Method for acquiring an image of a biological sample in a cuvette

Abstract

The present invention relates to a method for acquiring an image of a biological sample in a cuvette.

Description

[Technical field]

[0001] The present invention relates to a method for acquiring an image of a biological sample in a cuvette. [Background technology]

[0002] Analytical devices are known for determining the optical properties of a sample contained in a cuvette. In such devices, the cuvette is at least partially filled with the sample for analysis and then placed into the device. By passing optical (or other) radiation through the sample, properties of the sample, such as the presence of a particular component, can be measured.

[0003] A typical sample examined by an analytical device is a biological sample, such as a blood sample. Blood analysis is used in a wide range of medical monitoring methods, such as monitoring the progress of systemic anti-cancer therapies. For example, an optical analytical device can analyze a blood sample to determine the presence and / or amount of white blood cells, such as granulocytes and agranulocytes, which are often used in monitoring the progress of systemic anti-cancer therapies. Analysis of a blood sample typically requires first centrifuging the sample. During centrifugation, the blood is separated into multiple layers. These layers are: (a) a red blood cell layer containing red blood cells, (b) a buffy coat layer containing white blood cells (such as granulocytes and agranulocytes), (c) a platelet (thrombocytocyte) layer, and (d) a plasma layer. The blood is then optically (or with other radiation) analyzed to obtain images of the layers. A typical optical radiation source is an LED light. The number of pixels in each layer can then be calculated from the image to determine the number of cells in each layer.

[0004] The cuvette is a disposable part of the analytical device, and a new cuvette is usually required for each measurement. It is important that the user can quickly and easily load the sample into the cuvette. It is also important that the cuvette can be manufactured at low cost, and that the sample can be easily loaded and fixed into the holder of the optical analytical device, and that it can be easily removed from the holder once the analysis is completed. Examples of known types of cuvettes are described, for example, in GB 2555403, EP 2096444, EP 1055112, WO 2007 / 008137, US 6365104, and WO 2018 / 078324, the disclosures of each of which are incorporated herein by reference.

[0005] A cuvette containing a sample can be coated with a composition. This composition has three purposes: (a) it aids in the collection of the sample by the cuvette, (b) it ensures that the sample remains in a processable state after collection, and (c) it aids in the production of a clear image of the sample. Conventional compositions for coating cuvettes include potassium oxalate, acridine orange, sodium heparin, and dipotassium ethylenediaminetetraacetic acid dehydrate. Examples of known compositions for use in cuvettes are described, for example, in U.S. Pat. No. 6,365,104, the disclosure of which is incorporated herein by reference.

[0006] There is a need for an improved method for acquiring an image of a sample in a cuvette, and in particular an improved method for acquiring an image of a blood sample in a cuvette that results in an image that clearly shows the buffy coat layer. Summary of the Invention

[0007] The present invention relates to a method for acquiring an image of a biological sample in a cuvette.

[0008] Representative features of the present invention are described below, which may be present alone or in any combination with one or more features disclosed in the text and / or figures of this specification. They can be combined in various ways.

[0009] The present invention is described in the following items. 1. A method for obtaining an image of a sample in a cuvette, comprising the steps of: (a) placing the sample in the cuvette; (b) mixing the sample with a collection solution; (c) introducing the mixture of step (b) into an imaging solution; (d) subjecting the mixture of step (c) to a centrifugal force; (e) exposing the centrifuged mixture of step (d) to optical radiation; and (f) obtaining an optical image of the mixture; A method comprising:

[0010] 2. The method of claim 1, wherein the mixture is subjected to centrifugal force in an Entia Liberty™ device in step (d).

[0011] 3. The method according to item 1 or 2, wherein the mixture is subjected to a centrifugal force of 1000 to 2000 rpm or 1500 rpm.

[0012] 4. The method according to any one of items 1 to 3, wherein the mixture is subjected to centrifugal force for up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, or up to 10 minutes.

[0013] 5. The method according to any one of items 1 to 4, wherein the mixture is subjected to centrifugal force at room temperature.

[0014] 6. The method according to any one of items 1 to 5, wherein the mixture is exposed to optical radiation from an LED light source, optionally operating at 400-700 nm, 425-600 nm, 450-500 nm, or 460 nm.

[0015] 7. The method according to any one of items 1 to 6, wherein the mixture is exposed to optical radiation for up to 15 minutes, up to 10 minutes, or up to 5 minutes.

[0016] 8. The method according to any one of items 1 to 7, wherein the mixture is exposed to optical radiation at room temperature or at a temperature of 15 to 40°C, 16 to 30°C, 18 to 25°C, 20 to 22°C, or 20°C.

[0017] 9. The method according to any one of items 1 to 8, wherein steps (c) and (d) occur simultaneously by centrifugal force causing the mixture from step (b) to mix with the imaging solution of step (c).

[0018] 10. The method according to any one of items 1 to 9, wherein the optical image of step (f) is acquired by a camera.

[0019] 11. The method according to any one of items 1 to 10, wherein the sample is a biological sample, optionally wherein the biological sample is a blood sample.

[0020] 12. The collection solution is Anticoagulants, Flocculants, and 12. The method according to any one of items 1 to 11, comprising a wetting agent.

[0021] 13. The collection solution is 5–25 mg / mL of anticoagulant; 5-25 mg / mL of flocculant, and 13. The method according to any one of items 1 to 12, comprising 0.05 to 0.25 mg / mL of a wetting agent.

[0022] 14. The method according to any one of items 1 to 13, wherein the collection solution further comprises water (optionally, the water is distilled water) or an alcohol solvent.

[0023] 15. The method according to any one of items 1 to 14, wherein the collection solution comprises a first anticoagulant and a second anticoagulant, and the first anticoagulant and the second anticoagulant are different.

[0024] 16. The method according to any one of items 12 to 15, wherein the anticoagulant is one or more of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, and any combination thereof.

[0025] 17. The method according to any one of items 12 to 16, wherein the flocculating agent is one or more of polyvinylpyrrolidone, proteolytic enzymes (bromelain, pepsin, and / or trypsin), polyethylene glycol, and any combination thereof.

[0026] 18. The method according to any one of items 12 to 17, wherein the wetting agent is one or more of well-known surfactants (i.e., ionic, nonionic, and / or cationic), Pluronic P-123, SILWET L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid alkoxylates, and any combination thereof.

[0027] 19. The collection solution is (a) 5 to 25 mg / mL, 7.5 to 20 mg / mL, 10 to 15 mg / mL, or 13 mg / mL of an anticoagulant; and / or (b) 5 to 25 mg / mL, 7.5 to 20 mg / mL, 9 to 11 mg / mL, or 10 mg / mL of a flocculant; and / or (c) The method according to any one of items 1 to 18, comprising 0.050 to 0.250 mg / mL, 0.075 to 0.120 mg / mL, 0.900 to 0.125 mg / mL, or 0.1 mg / mL of a wetting agent.

[0028] 20. The method according to any one of items 1 to 19, wherein the collection solution comprises a first anticoagulant at 2 to 10 mg / mL, 4 to 8 mg / mL, 5 to 7 mg / mL, or 6 mg / mL, and a second anticoagulant at 3 to 11 mg / mL, 5 to 9 mg / mL, 6 to 8 mg / mL, or 7 mg / mL, and the first anticoagulant and the second anticoagulant are different.

[0029] 21. The method according to any one of items 1 to 20, wherein the collection solution comprises or consists of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, polyvinylpyrrolidone, Pluronic® P-123, and water, optionally wherein the water is distilled water.

[0030] 22. The collection solution is (a) 3-11 mg / mL, 5-9 mg / mL, 6-8 mg / mL, or 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrating agent; (b) 2-10 mg / mL, 4-8 mg / mL, 5-7 mg / mL, or 6 mg / mL potassium oxalate; (c) 5 to 25 mg / mL, 7.5 to 20 mg / mL, 9 to 11 mg / mL, or 10 mg / mL of polyvinylpyrrolidone; and (d) 0.050 to 0.250 mg / mL, 0.075 to 0.120 mg / mL, 0.900 to 0.125 mg / mL, or 0.1 mg / mL of Pluronic® P-123; 22. The method according to any one of items 1 to 21, wherein the balance is water, optionally wherein the water is distilled water.

[0031] 23. The method according to any one of items 1 to 22, wherein the collection solution comprises 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrating agent, 6 mg / mL of potassium oxalate, 10 mg / mL of polyvinylpyrrolidone, and 0.1 mg / mL of Pluronic® P-123, with the remainder being water, and the water being distilled water.

[0032] 24. The method according to any one of items 1 to 23, wherein the imaging solution comprises an imaging agent.

[0033] 25. The method according to item 24, wherein the imaging solution further comprises water (optionally, the water is distilled water) or an alcohol solvent.

[0034] 26. The method according to item 24 or 25, wherein the imaging agent is one or more of acridine orange, quaternary cationic metachromatic dyes (such as Greifswalder's blue, blue borrel, rhodanile blue, toluylene blue, night blue, Hofmann's violet, basic orange 21), permanent dyes (such as cytocyanine permanent dyes, SYTO dyes, oxaline dyes, phenanthridine (intercalation) dyes, indole dyes, imidazole dyes, and any combination thereof).

[0035] 27. The method according to any one of items 24 to 25, wherein the concentration of the imaging agent is 0.01 to 0.030 mg / mL, 0.015 to 0.025 mg / mL, 0.019 to 0.021 mg / mL, or 0.020 mg / mL.

[0036] 28. The method according to any one of items 1 to 27, wherein the imaging solution comprises or consists of acridine orange and water, optionally wherein the water is distilled water.

[0037] 29. The method according to any one of items 1 to 28, wherein the imaging solution contains 0.01 to 0.030 mg / mL, 0.015 to 0.025 mg / mL, 0.019 to 0.021 mg / mL, or 0.020 mg / mL of acridine orange, and the remainder is water, optionally wherein the water is distilled water.

[0038] 30. The method according to any one of items 1 to 29, wherein the imaging solution comprises 0.020 mg / mL acridine orange, the remainder being water, optionally wherein the water is distilled water.

[0039] 31. The method according to any one of items 1 to 30, wherein the imaging solution has a pH of 3 to 6, 3.5 to 4.5, or 4.

[0040] 32. The method according to any one of items 1 to 31, wherein the cuvette comprises a collection chamber and an analysis chamber, the collection chamber being coated (fully or partially) with the collection solution, and step (b) of item 1 is carried out in the collection chamber.

[0041] 33. The method according to any one of items 1 to 32, wherein the cuvette comprises a collection chamber and an analysis chamber, the analysis chamber is coated (wholly or partially) with the imaging solution, and step (c) of item 1 is carried out in the analysis chamber.

[0042] 34. The method according to any one of items 1 to 33, wherein step (e) and step (f) of item 1 are carried out while the sample is in the cuvette. [Brief description of the drawings]

[0043] [Figure 1] FIG. 13 shows acceptable and unacceptable coatings of collection solution on the collection chamber half of a cuvette. [Diagram 2] FIG. 1 shows the GRIPTIP spacing used in multichannel pipettes. [Diagram 3] FIG. 13 illustrates a preferred method of placing the imaging solution over the analytical chamber half of the cuvette. [Figure 4] FIG. 1 shows images obtained when analyzing the effect of agglutinating agent concentration on the quality of images obtained from blood samples. [Diagram 5]FIG. 1 shows images obtained from a blood sample in a cuvette coated with a composition of the present invention and a blood sample in a cuvette coated with a different composition (not according to the present invention). [Figure 6] FIG. 13 shows an image obtained when the entire length of the middle wall of the analytical chamber of one half of the cuvette was coated with the imaging solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Embodiments of the present disclosure are described in more detail below with reference to the accompanying drawings, in which like numerals represent like elements throughout the several views and in which exemplary embodiments are illustrated. However, the claimed embodiments may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0045] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. The accompanying drawings illustrate various embodiments of the system and method, as well as embodiments of various other aspects of the present disclosure. Those skilled in the art will appreciate that the element boundaries (e.g., boxes, groups of boxes, or other shapes) shown in the figures represent examples of those boundaries. In some examples, an element may be designed as multiple elements, or multiple elements may be designed as an element. In some examples, an element shown as an internal component of one element may be implemented as an external component of another element, and vice versa. Furthermore, elements may not be drawn to scale. A non-limiting and non-exhaustive description is provided with reference to the following drawings. The components in the figures are not necessarily to scale, with emphasis instead being placed on illustrating the principles.

[0046] Words such as "comprising," "having," "containing," "including," and other forms thereof, are intended to be open-ended in that the items following any of these words are not intended to be an exhaustive list of such items, nor are they intended to be limited to only the listed items. It should also be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Although any systems and methods similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure, the preferred systems and methods are described herein.

[0047] Some of the terms used to describe the present invention are listed below.

[0048] "Acridine orange" refers to an imaging dye. Acridine orange causes cells to fluoresce upon exposure to optical radiation. Typically, acridine orange is excited at wavelengths between 200 and 460 nm. The mechanism by which acridine orange differentiates cells is called metachromasia. Through metachromasia, acridine orange binds to different components within the sample upon excitation, producing peaks at different wavelengths in the spectrum. For example, when acridine orange binds to DNA, the cell will fluoresce green up to 530 nm when excited with blue light. When acridine orange binds to RNA, the resulting combination condenses (i.e., transitions to a solid state) and then precipitates. The resulting components phosphoresce or emit light red up to 640 nm when excited with blue light. The concentration of acridine orange affects the form of available genetic material and therefore also the color emission of the sample. It is well known that acridine orange is less sensitive for RNA selection than for DNA selection. For example, a high concentration of acridine orange denatures DNA and simultaneously stains it red. This is because a high concentration of acridine orange causes DNA to become single-stranded. Thus, a high concentration of acridine results in a high amount of red light emission. Conversely, a low concentration of acridine orange results in incomplete denaturation of RNA, which stains part of the RNA green. Thus, a low concentration of acridine orange results in a high amount of green light emission. Furthermore, the coloration produced by using acridine orange to bind to cells depends on the structure of the cells and can assist in the differentiation of different cells. For example, granulocytic cells emit light prominently in the red spectrum, as their granules are stained orange and their nuclei are stained red, whereas agranulocytic cells emit light in the green spectrum, as their nuclei are stained yellow-green and their cytoplasm is stained red.

[0049] "Aggregating agent" refers to a substance that aids in the aggregation of particles. For example, aggregating agents aid in the aggregation of cells, such as bacteria and red blood cells. In particular, aggregating agents can act directly on red blood cells to aggregate them, for example, by reducing the repulsive forces between the red blood cells. Examples of aggregating agents include, but are not limited to, lectin proteins, hemagglutinin proteins, polyvinylpyrrolidone, solutions containing polyvinylpyrrolidone K12, proteolytic enzymes (such as bromelain, pepsin, and / or trypsin), and polyethylene glycol.

[0050] "Agranulocyte" refers to a type of white blood cell that does not have distinct granules. Examples of agranulocytes include, but are not limited to, lymphocytes and monocytes.

[0051] "Alcohol solvent" refers to an alcohol-based solvent, including, but not limited to, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butyl alcohol, and ethanol.

[0052] "Anticoagulant" refers to a substance that prevents blood from clotting. Anticoagulants ensure that a blood sample is not significantly altered before it is analyzed. Examples of anticoagulants include, but are not limited to, ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, and hirudin.

[0053] "Buffy coat" refers to a fraction of an anticoagulated blood sample. In a centrifuged blood sample, the buffy coat contains the majority of the white blood cells and platelets.

[0054] "Collection solution" refers to a solution containing an anticoagulant, a flocculating agent, and a wetting agent. The collection solution is typically intended to increase the ease and efficiency of collection of a sample by an analytical device such as a cuvette.

[0055] "Cuvette" refers to a container used in an analytical device, especially a container used for analysis of a sample by centrifugation. Examples of cuvettes are described in GB 2555403 and GB 2108004.9, the disclosures of which are incorporated herein by reference.

[0056] "Entia Liberty™ Device" refers to a device manufactured by Entia that uses optical radiation to measure the concentration of a component in a sample. Specifically, the device is used to measure the presence (quantity) of white blood cells in blood samples taken from patients who are being monitored for progress of systemic anti-cancer therapy.

[0057] "Fluid" refers to a substance that does not have a fixed shape and is easily deformed by external pressure. Examples of "fluids" include, but are not limited to, a blood sample.

[0058] "Granulocyte" refers to a type of white blood cell that has small granules. These granules contain proteins. Examples of granulocytes include neutrophils, eosinophils, and basophils.

[0059] "Homogeneous" refers to a material having uniform composition and properties throughout.

[0060] "Imaging solution" refers to a solution containing an imaging agent. Examples of imaging agents include, but are not limited to, acridine orange, quaternary cationic metachromatic dyes (such as Greifswalder's blue, blue borrel, rhodanile blue, toluylene blue, night blue, Hofmann's violet, basic orange 21, etc.), permanent dyes (such as cytocyanine permanent dyes, SYTO dyes, oxaline dyes, phenanthridine (intercalation) dyes, indole dyes, or imidazole dyes, etc.). The imaging solution may further comprise water. The imaging solution is intended to allow an image of the sample being analyzed to be obtained by radiation of light (or another source).

[0061] "Multichannel pipette" refers to an electronic device used in laboratories to accurately measure and fill multiple vials with liquid at once. Examples of multichannel pipettes include, but are not limited to, Eppendorf Research plus multichannel pipettes, CappAero multichannel pipettes, and VIAFLO multichannel pipettes. Multichannel pipettes with a tip distance of 4 mm or less and capable of dispensing at least 0.5 μL of liquid may be preferably used.

[0062] "Mixture" means a material made by combining two or more distinct components. A mixture may or may not be homogeneous throughout.

[0063] "Room temperature" refers to a temperature of 18 to 25°C, preferably 20 to 22°C, and more preferably 20°C.

[0064] "Sample" refers to the substance to be analyzed. The sample is analyzed in the fluid phase.

[0065] "Wetting agent" refers to a substance that reduces the surface tension of a liquid, thereby increasing the spreading and penetrating properties of the liquid. Examples of wetting agents include the well-known surfactants (ionic, nonionic, and / or surfactants). or cationic), Pluronic® P-123 (also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)), Silwet L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, or fatty acid alkoxylates.

[0066] composition The present invention provides a composition. In particular, the composition is for coating all or part of a cuvette. The composition is an improvement over existing compositions for coating a cuvette. The composition of the present invention has at least the following advantages: improved ease and efficiency of sample uptake by the cuvette; improved imaging of a sample held in the cuvette; improved imaging of a sample held in the cuvette by allowing visualization of the different layers of the buffy coat of a blood sample.

[0067] The compositions include a collection solution and an imaging solution.

[0068] The collection solution includes an anticoagulant, a flocculating agent, and a wetting agent. The collection solution may also include water or any suitable organic or inorganic solvent, such as an alcoholic solvent.

[0069] The anticoagulant may include one or more of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, and any combination thereof.

[0070] The flocculating agent may include one or more of polyvinylpyrrolidone, a solution containing polyvinylpyrrolidone K12, proteolytic enzymes (bromelain, pepsin, and / or trypsin), polyethylene glycol, and any combination thereof. Advantageously, the inclusion of a flocculating agent in the collection solution reduces blood relaxation after centrifugation of a blood sample in a cuvette containing the collection solution. This has the advantage of increasing the time that the sample can be used for imaging. Further advantageously, the flocculating agent acts as a plasma expander for the blood sample, increasing the plasma volume and sedimentation rate, and decreasing the hematocrit.

[0071] The wetting agents include one or more of Pluronic® P-123, Silwet L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid alkylates, and any combination thereof.

[0072] Preferably, the pH of the collection solution is acidic. Preferably, the pH of the collection solution is 3 to 6, 3.5 to 4.5, or 4 (±0.2).

[0073] The imaging solution includes an imaging agent. The imaging solution may include water or any suitable organic or inorganic solvent, such as an alcoholic solvent.

[0074] The imaging agent is one or more of acridine orange, quaternary cationic metachromatic dyes (such as Greifswalder's blue, blue borrel, rhodanile blue, toluylene blue, night blue, Hofmann's violet, basic orange 21, etc.), permanent dyes (such as cytocyanine permanent dyes, SYTO dyes, oxaline dyes, phenanthridine (intercalation) dyes, indole dyes, imidazole dyes, etc.), and any combination thereof. Advantageously, the dyes are positively charged and therefore capable of passing through the cell membrane of blood cells. Thus, the dyes chemically bind blood cells. Blood cells can be stained dynamically. By visually staining blood cells, blood cells can be visualized. Further advantageously, the dyes can be used over a wide pH range, temperature range, and the staining properties of the dyes are not affected by changes in concentration. The dyes are fixed in a fixed position (as the dye dries on the surface of the cuvette), and there is no need to fix cells in a fixed position. Instead, cells are allowed to flow freely. Freely flowing cells are desirable for imaging, since fixing cells tends to lose important information, such as biochemical information.

[0075] Preferably, the imaging solution has an acidic pH, preferably, the imaging solution has a pH of 3 to 6, 3.5 to 4.5, or 4 (±0.2).

[0076] Cuvettes In some examples, the present invention also relates to a cuvette that is fully or partially coated with the composition.

[0077] In some examples, the cuvette is a cuvette described in UK Patent Application Publication No. 2108004.9, filed June 4, 2021, the disclosure of which is incorporated herein by reference.

[0078] In some examples, the cuvette is formed of two halves (hereafter referred to as cuvette halves), which are manufactured separately and then joined together. One half includes the top surface of the cuvette and the other half includes the bottom surface of the cuvette. A recess is formed on the inner surface of one or both halves, and when the two halves are secured together, one or more chambers are present within the body of the cuvette. Preferably, the body of the cuvette includes a sample chamber formed therein, the sample chamber communicating with the exterior of the cuvette by an opening formed on the exterior of the body, the sample chamber including a collection chamber and an analysis chamber. Figure 3 shows a cuvette half (300) having a collection chamber (340) and an analysis chamber (350). The collection chamber has a first end in fluid communication with an opening and a second end in fluid communication with the analysis chamber. The analysis chamber has a first end in fluid communication with the collection chamber and a second (closed) end. The analysis chamber is in fluid communication with the exterior of the cuvette only through the collection chamber. However, it should be understood that the invention is not limited to this method of manufacturing the cuvette and the cuvette may be formed in any other suitable or convenient manner.

[0079] In some instances, a recess is formed in the inner surface of one cuvette half, and the inner surface of the other half is substantially smooth and / or flat. In other instances, recesses may be formed in the inner surfaces of both halves, and when the cuvette is assembled, at least a portion of the recesses align with one another to form chambers. As previously mentioned, various chambers may be created by recesses formed in the interior of one or both cuvette halves. The skilled reader will appreciate that there are many other possibilities.

[0080] In some instances, the depth of the collection chamber and the analysis chamber are the same or approximately the same.

[0081] In some examples, the collection chamber has four opposing parallel sidewalls. The collection chamber extends from a first end in fluid communication with an opening formed in the exterior of the body and a second end in fluid communication with the analysis chamber. In some examples, the analysis chamber preferably has a constant width along its length and has four opposing parallel sidewalls. Preferably, the cuvette is comprised of two cuvette halves, one cuvette half having a recess to form a chamber and the other cuvette half having a recess to form a chamber. If the cuvette halves are substantially smooth or flat, the recesses of one cuvette half form three of the four opposing side walls of the collection and analysis chambers, and the substantially smooth or flat surface of the second cuvette half forms the fourth opposing side wall. The analysis chamber is preferably of constant depth along its length. The analysis chamber extends towards the opposite end of the cuvette to the collection chamber and terminates in a closed end that does not allow any communication with the exterior of the cuvette. The closed end of the analysis chamber preferably has a rectangular shape with an end face that is perpendicular or substantially perpendicular to the length of the analysis chamber. The closed end may also be referred to as a closed wall.

[0082] In some examples, the cuvette includes a vent to allow air to escape from the cuvette.

[0083] In some instances, the cuvette includes a transfer chamber between the collection chamber and the analysis chamber, the function of which is to prevent overfilling of the cuvette while at the same time avoiding cell damage and / or activation.

[0084] In some instances, the collection chamber is coated (wholly or partially) with a collection solution of the present invention. In other instances, the analysis chamber is coated (wholly or partially) with an imaging solution of the present invention. In other instances, the collection chamber is coated (wholly or partially) with the collection solution and the analysis chamber is coated (wholly or partially) with the imaging solution.

[0085] Method for producing the composition The present invention also provides a method for producing a composition for coating (wholly or partially) a cuvette, the method comprising the steps of: (a) preparing a collection solution; and (b) Preparing an imaging solution.

[0086] The present invention also provides a method of producing the collection solution, the method comprising the steps of: (a) preparing an anticoagulant, a flocculating agent, and a wetting agent; (b) preparing stock solutions of the anticoagulant, the flocculant, and the wetting agent; and (c) combining said stock solutions.

[0087] In step (b), the stock solutions are prepared separately.

[0088] In some examples, the anticoagulant, the flocculant, and the wetting agent are weighed separately into separate containers. An example of a preferred separate container is a disposable tray. Each container is washed with a cleaning agent such as isopropyl alcohol before use.

[0089] In some examples, the anticoagulant, the flocculant, and the wetting agent are transferred separately to another vial, and a solvent is added to the vial. Preferably, the solvent used is water. Preferably, any suitable organic or inorganic solvent, such as an alcoholic solvent, is used. Preferably, the anticoagulant, the flocculant, and the wetting agent are dissolved in the solvent by stirring the solutions at 700 rpm to 900 rpm, or 800 rpm. Preferably, the anticoagulant, the flocculant, and the wetting agent are dissolved in the solvent by stirring the solutions for 5 to 15 minutes, or 10 minutes. Preferably, the anticoagulant, the flocculant, and the wetting agent are dissolved in the solvent at a temperature of 10 to 30°C or 15 to 25°C.

[0090] In some instances, the collection solution contains two anticoagulants, a first anticoagulant and a second anticoagulant. The stock solution containing the first anticoagulant includes a coagulant. Preferably, the first anticoagulant and the second anticoagulant are different. Preferably, the stock solution containing the first anticoagulant is prepared with a first anticoagulant concentration of 25 to 75 mg / mL, 35 to 65 mg / mL, 45 to 55 mg / mL, or 50 mg / mL. Preferably, the stock solution containing the second anticoagulant is prepared with a second anticoagulant concentration of 50 to 150 mg / mL, 75 to 125 mg / mL, 90 to 110 mg / mL, or 100 mg / mL.

[0091] In some examples, the stock solution containing the flocculant is prepared so that the concentration of the flocculant is 25 to 75 mg / mL, 35 to 65 mg / mL, 45 to 55 mg / mL, or 50 mg / mL.

[0092] In some examples, the stock solution containing the wetting agent is prepared so that the concentration of the wetting agent is 0.005 mg / mL to 0.015 mg / mL, 0.0075 mg / mL to 0.0125 mg / mL, 0.009 to 0.011 mg / mL, or 0.01 mg / mL.

[0093] In some examples, the stock solution containing the first anticoagulant is prepared with a first anticoagulant concentration of 50 mg / mL, the stock solution containing the second anticoagulant is prepared with a second anticoagulant concentration of 100 mg / mL, the stock solution containing the flocculant is prepared with a flocculant concentration of 50 mg / mL, and the stock solution containing the wetting agent is prepared with a wetting agent concentration of 0.01 mg / mL. Preferably, the stock solution containing potassium oxalate is prepared with a potassium oxalate concentration of 50 mg / mL, the stock solution containing ethylenediaminetetraacetic acid dipotassium salt dehydrate is prepared with a ethylenediaminetetraacetic acid dipotassium salt dehydrate concentration of 100 mg / mL, the stock solution containing polyvinylpyrrolidone is prepared with a polyvinylpyrrolidone concentration of 50 mg / mL, and the stock solution containing Pluronic® P-123 is prepared with a Pluronic® P-123 concentration of 0.01 mg / mL.

[0094] In some instances, each stock solution may be stored for up to four weeks from the date the stock solution was prepared.

[0095] In step (c), the stock solutions are combined to form a harvest solution.

[0096] In some examples, the collection solution includes two anticoagulants, a first anticoagulant and a second anticoagulant. Preferably, the first anticoagulant and the second anticoagulant are different. Preferably, the collection solution includes a first anticoagulant at 2-10 mg / mL, 4-8 mg / mL, 5-7 mg / mL, or 6 mg / mL. Preferably, the collection solution includes a second anticoagulant at 3-11 mg / mL, 5-9 mg / mL, 6-8 mg / mL, or 7 mg / mL.

[0097] In some examples, the collection solution contains the flocculant at 5-25 mg / mL, 7.5-20 mg / mL, 9-11 mg / mL, or 10 mg / mL.

[0098] In some examples, the collection solution contains the wetting agent at 0.050-0.250 mg / mL, 0.075-0.120 mg / mL, 0.900-0.125 mg / mL, or 0.1 mg / mL.

[0099] In some examples, the collection solution comprises 6 mg / mL of a first anticoagulant, 7 mg / mL of a second anticoagulant, 10 mg / mL of a flocculating agent, and 0.1 mg / mL of a wetting agent. Preferably, the collection solution comprises 6 mg / mL of potassium oxalate, 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate, 10 mg / mL of polyvinylpyrrolidone, and 0.1 mg / mL of Pluronic® P-123.

[0100] In some instances, the storage period of the harvesting solution is 7 days. After 7 days, a new harvesting solution is prepared.

[0101] The present invention also provides a method for producing said imaging solution, the method comprising the steps of: (a) providing an imaging agent; and (b) preparing a stock solution for said imaging agent.

[0102] In step (b), the stock solution is prepared. In some examples, the imaging agent is a solid and is measured into a container. An example of a preferred container is a disposable tray. The container is washed with a cleaning agent, such as isopropyl alcohol, before use. In another example, the imaging agent is a liquid and the required amount of imaging agent is measured into a graduated cylinder. The graduated cylinder is washed with a cleaning agent, such as isopropyl alcohol, before use.

[0103] In some examples, the imaging agent is transferred to a vial and a solvent is added to the vial. Preferably, the solvent used is water. Preferably, any suitable organic or inorganic solvent is used, such as an alcoholic solvent. Preferably, the imaging agent is dissolved in the solvent by stirring the solution at 700 rpm to 900 rpm, or 800 rpm. Preferably, the imaging agent is dissolved in the solvent by stirring the solution for 5 to 15 minutes, or 10 minutes. Preferably, the imaging agent is dissolved in the solvent at a temperature of 10 to 30°C, or 15 to 25°C.

[0104] In some examples, the stock solution containing the imaging agent is prepared so that the concentration of the imaging agent is 0.01 to 0.030 mg / mL, 0.015 to 0.025 mg / mL, 0.019 to 0.021 mg / mL, or 0.020 mg / mL.

[0105] In some examples, the stock solution containing the imaging agent is prepared so that the concentration of the imaging agent is 0.020 mg / mL. Preferably, the stock solution containing acridine orange is prepared so that the concentration of acridine orange is 0.020 mg / mL.

[0106] In some examples, the imaging solution has a shelf life of up to one month. After one month, a new imaging solution needs to be prepared. Preferably, the imaging solution is stored protected from light. Preferably, the imaging solution is stored at a temperature of 10° C. or less, 5° C. or less, or 4° C. or less.

[0107] In some instances, the collection solution and the imaging solution are combined to form one solution.

[0108] Method for coating a cuvette with a composition The present invention also includes a method of coating a cuvette with said composition, the method comprising the steps of: (a) providing a cuvette comprising a collection chamber and an analysis chamber; (b) preparing a collection solution; (c) preparing an imaging solution; (d) coating (wholly or partially) the collection chamber of the cuvette with the collection solution; (e) coating (wholly or partially) the analysis chamber of the cuvette with the imaging solution; (f) drying the coating of the collection solution on the collection chamber; and (g) drying the coating of imaging solution on the analysis chamber.

[0109] Before coating the cuvette with the collection or imaging solutions, check the shelf life of each solution.

[0110] In some examples, the collection solution is mixed thoroughly prior to coating the collection chamber of the cuvette with the collection solution. Preferably, the collection solution is mixed until homogenous. The collection solution is then dispensed into the collection chamber with a pipette. Preferably, 10-20 μL, 12.5-17.5 μL, or 15 μL of the collection solution is dispensed into the collection chamber. Preferably, the collection solution dispensed into the collection chamber of the cuvette is collected from the center of the collection solution.

[0111] In some examples, the imaging solution is mixed thoroughly prior to coating the analysis chamber of the cuvette with the imaging solution. Preferably, the imaging solution is mixed until homogenous. The imaging solution is then dispensed into the analysis chamber with a pipette. Preferably, 10-20 μL, 12.5-17.5 μL, or 15 μL of the imaging solution is dispensed into the analysis chamber. Preferably, the imaging solution dispensed into the analysis chamber of the cuvette is taken from the center of the imaging solution.

[0112] In some examples, a multichannel pipette is used to dispense the imaging solution into the analysis chamber. Preferably, the multichannel pipette is adjusted such that 1-60% of the surface area, 10-50% of the surface area, 20-40% of the surface area, 30-40% of the surface area, or 35% of the surface area of ​​the second chamber of the cuvette is coated with the imaging solution. Preferably, the multichannel pipette is adjusted such that 40-99% of the surface area, 50-90% of the surface area, 80-40% of the surface area, 70-60% of the surface area, or 65% of the surface area of ​​the analysis chamber is not coated with the imaging solution. Preferably, the multichannel pipette is adjusted such that the imaging solution is disposed at the first end of the analysis chamber and the second (closed) end of the analysis chamber, and there is a portion of the analysis chamber between the first end of the analysis chamber and the second (closed) end of the analysis chamber where the imaging solution is not dispensed. Preferably, the proportion of the imaging solution present at the first end of the analysis chamber is greater than the proportion of the imaging solution present at the second (closed) end of the analysis chamber. Preferably, the ratio of the imaging solution present at the first end of the analysis chamber to the imaging solution present at the second (closed) end of the analysis chamber is 5:3 to 5:4. Preferably, the analysis chamber has four opposing side walls, and the imaging solution is present on at most one of the four opposing side walls. Preferably, the analysis chamber has four opposing side walls, and the imaging solution is present on at most one of the four opposing side walls, and the imaging solution is present on the side wall of the first (open) end of the analysis chamber, on the side wall of the second (closed) end of the analysis chamber, and not in an intermediate region of the side wall between the first (open) end and the second (closed) end of the analysis chamber.

[0113] In another example, a single pipette is used to dispense the imaging solution into the analysis chamber. Preferably, the single pipette is adjusted such that 1-60% of the surface area, 10-50% of the surface area, 20-40% of the surface area, 30-40% of the surface area, or 35% of the surface area of ​​the second chamber of the cuvette is coated with the imaging solution. Preferably, the single pipette is adjusted such that 40-99% of the surface area, 50-90% of the surface area, 80-40% of the surface area, 70-60% of the surface area, or 65% of the surface area of ​​the analysis chamber is not coated with the imaging solution. Preferably, the single pipette is adjusted such that the imaging solution is disposed at the first end of the analysis chamber and at the second (closed) end of the analysis chamber and at the second (closed) end of the analysis chamber. The imaging solution is adjusted so that there is a portion of the analysis chamber between the first end of the analysis chamber and the second (closed) end of the analysis chamber, where the imaging solution is not distributed. Preferably, the proportion of the imaging solution present at the first end of the analysis chamber is greater than the proportion of the imaging solution present at the second (closed) end of the analysis chamber. Preferably, the ratio of the imaging solution present at the first end of the analysis chamber to the imaging solution present at the second (closed) end of the analysis chamber is 5:3 to 5:4. Preferably, the analysis chamber has four opposing side walls, and the imaging solution is present on at most one of the four opposing side walls. Preferably, the analysis chamber has four opposing side walls, and the imaging solution is present on at most one of the four opposing side walls, and the imaging solution is present on the side wall of the first (open) end of the analysis chamber, on the side wall of the second (closed) end of the analysis chamber, and not in the intermediate region of the side wall between the first (open) end and the second (closed) end of the analysis chamber.

[0114] In some examples, after the collection solution is dispensed onto the collection chamber, the collection solution and the collection chamber are dried. Preferably, drying is accomplished by exposing the collection solution and the collection chamber to heat. Preferably, the collection solution and the collection chamber are exposed to heat at a temperature of 40-80° C., 50-70° C., or 60° C. Preferably, the collection solution and the collection chamber are exposed to heat for 1-20 minutes, 5-15 minutes, or 10 minutes.

[0115] In some examples, after dispensing the imaging solution onto the analysis chamber, the imaging solution and the analysis chamber are dried. Preferably, drying is accomplished by exposing the imaging solution and the analysis chamber to heat. Preferably, the imaging solution and the analysis chamber are exposed to heat at a temperature of 40-80° C., 50-70° C., or 60° C. Preferably, the imaging solution and the analysis chamber are exposed to heat for 1-20 minutes, 5-15 minutes, or 10 minutes.

[0116] In some examples, the collection solution and the imaging solution are dried simultaneously on the collection chamber and the analysis chamber. Preferably, drying is performed by exposing the collection solution, the imaging solution, the collection chamber, and the analysis chamber to heat. Preferably, the collection solution, the imaging solution, the collection chamber, and the analysis chamber are exposed to heat at a temperature of 40-80° C., 50-70° C., or 60° C. Preferably, the collection solution, the imaging solution, the collection chamber, and the analysis chamber are exposed to heat for 1-20 minutes, 5-15 minutes, or 10 minutes.

[0117] In some examples, the coated cuvettes can be stored in a dust-free environment. Preferably, the storage temperature is 10-30° C. or 15-25° C. Preferably, the storage relative humidity is 20-70% relative humidity or 30-60% relative humidity.

[0118] In some examples, the cuvette is formed from two halves that are fastened together to form a cuvette. Splitting the cuvette into two halves allows for easy access to the collection chamber and the analysis chamber during the coating process of the collection chamber and the analysis chamber. Advantageously, this allows for the formation of a uniform coating of the imaging solution in the analysis chamber.

[0119] Methods of using cuvettes coated with the composition The cuvette coated with the composition can be used for optical analysis of a sample, such as a blood sample. To optically analyze a sample, the following steps are followed: (a) placing a sample in a cuvette; (b) mixing the sample with a collection solution; (c) introducing the mixture of step 1 into an imaging solution; (d) subjecting the mixture of step 2 to centrifugal force; (e) exposing the mixture to optical radiation; and (f) obtaining an optical image of the mixture.

[0120] In some instances, a portion of the sample is taken from the center of the sample prior to step (b).

[0121] In some examples, in step (b), the collection solution is present on a collection chamber of the cuvette and step (b) is performed in the collection chamber of the cuvette. In some examples, in step (c), the imaging solution is present on an analysis chamber of the cuvette and step (c) is performed in the analysis chamber of the cuvette.

[0122] In some examples, the mixture is subjected to centrifugation in an Entia Liberty™ device in step (d). Preferably, the mixture is subjected to a centrifugation force of 1000-2000 rpm or 1500 rpm. Preferably, the mixture is subjected to centrifugation at room temperature. Preferably, the mixture is subjected to centrifugation at 15-40° C., 16-30° C., 18-25° C., 20-22° C., or 20° C. Preferably, the mixture is subjected to centrifugation for up to 30 minutes, up to 25 minutes, up to 20 minutes, up to 15 minutes, or up to 10 minutes. Preferably, the mixture is subjected to centrifugation in an Entia Liberty™ device providing a centrifugation force of 1500 rpm for up to 20 minutes at room temperature.

[0123] In some examples, the mixture is exposed to optical radiation from an LED light source having a wavelength of 460 nm during step (e). Preferably, the mixture is exposed to optical radiation at room temperature. Preferably, the mixture is exposed to optical radiation for up to 15 minutes, up to 10 minutes, or up to 5 minutes. Preferably, the mixture is exposed to optical radiation from an LED light source having a wavelength of 460 nm for up to 5 minutes at room temperature.

[0124] In some examples, steps (e) and (f) are performed while the sample is in the cuvette.

[0125] In some examples, the optical image is analyzed and the pixel counts of the image obtained in step (f) are converted into cell counts. EXAMPLES

[0126] Below are non-limiting examples that illustrate at least some of the advantages of the present invention, with reference to tables and figures. The examples described herein are not limiting, but merely examples among other possible examples.

[0127] Example 1 Preparation of the composition In the following non-limiting examples, compositions were made that included a collection solution and an imaging solution. The collection solution and the imaging solution were made separately.

[0128] In this non-limiting example, a collection solution was made containing polyvinylpyrrolidone, potassium oxalate, ethylenediaminetetraacetic acid dipotassium salt dehydrate, Pluronic® P-123, and water. The collection solution was made from stock solutions of each component.

[0129] In this non-limiting example, an imaging solution was made containing acridine orange and water. The imaging solution was made from a stock solution of acridine orange.

[0130] Stock solutions of each component used in the collection and imaging solutions were made separately. To make the stock solutions, each component of the collection and imaging solutions was weighed separately in separate disposable trays and then mixed with the respective amount of water in separate clean, empty vials. After each component of the collection and imaging solutions was added to the water, the trays were weighed to verify the concentration of each stock solution and to ensure that no residue remained (the maximum amount of residue was 0.005 g).

[0131] The powdered reagent was completely dissolved in water by thorough stirring.

[0132] The component, Pluronic® P-123, was initially in the form of a gel block. To dissolve the Pluronic® P-123 in water, the Pluronic® P-123 was stirred at 800 rpm for 10 minutes at room temperature (15-25°C) and relative humidity (RH) of 30-60%. The temperature was carefully monitored to ensure it did not exceed 35°C, but the degree of dilution increased with increasing temperature.

[0133] Table 1 shows the concentrations of each stock solution prepared.

[0134] Table 1: Concentrations of each stock solution used to prepare the collection and imaging solutions. [Table 1]

[0135] The collection solution was made by mixing stock solutions of polyvinylpyrrolidone, potassium oxalate, ethylenediaminetetraacetic acid dipotassium salt dehydrate, Pluronic® P-123, and water to the concentrations shown in Table 2. The stock solutions were mixed and the solution was mixed until the resulting collection solution was homogenous throughout.

[0136] The imaging solution was made by diluting a stock solution of acridine orange with water to the desired concentration. Once the stock solution was diluted, the solution was mixed so that the resulting imaging solution was homogenous throughout.

[0137] The concentrations of each component in the collection solution and imaging solution are shown in Table 2.

[0138] Table 2: Concentrations of each component used in the collection and imaging solutions. Each component was supplied from its stock solution. [Table 2]

[0139] After preparation, the collection solution and the imaging solution were stored separately (i.e., without mixing) until use.

[0140] Example 2 The collection chamber of the cuvette is coated with the collection solution. In the following non-limiting examples, the collection chamber of a cuvette was coated with the collection solution. The cuvette was formed from two halves that were fastened together to form the cuvette.

[0141] In this non-limiting example, the following procedure was followed to coat the collection chamber of a cuvette half in collection solution: (a) The collected solution was stirred; (b) 15 μL of collection solution was removed from the center of the collection solution with a pipette; (c) 15 μL of the collection solution from step (b) was dispensed onto the surface of the collection chamber of the cuvette half within the tolerance limits shown in FIG. 1 and described below; and (d) The collection solution in half the cuvette and the collection chamber were allowed to dry.

[0142] FIG. 1 shows the collection chamber (160) of a half cuvette coated with collection solution (150). FIG. 1 shows two images (110, 120) with an acceptable coating of collection solution. As shown in FIG. 1, the acceptable limit for dispensing collection solution onto the collection chamber is either the entire collection chamber is coated (110) or the entire length of the collection chamber is coated (120). Placing the collection solution within the acceptable limit ensures easy collection of the sample. FIG. 1 also shows two images (130, 140) with an unacceptable coating of collection solution. As shown in FIG. 1, the unacceptable limit is for the collection solution to not cover the first and second ends of the collection chamber (130). The collection solution needs to be in an area where it can assist in the collection of the sample and maintain the integrity of the sample. If the collection solution does not cover the first and / or second ends of the collection chamber, the collection solution cannot perform this function. The second unacceptable limit is the spread of the collection solution beyond the second end of the collection chamber (140), which causes the collection solution to block the vent in the cuvette and prevent the sample from entering the analysis chamber, but only into the collection chamber.

[0143] To dry the collection solution and the collection chamber, they were exposed to a temperature of 60° C. for 10 min in a universal oven. During the drying process, the cuvettes were not stacked on top of other cuvettes and were protected from contamination by dust particles.

[0144] The collection solution can be coated onto the collection chamber of the cuvette half, followed by coating the imaging solution onto the analysis chamber of the cuvette half. Alternatively, the imaging solution can be coated onto the analysis chamber of the cuvette half before coating the collection solution onto the collection chamber of the cuvette half. After the collection and imaging solutions are coated onto the collection and analysis chambers of the cuvette halves, respectively, the two cuvette halves are clamped together to complete the cuvette assembly.

[0145] Example 3 The analytical chamber of the cuvette is coated with the imaging solution. In the following non-limiting examples, the analysis chamber of a cuvette was coated with the imaging solution. The cuvette was formed from two halves that were fastened together to form a cuvette. The inner surface of one half had recesses that formed three of the four opposing side walls of the analysis chamber, and only the middle wall of the three side walls was partially coated with the imaging solution. The other two walls and the other half of the cuvette were not coated. The other half of the cuvette was substantially smooth or flat. Due to the recesses on the inner surface of one half of the cuvette, the collection chamber and the analysis chamber were present within the cuvette body when the two halves were fastened together. Splitting the cuvette into two halves allowed easy access to the analysis chamber during the coating process.

[0146] In this non-limiting example, the following method was followed to coat the analysis chamber of the cuvette in the imaging solution: (a) the imaging solution was stirred; (b) The multichannel pipette (VIAFLO multichannel pipette) was turned on; (c) placing one half of the cuvette to be coated in the coating jig of the multichannel pipette (the area of ​​the multichannel pipette designated for placing the surface to be coated); (d) GRIPTIPS tips were inserted into the multichannel pipette in the following order ((4) closest to the first end of the analysis chamber, (1) closest to the second (closed) end of the analysis chamber), for a total of eight tips; the following GRIPTIPS order is shown in FIG. 2: (1) 2 chips (210), (2) 3 spaces (220), (3) 6 chips (230), (4) 5 spaces (240); (e) GRIPTIPS were immersed in the imaging solution and 3 μL of the imaging solution was collected on the GRIPTIPS; (f) visually inspecting the GRIPTIPS to ensure that the amount of imaging solution dispensed was uniform across all GRIPTIPS; (g) GRIPTIPS were aligned over the coating jig and lowered so that they lightly touched the surface of the middle of the three side walls of the analysis chamber of the cuvette half; (h) For each GRIPTIPS, a first droplet containing 0.94 μL of the imaging solution was dispensed onto the surface; (i) The GRIPTIPS were realigned by moving them 2 mm towards the second (closed) end of the analysis chamber; (j) each GRIPTIPS was subjected to ejection of a second droplet containing 0.94 μL of the imaging solution onto the surface; and (k) drying the imaging solution in the cuvette half and the analysis chamber; Ta.

[0147] After the pipette was allowed to dispense a second drop (step (j)), the cuvette halves and GRIPTIPS were removed from the coating jig. The GRIPTIPS were placed on absorbent tissue and any remaining sample was removed from the pipette.

[0148] FIG. 3 shows an image of a cuvette half (300) with a collection chamber (340) and an analysis chamber (350). In FIG. 3, the area of ​​the analysis chamber (350) that is coated with the imaging solution is highlighted. As shown in FIG. 3, the portions of the analysis chamber (350) that are coated with the imaging solution are labeled 310 and 330, and the portion of the analysis chamber that is not coated with the imaging solution is labeled 320. The lengths of the two portions of the analysis chamber that are coated with the imaging solution are 22 mm (310) and 10 mm (330). The total area of ​​the analysis chamber that is coated with the imaging solution is 35% of the surface area with 15 μL of imaging solution.

[0149] To dry the imaging solution and the analysis chamber, the imaging solution and the analysis chamber were exposed to a temperature of 60° C. for 10 minutes in a universal oven. During the drying process, the cuvettes were not stacked on top of other cuvettes and were protected from contamination by dust particles. Advantageously, coating the imaging solution on the analysis chamber ensures that the imaging solution is in contact with the sample during the entire time of centrifugation. As a result, coating the imaging solution on the analysis chamber reduces the variability of the absorption of the imaging solution by the sample. The imaging solution is present in the analysis chamber and dries on the surface of the analysis chamber, so it does not enter the collection chamber.

[0150] The imaging solution can be coated onto the analysis chamber of the cuvette half, and then the collection solution can be coated onto the collection chamber of the cuvette half. Alternatively, the collection solution can be coated onto the collection chamber of the cuvette half before the imaging solution is coated onto the analysis chamber of the cuvette half. After the collection and imaging solutions are coated onto the collection and analysis chambers of the cuvette halves, respectively, the two cuvette halves are clamped together to complete the cuvette set.

[0151] Example 4 The analytical chamber of the cuvette is coated with the imaging solution. In the following non-limiting examples, the analysis chamber of a cuvette was coated with the imaging solution. The cuvette was formed from two halves that were fastened together to form a cuvette. The inner surface of one half had recesses that formed three of the four opposing side walls of the analysis chamber, and only the middle wall of the three side walls was partially coated with the imaging solution. The other two walls and the other half of the cuvette were not coated. The other half of the cuvette was substantially smooth or flat. Due to the recesses on the inner surface of one half of the cuvette, the collection chamber and the analysis chamber were present within the cuvette body when the two halves were fastened together. Splitting the cuvette into two halves allowed easy access to the analysis chamber during the coating process.

[0152] In this non-limiting example, the following method was followed to coat the analysis chamber of the cuvette in the imaging solution: (a) the imaging solution was stirred; (b) A single pipette was prepared; (c) immersing the single pipette in the imaging solution and drawing 15 μL of the imaging solution into the single pipette; (d) placing the single pipette on the surface of the middle wall of the three side walls of the analysis chamber of the cuvette half and dropping 15 μL of the imaging solution onto the entire surface to achieve coating of the analysis chamber (the total area of ​​the analysis chamber coated with the imaging solution was 35% of the surface area, and the coating was applied to one of the four side walls forming the analysis chamber, and the coating of the imaging solution was only present at the edge of the side wall of the first (open) end of the analysis chamber and the edge of the side wall of the second (closed) end of the analysis chamber, and was not present in the intermediate region between the first (open) end and the second (closed) end of the analysis chamber); and (f) The imaging solution in the cuvette half and the analysis chamber were allowed to dry.

[0153] To dry the imaging solution and the analysis chamber, the imaging solution and the analysis chamber were exposed to a temperature of 60° C. for 10 minutes in a universal oven. During the drying process, the cuvettes were not stacked on top of other cuvettes and were protected from contamination by dust particles. Advantageously, coating the imaging solution on the analysis chamber ensures that the imaging solution is in contact with the sample during the entire time of centrifugation. Coating the imaging solution on the analysis chamber reduces the variability of the absorption of the imaging solution by the sample. The imaging solution is present in the analysis chamber and dries on the surface of the analysis chamber, so it does not enter the collection chamber.

[0154] The imaging solution can be coated onto the analysis chamber of the cuvette half, and then the collection solution can be coated onto the collection chamber of the cuvette half. Alternatively, the collection solution can be coated onto the collection chamber of the cuvette half before the imaging solution is coated onto the analysis chamber of the cuvette half. After the collection and imaging solutions are coated onto the collection and analysis chambers of the cuvette halves, respectively, the two cuvette halves are clamped together to complete the cuvette set.

[0155] Example 5 The effect of the concentration of the flocculant in the solution is analyzed. In the following non-limiting examples, the effect of a flocculant present in the solution on the resulting image was analyzed. The flocculant analyzed in this example was polyvinylpyrrolidone.

[0156] In this non-limiting example, a solution was prepared containing polyvinylpyrrolidone, acridine orange, and water. The solution was prepared from a stock solution of polyvinylpyrrolidone and acridine orange. The stock solutions were prepared as described in Example 1.

[0157] Three solutions with different concentrations of polyvinylpyrrolidone were made. The three different concentrations of polyvinylpyrrolidone were 10.00 mg / mL, 20.00 mg / mL, and 30.00 mg / mL. The solution with a concentration of 10.00 mg / mL of polyvinylpyrrolidone was labeled solution 1, the solution with a concentration of 20.00 mg / mL of polyvinylpyrrolidone was labeled solution 2, and the collected solution with a concentration of 30.00 mg / mL of polyvinylpyrrolidone was labeled solution 3. Stock solutions of polyvinylpyrrolidone, acridine orange, and water were then combined to prepare solutions with the concentrations shown in Table 3. The stock solutions were mixed and the solutions were mixed so that the resulting solutions were homogenous throughout.

[0158] Table 3: Concentration of each component in solution. [Table 3]

[0159] Glass capillaries (microslide 0.3x0.5mm glass capillaries) were coated with the solutions. Different glass capillaries were used for different solutions. The glass capillaries were coated in the following way: (a) stirring the solution; (b) 50 μL of the solution was removed from the center of the solution with a pipette; (c) dispensing 50 μL of the solution from step (b) into the glass capillary tube; and (d) The solution and the glass capillary were allowed to dry.

[0160] To dry the solution on the glass capillary tube, the solution and the glass capillary tube were exposed to a temperature of 60° C. for 10 minutes in a universal oven.

[0161] After coating the glass capillary with the solutions, a venous blood sample was inserted into the glass capillary coated with solution 1. The glass capillary with the blood sample was placed in a laboratory centrifuge and run at 3000G for 5 minutes at room temperature. Upon centrifugation, the blood sample separated into a platelet layer, an agranulocyte layer, a granulocyte layer, and a red blood cell layer. Images of the separated blood sample were taken with a Nikon D5100 camera. The images were generated using an LED light operating at a wavelength of 460 nm at room temperature. The same procedure was performed for each glass capillary coated with solution 2 or solution 3. The same experiment was then repeated for 16 samples.

[0162] The results of the analysis are shown in Figure 4. The image labeled 410 corresponds to solution 1, the image labeled 420 corresponds to solution 2, and the image labeled 430 corresponds to solution 3.

[0163] Example 6 The effect of the concentration of anticoagulant in the solution is analyzed. In the following non-limiting examples, the effect of anticoagulants in solution on the resulting images was analyzed. The anticoagulant analyzed in this example was the dipotassium salt of ethylenediaminetetraacetic acid.

[0164] In this non-limiting example, a solution was prepared containing dipotassium ethylenediaminetetraacetic acid dehydrate, polyvinylpyrrolidone, acridine orange, and water. The solution was prepared from a stock solution of each component. The stock solution was prepared as described in Example 1. The concentration of the stock solution containing polyvinylpyrrolidone was adjusted so that polyvinylpyrrolidone was present at a concentration of 3.0 mg / mL, and the concentration of the stock solution containing acridine orange was adjusted so that acridine orange was present at a concentration of 0.02 mg / mL.

[0165] Two solutions with different concentrations of ethylenediaminetetraacetic acid dipotassium salt dehydrate were analyzed. The two different concentrations of ethylenediaminetetraacetic acid dipotassium salt dehydrate were 7.00 mg / mL and 19.00 mg / mL. The solution with the concentration of ethylenediaminetetraacetic acid dipotassium salt dehydrate of 7.00 mg / mL was labeled solution 4, and the solution with the concentration of ethylenediaminetetraacetic acid dipotassium salt dehydrate of 19.00 mg / mL was labeled solution 5. Next, ethylenediaminetetraacetic acid dipotassium salt dehydrate was analyzed. Stock solutions of dipotassium diaminetetraacetate dehydrate, polyvinylpyrrolidone, acridine orange, and water were mixed to prepare solutions with the concentrations shown in Table 4. The stock solutions were mixed and the solution was mixed until the resulting solution was homogenous throughout.

[0166] Table 4: Concentration of each component in solution. [Table 4]

[0167] Glass capillaries (microslide 0.3x0.5mm glass capillaries) were coated with the solutions. Different glass capillaries were used for different solutions. The glass capillaries were coated in the following way: (a) stirring the solution; (b) 50 μL of the solution was removed from the center of the solution with a pipette; (c) dispensing 50 μL of the solution from step (b) into the glass capillary tube; and (d) The solution and the glass capillary were allowed to dry.

[0168] To dry the solution on the glass capillary tube, the solution and the glass capillary tube were exposed to a temperature of 60° C. for 10 minutes in a universal oven.

[0169] After coating the glass capillary with the solution, a venous blood sample was inserted into the glass capillary coated with solution 4. The glass capillary with the blood sample was placed in a laboratory centrifuge and run at 3000G for 5 minutes at room temperature. Upon centrifugation, the blood sample separated into a platelet layer, an agranulocyte layer, a granulocyte layer, and a red blood cell layer. Images of the separated blood sample were taken with a Nikon D5100 camera. The images were generated using an LED light operating at a wavelength of 460 nm at room temperature. The same procedure was performed for each glass capillary coated with solution 5. The experiment was then repeated for 12 samples.

[0170] The results showed that solutions containing high concentrations of ethylenediaminetetraacetic acid dipotassium salt dehydrate dehydrated damaged cells in the samples analyzed.

[0171] Example 7: Comparing a composition according to the invention with a different composition (not according to the invention) In this non-limiting example, a solution similar to the composition of the present invention coated onto a cuvette is compared to a cuvette coated with a different composition (not according to the present invention).

[0172] The non-inventive solution was the solution described in U.S. Patent No. 6,365,104. The solution described in U.S. Patent No. 6,365,104 contains sodium heparin, dipotassium ethylenediaminetetraacetic acid dehydrate, acridine orange, and potassium oxalate. Table 5 shows the mass and corresponding concentration of each component in the U.S. Patent No. 6,365,104 solution.

[0173] Table 5: Mass of each component present in the solution of US Pat. No. 6,365,104 and the corresponding concentration. [Table 5]

[0174] Next, a solution replicating the solution of US Patent No. 6,365,104 was made with the following concentrations: 6.000 mg / mL potassium oxalate, 76.0 USP / mL sodium heparin, 7.0 mg / mL ethylenediaminetetraacetic acid dipotassium salt dehydrate, 10 mg / mL polyvinylpyrrolidone, and 0.110 mg / mL acridine orange. Because the composition of the present invention contained polyvinylpyrrolidone, the composition replicating the composition of US Patent No. 6,365,104 also contained polyvinylpyrrolidone.

[0175] A solution similar to the composition of the present invention was then made with the following concentrations in solution: 6.000 mg / mL potassium oxalate, 7.0 mg / mL ethylenediaminetetraacetic acid dipotassium salt dehydrate, 10 mg / mL polyvinylpyrrolidone, and 0.110 mg / mL acridine orange.

[0176] The solutions were made from the stock solutions described in Example 1. The stock solutions of each component were then combined to prepare solutions at the concentrations listed above. The stock solutions were mixed and the solution was mixed such that the resulting harvest solution was homogenous throughout.

[0177] Glass capillaries (microslide 0.3x0.5mm glass capillaries) were then coated with the solution. Different glass capillaries were used for different solutions. The glass capillaries were coated in the following manner: (a) stirring the solution; (b) 50 μL of the solution was removed from the center of the solution with a pipette; (c) dispensing 50 μL of the solution from step (b) into the glass capillary tube; and (d) The solution and the glass capillary were allowed to dry.

[0178] To dry the solution on the glass capillary tube, the solution and the glass capillary tube were exposed to a temperature of 60° C. for 10 minutes in a universal oven.

[0179] After coating the glass capillary with the solution, a venous blood sample was inserted into the glass capillary coated with the solution described in US Pat. No. 6,365,104. The glass capillary together with the blood sample was placed in a laboratory centrifuge and run at 3000G for 5 minutes at room temperature. Upon centrifugation, the blood sample separated into a platelet layer, an agranulocyte layer, a granulocyte layer, and a red blood cell layer. Images of the separated blood sample were taken with a Nikon D5100 camera. The images were generated using an LED light operating at a wavelength of 460 nm at room temperature. The same procedure was also performed on glass capillary coated with a solution similar to the composition of the present invention.

[0180] The results of the analysis are shown in Figure 5. The solution described in U.S. Patent No. 6,365,104 is labeled 510, and a solution similar to the composition of the present invention is labeled 520. As shown in Figure 5, the solution similar to the composition of the present invention improves the clarity of the image obtained from the blood sample.

[0181] Example 8 The effect of placing the imaging solution at the first end and the second (closed) end (but not the central portion) of the analysis chamber is analyzed. In this non-limiting example, the effects of placing the imaging solution toward or at the first (open) end of the analysis chamber and toward or at the second (closed) end (but not in the center) were analyzed.

[0182] In this non-limiting example, collection and imaging solutions were prepared as described in Example 1, and cuvettes were coated as described in Examples 2 and 3. The cuvettes were formed from two halves that were fastened together to form the cuvette.

[0183] A further cuvette was coated with the collection solution and the imaging solution. The cuvette was formed from two halves that were fastened together to form a cuvette. The inner surface of one half had a recess that formed three of the four opposing side walls of the collection chamber and the analysis chamber. The other half was substantially smooth or flat and formed the fourth opposing side wall of the collection chamber and the analysis chamber. The collection chamber was coated with the collection solution as described in Example 2. The analysis chamber of the cuvette was coated with the imaging solution. The coating of the imaging solution on the cuvette half having the recess was not restricted to being present only at the first (open) end and the second (closed) end of the middle wall of the three side walls of the analysis chamber of the cuvette half, but not in the middle portion (as described in Example 3). By not restricting the location of the coating of the imaging solution on the cuvette half, a portion of the imaging solution could be trapped between the two halves of the cuvette when they were fastened. Thus, when the blood sample entered the analysis chamber, a portion of the captured imaging solution could not enter the blood sample but instead remained trapped between the two halves of the cuvette.

[0184] A venous blood sample was then inserted into each assembled cuvette, and each cuvette and blood sample was separately placed in the Entia Liberty™ device. For each cuvette and blood sample, the device was operated at 1500 rpm for 20 minutes at room temperature. The blood sample was separated into a platelet layer, agranulocyte layer, a granulocyte layer, and a red blood cell layer. The blood sample was then exposed to an LED light source operating at a wavelength of 460 nm in the Entia Liberty™ device for 5 minutes at room temperature, and an image of the sample was obtained.

[0185] Figure 6 shows images (610, 620, and 630) acquired on cuvettes where the coating of imaging solution was not restricted to only the first (open) and second (closed) ends of the middle of the three side walls of the analytical chamber of the cuvette half, but not the central portion. As can be seen in Figure 6, light rays are visible in the acquired images.

[0186] Advantageously, by placing the imaging solution towards or at the first (open) end and towards or at the second (closed) end (but not at the center) of the middle of the three side walls of the analysis chamber of the cuvette half, light lines are eliminated from the desired image of the buffy coat. The portion of the analysis chamber that is not coated with imaging solution corresponds to the location of the buffy coat layer of the separated blood sample. The location of the imaging solution on the middle of the three side walls was limited because the coating process of the cuvette was manual and could not ensure that the side walls were free of coating. The light lines (610, 620, and 630) correspond to the imaging solution that was captured by the adhesive during the fixation process used to fix the two cuvette halves. By (a) placing the imaging solution only on the middle of the three side walls of the analysis chamber of the cuvette half having the recess, as well as (b) not placing the imaging solution on the center part of the middle wall of the analysis chamber, light lines can be eliminated or reduced in the image obtained from the sample.

[0187] More advantageously, the imaging solution is coated onto the analysis chamber, so that it is in contact with the biological sample throughout the centrifugation and imaging process, allowing the mechanism by which the imaging agent acts to function efficiently.

[0188] Further advantageously, by not coating the collection chamber with imaging solution, no imaging solution residue is left behind within the collection chamber, reducing variability between images obtained.

[0189] The features disclosed in the foregoing description, the following claims, or the accompanying drawings may be expressed, as appropriate, in their specific form, or as means for performing a disclosed function, or in a method or process for achieving a disclosed result, and such features may be utilized individually or in any combination to realize the invention in its various forms.

[0190] Although certain exemplary embodiments of the present invention have been described, the appended claims are not intended to be limited to only these examples. The claims are intended to be interpreted literally, intentionally, and / or to encompass equivalents.

Claims

1. A method for obtaining an image of a sample in a cuvette, comprising the following steps: (a) The step of placing the sample in the cuvette; (b) A step of mixing the sample with the collection solution; (c) A step of introducing the mixture from step (b) into the imaging solution; (d) A step of applying centrifugal force to the mixture from step (c); (e) Exposing the centrifugal mixture from step (d) to optical radiation; and (f) Steps to obtain an optical image of the mixture. Methods that include...

2. The method according to claim 1, wherein the mixture is subjected to centrifugal force in an Entia Liberty™ device in step (d).

3. The method according to claim 1 or 2, wherein the mixture is subjected to a centrifugal force of 1,000 to 2,000 rpm or 1,500 rpm.

4. The method according to claim 1 or 2, wherein the mixture is subjected to centrifugal force for a maximum of 30 minutes, a maximum of 25 minutes, a maximum of 20 minutes, a maximum of 15 minutes, or a maximum of 10 minutes.

5. The mixture is subjected to centrifugal force at room temperature, and / or The method according to claim 1 or 2, wherein the mixture is exposed to optical radiation from an LED light source, and optionally the LED light source operates at 400-700 nm, 425-600 nm, 450-500 nm, or 460 nm.

6. The mixture is exposed to optical radiation for a maximum of 15 minutes, a maximum of 10 minutes, or a maximum of 5 minutes, and / or The method according to claim 1 or 2, wherein the mixture is exposed to optical radiation at room temperature, or at a temperature of 15-40°C, 16-30°C, 18-25°C, 20-22°C, or 20°C.

7. Steps (c) and (d) occur simultaneously by centrifugal force that mixes the mixture from step (b) with the imaging solution in step (c), and / or The optical image of step (f) is acquired by a camera, as described in claim 1 or 2. method.

8. The method according to claim 1 or 2, wherein the sample is a biological sample, and optionally the biological sample is a blood sample.

9. The aforementioned sampled solution Anticoagulants, Coagulants, and containing a wetting agent, and / or The aforementioned sampled solution Anticoagulants in doses of 5-25 mg / mL, A coagulant in a concentration of 5-25 mg / mL, and The method according to claim 1 or 2, comprising 0.05 to 0.25 mg / mL of a wetting agent.

10. The method according to claim 1 or 2, wherein the collected solution further comprises water (preferably distilled water) or an alcohol solvent.

11. The collected solution comprises a first anticoagulant and a second anticoagulant, wherein the first anticoagulant and the second anticoagulant are different, and / or The method according to claim 1 or 2, wherein the anticoagulant is one or more of ethylenediaminetetraacetate dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, and any combination thereof.

12. The flocculant is one or more of polyvinylpyrrolidone, proteolytic enzymes (bromelain, pepsin, and / or trypsin), polyethylene glycol, and any combination thereof, and / or The method according to claim 9, wherein the wetting agent is one or more of well-known surfactants (i.e., ionic, nonionic, and / or cationic), Pluronic® P-123, Silwet L600, aliphatic alcohol ethoxylates, alkylphenol ethoxylates, fatty acid alkoxylates, and any combination thereof.

13. The aforementioned sampled solution (a) Anticoagulants in amounts of 5-25 mg / mL, 7.5-20 mg / mL, 10-15 mg / mL, or 13 mg / mL; and / or (b) A coagulant in amounts of 5–25 mg / mL, 7.5–20 mg / mL, 9–11 mg / mL, or 10 mg / mL; and / or (c) The method according to claim 1 or 2, comprising a wetting agent in an amount of 0.050 to 0.250 mg / mL, 0.075 to 0.120 mg / mL, 0.900 to 0.125 mg / mL, or 0.1 mg / mL.

14. The collected solution comprises a first anticoagulant in a concentration of 2-10 mg / mL, 4-8 mg / mL, 5-7 mg / mL, or 6 mg / mL, and a second anticoagulant in a concentration of 3-11 mg / mL, 5-9 mg / mL, 6-8 mg / mL, or 7 mg / mL, wherein the first and second anticoagulants are different, and / or The method according to claim 1 or 2, wherein the collected solution contains or consists of dipotassium ethylenediaminetetraacetate dehydrated product, potassium oxalate, polyvinylpyrrolidone, Pluronic® P-123, and water, and optionally the water is distilled water.

15. The aforementioned sampled solution (a) Dipotassium ethylenediaminetetraacetate dehydrating agent in concentrations of 3-11 mg / mL, 5-9 mg / mL, 6-8 mg / mL, or 7 mg / mL; (b) potassium oxalate in concentrations of 2-10 mg / mL, 4-8 mg / mL, 5-7 mg / mL, or 6 mg / mL; (c) Polyvinylpyrrolidone in amounts of 5-25 mg / mL, 7.5-20 mg / mL, 9-11 mg / mL, or 10 mg / mL; and (d) comprising Pluronic® P-123 in a concentration of 0.050–0.250 mg / mL, 0.075–0.120 mg / mL, 0.900–0.125 mg / mL, or 0.1 mg / mL, The method according to claim 1 or 2, wherein the remainder is water, and optionally the water is distilled water.

16. The method according to claim 1 or 2, wherein the collected solution comprises 7 mg / mL of dipotassium ethylenediaminetetraacetate salt dehydrating agent, 6 mg / mL of potassium oxalate, 10 mg / mL of polyvinylpyrrolidone, and 0.1 mg / mL of Pluronic® P-123, with the remainder being water, the water being distilled water.

17. The method according to claim 1 or 2, wherein the imaging solution comprises an imaging agent.

18. The imaging solution further comprises water (preferably distilled water) or an alcohol solvent, and / or The method according to claim 17, wherein the imaging agent is one or more of acridine orange, quaternary cationic heteroplasty dyes (such as Greifswalder's blue, blue borrel, rhodanile blue, toluylene blue, night blue, Hofmann's violet, basic orange 21, etc.), permanent dyes (such as cell cyanine permanent dyes, SYTO dyes, oxane dyes, phenanthridine (intercalation) dyes, indole dyes, imidazole dyes, etc.), and any combination thereof.

19. The method according to claim 18, wherein the concentration of the imaging agent is 0.01 to 0.030 mg / mL, 0.015 to 0.025 mg / mL, 0.019 to 0.021 mg / mL, or 0.020 mg / mL.

20. The method according to claim 1 or 2, wherein the imaging solution comprises or consists of acridine orange and water, and optionally the water is distilled water.

21. The method according to claim 1 or 2, wherein the imaging solution contains 0.01 to 0.030 mg / mL, 0.015 to 0.025 mg / mL, 0.019 to 0.021 mg / mL, or 0.020 mg / mL of acridine orange, with the remainder being water, and optionally the water being distilled water.

22. The method according to claim 1 or 2, wherein the imaging solution contains 0.020 mg / mL of acridine orange, the remainder being water, and optionally the water being distilled water.

23. The method according to claim 1 or 2, wherein the pH of the imaging solution is 3 to 6, 3.5 to 4.5, or 4.

24. The cuvette comprises a sampling chamber and an analysis chamber, the sampling chamber is (completely or partially) coated with the sampling solution, and step (b) of claim 1 is performed in the sampling chamber, and / or The method according to claim 1 or 2, wherein the cuvette comprises a sampling chamber and an analysis chamber, the analysis chamber is coated (whole or partially) with the imaging solution, and step (c) of claim 1 is performed inside the analysis chamber.

25. The method according to claim 1 or 2, wherein steps (e) and (f) of claim 1 are performed while the sample is in the cuvette.

Images