Pentra 60C. 9 uL whole blood was dropped onto plate 2, and pressed by plate 1. The card is then read by smartphone based optical system as shown in Fig. 8. The cells are counted by local software using both OpenCV and machine learning algorithm.

[0114] Fig. 9 shows the example HgB, WBC, RBC, PLT analyze results of whole blood samples using QMAX device and compared with commercial hemocytometer as Horiba Pentra 60C. The results show good accuracy of the device and method compared with commercial machine.

[0115] In details, compared with Horiba Pentra 60C, the HgB reading has R2 = 98.5% with commercial machine over measured range of 7 g / dL to 20 g / dL, the WBC reading has R2 = 99.3% with commercial machine over measured range of 0.4 x 10 3Example 2 QMAX device measure WBC and WBC differentiate

[0116] One example result using QMAX device to measure WBC and WBC differentiate is shown in Fig. 10. The device is able to measure all the WBC and three sub-types (granulocyte, monocyte, lymphocyte) without dilution. The preliminary test shows the results using such device is accurate compared with commercial machine.

[0117] Fig. 10 shows the (a) the fluoresence photo take by smartphone optical system of WBCs in one QMAX device and (b) the statistic counting summary of WBCs in the device, ploting WBC count vs. Green channel intensity over red channel intensity of each WBC.

[0118] The device was same as the WBC device in example-1.

[0119] The acridine orange dye for staining WBC was coated on the plate 1. When binding to DNA, AO intercalates with DNA as a monomer and yields intense green fluorescence under blue excitation. When binding to RNA and proteins it forms an electrostatic complex in a polymeric form that yields red fluorescence under blue excitation. Since the three sub-types (granulocyte, monocyte, lymphocyte) have different DNA / RNA ratio, by analyzing the green and red fluorescence ratio of each WBC, the WBC differenciate can be achieved.

[0120] The acridine orange was coated on the plate with an area concentration of 10 to 80 ng / mm 2< and Zwittergent was coated on the plate with an area concentration of 20 to 130 ng / mm 2< .

[0121] When measuring and analyzing whole blood sample using such device, comprising following steps: (a) obtaining a whole blood sample (can be finger prick fresh blood or K 2 EDTA venous whole blood) and a device; (b) depositing the sample on one or both of the plates when the plates are configured in the open configuration, (c) after (b), forcing the two plates into a closed configuration; and (d) illuminating the light on the device and capturing images of sample in the device while the plates are the closed configuration; and (e) analyzing the images to analyze complete blood count in the device.

[0122] The excitation illumination is at the wavelength 450 nm to 480 nm, the emission is long pass with cut off at around 520 nm, thus both green (550 nm) and red color (650 nm) fluorescence of each WBC can be observed from camera.

[0123] From the result, the white blood cell is stained with AO dye and is colorful dots in the fluorescence image with clearly three color (green, yellow and red) as shown in Fig. 10(a), which corresponding to lymphocyte (more DNA), monocyte (balance DNA, RNA) and granulocyte (more RNA). The color of each WBC was analyzed with machine learning and software, and distinguished into 3 clusters as shown in Fig. 10(b).iMOST HgB + WBC + WBC differentiate QMAX card example:

[0124] The spacer height, the spacing between the plates, and / or sample thickness is around 30 um.

[0125] The spacer height, the spacing between the plates, and / or sample thickness is 20 um to 40 um.

[0126] The spacer is rectangle shape with round corners.

[0127] The lateral dimension of a spacer is around 30 µm by 40 um.

[0128] The lateral dimension of a spacer is 10 um to 40 um.

[0129] The round corners of spacer has a diameter of 10 um.

[0130] The spacer is in a rectangular lattice array.

[0131] The inter-spacer spacing of spacers is around 80 µm.

[0132] The inter-spacer spacing of spacers is 70 µm to 150 um.

[0133] The length of one plate of Q-Card is 27 mm and the width of this plate is 22 mm.

[0134] The length of one plate is Q-Card is 32 mm and the width of this plate is 24 mm.

[0135] The area of one plate is around 600 mm 2< and the area of another plate is around 750 mm 2< .

[0136] The thickness of one plate of Q-Card is around 175 um.

[0137] The thickness of one plate of Q-Card is around 1 mm.

[0138] The area of the notch on the QMAX card is in the range of 10 to 30 mm 2< .

[0139] The notch is half-round shape with a diameter of 3 to 6 mm.

[0140] The notch has a width of 3 mm and a length of 6 mm.

[0141] The width of the hinge joint is around 6 mm.

[0142] The length of the hinge joint is around 20 mm.

[0143] The hinge has a thickness around 70 µm.

[0144] The reagent is coated by droplet printing into an array.

[0145] The reagent is coated by spray.

[0146] The acridine orange or other staining reagents is coated onto the first plate, or the second plate or both.

[0147] The Zwittergent or other detergent is coated onto the first plate, or the second plate or both.

[0148] The acridine orange is coated on the plate with an area concentration of 10 to 60 ng / mm 2< and Zwittergent is coated on the plate with an area concentration of 20 to 130 ng / mm 2< .

[0149] The material of first place and second plate is Poly(methyl methacrylate).

[0150] A landing mark for blood droplet is on the outside surface of first plate or second plate.

[0151] A landing mark for blood droplet is a small dot or a small cross.

[0152] A landing mark for blood droplet is outside the field of view of the image.

[0153] A landing mark for blood droplet is near the center of the card.

[0154] At least one of the plates is transparent.iMOST RBC + PLT QMAX card example:

[0155] Same as above 1, except: The spacer height, the spacing between the plates, and / or sample thickness is around 5 um.

[0156] The spacer height, the spacing between the plates, and / or sample thickness is 2 um to 7 um.

[0157] The lateral dimension of a spacer is around 30 µm by 40 um.

[0158] The lateral dimension of a spacer is 5 um to 40 um.

[0159] The acridine orange or other staining reagents is coated onto the first plate, or the second plate or both.

[0160] The Zwittergent or other detergent is coated onto the first plate, or the second plate or both.

[0161] The acridine orange is coated on the plate with an area concentration of 10 to 60 ng / mm 2< and Zwittergen is coated on the plate with an area concentration of 20 to 130 ng / mm 2< .

Claims

1. A method for analyzing an analyte in a liquid sample, comprising: (a) obtaining the liquid sample and providing a device; (b) compressing at least part of the sample into a layer of uniform thickness, (c) capturing images of the sample in the layer of uniform thickness with a camera, wherein the images show the analyte; and (d) analyzing the images to enumerate the analyte in the images, (e) calculating the concentration of the analyte in the sample based on the uniform thickness, a 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 in which the camera captures the images; wherein the correction factor is determined by a miscount ratio, which is dependent on the field of view, the uniform thickness, and properties of the analyte; and characterized in that the device comprises: (i) an attachment member (2) configured to attach the device to an apparatus (1) that comprises a light source and the camera; (ii) a card slot (4) configured to accommodate a sample card, wherein the sample card comprises two plates capable of sandwiching the liquid sample into the layer of uniform thickness, wherein when the sample card is inserted into the card slot, the sample is positioned under the view of the camera and the light source; (iii) an optical filter (15) configured to filter light from the light source to form a first beam with a specific wavelength range, wherein a part of the first beam illuminates on the edge of the sample card and travels inside the sample card to illuminate the sample; (iv) a mirror (10) configured to deflect a second part of the first beam to form a second beam that back-illuminates the sample in an oblique angle; and (v) an absorber (9) configured to absorb a remaining part of the first beam that has a divergence angle.

2. The method of claim 1, wherein the analyte is platelet.

3. The method of claim 1, wherein the analyte is white blood cell comprising neutrophils, eosinophils, basophils, lymphocytes, and monocytes.

4. The method of claim 1, wherein the analyte is while blood cell, and the method further comprising staining the white blood cell to provide fluorescence color, structure and dimension, which are used to distinguish the white blood cell and differentiate subtypes of the white blood cell.

5. The method of claim 1, wherein the analyte is WBC (White Blood Cell), and capturing images includes a step of capturing a fluorescence image for analyzing the count of the WBC and differentiating subtypes of the WBC.

6. The method of claim 1, wherein the analyte is WBC, and wherein both the color, being the emission wavelength range, and the structure of the WBC are used for counting the WBC and differentiating subtypes of the WBC.

7. The method of claim 11, wherein the analyzing uses the images' Red, Green, Blue channel.

8. The method of claim 1, wherein the device further comprises, on one or both plates, multi reagent layers including anti-conglutination, cell lysing, cell staining, release time control layers, or their combinations.

9. The method of claim 1, wherein the device further comprises a housing member (3).

10. The method of claim 1, wherein the device further comprises a lever (8) capable of being inserted into or extracted from the housing member.

11. The method of claim 1, wherein the mirror and the absorber are mounted on the lever.

12. The method of claim 1, wherein the card slot has a secured opening (16) that allows the insertion of the sample card and prevents ambient light from entering the card slot.

13. The method of claim 1, wherein the analyte is marked with fluorescence.