A staining reagent combination, method of preparation and application thereof

Abstract

The present disclosure relates to a staining reagent combination comprising a reagent A and a reagent B. The staining reagent combination is capable of simultaneously assessing total count of WBC, differential count of WBC and haemoglobin concentration from a biological sample in suspension form. The staining reagent combination is ideal for use in microfluidic platforms due to its high viscosity and lower volume required for measurement. The staining reagent combination is economical, non-toxic, has quick reaction and long working time.

Description

[0001] A STAINING REAGENT COMBINATION, METHOD OF PREPARATION AND APPLICATION THEREOF

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to the field of haematology. The present disclosure particularly relates a staining reagent combination, method of preparation of said staining reagent combination, and application thereof.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] Staining of blood cells using commercially available staining compositions or staining kits require two separate sample preparation steps for analysing WBC and haemoglobin separately. Thus, a large volume of biological samples is required to individually analyse WBC and haemoglobin. Moreover, it may be exhausting and / or painful to the subject while extracting the biological fluid multiple times and in large amounts from the subject for analysis of WBC and haemoglobin. Further preparation of multiple samples by an operator is time-consuming and may involve operator errors.

[0006] The staining solutions or reagents known in the state of the art for analysing blood cells contain alcohol and water. Presence of alcohol in the staining solution of reagent causes coagulation of red blood cells and presence of water is known to induce precipitation of oppositely charged dyes. Water is not suitable as a solvent for charged dye molecules like methylene blue and eosin Y due to its propensity for extensive hydrogen bonding, which can interfere with the electrostatic stabilization of charged species. This can lead to reduced solubility, poor stability, and potential precipitation or aggregation of the dye molecules, compromising the performance of the solution. Thus, staining solutions comprising alcohol and / or water is not suitable for analysing haemoglobin in biological samples.

[0007] Further, the staining solutions or reagents used in traditional blood smear staining is not suitable in microfluidic platforms due to spillage problems and prolonged exposure of polymethyl methacrylate (PMMA) to alcoholic solvents present in the staining solutions or reagents causes swelling, deformation, or even cracking of the commonly used PMMA based microfluidic platforms. Additionally, the staining methods known in the art for identification of white blood cells require preparation of peripheral blood smear, which involves several processing steps, such as fixing and drying the biological sample on to a slide before staining the blood cells. In addition, it also requires a specialized professional to perform these activities.

[0008] Thus, there is a need for staining reagents which mitigates the limitations known in the art and that allow differential staining of white blood cells (WBC) in microfluidic platforms. The present disclosure herein describes a staining reagent combination which addresses above noted issues and provides for simultaneously assessing WBC total count, WBC differential count and concentration of haemoglobin from biological samples.

[0009] SUMMARY OF THE DISCLOSURE

[0010] Accordingly, the present disclosure relates to a staining reagent combination composed of: a. a Reagent A comprising: i. about 0.01 wt.% to 0.9 wt.% of cationic dye selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof; ii. about 0.8 wt.% to 1.8 wt.% of surfactant; and iii. about 70 wt.% to 98.5 wt.% of a first solvent; and iv. about 0.5 wt.% to 30 wt.% of a second solvent, and b. a Reagent B comprising: i. about 0.05 wt.% to 0.3 wt.% of anionic dye selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof; ii. about 0.5 wt.% to 3 wt.% of surfactant; iii. about 70 wt.% to 99 wt.% of first solvent; iv. about 0.05 wt.% to 30 wt.% of second solvent; and v. about 0.3 wt.% to 1.5 wt.% of stabilizing agent.

[0011] The staining reagent combination of the present disclosure is capable of staining and simultaneously assessing total count of white blood cells (WBC), differential count of WBC and haemoglobin concentration from biological samples in suspension form. The staining reagent combination of the present disclosure is ideal for use in microfluidic platforms due to its high viscosity, lower volume requirement for measurement and compatibility of solvents used in the reagents with polymethyl methacrylate (PMMA) based microfluidic platforms. The staining reagent combination is economical, non-toxic, has quick reaction time and long working time, respectively.

[0012] The present disclosure further relates to a method of preparing the staining reagent combination, said method comprises - a) preparing the Reagent A, comprising- mixing the cationic dye, the surfactant, the first solvent and the second solvent to obtain the Reagent A; and b) preparing the Reagent B, comprising- mixing the anionic dye, the surfactant, the first solvent, the second solvent and the stabilizing agent to obtain the Reagent B.

[0013] The present disclosure further relates to a method of staining cells in a biological sample, said method comprises: diluting the biological sample with a buffer; contacting the diluted biological sample with the staining reagent combination, followed by mixing to obtain a mixture; and incubating the mixture to obtain stained cells.

[0014] The present disclosure further relates to a kit comprising:

[0015] - the staining reagent combination; and an instruction manual comprising details of using the staining reagent combination for staining cells in a biological sample.

[0016] The present disclosure further relates to use of the staining reagent combination for staining white blood cells (WBC), measuring total count of WBCs and differential count of WBCs, and identifying haemoglobin content.

[0017] BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

[0018] In order that the present disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where: FIGURE 1 provides a schematic representation of determination of total count of white blood cells (WBC), differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination of the present disclosure, wherein the sample preparation step is carried out by manual mixing using vial.

[0019] FIGURE 2 provides a schematic representation of determination of total count of white blood cells (WBC), differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination, wherein the sample preparation step is carried out by mixing using microfluidic cartridge.

[0020] FIGURE 3 provides a graph depicting measurement of haemoglobin in a blood sample by spectrophotometer using the staining reagent combination of the present disclosure.

[0021] FIGURE 4 provides a graph depicting measurement of total count (TC) of WBC in a blood sample using the staining reagent combination of the present disclosure.

[0022] FIGURE 5 provides images of stained white blood cells (WBC) in a blood sample using the staining reagent combination of the present disclosure.

[0023] FIGURE 6A provides image of stained white blood cells (WBC) using a staining reagent without anionic dyes.

[0024] FIGURE 6B provides image of stained white blood cells (WBC) using staining reagent with cationic dye and anionic dye in a ratio of 1: 1 (outside the ratio described in the present disclosure).

[0025] FIGURE 6C provides image of stained white blood cells (WBC) using staining reagent comprising DNA binding dye, such as Acridine Orange.

[0026] FIGURE 7A provides image of stained white blood cells (WBC) using staining reagent with surfactant in an amount of 2.0 wt.% (the amount is outside the range of surfactant employed in the staining reagent combination of the present disclosure). FIGURE 7B provides image of stained white blood cells (WBC) using staining reagent with surfactant in an amount of 4.0 wt.% (the amount is outside the range of surfactant employed in the staining reagent combination of the present disclosure).

[0027] FIGURE 7C provides image of stained white blood cells (WBC) using staining reagent without surfactant.

[0028] FIGURE 7D provides image of stained white blood cells (WBC) using staining reagent with surfactant in an amount of about 0.2 % (the amount outside the range of surfactant employed in the staining reagent combination of the present disclosure).

[0029] FIGURE 8A provides image of stained white blood cells (WBC) using staining reagent prepared using Isopropyl alcohol as solvent (conventional staining reagent comprising alcohol).

[0030] FIGURE 8B provides image of stained white blood cells (WBC) using staining reagent prepared using water as solvent (conventional staining reagent comprising water).

[0031] FIGURE 9 provides image of stained white blood cells (WBC) using staining reagent without stabilizing agent.

[0032] DETAIEED DESCRIPTION OF THE DISCEOSURE

[0033] As used herein, the term ‘Triton X 100’ refers to a surfactant having chemical names ‘Polyoxyethylene (10) isooctylcyclohexyl ether’ or ‘octyl phenoxypolyethoxylethanol’.

[0034] As used herein, the term ‘Brij L23’ refers to a surfactant having chemical name ‘Polyoxyethylene (23) lauryl ether’.

[0035] The present disclosure relates to staining reagent combinations. The staining reagent combination of the present disclosure is capable of simultaneously assessing total count of white blood cells (WBC), differential count of WBC and haemoglobin concentration from biological samples in a suspension form without any limitations. The staining reagent combination of the present application can be used in microfluidic platforms due to its high viscosity and lower volume required for measurement. The staining reagent combination is economical, non-toxic, has quick reaction and long working time.

[0036] In some embodiments of the present disclosure, the staining reagent combination is composed of: a. a Reagent A comprising: i. about 0.01 wt.% to 0.9 wt.% of cationic dye selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof; ii. about 0.8 wt.% to 1.8 wt.% of surfactant; and iii. about 70 wt.% to 98.5 wt.% of first solvent; and iv. about 0.5 wt.% to 30 wt.% of second solvent, and b. a reagent B comprising: i. about 0.05 wt.% to 0.3 wt.% of anionic dye selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof; ii. about 0.5 wt.% to 3 wt.% of surfactant; iii. about 70 wt.% to 99 wt.% of first solvent; iv. about 0.05 wt.% to 30 wt.% of second solvent; and v. about 0.3 wt.% to 1.5 wt.% of stabilizing agent.

[0037] In some embodiments of the present disclosure, the Reagent A comprises cationic dye in an amount ranging from about 0.01 wt.% to about 0.9 wt.%, including all the values in the range, for instance, about 0.01 wt.%, about 0.02 wt.%, about 0.03 wt.%, about 0.04 wt.%, and so on and so forth, up until about 0.9 wt.%, and including subranges of the range about 0.01 wt.% to about 0.9 wt.%. In an embodiment, the cationic dye is selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof.

[0038] In some embodiments of the present disclosure, the Reagent A comprises methylene blue, Azure B and Toluidine Blue in an amount ranging from about 0.04 wt.% to about 0.9 wt.%. In some embodiments of the present disclosure, the Reagent A comprises methylene blue in an amount ranging from about 0.05 wt.% to about 0.42 wt.%, Azure B blue in an amount ranging from about 0.05 wt.% to about 0.3 wt.% and Toluidine Blue in an amount ranging from about 0.02 wt.% to about 0.15 wt.%.

[0039] In some embodiments of the present disclosure, the Reagent A comprises Crystal Violet in an amount ranging from about 0.01 wt.% to about 0.3 wt.%.

[0040] In some embodiments of the present disclosure, the Reagent A comprises Neutral Red in an amount ranging from about 0.02 wt.% to about 0.1 wt.%.

[0041] In some embodiments of the present disclosure, the Reagent A comprises Nile blue sulfate in an amount ranging from about 0.05 wt.% to about 0. 1 wt.%.

[0042] In some embodiments of the present disclosure, the Reagent A comprises Safranin O in an amount ranging from about 0.02 wt.% to about 0.1 wt.%.

[0043] In some embodiments of the present disclosure, the Reagent A comprises surfactant in an amount ranging from about 0.8 wt.% to about 1.8 wt.%, including all the values in the range, for instance, about 0.9 wt.%, about 0.10 wt.%, about 1.0 wt.%, about 1.1 wt.%, and so on and so forth, up until about 1.8 wt.%, and including subranges of the range about 0.8 wt.% to about 1.8 wt.%. In an embodiment, the surfactant of the Reagent A is selected from a group comprising Triton X 100, Brij L23, Saponin and combinations thereof. In an embodiment, the surfactant in the Reagent A provides for complete lysis of red blood cells (RBC) and thus helps to prevent interference of RBC during WBC staining.

[0044] In some embodiments of the present disclosure, the Reagent A comprises Triton X 100 in an amount ranging from about 0.8 wt.% to about 1.8 wt.%.

[0045] In some embodiments of the present disclosure, the Reagent A comprises first solvent in an amount ranging from about 70 wt.% to about 98.5 wt.% and including all the values in the range, for instance, about 71 wt.%, about 72 wt.%, about 73 wt.%, about 74 wt.%, about 75 wt.%, and so on and so forth, up until about 98.5 wt. %, and including subranges of the range about 70 wt.% to about 98.5 wt.%. In an embodiment, the first solvent of the reagent A is selected from a group comprising glycerol, ethylene glycol, polyethylene glycol and combinations thereof.

[0046] In some embodiments of the present disclosure, the Reagent A comprises glycerol in an amount ranging from about 70 wt.% to about 98.5 wt.%.

[0047] In some embodiments of the present disclosure, the Reagent A comprises second solvent in an amount ranging from about 0.5 wt.% to about 30 wt.%, including all the values in the range, for instance, about 0.6 wt.%, 0.7 wt.%, 0.8 wt.%, 0.9 wt.% and so on and so forth, up until about 30 wt.%, and including subranges of the range about 0.5 wt.% to about 30 wt.%. In an embodiment, the second solvent of the Reagent A includes but not limited to dimethyl sulfoxide (DMSO).

[0048] In some embodiments of the present disclosure, the Reagent A comprises DMSO in an amount ranging from about 0.5 wt.% to about 30 wt.%.

[0049] In some embodiments of the present disclosure, the Reagent B comprises anionic dye in an amount ranging from about 0.05 wt.% to 0.3 wt.%, including all the values in the range, for instance, about 0.06 wt.%, 0.07 wt.%, 0.08 wt.%, 0.09 wt.% and so on and so forth, up until 0.3 wt.%, and including subranges of the range 0.05 wt.% to 0.3 wt.%. In an embodiment, the anionic dye of the Reagent B is selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof.

[0050] In some embodiments of the present disclosure, the Reagent B comprises Eosin Y in an amount ranging from about 0.05 wt.% to 0.3 wt.%.

[0051] In some embodiments of the present disclosure, the Reagent B comprises Eosin Y in an amount ranging from about 0.08 wt.% to 0.2 wt.%.

[0052] In some embodiments of the present disclosure, the Reagent B comprises Acid Fuchsin in an amount ranging from about 0.05 wt.% to about 0.15 wt.%.

[0053] In some embodiments of the present disclosure, the Reagent B comprises Indigo carmine in an amount ranging from about 0.05 wt.% to about 0.2 wt.%. In some embodiments of the present disclosure, the Reagent B comprises surfactant in an amount ranging from about 0.5 wt.% to about 3 wt.%, including all the values in the range, for instance, about 0.6 wt.%, 0.7 wt.%, 0.8 wt.%, 0.9 wt.% and so on and so forth, up until 3 wt.% and including subranges of the range about 0.5 wt.% to about 3 wt.%. In an embodiment, the surfactant of the Reagent B is selected from a group comprising Triton X 100, Brij L23, Saponin and combinations thereof. In an embodiment, the surfactant in the Reagent B provides for complete lysis of red blood cells (RBC) and thus helps to prevent interference of RBC during while blood cell (WBC) staining.

[0054] In some embodiments of the present disclosure, the Reagent B comprises Triton X 100 and Brij L23 in an amount ranging from about 0.5 wt.% to about 3 wt.%, respectively.

[0055] In some embodiments of the present disclosure, the reagent B comprises Triton X 100 in an amount ranging from about 0.8 wt.%to 1.5 wt.% and Brij L23 in an amount ranging from about 0.1 wt.% to 1.2 wt.%.

[0056] In some embodiments of the present disclosure, the Reagent B comprises first solvent in an amount ranging from about 70 wt.% to about 99 wt.% and including subranges of the range about 70 wt.% to about 99 wt.%. In an embodiment, the first solvent is in an amount of about 71 wt.%, about 72 wt.%, about 73 wt.%, about 74 wt.%, about 75 wt.%, about 78 wt.%, about 79 wt.%, about 80 wt.%, about 81 wt.%, about 82 wt.%, about 83 wt.%, about 84 wt.%, about 85 wt.%, about 86 wt.%, about 87 wt.%, about 88 wt.%, about 89 wt.%, about 90 wt.%, about 91 wt.%, about 92 wt.%, about 93 wt.%, about 94 wt.%, about 95 wt.%, about 96 wt.%, about 97 wt.%, about 98 wt.% or about 99 wt.%. In an embodiment, the first solvent of the reagent B is selected from a group comprising glycerol, ethylene glycol, polyethylene glycol and combinations thereof.

[0057] In some embodiments of the present disclosure, the Reagent B comprises glycerol in an amount ranging from about 70 wt.% to about 99 wt.%.

[0058] In some embodiments of the present disclosure, the Reagent B comprises second solvent in an amount ranging from about 0.05 wt.% to about 30 wt.%, including all the values in the range, for instance, about 0.06 wt.%, 0.07 wt.%, 0.08 wt.%, 0.09 wt.% and so on and so forth, up until about 30 wt.%, and including subranges of the range about 0.05 wt.% to about 30 wt.%. In an embodiment the second solvent of the Reagent B includes but not limited to dimethyl sulfoxide (DMSO).

[0059] In some embodiments of the present disclosure, the reagent B comprises dimethyl sulfoxide (DMSO) in an amount ranging from about 0.05 wt.% to about 30 wt.%.

[0060] In some embodiments of the present disclosure, the Reagent B comprises stabilizing agent in an amount ranging from about 0.3 wt.% to about 1.5 wt.% and including subranges of the range about 0.3 wt.% to about 1.5 wt.%. In an embodiment, the stabilizing agent is in an amount of about 0.4 wt.%, about 0.5 wt.%, about 0.6 wt.%, about 0.7 wt.%, about 0.8 wt.%, about 0.9 wt.%, about 1.0 wt.%, about 1.1 wt.%, about 1.2 wt.%, about 1.3 wt.%, about 1.4 wt.% or about 1.5 wt.%. In an embodiment, the stabilizing agent of the Reagent B is selected from a group comprising dimethyl ammonium chloride, diethyl ammonium chloride, N-Ethylmethylamine, N-Methylethylamine hydrochloride and combinations thereof. The stabilizing agent helps in preventing precipitation of oppositely charged dyes i.e., cationic and anionic dyes of the staining reagent combination.

[0061] In some embodiments of the present disclosure, the Reagent B comprises dimethyl ammonium chloride in an amount ranging from about 0.3 wt.% to about 1.5 wt.%.

[0062] In some embodiments of the present disclosure, the R B comprises dimethyl ammonium chloride in an amount ranging from about 0.4 wt.% to about 1.2 wt.%.

[0063] In some embodiments of the present disclosure, the staining reagent combination is composed of:

[0064] - the Reagent A comprising- about 0.01 wt.% to 0.9 wt.% of methylene blue, Azure B and Toluidine Blue; about 0.8 wt.% to 1.8 wt.% of Triton X 100; about 70 wt.% to 98.5 wt.% of glycerol; and about 0.5 wt.% to 30 wt.% of DMSO; and

[0065] - the Reagent B comprising- about 0.05 wt.% to 0.3 wt.% of Eosin Y; about 0.5 wt.% to 3 wt.% of Triton X 100 and Brij L23; about 70 wt.% to 99 wt.% of glycerol; about 0.05 wt.% to 30 wt.% of DMSO; and about 0.3 wt.% to 1.5 wt.% of dimethyl ammonium chloride. In some embodiments of the present disclosure, the staining reagent combination is composed of:

[0066] - the Reagent A comprising- about 0.05 wt.% to 0.42 wt.% of methylene blue, about 0.05 wt.% to 0.3 wt.% Azure B, about 0.02 wt.% to 0.15 wt.% of Toluidine Blue, about 70 wt.% to 98.5 wt.% of glycerol, about 0.8 wt.% to 1.8 wt.% of Triton X 100 and about 0.5 wt.% to 30 wt.% of DMSO; and

[0067] - the Reagent B comprising- about 0.08 wt.% to 0.2 wt.% of eosin Y, about 0. 1 wt.% to 1.2 wt.% of Brij L23, about 70 wt.% to 99 wt.% of glycerol, about 0.8 wt.% to 1.5 wt.% of Triton X 100, about 0.05 wt.% to 30 wt.% of DMSO, and 0.4 wt.% to 1.2 wt.% of dimethyl ammonium chloride.

[0068] In some embodiments of the present disclosure, the staining reagent combination is devoid of alcohol and water. In an embodiment, the Reagent A is devoid of both alcohol and water. In an embodiment, the Reagent B is devoid of both alcohol and water. In an embodiment, the staining reagent combination does not cause coagulation of red blood cells and hence is suitable for analysing haemoglobin in biological samples. The staining reagent combination of the present disclosure addresses the drawbacks noted in conventionally available staining compositions / reagents that employ alcohol and / or water for staining. The staining reagent combination provides for simultaneously assessing total count of white blood cells (WBC), differential count of WBC and haemoglobin concentration in biological samples.

[0069] The staining reagent combination of the present disclosure comprises anionic dye to cationic dye in a ratio ranging from about 1:3 to about 1: 16. The specific ratio of cationic dye and anionic dye in the staining reagent combination provides for effective staining of WBCs and thus helps in differentiating neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells, nucleated red blood cells (NRBCs). In an embodiment, the cationic dye and anionic dye in the specific ratio provides for negligible absorbance around 407 nm and thus provides for efficient measurement of haemoglobin in blood sample. In an embodiment, the cationic dye and anionic dye in a specific ratio in the staining reagent combination prevents precipitation of reagent components after mixing with the biological sample.

[0070] In some embodiments of the present disclosure, the staining reagent combination has high absolute viscosity ranging from about 250 cp to about 950 cp at 20 °C. Higher viscosity of the staining reagent combination aids in preventing spillage or leakage issues associated with traditional staining compositions when applied in microfluidic platforms. Thus, the staining reagent combination is ideal for use in microfluidic platforms.

[0071] In some embodiments of the present disclosure, the staining reagent combination is in the form of a gel.

[0072] In some embodiments of the present disclosure, the staining reagent combination is non-toxic and compatible with plastic materials without any limitations.

[0073] The present disclosure further relates to a method of preparing the staining reagent combination.

[0074] While the subsequent embodiments focus on the method of preparing the staining reagent combination, the feature and characteristics of the staining reagent combination are as described by any of the embodiments above. For the sake of brevity and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the cited embodiments completely fall within the purview of the method of preparing the staining reagent combination.

[0075] In some embodiments of the present disclosure, the method of preparing the staining reagent combination comprises - a) preparing the Reagent A, comprising- mixing the cationic dye, the surfactant, the first solvent and the second solvent to obtain the Reagent A; and b) preparing the reagent B, comprising- mixing the anionic dye, the surfactant, the first solvent, the second solvent and the stabilizing agent to obtain the Rreagent B.

[0076] In some embodiments of the present disclosure, preparing the Reagent A comprises - a) mixing the surfactant and the second solvent under stirring at a speed ranging from about 50 rpm to 650 rpm for a duration ranging from about 1 hours to 4 hours at room temperature (20 °C to 40 °C); b) adding the cationic dye and further mixing under stirring at a speed ranging from about 50 rpm to 650 rpm for a duration ranging from about 1 hours to 4 hours at room temperature (20 °C to 40 °C); and c) adding first solvent and further mixing under stirring at a speed ranging from about 50 rpm to 650 rpm for about 4 hours to 16 hours at room temperature (20 °C to 40 °C) to obtain the Reagent A.

[0077] In an exemplary embodiment, the mixing of the surfactant and the second solvent is carried out under stirring at a speed of about 500 rpm for a duration of about one hour at room temperature (20 °C to 40 °C). In an exemplary embodiment, the further mixing after adding the cationic dye is carried out under stirring at a speed of about 500 rpm for a duration of about 2 hours at room temperature (20 °C to 40 °C). In an embodiment, further mixing after adding the first solvent is carried out under stirring at a speed of about 500 rpm for a duration of about 24 hours at room temperature (20 °C to 40 °C).

[0078] In some embodiments of the present disclosure, preparing the Reagent B comprises- a) mixing the stabilizing agent, the surfactant and the second solvent under stirring at a speed ranging from about 50 rpm to 650 rpm for a duration ranging from about 1 hours to 4 hours at room temperature (20 °C to 40 °C); b) adding the anionic dyes and further mixing under stirring speed ranging from about 50 rpm to 650 rpm for a duration ranging from about 1 hours to 4 hours at room temperature (20 °C to 40 °C); and c) adding the surfactant and the first solvent and further mixing under stirring at a speed of about 50 rpm to 650 rpm for a duration ranging from about 4 hours to 16 hours at room temperature (20 °C to 40 °C) to obtain the Reagent B.

[0079] In an exemplary embodiment, the mixing of the stabilizing agent, the surfactant and the second solvent is carried out under stirring at a about 500 rpm for a duration of about Ihour at room temperature (20 °C to 40 °C). In an exemplary embodiment, further mixing after adding the anionic dye is carried out under stirring at a speed of about 500 rpm for a duration of about 1 hour at room temperature (20 °C to 40 °C). In an exemplary embodiment, further mixing after adding the surfactant and the first solvent is carried out under stirring speed of about 500 rpm for a duration of about 24 hours at room temperature (20 °C to 40 °C).

[0080] The present disclosure further relates to a method of staining cells in a biological sample. While the subsequent embodiments focus on the method of staining cells in a biological sample, the feature and characteristics of the staining reagent combination and method of preparing the staining reagent combination are as described by any of the embodiments above. For the sake of brevity and avoiding repetition, each of those embodiments are not being reiterated here again. However, each of the cited embodiments completely fall within the purview the method of staining cells in a biological sample.

[0081] In some embodiments of the present disclosure, the method of staining cells in a biological sample comprises: diluting the biological sample with a buffer; contacting the diluted biological sample with the staining reagent combination described above; and

[0082] - mixing and incubating the biological sample to obtain stained cells.

[0083] In some embodiments of the present disclosure, in the method of staining cells, the buffer for diluting the biological sample has pH ranging from about 6 to about 8, including all the values in the range, for instance, 6.1, 6.2, 6.3, 6.4 and so on and so forth, up until 8, and including subranges of the range about 6 to about 8. In an embodiment, the buffer is selected from a group comprising phosphate buffered saline, 4-(2 -hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES) Saline, Tris(hydroxymethyl)aminomethane Ethylenediaminetetraacetic acid (TE) buffer and combinations thereof.

[0084] In some embodiments of the present disclosure, in the method of staining cells, the dilution ratio of the biological sample and the buffer is ranging from about 1 : 10 to about 1 :25, including all the values in the range, for instance, about 1: 11, about 1: 12, about 1: 13, about 1: 14, and including subranges of the range about 1: 10 to about 1:25.

[0085] In some embodiments of the present disclosure, in the method of staining cells, the incubation is carried out at a temperature ranging from about 18 °C to about 32 °C, and including subranges of the range about 18 °C to about 32 °C. In an embodiment, the incubation is carried out at a temperature of 18 °C, about 19 °C, about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C, about 30 °C, about 31 °C or about 32 °C, . In an embodiment, the incubation is carried out at a temperature ranging from about 90 seconds to about 2100 seconds, including all the values in the range, for instance, 91 seconds, 92 seconds, 93 seconds, 94 seconds, and so on and so forth, up until 2100 seconds, and including subranges of the range about 90 seconds to about 2100 seconds.

[0086] In some embodiments of the present disclosure, the biological sample employed for staining is selected from a group comprising blood, urine, saliva, mucus, sputum, cerebrospinal fluid. In an embodiment, the biological sample employed for staining is blood.

[0087] In some embodiments of the present disclosure, the biological sample comprising the cells are stained in suspension form upon contacting the diluted biological sample with the staining reagent combination. In an embodiment, the stained cells are white blood cells (WBCs). In an embodiment, the stained cells are selected from a group comprising neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells, nucleated red blood cells (NRBCs) and combinations thereof.

[0088] In some embodiments of the present disclosure, the method of staining cells in a biological sample further comprises measuring and identifying the stained cells. In an embodiment, measuring and identifying the stained cells comprises total count of WBCs and differential count of WBCs. In an embodiment, the differential count of WBCs is 5 to 7 parts differential count of WBCs. In an embodiment, the total count of WBCs and the differential count of WBCs is carried out by technique selected from a group comprising hemocytometer, artificial intelligence based automated microscopy, Fluorimetry, vision based cell counters and combinations thereof.

[0089] In some embodiments of the present disclosure, the method further comprises measuring haemoglobin content. In an embodiment, measuring hemoglobin content is carried out by techniques selected from a group comprising spectrophotometric, image based quantification and combinations thereof.

[0090] In some embodiments of the present disclosure, Figure 1 of the present disclosure provides a schematic representation of determination of total count of WBC, differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination, wherein the sample preparation step is carried out by manual mixing using a vial.

[0091] In some embodiments of the present disclosure, Figure 2 of the present disclosure provides a schematic representation of determination of total count of WBC, differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination, wherein the sample preparation step is carried out by mixing using microfluidic cartridge.

[0092] In some embodiments of the present disclosure, the method of staining of cells in a biological sample using the staining reagent combination is simple with minimal steps and allows simultaneous determination of WBCs and haemoglobin from the same blood sample processed in a single sample preparation step.

[0093] In an embodiment, by employing the staining reagent combination of the present disclosure, low volume of biological sample is sufficient for the measurement of WBCs and haemoglobin. In an embodiment, the volume of the biological sample is ranging from about 5 pl to about 100 pl. In an embodiment, low volume of the biological sample is sufficient to measure 3 parameters -i. total count of WBC, ii. differential count of WBC; and iii. haemoglobin.

[0094] In some embodiments of the present disclosure, determination of WBCs and haemoglobin in biological sample requires low staining reagent combination to sample ratio of about 1: 10 to 1:30 depending on HCT.

[0095] In some embodiments of the present disclosure, in the method of staining of cells in a biological sample, the staining reagent combination provides for low turn-around time or quick reaction time of less than two minutes and long working time of upto six hours. In an embodiment, lysis of RBCs and staining of WBCs happen within 2 minutes. In an embodiment, the measurement time window for haemoglobin is ranging from about 2 minutes to about 6 hours, including all the values in the range, for instance, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, and so on and so forth up until about 6 hours, and including subranges of the range 2 minutes to about 6 hours.

[0096] In an embodiment, the measurement time window for total count of WBC is ranging from about 2 minutes to about 4 hours, for instance, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes and so on and so forth up until about 4 hours, and including subranges of the range 2 minutes to about 4 hours. In an embodiment, the measurement time window for differential count of WBC is ranging from about 2 minutes to about 45 minutes, for instance, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes and so on and so forth up until about 45 minutes and including subranges of the range 2 minutes to about 45 minutes.

[0097] In an embodiment, Figure 5 provides images of stained WBCs in a blood sample by using the staining reagent combination of the present disclosure. The cells stained by the staining reagent combination include neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells, nucleated red blood cells (NRBCs). The stained cells appear to be similar to that of the cells stained using traditional blood smear staining.

[0098] In an embodiment, Figures 6 to 9 of the present disclosure provides images of stained WBC cells by employing comparative staining composition. The staining composition not prepared in accordance with the present disclosure is not in efficient in determination of WBCs, haemoglobin in blood samples and / or not suitable for application in microfluidic platforms.

[0099] The present disclosure further relates to a kit.

[0100] In some embodiments of the present disclosure, the kit comprises:

[0101] - the staining reagent combination; and an instruction manual comprising details of staining cells in a biological sample.

[0102] In some embodiments of the present disclosure, the kit comprises buffer for diluting the blood sample. In an embodiment the buffer is selected from a group comprising phosphate buffered saline, 4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid (HEPES) Saline, Tris(hydroxymethyl)aminomethane Ethylenediaminetetraacetic acid (TE) buffer and combinations thereof. In an embodiment the buffer has pH ranging from about 6 to about 8.

[0103] In some embodiments of the present disclosure, the kit includes a container comprising the Reagent A comprising- i. about 0.01 wt.% to 0.9 wt.% of cationic dye selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof; ii. about 0.8 wt.% to 1.8 wt.% of surfactant; and iii. about 70 wt.% to 98.5 wt.% of a first solvent; and iv. about 0.5 wt.% to 30 wt.% of a second solvent

[0104] In some embodiments of the present disclosure, the kit includes a container comprising the Reagent B comprising- i. about 0.05 wt.% to 0.3 wt.% of anionic dye selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof; ii. about 0.5 wt.% to 3 wt.% of surfactant; iii. about 70 wt.% to 99 wt.% of first solvent; iv. about 0.05 wt.% to 30 wt.% of second solvent; and v. about 0.3 wt.% to 1.5 wt.% of stabilizing agent.

[0105] In some embodiments of the present disclosure, the kit comprises- a container comprising the Reagent A comprising- i. about 0.01 wt.% to 0.9 wt.% of cationic dye selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof; ii. about 0.8 wt.% to 1.8 wt.% of surfactant; iii. about 70 wt.% to 98.5 wt.% of a first solvent; and iv. about 0.5 wt.% to 30 wt.% of a second solvent; a container comprising the Reagent B comprising- i. about 0.05 wt.% to 0.3 wt.% of anionic dye selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof; ii. about 0.5 wt.% to 3 wt.% of surfactant; iii. about 70 wt.% to 99 wt.% of first solvent; iv. about 0.05 wt.% to 30 wt.% of second solvent; and v. about 0.3 wt.% to 1.5 wt.% of stabilizing agent; and instructions manual comprising details of staining cells in a biological sample.

[0106] The present disclosure further relates to use of the staining reagent combination for staining white blood cells, measuring total count of WBCs and differential count of WBCs, and identifying haemoglobin content.

[0107] Whde the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples (and drawings) described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is therefore further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.

[0108] 5

[0109] EXAMPLES

[0110] The present disclosure is further described with reference to the following example, which is only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner. 0

[0111] Example 1: Preparation of the staining reagent combination a) Preparing the Reagent A

[0112] 1. 1.34 wt.% of Triton X 100 and 1.33 wt.% of DMSO was combined and mixed with a magnetic stirrer at 500 rpm for one hour at room temperature. 5 2. Subsequently, 0.19 w% of Methylene Blue, 0.12 w% Azure B, and 0.02 w% Toluidine Blue O dyes was added while stirring to obtain a mixture. Stirring of the mixture was continued for an additional two hours at room temperature.

[0113] 3. Following this, a 97.01 w% of glycerol was added under stirring conditions and stirred for another 24 hours at room temperature. (b) Preparing the Reagent B

[0114] 1. About 0.45 wt.% of dimethyl ammonium chloride, 1.07 wt.% Triton X 100, and 7.59 wt.% DMSO were added and mixed using a magnetic stirrer at 500 rpm for one hour at room temperature.

[0115] 2. To this mixture, 0.18 wt.% ofEosin Y was added and stirred for an additional hour at room5 temperature.

[0116] 3. Then, 0.20 wt.% of Brij L23 and 97.01 wt.% glycerol was added under stirring conditions and stirred for another 24 hours at room temperature.

[0117] Table 1 provides the CAS. No, Name, moles, weight & weight % of chemicals used to0 prepare the Reagent A of the staining reagent combination.

[0118] Table 2 provides the CAS. No, Name, moles, weight & weight % of chemicals used to prepare the Reagent B of the staining reagent combination.

[0119] Example 2: Preparation of the staining reagent combination a) Preparing the Reagent A

[0120] 1. 0.9 wt.% of Triton X 100 and 12 wt.% DMSO was combined and mixed with a magnetic stirrer at 500 rpm for one hour at room temperature. 2. Subsequently, 0.07 wt.% of Methylene Blue, 0.07 wt.% Azure B, and 0.05 wt.% Toluidine Blue O dyes was added while stirring to obtain a mixture. Stirring of the mixture was continued for an additional two hours at room temperature.

[0121] 3. Following this, 88.91 w% of glycerol was added under stirring conditions and stirred for another 24 hours at room temperature. b) Preparing the Reagent B

[0122] 1. About 0.8 wt.% of dimethyl ammonium chloride, 0.9 wt.% Triton X 100, and 1.33 w% DMSO were added and mixed using a magnetic stirrer at 500 rpm for one hour at room temperature. 2. To this mixture, 0.09 wt.% of Eosin Y was added and stirred for an additional hour at room temperature.

[0123] 3. Then, 0.6 wt.% of Brij L23 and 96.28 wt.% glycerol was added under stirring conditions and stirred for another 24 hours at room temperature.

[0124] Example 3: Preparation of the staining reagent composition a) Preparing the Reagent A

[0125] 1. 1.7 wt.% ofTriton X 100 and 25 wt.% DMSO was combined and mixed with a magnetic stirrer at 500 rpm for one hour at room temperature.

[0126] 2. Subsequently, 0.3 wt.% of Methylene Blue, 0.22 wt.% Azure B, and 0.1 wt.% Toluidine Blue O dyes was added while stirring to obtain a mixture. Stirring of the mixture was continued for an additional two hours at room temperature.

[0127] 3. Following this, 72.68 wt.% of glycerol was added under stirring conditions and stirred for another 24 hours at room temperature. b) Preparing the Reagent B 1. About 1.1 wt.% of dimethyl ammonium chloride, 1.33 wt.% Triton X 100, and 25 wt.%

[0128] DMSO were added and mixed using a magnetic stirrer at 500 rpm for one hour at room temperature.

[0129] 2. To this mixture, 0.25 wt.% of Eosin Y was added and stirred for an additional hour at room temperature. 3. Then, 1.1 wt.% of Brij L23 and 70.35 wt.% glycerol was added under stirring conditions and stirred for another 24 hours at room temperature.

[0130] Example 4: Determination of total count of WBC, differential count of WBC and concentration of haemoglobin in blood samples.

[0131] Any of the staining reagent combination prepared in the examples 1 to 3 was employed for obtaining total count of WBC, differential count of WBC and concentration of haemoglobin in blood samples.

[0132] 5 pl of blood sample was taken. - The blood sample was diluted with 125 pl of phosphate buffered saline having pH 7.2. i.e, a dilution ratio of 1:25 (blood: buffer).

[0133] - About 125 pl of diluted blood was mixed with about 4 pl of Reagent Band about 8 pl of Reagent A.

[0134] - The mixture containing the blood sample and the reagent combination was incubated at room temperature for about 2 minutes.

[0135] The haemoglobin content of the blood sample was measured using a spectrophotometer and the WBC TC and WBC DC was measured using manual microscopy.

[0136] Figure 1 provides a schematic representation of determination of total count of WBC, differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination, wherein the sample preparation step is carried out by manual mixing using vial.

[0137] Figure 2 provides a schematic representation of determination of total count of WBC, differential count of WBC and concentration of haemoglobin in a blood sample by employing the staining reagent combination, wherein the sample preparation step is carried out by mixing using microfluidic cartridge.

[0138] Figure 3 provides a graph depicting haemoglobin measurement using spectrophotometer by using the staining reagent combination of the present disclosure.

[0139] The concentration of haemoglobin in the blood sample was found to be about 8.2 g / dL.

[0140] Figure 4 provides a graph depicting WBC measurement using a microfluidic cartridge by using the staining reagent combination of the present disclosure.

[0141] The total count (TC) of WBC in the blood sample was found to be 6.43 x 103cells / pL.

[0142] Table 3A, Table 3B and Table 3C shows differential count (DC) of WBC in the blood samples Table 3A:

[0143] Table 3B:

[0144] Table 3C:

[0145] Thus, the staining reagent combination of the present disclosure enables simultaneous measurement of both WBC and Haemoglobin using the same blood sample processed in a single sample preparation step. Thus, analysing WBC and haemoglobin is possible with less volume of biological sample and staining reagent.

[0146] EXAMPLE 5: Staining of WBCs in biological samples

[0147] Any of the staining reagent combination prepared in the examples 1 to 3 was employed for identification of WBCs in blood samples using the method described in Example 4.

[0148] FIGURE 5 provides images of stained WBC cells in a blood sample by using the staining reagent combination of the present disclosure.

[0149] As can be seen from Figure 5 the staining reagent combination of the present disclosure efficiently stains WBCs in the blood samples. The cells stained by the staining reagent combination include neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells, nucleated red blood cells (NRBCs). Based on the staining of WBCs the total count of WBC, 5 to 7 parts differential count of WBC in a biological sample can be determined. The stained cells appear to be similar to that of the cells stained using traditional blood smear staining. Table 4 is the evaluation of staining quality and the rating for ease of identification of WBCs by employing the staining reagent combination of the present disclosure.

[0150] Table 4:

[0151] Data represented in Table 4 shows that the staining reagent combination of the present disclosure enables efficient determination of total count of WBC, 5 to 7 parts differential count ofWBC.

[0152] Comparative Example: Comparison of different staining reagent compositions

[0153] Table 5 shows the comparison of the staining reagent combination of the present disclosure over other comparative staining reagent compositions not prepared in accordance with the staining reagent combination of the present disclosure.

[0154] Table 5:

[0155] The data provided in Table 5 shows that the staining reagent combination of the present disclosure is highly effective in for measurement of WBCs and haemoglobin in blood samples when compared to comparative staining composition not prepared in accordance with the staining reagent combination of the present disclosure.

[0156] The staining reagent combination of the present disclosure provides for simultaneously assessing total count of WBC, differential count of WBC and concentration of haemoglobin from biological samples. As the staining reagent combination can access both WBCs and haemoglobin in a biological sample, single sample preparation step is sufficient and thus requires less volume of biological sample.

[0157] The staining reagent combination is ideal for use in microfluidic platforms due to its high viscosity and lower volume required for measurement. The staining reagent combination has quick reaction and long working time. Example 7: Staining kit

[0158] A kit was prepared by including the Reagent A and Reagent B prepared in any of the examples 1 to 3 in separate containers. A buffer was included in a separate container for diluting the blood sample. The kit was further provided with an instruction manual comprising details of staining cells in a biological sample.

[0159] The staining reagent combination and the method of staining the cells of the present disclosure provides for following advantages:

[0160] The staining reagent combination is devoid of alcohol and water and hence does not cause coagulation of red blood cells and therefore is suitable for analysing the concentration of haemoglobin in biological samples.

[0161] The specific ratio of cationic dye and anionic dye in the staining reagent combination helps to prevent precipitation of reagent components after mixing with biological sample.

[0162] The staining reagent combination has negligible absorbance around 400 nm to 420 nm where haemoglobin is measured thus provides for efficient measurement of haemoglobin in blood sample.

[0163] The staining reagent combination provides for effective staining of WBCs which helps in differentiating neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells and nucleated red blood cells (NRBCs).

[0164] The staining reagent combination has high viscosity and thus does not lead to spillage or leakage from the reagent chamber when used in microfluidic platforms. Thus, the staining reagent combination is ideal for use in microfluidic platforms or for storage without an enclosed chamber.

[0165] The surfactants in the staining reagent combination provides for complete lysis of RBC’s and thus there is no interference of RBCs during WBC staining.

[0166] The staining reagent combination is a non-toxic composition and compatible with plastic materials.

[0167] The staining reagent combination enables staining of cells in suspension form and thus allowing its visualization in a microscope or in point-of-care image processing devices. The method of staining of cells in a biological sample using the staining reagent combination is simple with minimal steps and allows simultaneous determination of WBCs and haemoglobin from the same blood sample processed in a single sample preparation step. The staining reagent combination can be used to differentiate up to 7 types of WBC in microfluidic cartridge.

[0168] The staining reagent combination provides for low turn-around time or quick reaction time of less than two minutes and long working time of up to six hours in determination biological of WBCs and haemoglobin in samples.

[0169] The method of staining cells requires low staining reagent combination to sample ratio of about 1: 10 to about 1:30 depending on HCT in determination of WBCs and haemoglobin.

[0170] Low volume of blood is sufficient to measure 3 parameters - i. total count of WBC, ii. differential count of WBC, and iii. haemoglobin.

[0171] Low cost: The tentative cost of the staining reagent combination is about 10 paise per sample.

[0172] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well- known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0173] The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

[0174] Thus, while considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

[0175] Throughout this specification, the word “comprise”, or variations such as “comprises” or “comprising” wherever used, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. Similarly, terms such as “include” or “have” or “contain” and all their variations are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0176] The terms "about" or “approximately” are used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical value / range, it modifies that value / range by extending the boundaries above and below the numerical value(s) set forth. In general, the term "about" is used herein to modify a numerical value(s) or a measurable value(s) such as a parameter, an amount, a temporal duration, and the like, above and below the stated value(s) by a variance of + / -20% or less, + / -10% or less, + / -5% or less, + / -1% or less, and + / -0. 1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention, and achieves the desired results and / or advantages as disclosed in the present disclosure. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0177] With respect to the use of substantially any plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. As used in this specification and the appended claims, the singular forms “a,” “an” and “the” includes both singular and plural references unless the content clearly dictates otherwise. The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0178] Numerical ranges stated in the form ‘from xto y’ include the values mentioned and those values that lie within the range of the respective measurement accuracy as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.

[0179] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I;

[0180] B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H;

[0181] C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0182] Any discussion of documents, acts, materials, devices, articles and the like that has been included in this specification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or all of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

[0183] All references, articles, publications, general disclosures etc. cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

WE CLAIM:

1. A staining reagent combination composed of: a. a Reagent-A comprising: i. about 0.01 wt.% to 0.9 wt.% of cationic dye selected from a group comprising Methylene Blue, Azure B, Toluidine Blue O, Crystal Violet, Neutral Red, Nile blue sulfate, Safranin O and combinations thereof; ii. about 0.8 wt.% to 1.8 wt.% of surfactant; and iii. about 70 wt.% to 98.5 wt.% of a first solvent; and iv. about 0.5 wt.% to 30 wt.% of a second solvent, and b. a Reagent-B comprising: i. about 0.05 wt.% to 0.3 wt.% of anionic dye selected from a group comprising Eosin Y, Acid Fuchsin, Indigo carmine and combinations thereof; ii. about 0.5 wt.% to 3 wt.% of surfactant; iii. about 70 wt.% to 99 wt.% of first solvent; iv. about 0.05 wt.% to 30 wt.% of second solvent; and v. about 0.3 wt.% to 1.5 wt.% of stabilizing agent.

2. The staining reagent combination as claimed in claim 1, wherein the surfactant of the Reagent A is selected from a group comprising Triton X 100, Brij L23, Saponin and combinations thereof.

3. The combination as claimed in claim 1, wherein the first solvent of the Reagent A is selected from a group comprising glycerol, ethylene glycol, polyethylene glycol and combinations thereof; and wherein the second solvent of the Reagent A is dimethyl sulfoxide (DMSO).

4. The combination as claimed in claim 1, wherein the surfactant of the Reagent B is selected from a group comprising Triton X 100, Brij L23, Saponin and combinations thereof.

5. The combination as claimed in claim 1, wherein the first solvent of the Reagent B is selected from a group comprising glycerol, ethylene glycol, polyethylene glycol andcombinations thereof; and wherein the second solvent of the Reagent B is dimethyl sulfoxide (DMSO).

6. The combination as claimed in claim 1, wherein the stabilizing agent of the Reagent B is selected from a group comprising dimethyl ammonium chloride, diethyl ammonium chloride, N-Ethylmethylamine, N-Methylethylamine hydrochloride and combinations thereof.

7. The staining reagent combination as claimed in claim 1, wherein the combination is composed of:- the Reagent A comprising- about 0.01 wt.% to 0.9 wt.% of methylene blue, Azure B and Toluidine Blue; about 0.8 wt.% to 1.8 wt.% of Triton X 100; about 70 wt.% to 98.5 wt.% of glycerol; and about 0.5 wt.% to 30 wt.% of DMSO; and- the Reagent B comprising- about 0.05 wt.% to 0.3 wt.% of Eosin Y; about 0.5 wt.% to 3 wt.% of Triton X 100 and Brij L23; about 70 wt.% to 99 wt.% of glycerol; about 0.05 wt.% to 30 wt.% of DMSO; and about 0.3 wt.% to 1.5 wt.% of dimethyl ammonium chloride.

8. The combination as claimed in claim 1, wherein the combination is composed of:- the Reagent A comprising- about 0.05 wt.% to 0.42 wt.% of methylene blue, about 0.05 wt.% to 0.3 wt.% Azure B, about 0.02 wt.% to 0.15 wt.% of Toluidine Blue, about 70 wt.% to 98.5 wt.% of glycerol, about 0.8 wt.% to 1.8 wt.% of Triton X 100 and about 0.5 wt.% to 30 wt.% of DMSO; and- the Reagent B comprising- about 0.08 wt.% to 0.2 wt.% of eosin Y, about 0. 1 wt.% to 1.2 wt.% of Brij L23, about 70 wt.% to 99 wt.% of glycerol, about 0.8 wt.% to 1.5 wt.% of Triton X 100, about 0.05 wt.% to 30 wt.% of DMSO, and 0.4 wt.% to 1.2 wt.% of dimethyl ammonium chloride.

9. The combination as claimed in any of claims 1 to 8, wherein the Reagent A is devoid of alcohol and water; and wherein the Reagent B is devoid of alcohol and water.

10. The combination as claimed in claim 1, wherein the ratio of anionic dye to cationic dye is ranging from about 1:3 to about 1: 16.

11. A method of preparing the staining reagent combination as claimed in any of claims 1 to 10, the method comprising - a) Preparing the Reagent A, comprising- mixing the cationic dye, the surfactant, the first solvent and the second solvent to obtain the Reagent A; and b) Preparing the Reagent B, comprising- mixing the anionic dye, the surfactant, the first solvent, the second solvent and the stabilizing agent to obtain the Reagent B.

12. The method as claimed in claim 11, wherein in preparing the Reagent A, the mixing is carried out under stirring conditions of about 50 rpm to about 650 rpm for about 1 hour to about 24 hours at room temperature; and wherein in preparing the Reagent B, the mixing is carried out under stirring conditions of about 50 rpm to about 650 rpm for about 1 hour to about 24 hours at room temperature.

13. A method of staining cells in a biological sample, said method comprising:- diluting the biological sample with a buffer;- contacting the diluted biological sample with the staining reagent combination as claimed in any of claims 1 to 10; and- mixing and incubating the biological sample to obtain stained cells.

14. The method as claimed in claim 13, wherein the buffer is selected from a group comprising phosphate buffered saline, 4-(2 -hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES) Saline, Tris(hydroxymethyl)aminomethane Ethylenediaminetetraacetic acid (TE) buffer and combinations thereof; and wherein the buffer has pH ranging from about 6 to 8.

15. The method as claimed in claim 13, wherein dilution ratio of the biological sample and the buffer is ranging from about 1: 10 to 1:25.

16. The method as claimed in claim 13, wherein the incubation is carried out at a temperature ranging from about 18 to 32 °C, for a duration ranging from about 90 s to 35 min.

17. The method as claimed in claim 13, wherein the biological sample is selected from a group comprising blood, urine, saliva, mucus, sputum, cerebrospinal fluid.

18. The method as claimed in claim 13, wherein the biological sample comprising the cells are stained in suspension form upon contacting the diluted biological sample with the staining reagent combination.

19. The method as claimed in claim 13, wherein the stained cells are white blood cells (WBCs).

20. The method as claimed in claim 13, wherein the stained cells are selected from a group comprising neutrophils, eosinophils, basophils, monocytes, lymphocytes, Immature Granulocytes (IGs), blast cells, nucleated red blood cells (NRBCs) and combinations thereof.

21. The method as claimed in claim 13, wherein the method further comprises measuring and identifying the stained cells.

22. The method as claimed in claim 21, wherein the measuring and identifying the stained cells comprises total count of WBCs and differential count of WBCs.

23. The method as claimed in claim 22, wherein the total count of WBCs and the differential count of WBCs is carried out by technique selected from a group comprising hemocytometer, artificial intelligence based automated microscopy, Fluorimetry, vision based cell counters and combinations thereof.

24. The method as claimed in claim 13, wherein the method further comprises measuring haemoglobin content, carried out by techniques selected from a group comprising spectrophotometric, image based quantification and combinations thereof.

25. A kit comprising: a staining reagent combination as claimed in any of claims 1 to 10; and an instruction manual comprising details of staining cells in a biological sample.

6. Use of the staining reagent combination as claimed in any of the claims 1 to 10 for staining white blood cells, measuring total count of WBCs and differential count of WBCs, and identifying haemoglobin content.

Images