Method for obtaining image of biological sample in cuvette

Abstract

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

Description

Technical Field

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

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

[0003] Common samples examined in analytical devices are biological samples, such as blood samples. Blood analysis is used in a wide range of medical monitoring methods, such as monitoring the progress of systemic anti-cancer therapy. For example, an optical analysis device can analyze a blood sample to determine the presence, absence, and / or amount of white blood cells (e.g., granulocytes and agranulocytes), which are often used to monitor the progress of systemic anti-cancer therapy. Analysis of blood samples usually requires centrifugation of the sample first. During the centrifugation process, the blood is separated into layers. The layers are: (a) an erythrocyte layer containing red blood cells; (b) a buffy coat containing white blood cells (e.g., granulocytes and agranulocytes); (c) a platelet (thrombocyte) layer; and (d) a plasma layer. The blood is then analyzed optically (or other radiation) to obtain an image of the layer. A typical optical radiation source is an LED light. The pixel count of each layer can then be calculated from the image to determine the cell count of each layer.

[0004] The cuvette is a disposable component 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, can be easily loaded with samples and fixed on the support of the optical analysis device, and can be easily removed from the support once the analysis is completed. Examples of known types of cuvettes can be seen in, for example, GB2555403A, EP2096444A1, EP1055112A1, WO2007 / 008137, US6365104B1 and WO2018078324A1 (the disclosures of which are each incorporated herein by reference).

[0005] The cuvette containing the sample therein may be coated with a composition. The composition has three purposes. The composition (a) assists the cuvette in collecting the sample; (b) ensures that once the sample is collected, the sample remains processable; and (c) assists in producing a clear image of the sample. Traditionally, the composition coating the cuvette comprises potassium oxalate, acridine orange, sodium heparin, and ethylenediaminetetraacetic acid dipotassium salt dehydrate. Examples of known compositions for cuvettes can be seen, for example, in US6365104B1 (the disclosure of which is incorporated herein by reference).

[0006] There is a need for an improved method to obtain an image of a sample in a cuvette. In particular, there is a need for an improved method to obtain an image of a blood sample in a cuvette, wherein the image clearly shows the buffy coat. Summary of the invention

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

[0008] Representative features of the invention are set out in the following clauses, which stand alone or may be combined in any combination with one or more features disclosed in the text of this specification and / or the drawings.

[0009] The present invention is as set forth in the following clauses:

[0010] 1. A method for obtaining an image of a sample in a cuvette, the method comprising the following steps:

[0011] (a) placing the sample in the cuvette;

[0012] (b) mixing the sample with a collection solution;

[0013] (c) introducing the mixture from step (b) into an imaging solution;

[0014] (d) subjecting the mixture from step (c) to centrifugal force;

[0015] (e) exposing the centrifuged mixture from step (d) to light radiation; and

[0016] (f) Obtaining an optical image of the mixture.

[0017] 2. The process according to clause 1, wherein during step (d) the mixture is heated at Entia Liberty TM The device is subjected to centrifugal force.

[0018] 3. A method according to clause 1 or clause 2, wherein the mixture is subjected to a centrifugal force of 1000 to 2000 rpm, or 1500 rpm.

[0019] 4. A method according to any one of clauses 1 to 3, wherein the mixture is subjected to centrifugal force for at most 30 minutes, or at most 25 minutes, or at most 20 minutes, or at most 15 minutes, or at most 10 minutes.

[0020] 5. A method according to any one of clauses 1 to 4, wherein the mixture is subjected to centrifugal force at room temperature.

[0021] 6. A method according to any one of clauses 1 to 5, wherein the mixture is exposed to light radiation from an LED light source; optionally, the LED light source operates at 400 to 700 nm, or 425 to 600 nm, or 450 to 500 nm, or 460 nm.

[0022] 7. A method according to any one of clauses 1 to 6, wherein the mixture is exposed to light radiation for at most 15 minutes, or at most 10 minutes, or at most 5 minutes.

[0023] 8. A method according to any one of clauses 1 to 7, wherein the mixture is exposed to light radiation at room temperature or at a temperature of 15 to 40°C, or 16 to 30°C, or 18 to 25°C, or 20 to 22°C, or 20°C.

[0024] 9. A method according to any one of clauses 1 to 8, wherein steps (c) and (d) occur simultaneously using centrifugal force, which forces the mixture from step (b) to mix with the imaging solution of step (c).

[0025] 10. The method according to any one of clauses 1 to 9, wherein the optical image of step (f) is obtained by a camera.

[0026] 11. A method according to any one of clauses 1 to 10, wherein the sample is a biological sample; optionally wherein the biological sample is a blood sample.

[0027] 12. The method according to any one of clauses 1 to 11, wherein the collection solution comprises:

[0028] Anticoagulants;

[0029] agglutinating agents; and

[0030] Wetting agent.

[0031] 13. The method according to any one of clauses 1 to 12, wherein the collection solution comprises:

[0032] 5 to 25 mg / mL of anticoagulant;

[0033] 5 to 25 mg / mL of agglutinating agent; and

[0034] 0.05 to 0.25 mg / mL of wetting agent.

[0035] 14. The method according to any one of clauses 1 to 13, wherein the collection solution further comprises water (optionally wherein the water is distilled water) or an alcohol solvent.

[0036] 15. The method according to any one of clauses 1 to 14, wherein the collection solution comprises a first anticoagulant and a second anticoagulant, wherein the first anticoagulant and the second anticoagulant are different.

[0037] 16. The method according to any one of clauses 12 to 15, wherein the anticoagulant is one or more of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, or any combination thereof.

[0038] 17. The method according to any one of clauses 12 to 16, wherein the agglutinating agent is one or more of polyvinyl pyrrolidone, a proteolytic enzyme such as bromelain, pepsin and / or trypsin, polyethylene glycol, or any combination thereof.

[0039] 18. The method according to any one of clauses 12 to 17, wherein the wetting agent is one or more of the well-known surfactants (i.e., ionic, nonionic and / or cationic), Pluronic P-123, Silwet L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid alkoxylates, or any combination thereof.

[0040] 19. The method according to any one of clauses 1 to 18, wherein the collection solution comprises:

[0041] (a) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 10 to 15 mg / mL, or 13 mg / mL of an anticoagulant; and / or

[0042] (b) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 9 to 11 mg / mL, or 10 mg / mL of an agglutinating agent; and / or

[0043] (c) 0.050 to 0.250 mg / mL, or 0.075 to 0.120 mg / mL, or 0.900 to 0.125 mg / mL, or 0.1 mg / mL of a wetting agent.

[0044] 20. A method according to any one of clauses 1 to 19, wherein the collection solution comprises 2 to 10 mg / mL, or 4 to 8 mg / mL, or 5 to 7 mg / mL, or 6 mg / mL of a first anticoagulant and 3 to 11 mg / mL, or 5 to 9 mg / mL, or 6 to 8 mg / mL, or 7 mg / mL of a second anticoagulant; wherein the first anticoagulant and the second anticoagulant are different.

[0045] 21. A method according to any one of clauses 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.

[0046] 22. The method according to any one of clauses 1 to 21, wherein the collection solution comprises:

[0047] (a) 3 to 11 mg / mL, or 5 to 9 mg / mL, or 6 to 8 mg / mL, or 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate reagent;

[0048] (b) 2 to 10 mg / mL, or 4 to 8 mg / mL, or 5 to 7 mg / mL, or 6 mg / mL of potassium oxalate;

[0049] (c) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 9 to 11 mg / mL, or 10 mg / mL of polyvinylpyrrolidone; and

[0050] (d) 0.050 to 0.250 mg / mL, or 0.075 to 0.120 mg / mL, or 0.900 to 0.125 mg / mL, or 0.1 mg / mL of Pluronic P-123;

[0051] The remainder is water; optionally wherein the water is distilled water.

[0052] 23. A method according to any one of clauses 1 to 22, wherein the collection solution comprises 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate reagent, 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; optionally wherein the water is distilled water.

[0053] 24. A method according to any one of clauses 1 to 23, wherein the imaging solution comprises:

[0054] Imaging agent.

[0055] 25. The method according to clause 24, wherein the imaging solution further comprises water (optionally wherein the water is distilled water) or an alcohol solvent.

[0056] 26. A method according to clause 24 or clause 25, wherein the imaging agent is one or more of acridine orange, quaternary ammonium cationic metachromatic dyes such as Greifswalder blue, bromocyanine blue, rhodanine blue, toluidine blue, night blue, Hofmann violet, basic orange 21, permanent dyes such as cell-persistent cyanine dyes, SYTO dyes, oxayne dyes, phenanthridine (intercalating) dyes, indole dyes, imidazole dyes, or any combination thereof.

[0057] 27. A method according to any one of clauses 24 to 25, wherein the imaging agent has a concentration of 0.01 to 0.030 mg / mL, or 0.015 to 0.025 mg / mL, or 0.019 to 0.021 mg / mL, or 0.020 mg / mL.

[0058] 28. A method according to any one of clauses 1 to 27, wherein the imaging solution comprises, or consists of, acridine orange and water; optionally wherein the water is distilled water.

[0059] 29. A method according to any one of clauses 1 to 28, wherein the imaging solution comprises 0.01 to 0.030 mg / mL, or 0.015 to 0.025 mg / mL, or 0.019 to 0.021 mg / mL, or 0.020 mg / mL of acridine orange; the remainder being water; optionally wherein the water is distilled water.

[0060] 30. A method according to any one of clauses 1 to 29, wherein the imaging solution comprises 0.020 mg / mL of acridine orange; the remainder being water; optionally wherein the water is distilled water.

[0061] 31. A method according to any one of clauses 1 to 30, wherein the imaging solution has a pH of 3 to 6, or 3.5 to 4.5, or 4.

[0062] 32. A method according to any one of clauses 1 to 31, wherein the cuvette comprises a collection compartment and an analysis compartment, and wherein the collection compartment is coated (completely or partially) in the collection solution, and step (b) of clause 1 is performed in the collection compartment.

[0063] 33. A method according to any one of clauses 1 to 32, wherein the cuvette comprises a collection compartment and an analysis compartment, and wherein the analysis compartment is coated (completely or partially) in the imaging solution, and step (c) of clause 1 is performed in the analysis compartment.

[0064] 34. A method according to any one of clauses 1 to 33, wherein steps (e) and (f) of clause 1 are performed while the sample is still in the cuvette. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Embodiments of the present invention are described below with reference to the accompanying drawings. The accompanying drawings illustrate various embodiments of the systems and methods of the present disclosure and embodiments of various other aspects of the present disclosure. It will be understood by those of ordinary skill in the art that the element boundaries (e.g., boxes, box groups, or other shapes) shown in the figures represent an example of the boundaries. In some instances, an element may be designed as multiple elements, or multiple elements may be designed as one element. In some instances, an element shown as an internal component of an element may be implemented as an external component in another element, and vice versa. In addition, the elements may not be drawn to scale. A non-restrictive and non-exhaustive description is described with reference to the following drawings. The components in the drawings are not necessarily drawn to scale, but emphasize the principles of explanation.

[0066] Figure 1 Acceptable and unacceptable coatings of the collection solution on the collection chamber portion of the cuvette are depicted.

[0067] Figure 2 Depicted is the GRIPTIP spacing used in a multichannel pipette.

[0068] Figure 3 Advantageous placement of the imaging solution on the analysis chamber portion of the cuvette is shown.

[0069] Figure 4 Shown are images obtained when analyzing the effect of agglutinating agent concentration on the quality of the resulting blood sample images.

[0070] Figure 5 The images obtained of a blood sample in a cuvette coated in a composition of the present invention and a blood sample in a cuvette coated with a different composition (not according to the present invention) are shown.

[0071] Figure 6 Shown are images obtained when the entire length of the middle wall of the analytical chamber portion of the cuvette was coated in imaging solution. DETAILED DESCRIPTION

[0072] Embodiments of the present disclosure will be described more fully below with reference to the accompanying drawings, wherein like numerals represent like elements in the several drawings, and wherein exemplary embodiments are shown. However, the embodiments of the claims may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0073] The words "comprising," "having," "containing," and "including," and other forms thereof, are intended to be equivalent in meaning and to be open ended in that one or more items following any of these words is not intended to be an exhaustive list of such items nor to be limited to the items listed. It must also be noted that references without a specific number as used herein and in the appended claims include plural referents unless the context clearly dictates 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, preferred systems and methods are now described.

[0074] Some terms used to describe the present invention are as follows:

[0075] "Acridine orange" refers to an imaging dye. Acridine orange causes cells to fluoresce when exposed to light radiation. Typically, acridine orange is excited at wavelengths between 200 and 460 nm. The mechanism by which acridine orange can distinguish between cells is known as metachromatism. With metachromatism, acridine orange binds to different components in a sample, producing peaks at different wavelengths in the spectrum upon excitation. For example, when acridine orange binds to DNA, the cell will emit green fluorescence with a maximum emission of 530 nm upon excitation with blue light. When acridine orange binds to RNA, the resulting combination condenses (i.e., turns to a solid state) and then precipitates. The resulting components will phosphoresce, or emit red light with a maximum emission of 640 nm, upon excitation with blue light. The concentration of acridine orange can also affect the color emission of the sample, as the concentration affects the form of available genetic material. It is well known that acridine orange is less sensitive in selecting RNA than DNA. For example, high concentrations of acridine orange cause DNA denaturation while also staining the DNA red. This is because high concentrations of acridine orange cause DNA to become single-stranded. Therefore, high concentrations of acridine will result in a lot of red light emission. In contrast, low concentrations of acridine orange result in incomplete denaturation of RNA and cause part of the RNA to be stained green. Therefore, low concentrations of acridine orange will result in a lot of green light emission. In addition, the colors produced using acridine orange in combination with cells depend on the structure of the cells and can help distinguish different cells. For example, granulocytes are stained orange in the granules and red in the nucleus, causing granulocytes to emit significantly in the red spectrum, while agranulocytes are stained yellow-green in the nucleus and red in the cytoplasm, causing agranulocytes to emit in the green spectrum.

[0076] "Agglutinating agent" refers to a material that helps particles to aggregate together. For example, agglutinating agents can help cells such as bacteria or red blood cells to aggregate. Specifically, agglutinating agents can aggregate red blood cells together by directly acting on the red blood cells, such as but not limited to by reducing the repulsive forces between the red blood cells. Examples of agglutinating agents include, but are not limited to, agglutinin proteins, or hemagglutinin proteins, or polyvinyl pyrrolidone, a solution containing polyvinyl pyrrolidone K12, a protease such as bromelain, pepsin and / or trypsin, or polyethylene glycol.

[0077] "Agranulocyte" refers to a leukocyte without distinct granules. Examples of agranulocytes include, but are not limited to, lymphocytes and monocytes.

[0078] "Alcohol solvent" refers to an alcohol-based solvent. Alcohol solvents include, but are not limited to, benzyl alcohol, 1,4-butanediol, 1,2,4-butanetriol, butanol, 1-butanol, 2-butanol, tert-butanol, and ethanol.

[0079] "Anticoagulant" refers to a material that prevents blood from clotting. Anticoagulants ensure that a blood sample does not change significantly before the sample 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.

[0080] "Buffy coat" refers to a fraction of a blood sample that is anticoagulated. The buffy coat contains most of the white blood cells and platelets in a centrifuged blood sample.

[0081] "Collecting solution" refers to a solution comprising an anticoagulant, a coagulant, and a wetting agent. The collecting solution may additionally comprise water. The purpose of the collecting solution is generally to increase the ease and efficiency with which the sample is taken up by an analytical device (e.g., a cuvette).

[0082] "Cuvette" refers to a container used in an analytical device, particularly a container for analyzing a sample under centrifugation. Examples of cuvettes are described in GB2555403A and British application GB2108004.9 (the disclosures of which are incorporated herein by reference).

[0083] "Entia Liberty TM "Device" refers to a device manufactured by Entia Ltd. for measuring the concentration of a component in a sample using optical radiation. Specifically, the device is used to measure the presence of leukocytes (by measuring the amount of leukocytes) in a blood sample obtained from a patient undergoing systemic anti-cancer therapy progress monitoring.

[0084] "Fluid" refers to any material that has no fixed shape and readily yields to external pressure. Examples of fluids include, but are not limited to, blood samples.

[0085] "Granulocyte" refers to a type of white blood cell that has small granules. These granules contain protein. Examples of granulocytes are neutrophils, eosinophils, and basophils.

[0086] "Homogeneous" refers to a substance having uniform composition and properties throughout.

[0087] "Imaging solution" refers to a solution comprising an imaging agent. Examples of imaging agents include, but are not limited to, acridine orange, quaternary ammonium cationic metachromatic dyes such as Greifswalder blue, ballast blue, rhodanine blue, toluidine blue, night blue, Hoffman violet, basic orange 21, permanent dyes such as cell-persistent cyanine dyes, SYTO dyes, oxayne dyes, phenanthridine (intercalating) dyes, indole dyes or imidazole dyes. The imaging solution may also comprise water. The purpose of the imaging solution is to ensure that an image of the sample to be analyzed can be obtained by optical (or other source) radiation.

[0088] "Multichannel pipette" refers to an electronic device used in laboratories to accurately measure liquids and fill multiple vials with liquids at once. Examples of multichannel pipettes include, but are not limited to, Eppendorf Research plus multichannel pipettes, CappAero multichannel pipettes, and VIAFLO multichannel pipettes. Preferably, a multichannel pipette having a distance between pipette tips of 4 mm or less and capable of dispensing at least 0.5 µL of liquid can be used.

[0089] A mixture is a material made by mixing two or more different components together. A mixture can be homogeneous or heterogeneous throughout.

[0090] "Room temperature" refers to a temperature of 18 to 25°C, preferably 20 to 22°C, preferably 20°C.

[0091] "Sample" refers to the material to be analyzed. The sample is analyzed in the fluid phase.

[0092] "Wetting agent" refers to a material that improves the diffusion and penetration properties of a liquid by reducing the surface tension of the liquid. Examples of wetting agents include, but are not limited to, well-known surfactants (i.e., ionic, nonionic and / or cationic), Pluronic P-123 (also known as polyethylene glycol-block polypropylene glycol-block polyethylene glycol), Silwet L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, or fatty acid alkoxylates.

[0093] Composition

[0094] The present invention provides a composition. Specifically, the composition is used to coat the whole or a part of a cuvette. The composition is improved compared to the existing composition for coating a cuvette. The composition of the present invention has at least the following advantages: improving the ease and efficiency of the cuvette to take a sample; improving the imaging of the sample held in the cuvette; and improving the imaging of the sample held in the cuvette by allowing the different layers of the buffy coat of the blood sample to be visualized.

[0095] The composition includes a collection solution and an imaging solution.

[0096] The collecting solution comprises an anticoagulant, a coagulant and a wetting agent. The collecting solution may also comprise water or any suitable organic or inorganic solvent such as an alcohol solvent.

[0097] The anticoagulant includes one or more of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, or any combination thereof.

[0098] The agglutinating agent comprises one or more of polyvinyl pyrrolidone, a solution containing polyvinyl pyrrolidone K12, a proteolytic enzyme such as bromelain, pepsin and / or trypsin, polyethylene glycol, or any combination thereof. Advantageously, the inclusion of an agglutinating agent in the collection solution reduces the relaxation of the blood after centrifugation of the blood sample in a cuvette containing the collection solution. This advantageously prolongs the time period that can be used to image the sample. More advantageously, the agglutinating agent can act as a plasma expander for the blood sample, increasing the plasma volume and sedimentation rate while reducing the hematocrit.

[0099] The wetting agent includes one or more of Pluronic P-123, Silwet L600, fatty alcohol ethoxylate, alkylphenol ethoxylate, fatty acid alkylate, or any combination thereof.

[0100] Preferably, the pH of the collection solution is acidic. Preferably, the pH of the collection solution is 3 to 6, or 3.5 to 4.5, or 4 (plus or minus 0.2).

[0101] The imaging solution comprises an imaging agent.The imaging solution may also comprise water or any suitable organic or inorganic solvent such as an alcohol solvent.

[0102] The imaging agent includes one or more of acridine orange, quaternary ammonium salt type cationic metachromatic dyes such as Greifswalder blue, Baorou blue, rhodanine blue, toluidine blue, night blue, Hoffman purple, basic orange 21, permanent dyes such as cell-persistent cyanine dyes, SYTO dyes, oxayne dyes, phenanthridine (intercalation) dyes, indole dyes, imidazole dyes, or any combination thereof. In an advantageous case, the dye is positively charged and is therefore able to pass through the cell membrane of blood cells. Therefore, the dye can chemically dye blood cells. Visual dyeing of blood cells can visualize blood cells. Further advantageously, the dye can be used in a large pH range and temperature range, and the dyeing properties of the dye are not affected by concentration changes. The dye is fixed in place (because the dye is dried on the surface of the cuvette) without the need to fix the cells in place. On the contrary, cells can flow freely. Freely flowing cells are ideal for imaging because when cells are fixed, cells tend to lose important information such as biochemical information.

[0103] Preferably, the pH of the imaging solution is acidic. Preferably, the pH of the imaging solution is 3 to 6, or 3.5 to 4.5, or 4 (plus or minus 0.2).

[0104] Cuvette

[0105] In some examples, the present invention also relates to a cuvette fully or partially coated in the composition.

[0106] In some instances, the cuvette is a cuvette described in UK patent application GB2108004.9 filed on June 4, 2021 (the disclosure of which is incorporated herein by reference).

[0107] In some examples, the cuvette is formed of two parts (hereinafter referred to as cuvette parts), which are manufactured separately and attached to each other. One part may include the upper surface of the cuvette and the other part includes its lower surface. One or both parts may be formed with a depression on its inner surface so that when the two parts are fixed together, one or more compartments are present in the body of the cuvette. Preferably, the body of the cuvette has a sample compartment formed therein, and the sample compartment is connected to the outside of the cuvette through an opening formed outside the body, wherein the sample compartment includes a collection compartment and an analysis compartment. Figure 3A cuvette portion (300) is shown having a collection chamber (340) and an analysis chamber (350). The collection chamber has a first end in fluid communication with the 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. It should be understood, however, that the present invention is not limited to such a method of manufacturing the cuvette, and the cuvette may be formed in any other suitable or convenient manner.

[0108] In some examples, depressions are formed on the inner surface of one portion of the cuvette, while the inner surface of the other portion is substantially smooth and / or flat. In other examples, both portions may have depressions formed on their inner surfaces, and when the cuvette is assembled, at least some of the depressions are aligned with each other to form the compartments. As discussed, various compartments may be created by depressions formed on the inner side of one or both portions of the cuvette. The skilled reader will appreciate that there are many other possibilities.

[0109] In some examples, the collection chamber and analysis chamber have the same or approximately the same depth.

[0110] In some examples, the collection chamber has four opposing side walls parallel to each other. The collection chamber extends from a first end in fluid communication with an opening formed on the outside of the body and a second end in fluid communication with the analysis chamber. In some examples, the analysis chamber preferably has a uniform width along its length, with four opposing side walls parallel to each other. Preferably, when the cuvette is made of two cuvette parts, one of which has a depression to form the chamber, and the second cuvette part is substantially smooth or flat, the depression on one cuvette part forms three of the four opposing side walls of the collection and analysis chamber, and the substantially smooth or flat surface of the second cuvette part forms the fourth opposing wall. The analysis chamber preferably has a constant depth along its length. The analysis chamber extends toward an end of the cuvette opposite to the collection chamber and terminates at a closed end, which does not allow any communication with the outside of the cuvette. The closed end of the analysis chamber preferably has a square shape, including an end face at right angles or substantially right angles to the length of the analysis chamber. The closed end may be referred to as an end wall.

[0111] In some examples, the cuvette includes a vent that allows air to escape from the cuvette.

[0112] In some examples, the cuvette includes a transition chamber between the collection chamber and the analysis chamber. The function of the transition chamber is to prevent overfilling of the cuvette while avoiding cell damage and / or activation.

[0113] In some examples, the collection compartment is coated (completely or partially) with a collection solution of the invention. In other examples, the analysis compartment is coated (completely or partially) with the imaging solution. In other examples, the collection compartment is coated (completely or partially) with the collection solution and the analysis compartment is coated (completely or partially) with the imaging solution.

[0114] Method for preparing the composition

[0115] The present invention also relates to a method for preparing a composition for coating a cuvette (completely or partially). The method comprises the following steps:

[0116] (a) providing a collection solution; and

[0117] (b) Providing imaging solution.

[0118] The present invention also provides a method for preparing the collection solution. The method comprises the following steps:

[0119] (a) providing anticoagulants, agglutinants and wetting agents;

[0120] (b) preparing stock solutions of the anticoagulant, agglutinating agent and wetting agent; and

[0121] (c) combining the stock solutions.

[0122] During step (b), the stock solutions are prepared separately.

[0123] In some examples, the anticoagulant, agglutinant and wetting agent are weighed into separate containers. An example of a preferred separate container is a disposable tray. Each container is cleaned with a cleaning agent such as isopropyl alcohol before use.

[0124] In some instances, the anticoagulant, agglutinant and wetting agent are transferred to separate vials, respectively, and a solvent is added to the vial. Preferably, the solvent used is water. Preferably, any suitable organic or inorganic solvent such as an alcohol solvent is used. Preferably, the anticoagulant, agglutinant and wetting agent are dissolved in the solvent by stirring the solution at 700 rpm to 900 rpm, or 800 rpm. Preferably, the anticoagulant, agglutinant and wetting agent are dissolved in the solvent by stirring the solution for 5 to 15 minutes, or 10 minutes. Preferably, the anticoagulant, agglutinant and wetting agent are dissolved in the solvent at a temperature of 10 to 30° C., or 15 to 25° C.

[0125] In some instances, the collection solution comprises two anticoagulants: a first anticoagulant and a second anticoagulant. Preferably, the first anticoagulant and the second anticoagulant are different. Preferably, the storage solution comprising the first anticoagulant is prepared to have a concentration of 25 to 75 mg / mL, or 35 to 65 mg / mL, or 45 to 55 mg / mL, or 50 mg / mL of the first anticoagulant. Preferably, the storage solution comprising the second anticoagulant is prepared to have a concentration of 50 to 150 mg / mL, or 75 to 125 mg / mL, or 90 to 110 mg / mL, or 100 mg / mL of the second anticoagulant.

[0126] In some examples, the stock solution comprising the agglutinating agent is prepared to have a concentration of 25 to 75 mg / mL, or 35 to 65 mg / mL, or 45 to 55 mg / mL, or 50 mg / mL of the agglutinating agent.

[0127] In some examples, a stock solution comprising the wetting agent is prepared to have a concentration of 0.005 mg / mL to 0.015 mg / mL, or 0.0075 mg / mL to 0.0125 mg / mL, or 0.009 to 0.011 mg / mL, or 0.01 mg / mL of the wetting agent.

[0128] In some examples, a reservoir solution comprising a first anticoagulant is prepared to have a concentration of 50 mg / mL of the first anticoagulant; a reservoir solution comprising a second anticoagulant is prepared to have a concentration of 100 mg / mL of the second anticoagulant; a reservoir solution comprising an agglutinating agent is prepared to have a concentration of 50 mg / mL of the agglutinating agent; and a reservoir solution comprising the wetting agent is prepared to have a concentration of 0.01 mg / mL of the wetting agent. Preferably, the stock solution comprising potassium oxalate is prepared to have a potassium oxalate concentration of 50 mg / mL; the stock solution comprising ethylenediaminetetraacetic acid dipotassium salt dehydrate is prepared to have a ethylenediaminetetraacetic acid dipotassium salt dehydrate concentration of 100 mg / mL; the stock solution comprising polyvinylpyrrolidone is prepared to have a polyvinylpyrrolidone concentration of 50 mg / mL; and the stock solution comprising Pluronic P-123 is prepared to have a Pluronic P-123 concentration of 0.01 mg / mL.

[0129] In some examples, the stock solution can be stored for up to 4 weeks from the day the stock solution is prepared.

[0130] In step (c), the reservoir solutions are combined to form the collection solution.

[0131] In some examples, the collection solution comprises two anticoagulants: a first anticoagulant and a second anticoagulant. Preferably, the first anticoagulant and the second anticoagulant are different. Preferably, the collection solution comprises 2 to 10 mg / mL, or 4 to 8 mg / mL, or 5 to 7 mg / mL, or 6 mg / mL of the first anticoagulant. Preferably, the collection solution comprises 3 to 11 mg / mL, or 5 to 9 mg / mL, or 6 to 8 mg / mL, or 7 mg / mL of the second anticoagulant.

[0132] In some examples, the collection solution comprises 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 9 to 11 mg / mL, or 10 mg / mL of the agglutinating agent.

[0133] In some examples, the collection solution comprises 0.050 to 0.250 mg / mL, or 0.075 to 0.120 mg / mL, or 0.900 to 0.125 mg / mL, or 0.1 mg / mL of the wetting agent.

[0134] 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 an agglutinating 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.

[0135] In some examples, the collection solution has a seven-day shelf life. Once seven days have passed, a fresh collection solution is prepared.

[0136] The present invention also provides a method for preparing the imaging solution. The method comprises the following steps:

[0137] (a) providing an imaging agent; and

[0138] (b) Preparing a stock solution of the imaging agent.

[0139] In step (b), the storage solution is prepared. In some examples, the imaging agent is a solid and is weighed into a container. An example of a preferred container is a disposable tray. The container is cleaned with a cleaning agent such as isopropyl alcohol before use. In another example, the imaging agent is a liquid and the required amount of the imaging agent is measured in a measuring cylinder. The measuring cylinder is cleaned with a cleaning agent such as isopropyl alcohol before use.

[0140] In some instances, 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 such as an alcohol solvent is used. 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.

[0141] In some examples, a stock solution comprising the imaging agent is prepared to have a concentration of 0.01 to 0.030 mg / mL, or 0.015 to 0.025 mg / mL, or 0.019 to 0.021 mg / mL, or 0.020 mg / mL of the imaging agent.

[0142] In some examples, the stock solution comprising the imaging agent is prepared to have a concentration of 0.020 mg / mL of the imaging agent. Preferably, the stock solution comprising acridine orange is prepared to have a concentration of 0.020 mg / mL of acridine orange.

[0143] In some examples, the imaging solution has a shelf life of up to one month. Once one month has passed, fresh imaging solution must be prepared. Preferably, the imaging solution is stored away from light. Preferably, the imaging solution is stored at a temperature of 10° C. or less, or 5° C. or less, or 4° C. or less.

[0144] In some examples, the collection solution and imaging solution are combined to form one solution.

[0145] Method for coating a cuvette in a composition

[0146] The present invention also includes a method for coating a cuvette in the composition. The method comprises the following steps:

[0147] (a) providing a cuvette comprising a collection compartment and an analysis compartment;

[0148] (b) providing a collection solution;

[0149] (c) providing an imaging solution;

[0150] (d) coating (completely or partially) the collection compartment of the cuvette with the collection solution;

[0151] (e) coating (completely or partially) the analysis compartment of the cuvette with the imaging solution;

[0152] (f) drying the coating of said collection solution on said collection compartment; and

[0153] (g) Drying the coating of the imaging solution on the analysis compartment.

[0154] Before coating the cuvette with the collection solution or imaging solution, check the shelf life of each solution.

[0155] In some examples, before the collection solution is coated on the collection compartment of the cuvette, the collection solution is thoroughly mixed. Preferably, the collection solution is mixed to homogeneity. The collection solution is then dispensed onto the collection compartment using a pipette. Preferably, 10 to 20 μL, or 12.5 to 17.5 μL, or 15 μL of the collection solution is dispensed onto the collection compartment. Preferably, the collection solution dispensed onto the collection compartment of the cuvette is taken from the middle of the collection solution.

[0156] In some examples, the imaging solution is mixed thoroughly before coating the analysis compartment of the cuvette with the imaging solution. Preferably, the imaging solution is mixed to homogeneity. The imaging solution is then dispensed onto the analysis compartment with a pipette, preferably 10 to 20 μL, or 12.5 to 17.5 μL, or 15 μL of the imaging solution is dispensed onto the analysis compartment. Preferably, the imaging solution dispensed onto the analysis compartment of the cuvette is taken from the middle of the imaging solution.

[0157] In some examples, the imaging solution is dispensed onto the analysis chamber using a multichannel pipette. Preferably, the multichannel pipette is adjusted so that 1 to 60% of the surface area, or 10 to 50% of the surface area, or 20 to 40% of the surface area, or 30 to 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 so that 40 to 99% of the surface area, or 50 to 90% of the surface area, or 80 to 40% of the surface area, or 70 to 60% of the surface area, or 65% of the surface area of ​​the analysis chamber is not coated in the imaging solution. Preferably, the multichannel pipette is adjusted so that the imaging solution is placed at the first end of the analysis chamber and the second closed end of the analysis chamber, while a portion of the analysis chamber that exists between the first end of the analysis chamber and the second closed end of the analysis chamber is not dispensed with the imaging solution. Preferably, the proportion of imaging solution present at the first end of the analysis chamber is greater than the proportion of imaging solution present at the second closed end of the analysis chamber. Preferably, the ratio of imaging solution present at the first end of the analysis chamber to 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, wherein the imaging solution is present on the side wall at the first (open) end of the analysis chamber, on the side wall at the second (closed) end of the analysis chamber, and not present at the intermediate region of the side wall between the first (open) end and the second (closed) end of the analysis chamber.

[0158] In an optional example, the imaging solution is dispensed onto the analysis chamber using a single pipette. Preferably, the single pipette is adjusted so that 1 to 60% of the surface area, or 10 to 50% of the surface area, or 20 to 40% of the surface area, or 30 to 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 so that 40 to 99% of the surface area, or 50 to 90% of the surface area, or 80 to 40% of the surface area, or 70 to 60% of the surface area, or 65% of the surface area of ​​the analysis chamber is not coated in the imaging solution. Preferably, the single pipette is adjusted so that the imaging solution is placed at the first end of the analysis chamber and the second closed end of the analysis chamber, while a portion of the analysis chamber that is between the first end of the analysis chamber and the second closed end of the analysis chamber is not dispensed with the imaging solution. Preferably, the proportion of imaging solution present at the first end of the analysis chamber is greater than the proportion of imaging solution present at the second closed end of the analysis chamber. Preferably, the ratio of imaging solution present at the first end of the analysis chamber to 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, wherein the imaging solution is present on the side wall at the first (open) end of the analysis chamber, on the side wall at the second (closed) end of the analysis chamber, and not present at the intermediate region of the side wall between the first (open) end and the second (closed) end of the analysis chamber.

[0159] In some examples, after dispensing the collection solution onto the collection compartment, the collection solution and collection compartment are dried. Preferably, the collection solution and collection compartment are dried by exposing them to heat. Preferably, the collection solution and collection compartment are exposed to heat at a temperature of 40 to 80° C., or 50 to 70° C., or 60° C. Preferably, the collection solution and collection compartment are exposed to heat for a period of 1 to 20 minutes, or 5 to 15 minutes, or 10 minutes.

[0160] In some examples, after dispensing the imaging solution onto the analysis compartment, the imaging solution and analysis compartment are dried. Preferably, the drying is performed by exposing the imaging solution and analysis compartment to heat. Preferably, the imaging solution and analysis compartment are exposed to heat at a temperature of 40 to 80° C., or 50 to 70° C., or 60° C. Preferably, the imaging solution and analysis compartment are exposed to heat for a period of 1 to 20 minutes, or 5 to 15 minutes, or 10 minutes.

[0161] In some examples, the collection solution and imaging solution are dried simultaneously on the collection chamber and analysis chamber. Preferably, the drying is performed by exposing the collection solution, imaging solution, collection chamber and analysis chamber to heat. Preferably, the collection solution, imaging solution, collection chamber and analysis chamber are exposed to heat at a temperature of 40 to 80° C., or 50 to 70° C., or 60° C. Preferably, the collection solution, imaging solution, collection chamber and analysis chamber are exposed to heat for a period of 1 to 20 minutes, or 5 to 15 minutes, or 10 minutes.

[0162] In some examples, the coated cuvette can be stored in a dust-free environment. Preferably, the storage temperature is 10 to 30° C., or 15 to 25° C. Preferably, the storage relative humidity is 20% to 70% relative humidity, or 30% to 60% relative humidity.

[0163] In some examples, the cuvette is composed of two parts that are fixed together to form the cuvette. By providing the cuvette as two parts, the collection chamber and analysis chamber can be easily accessed during the coating process of the collection chamber and analysis chamber. Advantageously, this allows a homogenous coating of the imaging solution to be formed in the analysis chamber.

[0164] Methods of using cuvettes coated with compositions

[0165] The cuvette coated with the composition can be used for optical analysis of samples such as blood samples. In order to perform optical analysis on the sample, the following steps will be followed:

[0166] (a) Place the sample in a cuvette;

[0167] (b) mixing the sample with a collection solution;

[0168] (c) introducing the mixture from step 1 into an imaging solution;

[0169] (d) applying centrifugal force to the mixture from step 2;

[0170] (e) exposing the mixture to light radiation; and

[0171] (f) Obtaining an optical image of the mixture.

[0172] In some examples, a portion of the sample is removed from the middle of the sample prior to step (b).

[0173] In some examples, during step (b), the collection solution is present on the collection chamber of the cuvette, and step (b) is performed in the collection chamber of the cuvette. In some examples, during step (c), the imaging solution is present on the analysis chamber of the cuvette, and step (c) is performed in the analysis chamber of the cuvette.

[0174] In some examples, during step (d), the mixture is TM Preferably, the mixture is subjected to centrifugal force at 1000 to 2000 rpm, or 1500 rpm. Preferably, the mixture is subjected to centrifugal force at room temperature. Preferably, the mixture is subjected to centrifugal force at 15 to 40°C, or 16 to 30°C, or 18 to 25°C, or 20 to 22°C, or 20°C. Preferably, the mixture is subjected to centrifugal force for up to 30 minutes, or up to 25 minutes, or up to 20 minutes, or up to 15 minutes, or up to 10 minutes. Preferably, the mixture is subjected to centrifugal force at room temperature in an Entia Liberty centrifuge providing a centrifugal force of 1500 rpm. TM The device is subjected to centrifugal force for up to 20 minutes.

[0175] In some examples, during step (e), the mixture is exposed to light radiation having a wavelength of 460 nm from an LED light source. Preferably, the mixture is exposed to light radiation at room temperature. Preferably, the mixture is exposed to light radiation for at most 15 minutes, or at most 10 minutes, or at most 5 minutes. Preferably, the mixture is exposed to light radiation having a wavelength of 460 nm from an LED light source at room temperature for at most 5 minutes.

[0176] In some examples, steps (e) and (f) are performed while the sample is in a cuvette.

[0177] In some examples, the optical image is analyzed to convert pixel counts of the image obtained in step (f) into cell counts.

[0178] Example

[0179] The following is a non-limiting embodiment that discusses at least some advantages of the present invention with reference to tables and figures.The embodiments set forth herein are non-limiting examples, being merely examples among other possible examples.

[0180] Example 1: Preparation of composition

[0181] In the following non-limiting examples, a composition comprising a collecting solution and an imaging solution was prepared. The collecting solution and the imaging solution were prepared separately.

[0182] In this non-limiting example, a collection solution comprising polyvinylpyrrolidone, potassium oxalate, ethylenediaminetetraacetic acid dipotassium salt dehydrate, Pluronic P-123 and water was prepared. The collection solution was prepared from stock solutions of each component.

[0183] In this non-limiting example, an imaging solution comprising acridine orange and water is prepared. The imaging solution is prepared from a stock solution of acridine orange.

[0184] Stock solutions of each component used in the collection and imaging solutions were prepared separately. To prepare the stock solutions, each component of the collection and imaging solutions was weighed separately in a separate disposable tray and then mixed with the corresponding volume of water in a separate clean empty vial. Once each component of the collection and imaging solutions was added to the water, the concentration of each stock solution was verified by weighing the tray to ensure that no residue was left (the maximum amount of residue left was 0.005 g).

[0185] By stirring thoroughly, any powdered reagents are completely dissolved in the water.

[0186] The component Pluronic P-123 is initially a gel mass. To dissolve Pluronic P-123 in water, stir Pluronic P-123 at 800 rpm for 10 minutes at room temperature of 15 to 25°C and relative humidity of 30 to 60% RH. The dilution increases with increasing temperature, however, the temperature is carefully monitored to ensure that it does not exceed 35°C.

[0187] Table 1 sets forth the concentrations of each stock solution prepared.

[0188] Table 1: Concentration (conc.) of each stock solution used to prepare collection solution and imaging solution.

[0189]

[0190] The collection solution was prepared by combining stock solutions of polyvinylpyrrolidone, potassium oxalate, ethylenediaminetetraacetic acid dipotassium salt dehydrate, Pluronic P-123 and water to obtain the concentrations set forth in Table 2. After combining the stock solutions, the solutions were mixed so that the resulting collection solution was homogeneous throughout.

[0191] The imaging solution was prepared by diluting a stock solution of acridine orange with water to the desired concentration. After the stock solution was diluted, the solutions were mixed so that the resulting imaging solution was homogeneous throughout.

[0192] The concentration of each component in the collection solution and imaging solution is listed in Table 2.

[0193] Table 2: Concentration (conc.) of each component used in the collection solution and imaging solution. Each component originated from a stock solution.

[0194]

[0195] After formation, the collection solution and imaging solution were kept separate (ie, not mixed) until use.

[0196] Example 2: Coating the collection compartment of a cuvette with a collection solution

[0197] In the following non-limiting example, the collection compartment of the cuvette is coated with the collection solution.The cuvette is formed of two parts that are fixed together to form the cuvette.

[0198] In this non-limiting example, the collection compartment of the cuvette portion is coated in the collection solution as follows:

[0199] (a) stirring the collection solution;

[0200] (b) using a pipette to draw 15 µL of the collection solution from the middle of the collection solution;

[0201] (c) dispensing 15 µL of the collection solution from step (b) onto the surface of the collection compartment of the cuvette portion, the surface being Figure 1 within the acceptable limits depicted in and described below; and

[0202] (d) The collection solution and the collection compartment of the cuvette are then dried.

[0203] Figure 1 The collection chamber (160) of the cuvette portion with a collection solution coating (150) is shown. Figure 1 Two images (110, 120) are shown with acceptable coatings of the collection solution. Figure 1 As shown in , the acceptable limits for dispensing the collection solution onto the collection chamber are the entire collection chamber being coated (110) or the entire length of the collection chamber being coated (120). By placing the collection solution within acceptable limits, easy collection of the sample is ensured. Figure 1 Also shown are two images (130, 140) with unacceptable collection solution coatings. Figure 1As shown in , an unacceptable limit is that the collection solution does not cover the first end and the second end (130) of the collection chamber. The collection solution needs to be present in an area that can help collect the sample and help maintain the integrity of the sample. When the collection solution does not cover the first end and / or the second end of the collection chamber, the collection solution cannot perform this function. The second unacceptable limit is that the collection solution extends beyond the second end (140) of the collection chamber. When the collection solution extends beyond the second end of the collection chamber, the collection solution blocks the vent in the cuvette, thereby preventing the sample from entering the analysis chamber: the sample will only enter the collection chamber.

[0204] To dry the collection solution and collection chamber, the collection solution and collection chamber were exposed to heat at a temperature of 60° C. for 10 minutes in a universal oven. During the drying step, the cuvettes were not stacked on top of other cuvettes and were protected from dust particles.

[0205] After the collection solution is coated on the collection compartment of the cuvette portion, the imaging solution can be coated on the analysis compartment of the cuvette portion. Alternatively, before the collection solution is coated on the collection compartment of the cuvette portion, the imaging solution can be coated on the analysis compartment of the cuvette portion. After the collection and analysis compartments of the cuvette portion have been coated with the collection and imaging solutions, respectively, the two cuvette portions are fixed together to form a complete cuvette.

[0206] Example 3: Coating the analytical compartment of a cuvette with an imaging solution

[0207] In the following non-limiting embodiment, the analysis compartment of the cuvette is coated with the imaging solution. The cuvette is formed of two parts, which are fixed together to form the cuvette. One part has a depression on its inner surface, forming three of the four opposing side walls of the analysis compartment; only the middle wall of these three side walls is partially coated with the imaging solution. The other two walls and the other cuvette part are not coated. The other cuvette part is substantially smooth or flat. The depression on the inner surface of the one cuvette part is such that when the two parts are fixed together, the collection chamber and the analysis compartment are present within the body of the cuvette. By providing the cuvette in two parts, the analysis compartment can be easily accessed during the coating process.

[0208] In this non-limiting example, the analytical compartment of the cuvette is coated in the imaging solution following the following method:

[0209] (a) stirring the imaging solution;

[0210] (b) Open the multichannel pipette VIAFLO multichannel pipette;

[0211] (c) placing the portion of the cuvette to be coated in the coating fixture of the multichannel pipette (the area of ​​the multichannel pipette designed to hold the surface to be coated);

[0212] (d) inserting GRIPTIP pipette tips into the multichannel pipette in the following order (with (4) closest to the first end of the analysis chamber and (1) closest to the second closed end of the analysis chamber), a total of 8 pipette tips; Figure 2 The following sequence of depictions of GRIPTIP is provided:

[0213] (1) 2 pipette tips (210);

[0214] (2) 3 vacancies (220);

[0215] (3) 6 pipette tips (230);

[0216] (4) 5 vacancies (240);

[0217] (e) immersing the GRIPTIPS in the imaging solution and collecting 3 µL of the imaging solution through the GRIPTIPS;

[0218] (f) visually inspecting the GRIPTIPS to ensure that the volume of imaging solution collected is uniform for all GRIPTIPS;

[0219] (g) aligning and lowering the GRIPTIPS over the coating fixture so as to lightly touch the surface of the middle wall of the three side walls of the analysis chamber of the cuvette portion;

[0220] (h) Each GRIPTIP dispenses a first droplet containing 0.94 µL of imaging solution onto the surface;

[0221] (i) realigning the GRIPTIPS by moving the GRIPTIPS 2 mm in the direction of the second closed end of the analysis chamber;

[0222] (j) each GRIPTIP dispenses a second droplet containing 0.94 µL of imaging solution onto the surface; and

[0223] (k) The imaging solution and the analytical compartment of the cuvette portion are then dried.

[0224] After the pipette has dispensed the second drop (step (j)), remove the cuvette portion and GRIPTIPS from the coating fixture. Place the GRIPTIPS over an absorbent paper towel to remove any remaining sample from the pipette.

[0225] Figure 3 An image of a cuvette portion (300) having a collection chamber (340) and an analysis chamber (350) is provided. Figure 3 In FIG. 1 , the area of ​​the analysis chamber ( 350 ) coated by the imaging solution is highlighted. Figure 3 As shown in FIG. 1 , the portions of the analysis chamber (350) coated with the imaging solution are labeled 310 and 330, and the portion of the analysis chamber not coated with the imaging solution is labeled 320. The lengths of the two portions of the analysis chamber coated with the imaging solution are 22 mm (310) and 10 mm (330). Using 15 μL of imaging solution, the total area of ​​the analysis chamber coated with the imaging solution is 35% of the surface area.

[0226] To dry the imaging solution and the analysis chamber, the imaging solution and the analysis chamber were exposed to heat at a temperature of 60° C. for 10 minutes in a universal oven. During the drying step, the cuvettes were not stacked on top of other cuvettes and were protected from 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 centrifugation time. 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 does not enter the collection chamber because the imaging solution is dried on the surface of the analysis chamber.

[0227] After the imaging solution is coated on the analysis compartment of the cuvette portion, the collection solution can be coated on the collection compartment of the cuvette portion. Alternatively, before the imaging solution is coated on the analysis compartment of the cuvette portion, the collection solution can be coated on the collection compartment of the cuvette portion. After the collection and analysis compartments of the cuvette portion have been coated with the collection and imaging solutions, respectively, the two cuvette portions are fixed together to form a complete cuvette.

[0228] Example 4: Coating the analytical compartment of a cuvette with an imaging solution

[0229] In the following non-limiting embodiment, the analysis compartment of the cuvette is coated with the imaging solution. The cuvette is formed of two parts, which are fixed together to form the cuvette. One part has a depression on its inner surface, forming three of the four opposing side walls of the analysis compartment; only the middle wall of these three side walls is partially coated with the imaging solution. The other two walls and the other cuvette part are not coated. The other cuvette part is substantially smooth or flat. The depression on the inner surface of the one cuvette part is such that when the two parts are fixed together, the collection chamber and the analysis compartment are present within the body of the cuvette. By providing the cuvette in two parts, the analysis compartment can be easily accessed during the coating process.

[0230] In this non-limiting example, the analytical compartment of the cuvette is coated in the imaging solution following the following method:

[0231] (a) stirring the imaging solution;

[0232] (b) providing a single pipette;

[0233] (c) immersing the single pipette in the imaging solution and collecting 15 µL of the imaging solution through the single pipette;

[0234] (d) placing the single pipette on the surface of a middle wall of the three side walls of the analysis chamber of the cuvette portion and dispensing 15 μL of the imaging solution in a droplet across the surface so as to achieve coating of the analysis chamber, wherein the total area of ​​the analysis chamber coated with the imaging solution is 35% of the surface area, and wherein the coating is on one of the four side walls forming the analysis chamber, and wherein the coating of the imaging solution is present only at the end of the side wall at the first (open) end of the analysis chamber and at the end of the side wall at the second (closed) end of the analysis chamber, and is not present in the middle area between the first (open) end and the second (closed) end of the analysis chamber;

[0235] (f) The imaging solution and the analytical compartment of the cuvette portion are then dried.

[0236] To dry the imaging solution and the analytical chamber, the imaging solution and the analytical chamber were exposed to heat at a temperature of 60°C for 10 minutes in a universal oven. During the drying step, the cuvettes were not stacked on top of other cuvettes and were protected from dust particles. Advantageously, coating the imaging solution on the analytical chamber ensured that the imaging solution was in contact with the sample during the entire centrifugation time: coating the imaging solution on the analytical chamber reduced the variability of the absorption of the imaging solution by the sample. The imaging solution was present in the analytical chamber and did not enter the collection chamber because it was dried on the surface of the analytical chamber.

[0237] After the imaging solution is coated on the analysis compartment of the cuvette portion, the collection solution can be coated on the collection compartment of the cuvette portion. Alternatively, before the imaging solution is coated on the analysis compartment of the cuvette portion, the collection solution can be coated on the collection compartment of the cuvette portion. After the collection and analysis compartments of the cuvette portion have been coated with the collection and imaging solutions, respectively, the two cuvette portions are fixed together to form a complete cuvette.

[0238] Example 5: Effect of the concentration of agglutinating agent in the analysis solution

[0239] In the following non-limiting examples, the effect of the presence of an agglutinating agent in the solution on the image obtained was analyzed. The agglutinating agent analyzed in this example was polyvinyl pyrrolidone.

[0240] In this non-limiting example, a solution comprising polyvinyl pyrrolidone, acridine orange and water is prepared. The solution is prepared from a stock solution of polyvinyl pyrrolidone and acridine orange. The stock solution is prepared as described in Example 1.

[0241] Three different solutions with three different concentrations of polyvinyl pyrrolidone were prepared. The three different concentrations of polyvinyl pyrrolidone were 10.00, 20.00 and 30.00 mg / mL. The solution with a polyvinyl pyrrolidone concentration of 10.00 mg / mL was labeled solution 1, the solution with a polyvinyl pyrrolidone concentration of 20.00 mg / mL was labeled solution 2, and the collected solution with a polyvinyl pyrrolidone concentration of 30.00 mg / mL was labeled solution 3. The solutions were then prepared by combining a stock solution of polyvinyl pyrrolidone, acridine orange and water to achieve the concentrations set forth in Table 3. After combining the stock solutions, the solutions were mixed so that the resulting solutions were homogeneous overall.

[0242] Table 3: Concentration of each component in the solution

[0243]

[0244] Glass capillaries (Microslide 0.3×0.5 mm glass capillaries) were coated in the solutions. Different solutions were used with different glass capillaries. The glass capillaries were coated by the following method:

[0245] (a) stirring the solution;

[0246] (b) using a pipette, draw out 50 µL of the solution from the middle of the solution;

[0247] (c) dispensing 50 µL of the solution from step (b) onto the glass capillary; and

[0248] (d) Drying the solution and the glass capillary.

[0249] To dry the solution on the glass capillary, the solution and the glass capillary were exposed to heat at a temperature of 60° C. for 10 minutes in a general purpose oven.

[0250] After the glass capillary has been coated in the solution, the venous blood sample is inserted into the glass capillary coated with solution 1. The glass capillary is placed in a laboratory centrifuge with the blood sample and run at 3000G for 5 minutes at room temperature. During the centrifugation, the blood sample is separated into platelets, agranulocytes, granulocytes and red blood cell layers. A Nikon D5100 camera is used to capture images of the separated blood samples. The images are generated using an LED lamp running at a wavelength of 460 nm at room temperature. The same procedure is performed on each glass capillary coated with solution 2 or solution 3. The experiment is then repeated for 16 samples.

[0251] The analysis results 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.

[0252] Example 6: Effect of Anticoagulant Concentration in Analytical Solution

[0253] In the following non-limiting example, the effect of an anticoagulant in solution on the image obtained was analyzed. The anticoagulant analyzed in this example was ethylenediaminetetraacetic acid dipotassium salt.

[0254] In this non-limiting example, a solution comprising ethylenediaminetetraacetic acid dipotassium salt dehydrate, polyvinylpyrrolidone, acridine orange and water was prepared. The solution was prepared from a stock solution of each component. The stock solutions were prepared as described in Example 1. A stock solution containing polyvinylpyrrolidone was prepared such that polyvinylpyrrolidone was present at a concentration of 3.0 mg / mL, and a stock solution containing acridine orange was prepared such that acridine orange was present at a concentration of 0.02 mg / mL.

[0255] Two different solutions with two different concentrations of ethylenediaminetetraacetic acid dipotassium salt dehydrate were analyzed. The two different concentrations of the ethylenediaminetetraacetic acid dipotassium salt dehydrate were 7.00 and 19.00 mg / mL. The solution with a concentration of 7.00 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate was labeled as solution 4, and the solution with a concentration of 19.00 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate was labeled as solution 5. The solutions were then prepared by combining stock solutions of ethylenediaminetetraacetic acid dipotassium salt dehydrate, polyvinylpyrrolidone, acridine orange, and water to achieve the concentrations set forth in Table 4. After combining the stock solutions, the solutions were mixed so that the resulting solutions were homogeneous throughout.

[0256] Table 4: Concentration of each component in the solution

[0257]

[0258] Glass capillaries (Microslide 0.3×0.5 mm glass capillaries) were coated in the solutions. Different solutions were used with different glass capillaries. The glass capillaries were coated by the following method:

[0259] (a) stirring the solution;

[0260] (b) using a pipette, draw out 50 µL of the solution from the middle of the solution;

[0261] (c) dispensing 50 µL of the solution from step (b) onto the glass capillary; and

[0262] (d) Drying the solution and the glass capillary.

[0263] To dry the solution on the glass capillary, the solution and the glass capillary were exposed to heat at a temperature of 60° C. for 10 minutes in a general purpose oven.

[0264] After the glass capillary has been coated in the solution, the venous blood sample is inserted into the glass capillary coated with solution 4. The glass capillary is placed in a laboratory centrifuge with the blood sample and run at 3000G for 5 minutes at room temperature. During the centrifugation, the blood sample is separated into platelets, agranulocytes, granulocytes and red blood cell layers. A Nikon D5100 camera is used to take images of the separated blood samples. The images are generated using an LED lamp running at a wavelength of 460 nm at room temperature. The same procedure is performed on each glass capillary coated with solution 5. The experiment is then repeated for 12 samples.

[0265] The results showed that solutions containing high concentrations of EDTA dipotassium salt dehydrate damaged cells in the analyzed samples.

[0266] Example 7: Comparison of a composition according to the invention with a different composition (not according to the invention)

[0267] In this non-limiting example, a solution of a composition similar to that of the present invention coated on a cuvette was compared to a cuvette coated with a different composition (not according to the present invention).

[0268] The solution not according to the invention is the solution described in US6365104B1. The solution described in US6365104B1 contains sodium heparin, ethylenediaminetetraacetic acid dipotassium salt dehydrate, acridine orange and potassium oxalate. Table 5 illustrates the mass and corresponding concentration of each component in the solution from US6365104B1.

[0269] Table 5: Mass and corresponding concentration of each component present in the solution of US6365104B1

[0270]

[0271] A solution replicating the solution in US6365104B1 was then prepared using the following concentrations: 6.000 mg / mL potassium oxalate, 76.0 USP / mL heparin sodium, 7.0 mg / mL ethylenediaminetetraacetic acid dipotassium salt dehydrate, and 10 mg / mL polyvinylpyrrolidone, and 0.110 mg / mL acridine orange. Polyvinylpyrrolidone was included in the composition replicating the composition in US6365104B1 because polyvinylpyrrolidone is present in the composition of the present invention.

[0272] A solution similar to the composition of the present invention was then prepared with the following concentrations present in the 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.

[0273] The solution was prepared from the stock solution described in Example 1. The solution was then prepared by combining the stock solutions of each component to achieve the above concentrations. After combining the stock solutions, the solutions were mixed so that the resulting collection solution was homogeneous throughout.

[0274] Then, glass capillaries (Microslide 0.3×0.5 mm glass capillaries) were coated in the solution. Different solutions were used with different glass capillaries. The glass capillaries were coated by the following method:

[0275] (a) stirring the solution;

[0276] (b) using a pipette, draw out 50 µL of the solution from the middle of the solution;

[0277] (c) dispensing 50 µL of the solution from step (b) onto the glass capillary; and

[0278] (d) Drying the solution and the glass capillary.

[0279] To dry the solution on the glass capillary, the solution and the glass capillary were exposed to heat at a temperature of 60° C. for 10 minutes in a general purpose oven.

[0280] After the glass capillary has been coated in the solution, the venous blood sample is inserted into the glass capillary coated with the solution from US6365104B1. The glass capillary is placed in a laboratory centrifuge with the blood sample and run at 3000G for 5 minutes at room temperature. During the centrifugation, the blood sample is separated into platelets, agranulocytes, granulocytes and red blood cell layers. A Nikon D5100 camera is used to take images of the separated blood samples. The images are generated using an LED lamp running at a wavelength of 460 nm at room temperature. The same procedure is performed on glass capillaries coated with a solution similar to the composition of the present invention.

[0281] The results of the analysis are as follows Figure 5 The solution from US6365104B1 is marked as 510, and the solution similar to the composition of the present invention is marked as 520. Figure 5 As shown in , solutions similar to the compositions of the present invention improve the clarity of the images obtained of blood samples.

[0282] Example 8: Analysis The imaging solution is placed at the first end and the second closed end of the analysis chamber (not in the middle The impact of

[0283] In this non-limiting example, the effect of placing the imaging solution towards or at the first (open) end and towards or at the second (closed) end of the analysis chamber (rather than in the middle) was analyzed.

[0284] In this non-limiting example, the collection and imaging solutions were prepared as described in Example 1, and the cuvette was coated as described in Examples 2 and 3. The cuvette was formed from two parts that were secured together to form the cuvette.

[0285] Another cuvette is coated with the collection and imaging solution. The cuvette is formed of two parts that are fixed together to form the cuvette. One part has a depression on its inner surface, which forms three of the four opposing side walls of the collection and analysis chamber. The other part is substantially smooth or flat and forms the fourth opposing side wall of the collection and analysis chamber. The collection chamber is coated with the collection solution as described in Example 2. Then, the analysis chamber of the cuvette is coated with the imaging solution. The imaging solution coating on the cuvette portion having the depression is not limited 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 portion, but not at the middle portion thereof (as described in Example 3). By not limiting the position of the imaging solution coating on the cuvette portion, a portion of the imaging solution may be trapped between the cuvette portions when the two portions of the cuvette are fixed together. Thus, when a blood sample enters the analysis chamber, the captured portion of the imaging solution cannot enter the blood sample but remains captured between the two parts of the cuvette.

[0286] The venous blood sample was then inserted into each assembled cuvette and each cuvette was placed in the Entia Liberty with the blood sample. TM The device was run at 1500 rpm for 20 minutes at room temperature for each cuvette and blood sample. The samples were separated into platelet, agranulocyte, granulocyte and erythrocyte layers. TM The blood sample in the device was exposed to an LED light source operating at a wavelength of 460 nm for five minutes at room temperature, and an image of the sample was acquired.

[0287] Figure 6 The images (610, 620 and 630) obtained for a cuvette showing that the coating of the imaging solution is limited 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 portion, but not at the middle portion thereof. As can be seen in Figure 6 As seen in the figure, there are light beams in the resulting image.

[0288] Advantageously, by placing the imaging solution toward or at a first (open) end of a middle wall of the three side walls of the analysis chamber of the cuvette portion and toward or at a second (closed) end (rather than a middle portion), light beams are removed from the desired image of the buffy coat. The portion of the analysis chamber not coated with the imaging solution corresponds to the location of the buffy coat of the separated blood sample. The process of coating the cuvette is a manual process that does not ensure that there is no coating on the side walls, so the location of the imaging solution on the middle wall of the three side walls is limited. Light beams (in 610, 620, and 630) correspond to imaging solution captured by the adhesive during the fixing process used to fix the two cuvette portions together. Light beams in the obtained sample image can be removed or reduced by (a) placing the imaging solution only on the middle wall of the three side walls of the analysis chamber of the cuvette portion having the recess, and (b) not placing the imaging solution in the middle portion of the middle wall of the analysis chamber.

[0289] It is further advantageous to coat the imaging solution on the analysis compartment.Thus, during the entire centrifugation and imaging process, the imaging solution is in contact with the biological sample, thereby allowing the working mechanism of the imaging agent to work efficiently.

[0290] It is a further advantage that by not coating the collection chamber with the imaging solution, no residue of the imaging solution is left in the collection chamber, thereby reducing the variability observed between images obtained.

[0291] The features disclosed in the above description or in the following claims or in the accompanying drawings, expressed in their specific form or in terms of means for performing the disclosed function or method or process for achieving the disclosed result, may be used alone or in any combination of these features to realize various forms of the invention, as the case may be.

[0292] Although certain exemplary aspects of the invention have been described, the scope of the appended claims is not intended to be limited to these examples. The claims should be interpreted literally, intentionally, and / or encompass equivalents.

Claims

1. A method for obtaining an image of a sample in a cuvette, the method comprising the following steps: (a) placing the sample in the cuvette; (b) mixing the sample with a collection solution; (c) introducing the mixture from step (b) into an imaging solution; (d) subjecting the mixture from step (c) to centrifugal force; (e) exposing the centrifuged mixture from step (d) to light radiation; and (f) Obtaining an optical image of the mixture.

2. The method of claim 1, wherein during step (d) the mixture is heated at Entia Liberty TM The device is subjected to centrifugal force.

3. A method according to claim 1 or claim 2, wherein the mixture is subjected to a centrifugal force of 1000 to 2000 rpm, or 1500 rpm.

4. The method of any one of claims 1 to 3, wherein the mixture is subjected to centrifugal force for at most 30 minutes, or at most 25 minutes, or at most 20 minutes, or at most 15 minutes, or at most 10 minutes.

5. The method according to any one of claims 1 to 4, wherein the mixture is subjected to centrifugal force at room temperature; and / or wherein the mixture is exposed to light radiation from an LED light source; optionally, the LED light source operates at 400 to 700 nm, or 425 to 600 nm, or 450 to 500 nm, or 460 nm.

6. The method according to any one of claims 1 to 5, wherein the mixture is exposed to light radiation for at most 15 minutes, or at most 10 minutes, or at most 5 minutes; and / or wherein the mixture is exposed to light radiation at room temperature or at a temperature of 15 to 40°C, or 16 to 30°C, or 18 to 25°C, or 20 to 22°C, or 20°C.

7. A method according to any one of claims 1 to 6, wherein steps (c) and (d) occur simultaneously using centrifugal force which forces the mixture from step (b) to mix with the imaging solution of step (c); and / or The optical image in step (f) is obtained by a camera.

8. The method according to any one of claims 1 to 7, wherein the sample is a biological sample; optionally wherein the biological sample is a blood sample.

9. The method according to any one of claims 1 to 8, wherein the collection solution comprises: Anticoagulants; agglutinating agents; and Wetting agents; and / or The collecting solution comprises: 5 to 25 mg / mL of anticoagulant; 5 to 25 mg / mL of agglutinating agent; and 0.05 to 0.25 mg / mL of wetting agent.

10. The method according to any one of claims 1 to 9, wherein the collection solution further comprises water (optionally wherein the water is distilled water) or an alcohol solvent.

11. The method according to any one of claims 1 to 10, wherein the collection solution comprises a first anticoagulant and a second anticoagulant, wherein the first anticoagulant and the second anticoagulant are different; and / or The anticoagulant is one or more of ethylenediaminetetraacetic acid dipotassium salt dehydrate, potassium oxalate, potassium ammonium oxalate, heparin, citrate, hirudin, or any combination thereof.

12. The method according to any one of claims 9 to 11, wherein the agglutinating agent is one or more of polyvinylpyrrolidone, a proteolytic enzyme such as bromelain, pepsin and / or trypsin, polyethylene glycol, or any combination thereof; and / or The wetting agent is one or more of known surfactants (i.e., ionic, nonionic and / or cationic), Pluronic P-123, Silwet L600, fatty alcohol ethoxylates, alkylphenol ethoxylates, fatty acid alkoxylates, or any combination thereof.

13. The method according to any one of claims 1 to 12, wherein the collection solution comprises: (a) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 10 to 15 mg / mL, or 13 mg / mL of an anticoagulant; and / or (b) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 9 to 11 mg / mL, or 10 mg / mL of an agglutinating agent; and / or (c) 0.050 to 0.250 mg / mL, or 0.075 to 0.120 mg / mL, or 0.900 to 0.125 mg / mL, or 0.1 mg / mL of a wetting agent.

14. The method of any one of claims 1 to 13, wherein the collection solution comprises 2 to 10 mg / mL, or 4 to 8 mg / mL, or 5 to 7 mg / mL, or 6 mg / mL of a first anticoagulant and 3 to 11 mg / mL, or 5 to 9 mg / mL, or 6 to 8 mg / mL, or 7 mg / mL of a second anticoagulant; wherein the first anticoagulant and the second anticoagulant are different; and / or The collecting 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.

15. The method according to any one of claims 1 to 14, wherein the collection solution comprises: (a) 3 to 11 mg / mL, or 5 to 9 mg / mL, or 6 to 8 mg / mL, or 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate reagent; (b) 2 to 10 mg / mL, or 4 to 8 mg / mL, or 5 to 7 mg / mL, or 6 mg / mL of potassium oxalate; (c) 5 to 25 mg / mL, or 7.5 to 20 mg / mL, or 9 to 11 mg / mL, or 10 mg / mL of polyvinylpyrrolidone; and (d) 0.050 to 0.250 mg / mL, or 0.075 to 0.120 mg / mL, or 0.900 to 0.125 mg / mL, or 0.1 mg / mL of Pluronic P-123; The remainder is water; optionally wherein the water is distilled water.

16. The method according to any one of claims 1 to 15, wherein the collection solution comprises 7 mg / mL of ethylenediaminetetraacetic acid dipotassium salt dehydrate reagent, 6 mg / mL of potassium oxalate, 10 mg / mL of polyvinylpyrrolidone and 0.1 mg / mL of Pluronic P-123, and the remainder is water; optionally wherein the water is distilled water.

17. The method of any one of claims 1 to 16, wherein the imaging solution comprises: Imaging agent.

18. The method of claim 17, wherein the imaging solution further comprises water (optionally wherein the water is distilled water) or an alcohol solvent; and / or The imaging agent is one or more of acridine orange, quaternary ammonium cationic metachromatic dyes such as Greifswalder blue, bromocyanine blue, rhodanine blue, toluidine blue, night blue, Hofmann violet, basic orange 21, permanent dyes such as cell-persistent cyanine dyes, SYTO dyes, oxayne dyes, phenanthridine (intercalating) dyes, indole dyes, imidazole dyes, or any combination thereof.

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

20. A method according to any one of claims 1 to 19, wherein the imaging solution comprises, or consists of, acridine orange and water; optionally wherein the water is distilled water.

21. The method of any one of claims 1 to 20, wherein the imaging solution comprises 0.01 to 0.030 mg / mL, or 0.015 to 0.025 mg / mL, or 0.019 to 0.021 mg / mL, or 0.020 mg / mL of acridine orange; the remainder being water; optionally wherein the water is distilled water.

22. The method of any one of claims 1 to 22, wherein the imaging solution comprises 0.020 mg / mL of acridine orange; the remainder being water; optionally wherein the water is distilled water.

23. The method of any one of claims 1 to 23, wherein the imaging solution has a pH of 3 to 6, or 3.5 to 4.5, or 4.

24. The method according to any one of claims 1 to 24, wherein the cuvette comprises a collection compartment and an analysis compartment, and wherein the collection compartment is coated (completely or partially) in the collection solution, and step (b) of claim 1 is performed in the collection compartment; and / or wherein the cuvette comprises a collection compartment and an analysis compartment, and wherein the analysis compartment is coated (completely or partially) in the imaging solution, and step (c) of claim 1 is performed in the analysis compartment.

25. The method of any one of claims 1 to 24, wherein steps (e) and (f) of claim 1 are performed while the sample is still within the cuvette.

Images