CN111487179A

CN111487179A - Portable blood cell counting kit

Info

Publication number: CN111487179A
Application number: CN201910087006.XA
Authority: CN
Inventors: 高婷娟; 李欣怡; 邓琼; 唐浴尘
Original assignee: Central China Normal University
Current assignee: Central China Normal University
Priority date: 2019-01-29
Filing date: 2019-01-29
Publication date: 2020-08-04

Abstract

The invention relates to a portable blood cell counting kit, a device containing the same and a blood cell counting analysis method. The kit comprises: 1) a solid mixture 1 comprising a surfactant 1 and a coloring agent 1; and/or 2) a solid mixture 2 comprising a surfactant 2 and a coloring agent 2. The kit is further applied to a blood cell counting device and a blood cell counting analysis method. By the kit, the device and the blood cell counting and analyzing method, trace quantity blood measurement and convenient operation can be realized, and the application requirements of economy, environmental protection and wide popularization can be met.

Description

Portable blood cell counting kit

Technical Field

The invention relates to the field of biochemical detection, in particular to a kit for detecting the number of blood cells in a blood sample and application thereof.

Background

Blood cell counting, which is one of the routine examinations for detecting the characteristic variables of blood cells, including the number, morphology, size, and cell structure, cell cycle, cell DNA, cell surface proteins, and specific proteins contained in cytoplasm, is widely used in biological and disease research.

The use of conventional blood tests by haemocytometer analysis is one of the tests commonly found in clinical laboratories, generally requiring the use of flow cytometers, such instruments and techniques are not readily available in remote areas where economy and traffic are not available, and therefore it is necessary to simplify the sample preparation steps and the instrument handling steps in order to ensure the accuracy of the results, however, these methods have their own drawbacks, such as the need to use multiple measuring instruments or complex sample preparation methods to obtain multiple parameters, and the simple sample preparation steps generally allow the detection of only one parameter, the recently reported automated blood routine test (HemoScreen by PixCell Medical) detects a series of blood routine parameters using flow-bound image analysis of cells in viscoelastic fluids, but this method requires 40 μ L, which cannot be matched with the standard fingertip blood sampling (the volume taken is about 10 μ L), the use of the simple blood sample preparation method to correct the sample counting of human blood sample No. 54, the sample preparation method does not require the use of the standard hemarten microscope 6754, No. 2 to correct the results of the blood sample obtained by human microtube

The foregoing research methods require the preparation of fresh reagents and the purchase of commercial sample cells. The used reagent is in a liquid state (for example, the reagent for staining the leucocytes is a dilute solution of a dye, cannot be stored for a long time and needs to be prepared for use; moreover, the process of preparing the solution needs to be performed step by step to avoid counting errors caused by mutual interference between the two reagents. Thus, this method is still not convenient enough when the audience is the public. Meanwhile, the higher cost of using commercial sample cells will affect the popularity of this research approach; furthermore, the commercial sample wells are made of plastic, and the current industrial field uses plastic in a large amount to make the recycling of plastic one of the main challenges in the environmental protection field. In order to solve the problems, the invention provides an environment-friendly blood cell imaging sample pool which is accurate in counting, low in cost and simple to prepare.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention mainly aims to provide a portable blood cell counting kit which can be used for preparing trace blood samples, is simple to use, is convenient to carry, has low cost, is economic and environment-friendly and is oriented to a wider application range.

A second object of the present invention is to provide a blood cell counting device comprising the above blood cell counting kit.

The third objective of the invention is to provide a blood cell counting analysis method, which only needs trace amount to measure blood, has simple sample preparation steps, and is suitable for being widely applied to biology and disease analysis.

The above purpose of the invention is realized by the following technical scheme:

in one aspect, the present invention provides a kit, comprising:

1) a solid mixture 1 comprising a surfactant 1 and a coloring agent 1;

and/or

2) A solid mixture 2 comprising a surfactant 2 and a coloring agent 2;

the

surfactants

1, 2 are independently selected from surfactants for sphering and/or lysing erythrocytes, wherein surfactant 1 is a solid amphoteric surfactant; the

staining agents

1, 2 are independently selected from staining agents for white blood cells and platelets, and may be the same or different.

According to an embodiment of the invention, the solid mixture 1 can be used for blood cell counting and white blood cell sorting, and the cuvette 2 and the mixture 2 can be used for white blood cell sorting. Preferably, the solid mixture 1 is used for blood cell counting.

According to an embodiment of the present invention, the surfactant 1 is a quaternary ammonium salt solid amphoteric surfactant. More preferably, the surfactant 1 is a solid amphoteric surfactant containing a sulfonic acid group and a quaternary ammonium salt, such as N, N-dimethyl-N- (3-sulfopropyl) -1-octaalkylammonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-dodecylammonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-octadecylammonium inner salt, and the like.

According to an embodiment of the present invention, the amount of the surfactant 1 and the coloring agent 1 contained in the solid mixture 1 is calculated by working concentration of 13 to 35 μ M and 30 to 50 μ M, respectively; the amount of the surfactant 2 and the amount of the coloring agent 2 contained in the solid mixture 2 are calculated by working concentration of 1-10 mM and 45-75 μ M, respectively.

As will be understood by those skilled in the art, the "working concentration" is the concentration of the reagent in the liquid used for a particular assay when the corresponding reagent in the kit is mixed with the target biological sample for the test assay by those skilled in the art.

It is further understood that according to embodiments of the present invention, the "working concentration" is the final concentration of the corresponding reagent after mixing the

solid mixture

1 or 2 with the diluted or undiluted blood cell sample, respectively.

Preferably, the surfactant 2 is selected from anionic surfactants, such as sodium lauryl sulfate.

Preferably, the

coloring agent

1 or 2 is selected from acridine orange and the like.

Preferably, the amount of the surfactant 1 and the amount of the stain 1 contained in the solid mixture 1 are calculated as 13 to 35. mu.M and 30 to 50. mu.M, more preferably 18 to 30. mu.M and 35 to 45. mu.M, and most preferably 22. mu.M and 37.5. mu.M, respectively, at the working concentration after the addition of the blood cell sample diluted with the diluent; preferably, the blood cell sample is diluted by a diluent by a factor of 40 to 80 times, more preferably by a factor of 50 to 70 times, and most preferably by a factor of 60 to 62 times.

Preferably, the amount of the surfactant 2 and the amount of the coloring agent 2 contained in the solid mixture 2 are calculated in working concentrations of 1 to 10mM and 45 to 75. mu.M, more preferably 2 to 8. mu.M and 50 to 65. mu.M, and most preferably 5.2mM and 52.5. mu.M, respectively, after the undiluted blood cell sample is added.

According to an embodiment of the present invention, when the solid mixture 1 and the solid mixture 2 exist at the same time, they are preferably placed in two different containers within the kit, respectively, for example, the solid mixture 1 is placed in the container 1, and the solid mixture 2 is placed in the container 2.

According to an embodiment of the present invention, the kit may further comprise one or more sample wells, and preferably, the kit comprises two sample wells. The thickness of the sample pool is preferably 60-150 mu m, and more preferably 100 mu m; the thickness of the other sample cell is preferably 10-50 μm, and more preferably 20 μm.

According to the invention, the kit further comprises a diluent; the diluent is preferably a phosphate buffer solution, and the diluent is preferably placed in a different container than the solid mixture 1, the solid mixture 2, for example in container 3.

According to the invention, the kit may also comprise one or more microsyrinths;

preferably, the microsyrinths can be three, and different volume specifications are adopted, such as the volumes of 1 μ L, 5 μ L and 50 μ L.

Preferably, the kit comprises: two sample cells; placing the

solid mixtures

1 and 2 in a container 1 and a container 2 respectively; the diluent is placed in the container 3; and 3 microsamplers of different volumes.

In another aspect, the present invention also provides a method of preparing the sample cell in the kit, comprising: the material is prepared by Soft lithography (Soft lithography) using a material such as a glass slide, glass cement, hair line, or the like. When the cell counting sample pool is manufactured, the thickness of the sample pool is controlled by hair, red blood cells at the edge are prevented from being gathered, and a prepared blood sample is transferred and flows into the cell counting sample pool under the action of capillary action and hydrophilic force. The leucocyte classification sample pool is manufactured without fixing hair, and the rest manufacturing steps are similar to those of the cell counting sample pool.

The method comprises the following steps:

(1) making a seal: making a stamp by using a mold, for example, making a PDMS (polydimethylsiloxane) stamp;

(2) optionally performing a hair setting step: when the sample cell is used for blood cell counting, a hair fixing step is performed: fixing two hairs on the surface of the glass slide, and keeping a certain distance between the two hairs; when the sample pool is used for leukocyte classification, hair is not fixed;

(3) manufacturing a sample cell: dipping the prepared seal with glass cement to adhere the seal to the PDMS seal; and pressing the stamp on the glass slide which is optionally subjected to the hair fixing step, and leaving the glass cement pattern on the stamp. And covering the glass cement pattern with a cover glass, and naturally airing the glass cement to form a sample cell.

According to an embodiment of the invention, the method comprises the steps of:

(1) preparing a PDMS (polydimethylsiloxane) seal: pouring PDMS and a curing agent (for example, the mass ratio of 8: 1-11: 1) onto a culture dish fixed with a mold, curing (for example, curing for 0.5-1.5 h at 75-85 ℃ in a constant temperature drying oven), forming a pattern at the bottom of the PDMS after curing, removing the pattern from the mold, and cutting the pattern into a cuboid to prepare a PDMS stamp; the mould is selected from a rectangle with the width of 6mm or 3 mm;

(2) optionally, a hair fixing step is carried out, wherein when the sample pool is used for blood cell counting, the hair fixing step is carried out, namely, the glass slide and the cover glass are dried, two hairs are fixed on the surface of the glass slide with the thickness of 75mm and × 25mm, the distance between the two hairs is 3mm, and when the sample pool is used for white blood cell classification, the hair fixing step is not carried out;

(3) the method comprises the steps of preparing a sample cell, dipping glass cement with a prepared PDMS stamp to adhere the glass cement to the PDMS stamp, pressing the PDMS stamp on a glass slide which is optionally subjected to a hair fixing step to leave a glass cement pattern of the stamp, covering and pressing an 18mm × 18mm cover glass sheet on the glass cement pattern, and naturally airing the glass cement to form the sample cell.

Preferably, the size of the sample pool for blood cell counting is 18mm × 3mm × 100 μm, and the size of the sample pool for leukocyte classification is 18mm × 3mm × 20 μm.

Preferably, the sample cell fabrication comprises a pretreatment step: the glass slide and the cover glass are wiped by using lens wiping paper and ethanol to remove impurities such as dust on the surfaces of the glass slide and the cover glass. And then placing the glass slide and the cover glass into a mixed solution of 98% concentrated sulfuric acid and 30% hydrogen peroxide (volume ratio is 7: 3), and carrying out ultrasonic treatment at 50-70 ℃ for 0.5-2 h to clean and hydrophilically treat the glass slide. Then, the glass slide and the cover glass are ultrasonically cleaned for 2-4 times at 25 ℃ by ultrapure water, and each time lasts for 10 min.

In another aspect, the invention further provides a blood cell counting device comprising the kit and a large-field imaging device, wherein the large-field imaging device comprises a bright-field light source, a fluorescent light source, an automatic filter wheel, a filter, a motorized translation stage, an objective lens and a CCD detector, the motorized translation stage is used for moving a sample pool to obtain the image of the whole sample pool, the objective lens is used for large-field imaging, the bright-field light source is preferably a red L ED lamp with the wavelength of 620nm, the fluorescent light source is preferably a blue L ED lamp with the wavelength of 470nm, more preferably, the imaging device adopts an objective lens of Nikon 4x and 0.2NA, the automatic filter wheel is used for converting the two fluorescent filters to obtain the fluorescent images of two channels (red: 685 +/-20 nm and green: 528 +/-19 nm), the images are recorded by a CCD camera (SBIG STF-8300M), and the software is written by BCB language.

In another aspect, the present invention further provides a method for blood cell count analysis, comprising the steps of:

(1) mixing a solid mixture 1 by adding a diluent and a blood cell sample, wherein the solid mixture 1 comprises a surfactant 1 and a staining agent 1;

and/or mixing the blood cell sample (whole blood) without adding a diluent with a solid mixture 2, wherein the solid mixture 2 comprises a surfactant 2 and a staining agent 2;

(2) flowing the mixture obtained in step (1) into a sample cell;

(3) and acquiring imaging information of the sample cell by adopting an imaging device.

According to an embodiment of the present invention, said

surfactants

staining agents

1 and 2 are independently selected from staining agents for staining white blood cells and platelets, and can be the same or different;

according to an embodiment of the present invention, the surfactant 1 is a quaternary ammonium salt solid amphoteric surfactant. More preferably, the surfactant 1 is a solid amphoteric surfactant containing a sulfonic acid group and a quaternary ammonium salt, such as N, N-dimethyl-N- (3-sulfopropyl) -1-octaalkylammonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-dodecylammonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-octadecylammonium inner salt, etc.; more preferably, the surfactant 1 is N, N-dimethyl-N- (3-sulfopropyl) -1-octadecamonium inner salt.

Preferably, the

coloring agent

1 or 2 is selected from acridine orange and the like.

Preferably, the diluent is Phosphate Buffered Saline (PBS).

Preferably, the above method uses the kit of the present invention and a blood cell counting device.

More preferably, the method specifically comprises the following steps:

(1) pre-storing a solid mixture 1 containing a surfactant 1 and a coloring agent in a container 1 and/or pre-storing a solid mixture 2 containing a surfactant 2 and a coloring agent in a container 2;

(2) transferring the diluent to a container 1 to be mixed with a pre-stored solid mixture 1, adding a whole blood sample, incubating for 1-10 min, measuring the obtained mixture, and enabling the mixture to flow into a sample cell; and/or, adding a sample of blood cells (whole blood) without diluent to the container 2, mixing it with the solid mixture 2 stored beforehand, and measuring the resulting mixture to flow into the sample cell;

(3) and acquiring the information of the sample pool by adopting an imaging device. Specifically, the process of acquiring information includes: the sample cell was placed on the motorized translation stage of the microscope. By moving the translation stage, several successive regions are imaged to obtain information of the entire sample cell.

The "blood cells" to which the present invention relates mainly include red blood cells, white blood cells and platelets. It will be appreciated by those skilled in the art that samples contemplated by the present invention may be referred to herein, depending on the context, as "blood samples," "blood cell-containing samples," "blood cell samples," "blood-containing samples," or "blood," and that samples suitable for use in the present invention may be derived from various types of bodily fluids, including whole blood, saliva, urine, spinal fluid, peritoneal fluid, synovial fluid, milk, sputum, and the like.

In the present invention, the "dilution ratio by a diluent" is generally understood to mean stock solution concentration/(stock solution concentration × removal volume/constant volume).

The invention has the beneficial effects that:

(1) the invention develops a user-friendly kit for preparing a trace blood cell-containing sample by aiming at a portable blood cell counting method, the kit has simple components, the reagent is stored in advance in a solid powder state, and a specially designed blood cell counting and classifying sample pool is manufactured. By using the kit provided by the invention in combination with a specially-constructed microscope and an automatic analysis method, the results of red blood cell counting, platelet counting, white blood cell counting and white blood cell three-classification are obtained and compared with the results of a clinical blood analyzer, and the results have better consistency;

(2) in the kit of the present invention, red blood cells are spheroidized using a solid amphoteric surfactant so that they do not affect the fluorescence of a cationic stain such as acridine orange and the subsequent staining effect of leukocytes and platelets, and thus the surfactant and the dye may be simultaneously mixed and dried, and then stored in a container in advance. Dissolving the pre-buried reagent by using a diluent such as PBS, adding a sample containing blood cells, and dyeing white blood cells and platelets while spheroidizing red blood cells, so that the preparation process is simplified without step-by-step operation;

(3) in the kit of the present invention, whole blood (undiluted blood sample) and a specific solid mixture stored in advance are mixed (5.2mM SDS, 52.5. mu.M AO) to ensure that the number of leukocytes for classifying leukocytes in a sample cell (preferably 20 μ M in thickness) is appropriate, thereby further improving the accuracy of leukocyte classification.

(4) The invention is used for manufacturing the materials of the slide, the glass cement, the hair and the like of the sample pool, and is economic and environment-friendly compared with the commercial sample pool made of plastics. Further, the present invention uses two sample wells of different thicknesses to count red blood cells, platelets, and white blood cells, respectively, and to classify white blood cells into three categories. The blood sample automatically flows into the sample cell under capillary and hydrophilic forces. By optimizing the dilution multiple of the blood sample and controlling the thickness of the sample pool, red blood cells, white blood cells and platelets form a monolayer in the sample pool, so that imaging is facilitated, and the accuracy of counting and classification is ensured.

Drawings

FIG. 1 is a flow chart of a procedure for preparing a trace human blood sample using a kit according to an embodiment of the present invention (a) the components of the kit according to an embodiment of the present invention; (b) a sample cell manufacturing scheme; (c) procedure for the preparation of blood samples using the kit of the invention in the specific examples.

FIG. 2 is a data collection and analysis process: (a) sample cell object diagram: cell a (approximately 100 μm thick) contained a diluted blood sample (dilution factor 61); (b) imaging (bright field, fluorescence overlay) and inset of sample well a under the microscope; (c) sample cell B (about 20 μm thick) contained whole blood (undiluted blood sample); (d) imaging of well B under the microscope (red and green fluorescence overlay) and inset.

FIG. 3 is a concentration optimization plot of DOPS sphered erythrocytes versus AO stained leukocytes: (a) chemical structures of AO, SDS, DOPS; (b) fluorescence spectra of surfactants with different concentrations mixed with AO; (c) - (f) bright field imaging of diluted blood samples (dilution factor 61) after exposure to 0. mu.M, 3.3. mu.M, 22. mu.M, 52. mu.M DOPS, scale 20 μ M; (g) - (i) is the red and green scatter plot of white blood cells stained with 9.38. mu.M, 52.5. mu.M, 225. mu.M AO for whole blood.

Fig. 4 is a comparison of image analysis results with clinical results. First behavioral correlation analysis (diagonal): the solid line is the condition that the experimental value is completely consistent with the clinical value, the region between the two dotted lines is a 95% confidence interval, and the region between the two dotted lines is an allowable error range of the industry standard; the vertical dashed line represents the clinical reference normal range for blood cell counts. Second behavior Bland-Altman analysis: the solid line represents the mean difference between experimental and clinical values for the samples tested (20), and the region between the two dashed lines is the 95% confidence interval.

Detailed Description

The preparation method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

Material, sample collection and instrumentation

Acridine orange (AO, 75% dye content, in the following experiments, AO concentrations are the effective concentrations converted) and Sodium Dodecyl Sulfate (SDS) were purchased from Sigma Aldrich trade, Inc. N, N-dimethyl-N- (3-sulfopropyl) -1-octadecanamine inner salt (DOPS) was purchased from Shanghai Bigdi medical science, Inc. Phosphate (PBS) buffer solution was purchased from Sammer Feishil science, Inc. Polydimethoxysilane (PDMS) was purchased from Dow Corning, dioxygen and 98% concentrated sulfuric acid were purchased from Jiangtai laboratory instruments, Inc. microscope slides (25mm × 75mm, 1-1.2mm thick) and coverslips (18mm × 18mm,0.13-0.16mm thick) were purchased from Jiangtai laboratory instruments, Inc. glass cement was purchased from Wacker Chemicals, Inc. clinical analysis was performed using Sysmex (4000 i).

The invention uses a microscope cell counter which is specially designed and built to measure all blood samples, and comprises an objective lens of Nikon 4x,0.2NA, two L ED lamps, an automatic filter wheel and filter, and other mechanical parts, a red L ED lamp (with the wavelength of 620nm) is used as a bright field light source, a blue L ED lamp (with the wavelength of 470nm) is used as a fluorescent light source, the automatic filter wheel is used to convert the two fluorescent filters to obtain fluorescent images of two channels (red: 685 +/-20 nm, green: 528 +/-19 nm), a computer-controlled electric transfer table is used to move a sample cell to obtain the image of the whole sample cell, the 4x objective lens is used for large-field imaging and high-flux counting cells, the detector is a CCD camera (SBIG STF-8300M), and software is written by BCB language.

The sample cell is placed on the motorized translation stage of the microscope. By moving the stage, we imaged 5-6 consecutive areas to obtain information on the entire sample cell. For each region, the bright field, red fluorescence, green fluorescence images were recorded. The entire pool of contiguous areas was connected to each other to form a complete picture, and the background was subtracted. Analysis of bright field imaging (using template matching) to count red blood cells, analysis of fluorescence imaging (using color coding) to count platelets and white blood cells, and classification of white blood cells.

EXAMPLE 1 kit preparation

1.1 sample cell fabrication

A mixture (10: 1 mass ratio) of PDMS (polydimethoxysilane) and a curing agent was poured into a petri dish on which a mold was fixed, and cured in a constant temperature drying oven at 80 ℃ for 1 hour. And forming a pattern matched with the mold at the bottom of the cured PDMS, slightly moving the PDMS out, and cutting off the edge part to obtain the PDMS stamp in the shape of a cuboid. The mould is divided into two types, namely a rectangle with the width of 6mm and a rectangle with the width of 3mm (the length is preferably slightly larger than 18 mm); the bottom patterns of the two PDMS stamps are respectively a sunken rectangle with the width of 6mm and a rectangle with the width of 3 mm.

When a hematology counting sample pool is manufactured, a glass slide and a cover glass are wiped by using lens wiping paper and ethanol to remove impurities such as dust on the surfaces of the glass slide and the cover glass, the glass slide and the cover glass are placed in concentrated sulfuric acid (98%): hydrogen peroxide (30%) at a volume ratio of 7: 3 for 1h under ultrasound at 65 ℃ to clean the glass slide and carry out hydrophilic treatment, then the glass slide and the cover glass are ultrasonically cleaned for 3 times at 25 ℃ by using ultrapure water, after the glass slide and the cover glass are dried for 10min each time, two hairs with a length of about 18mm (the diameter of about 100 mu m) are fixed on the surface of a clean hydrophilic glass slide (75mm × mm), the distance between the two hairs is 3mm, a PDMS stamp with a pattern of 6mm width is dipped in glass cement, the PDMS stamp with the bottom adhered with the glass cement is pressed on the glass slide with the fixed hair by fingers, the PDMS stamp is moved away, the pattern of the glass cement is slightly left on the glass slide, the cover glass is pressed, the glass stamp with a length of 18mm 6356 mm, the glass is slightly dried, the sample is formed, and the length of the sample pool is equal to a length of 100 mm, the sample pool, the sample is 3mm, the length of the sample pool, the length of the sample pool is equal to be.

When a leukocyte classification sample cell was prepared, the procedure was similar to the above except that there was no fixing step of hair, and a PDMS stamp with a pattern of 3mm width was used to finally prepare a 18mm × 3mm × 20 μm sample cell (FIG. 1B, sample cell B). The length was the length of the cover glass (18mm), the width was the width of the pattern at the bottom of the PDMS stamp (3mm), and the thickness was the thickness of the glass paste (20 μm).

1.2 reagent preparation

Pre-burying a surfactant and a dye in a centrifugal tube, and preparing two reagents: reagent a for red blood cell, white blood cell and platelet counting; reagent B, for leukocyte classification. The specific manufacturing steps are as follows. Reagent C was PBS buffer (1 ×) in the third centrifuge tube.

Mu. L AO stock solution (9.375mM) was diluted with 952. mu. L PBS to 1M L AO solution (450. mu.M). mu. L DOPS stock solution (26mM) was diluted with 990. mu. L PBS to 1M L DOPS solution (260. mu.M). mu. L AO solution (450. mu.M) was mixed with 500. mu. L DOPS solution (260. mu.M) to prepare a mixed solution of reagent A1: 225. mu.M AO and 130. mu.M DOPS.

Mu. L AO stock solution (9.375mM) was diluted with 900. mu. L PBS to a 1M L0 AO solution (937.5. mu.M), 112. mu. L AO solution (937.5. mu.M) was diluted with 888. mu. L PBS to a 1M L AO solution (105. mu.M), 400. mu. L SDS stock solution (26mM) was diluted with 600. mu. L PBS to a 1M L SDS solution (10.4mM), 500. mu. L AO solution (105. mu.M) was mixed with 500. mu. L SDS solution (10.4mM) to prepare a mixed solution of reagent B1: 52.5. mu.M AO with 5.2mM SDS.

Respectively weighing 10 mu L of the reagent A1 in one centrifuge tube, 5 mu L of the reagent B1 in the other centrifuge tube, placing the reagents in a constant temperature drying oven under the condition of keeping out of the sun and drying for 2h at 60 ℃, and obtaining the reagent A and the reagent B after drying (figure 1 a).

Comparative analysis of surfactants: to simplify the procedure, the amphoteric surfactant DOPS was used in the present invention to spherize erythrocytes (fig. 3 a); in contrast to anionic surfactants such as SDS, DOPS does not affect the fluorescence of AO, see in particular fig. 3 b. Wherein the fluorescence intensity curve of 37.5. mu.M AO was similar to the fluorescence intensity of 37.5. mu.M AO + 22. mu.M DOPS. FIGS. 3c-3f show the concentration optimization of DOPS sphered erythrocytes in the bright field, and the graphs (c) - (f) are for the bright field imaging of diluted blood samples (dilution factor 61) after exposure to 0 μ M, 3.3 μ M, 22 μ M, 52 μ M DOPS, respectively, with a scale of 25 μ M. We have found that a range of 13 μ M to 35 μ M is effective in sphering red blood cells without lysing the red blood cells. For blood samples with higher or lower numbers of red blood cells, too high or too low a concentration of DOPS may correspondingly lyse or globalize the red blood cells insufficiently.

EXAMPLE 2 preparation of blood samples Using the kit

2.1 preparation of samples for blood cell counting

For blood cell counting, as shown in FIG. 1c, 60 μ L PBS was pipetted with a micropipette into reagent A, 1 μ L whole blood was added to the reagent A after the reagent was dissolved, the final dilution factor of the blood was 61X, AO final concentration was 37.5 μ M, DOPS final concentration was 22 μ M, incubation was performed for 5min to ensure complete red cell spheronization, white cell and platelet staining, this sample was named sample A. 5 μ L sample A (5 μ L micropipette) was pipetted into sample well A, and the whole sample well was photographed for light field and fluorescence imaging.

As shown in fig. 2a, cell a (about 100 μm thick) contained a diluted blood sample (dilution factor 61 ×). FIG. 2b shows a superimposed image of fluorescence and bright field in the sample cell, and the inset shows the imaging details of the center and edge regions of the sample cell: the red blood cells present clear bright-field images, the white blood cells present clear fluorescent images, and the red blood cells do not aggregate at the edge of the sample pool to interfere with the identification and counting of the cells.

2.2 preparation of samples for leukocyte Classification

For the leukocyte classification, as shown in FIG. 1c, 5. mu. L of whole blood was pipetted into reagent B, the reagent was lysed, and the leukocyte staining was completed by incubating for 10min to obtain sample B, in which the blood was not diluted, the final concentration of AO was 52.5. mu.M, and the final concentration of SDS was 5.2 mM., 1. mu. L of sample B (1. mu. L microsyringe) was pipetted into sample cell B, and a fluorescence image of the entire sample cell was photographed.

As shown in fig. 2c, well B (about 20 μm thick) contains an undiluted blood sample. FIG. 2d shows a fluorescence image in the sample cell with the inset being a magnified fluorescence imaging detail of the gray solid box area. The thickness of the sample well of 20 μm ensures a morphology of the spread of the leukocyte monolayer, as well as a sufficient number for counting and sorting. FIGS. 3g-3i show scatter plots of the white blood cell triage at different AO concentrations. Increasing the staining concentration of AO appropriately to 52.5. mu.M increased the signal intensity of fluorescence compared to the AO concentration used for cytometry (37.5. mu.M); at the same time, 5.2mM SDS also significantly increased the fluorescence of AO (fig. 3 b).

Example 3 blood cell count and white blood cell classification: 20 groups of clinical blood samples were measured and compared with clinical results

The present invention tested 20 groups of clinical blood samples for their total number of red blood cells, total number of platelets, total number of white blood cells, and white blood cell classification, and compared the results of the testing according to the apparatus and method of the present invention with the clinical results.

Fig. 4 shows the comparison results, and the first and third rows of fig. 4 show the correlation (indicated by oblique lines) between the clinical values and the image analysis values, wherein the solid line shows the complete coincidence of the image analysis values and the clinical values, and the area between the two dotted lines is the 95% confidence interval of the clinical measurement, defined as ± 1.96 times the standard deviation, which is the basic parameter of the clinical blood analyzer. The area between the two dotted lines is the allowable error range of the industry standard; the vertical dashed line represents the clinical reference normal range for blood cell counts. As shown, most of the data points fall within the error bars. The second and fourth rows of FIG. 4 are for the Bland-Altman analysis, where the mean and difference between the experimental and clinical values are on the horizontal and vertical axes. The solid horizontal line represents the deviation of all 20 groups of data (defined as the mean of the difference between experimental and clinical values) and the area between the dashed horizontal lines is the 95% confidence interval.

The root mean square error, deviation and 95% confidence interval for each measured parameter for the methods and clinical methods of the invention (clinical automated hematology analyzer analysis) are listed in table 1.

Table 1 root mean square error, deviation and 95% confidence interval between the method of the invention and the clinical method.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit, comprising:

1) a solid mixture 1 comprising a surfactant 1 and a coloring agent 1;

and/or

2) A solid mixture 2 comprising a surfactant 2 and a coloring agent 2;

the surfactants 1, 2 are independently selected from surfactants for sphering red blood cells, wherein surfactant 1 is a solid amphoteric surfactant; the staining agents 1 and 2 are independently selected from staining agents for staining white blood cells and platelets, and can be the same or different; the solid mixture 1 can be used for blood cell counting and white blood cell classification, and the sample pool 2 and the mixture 2 can be used for white blood cell classification; preferably, the solid mixture 1 is used for blood cell counting.

2. The kit according to claim 1, characterized in that said surfactant 1 is a quaternary ammonium solid amphoteric surfactant; more preferably, the surfactant 1 is a solid amphoteric surfactant containing a sulfonic acid group and a quaternary ammonium salt, such as N, N-dimethyl-N- (3-sulfopropyl) -1-octadecamonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-dodecylammonium inner salt, N-dimethyl-N- (3-sulfopropyl) -1-octadecamonium inner salt, etc.; the surfactant 2 is selected from anionic surfactants such as sodium lauryl sulfate; the stain 1 or 2 is selected from acridine orange and the like; the amount of the surfactant 1 and the amount of the coloring agent 1 contained in the solid mixture 1 are calculated by working concentration of 13-35 mu M and 30-50 mu M respectively; the amount of the surfactant 2 and the amount of the coloring agent 2 contained in the solid mixture 2 are calculated by working concentration of 1-10 mM and 45-75 μ M, respectively.

3. The kit according to claim 1 or 2, characterized in that the amount of the surfactant 1 and the amount of the stain 1 contained in the solid mixture 1 are calculated in the working concentration of 13 to 35 μ M and 30 to 50 μ M, more preferably 18 to 30 μ M and 35 to 45 μ M, most preferably 22 μ M and 37.5 μ M, respectively, after the addition of the blood cell sample diluted with the diluent; preferably, the blood cell sample is diluted by a diluent by a factor of 40 to 80 times, more preferably by a factor of 50 to 70 times, and most preferably by a factor of 60 to 62 times.

The amounts of the surfactant 2 and the coloring agent 2 contained in the solid mixture 2 are in working concentrations of 1 to 10mM and 45 to 75. mu.M, more preferably 2 to 8. mu.M and 50 to 65. mu.M, and most preferably 5.2mM and 52.5. mu.M, respectively, after the undiluted blood cell sample is added thereto.

4.A kit according to any of claims 1 to 3, wherein the solid mixture 1 and the solid mixture 2 are present simultaneously, preferably in two separate containers within the kit, e.g. solid mixture 1 is placed in container 1 and solid mixture 2 is placed in container 2;

the kit further comprises a diluent; the diluent is preferably a phosphate buffer solution, and the diluent is preferably placed in a different container than the solid mixture 1, the solid mixture 2, for example, in container 3;

the kit can also comprise one or more microsamplers, preferably three microsamplers, which are used in different volume specifications, such as the volume of 1 mu L, 5 mu L and 50 mu L;

preferably, the kit comprises: two sample cells; placing the solid mixtures 1 and 2 in a container 1 and a container 2 respectively; the diluent is placed in the container 3; and 3 microsamplers of different volumes.

5. The kit according to any one of claims 1 to 4, wherein the kit further comprises one or more sample wells, preferably wherein the kit comprises two sample wells; the thickness of one sample cell is preferably 60-150 mu m, and the thickness of the other sample cell is preferably 10-50 mu m.

6. The kit according to claim 5, wherein the preparation of the sample cell comprises the steps of:

(1) manufacturing a PDMS stamp: pouring PDMS and a curing agent (for example, according to the mass ratio of 8: 1-11: 1) onto a culture dish fixed with a mold, curing (for example, curing at 75-85 ℃ for 0.5-1.5 h), and then removing the culture dish from the mold and cutting the culture dish into a cuboid to prepare a PDMS stamp; the width of the die is selected from a rectangle of 6mm or 3 mm;

(2) optionally, a hair fixation step is performed, wherein when the sample cell is used for blood cell counting, the hair fixation step is performed, wherein two hairs (about 18mm in length) are fixed on the surface of a glass slide (75mm × 25mm in length, for example) and the distance between the two hairs is 3 mm;

(3) dipping the prepared PDMS stamp with glass cement, pressing the PDMS stamp on a glass slide which is optionally subjected to a hair fixing step, and leaving a glass cement pattern, covering a cover glass (such as 18mm × 18mm) on the glass cement pattern, and pressing to form a sample cell;

(4) the size of the sample pool for blood cell counting is preferably 18mm × 3mm × 100 μm, and the size of the sample pool for leukocyte classification is preferably 18mm × 3mm × 20 μm.

7. A blood cell counting device, comprising:

(1) the kit according to any one of claims 1 to 6;

(2) the large-field-of-view imaging device comprises a bright-field light source, a fluorescent light source, an automatic filter wheel, a filter plate, an electric translation table, an objective lens and a CCD detector, wherein the electric translation table is used for moving a sample pool to obtain imaging of the whole sample pool, the objective lens is used for large-field-of-view imaging, the bright-field light source is preferably a red L ED lamp with the wavelength of 620nm, the fluorescent light source is preferably a blue L ED lamp with the wavelength of 470nm, more preferably, the imaging device adopts an Nikon 4x and 0.2NA objective lens, the automatic filter wheel is used for converting two kinds of fluorescent filter plates to obtain fluorescent imaging of two channels (red: 685 +/-20 nm and green: 528 +/-19 nm), images are recorded by a CCD camera (SBIG STF-8300M), and software is written by BCB language.

8. A method of blood count analysis comprising the steps of:

(1) mixing a solid mixture 1 and a blood cell sample added with a diluent, wherein the solid mixture 1 comprises a surfactant 1 and a staining agent 1;

and/or mixing a solid mixture 2 and a blood cell-containing sample (whole blood) without adding a diluent, wherein the solid mixture 2 comprises a surfactant 2 and a staining agent 2;

(2) flowing the mixture obtained in step (1) into a sample cell;

(3) acquiring imaging information of the sample pool by using an imaging device;

the solid mixture 1 can be used for blood cell counting and white blood cell classification, and the sample pool 2 and the mixture 2 can be used for white blood cell classification; preferably, the solid mixture 1 is used for blood cell counting.

9. The method according to claim 8, wherein the method uses the kit of any one of claims 1 to 6 or the blood cell counting device of claim 7; more preferably, the method specifically comprises the following steps:

(1) pre-storing a solid mixture 1 containing a surfactant 1 and a coloring agent 1 in a container 1 and/or pre-storing a solid mixture 2 containing a surfactant 2 and a coloring agent 2 in a container 2;

(2) transferring the diluent into a container 1 in which a solid mixture 1 is stored in advance, adding a whole blood sample, incubating for a certain time (such as 1-10 min), measuring the obtained mixture, and enabling the mixture to flow into a sample cell; and/or adding the blood cell sample without adding the diluent into a container 2 in which the solid mixture 2 is stored in advance, measuring the obtained mixture, and enabling the mixture to flow into the sample pool;

(3) the method comprises the following steps of acquiring sample pool information by adopting an imaging device, specifically, the information acquiring process comprises the following steps: the sample cell is placed on an electric translation stage of a microscope, and a plurality of continuous areas are imaged by moving the translation stage to acquire information of the whole sample cell.

10. The method of claim 9, wherein the diluent is a phosphate buffer solution; the blood cell sample in the sample cell is preferably a body fluid containing blood cells, and more preferably whole blood, saliva, urine, spinal fluid, peritoneal fluid, synovial fluid, milk, or sputum.