CN117467734A

CN117467734A - Blood analysis method and blood analyzer

Info

Publication number: CN117467734A
Application number: CN202210853485.3A
Authority: CN
Inventors: 刘宗俊; 陈庚文
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2024-01-30

Abstract

The present invention relates to a blood analysis method comprising:treating the same blood sample to be tested with a dye reagent comprising a first dye and a hemolytic agent for lysing erythrocytes to obtain a sample solution to be tested, the first dye being capable of staining parasites in the blood and comprising a compound having a structure according to formula I; making particles in the sample liquid to be detected pass through an optical detection area one by one and irradiating the particles flowing through the optical detection area with light so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid to be detected after the particles are irradiated with the light, wherein the fluorescence information comprises first fluorescence information from a first dye; and identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information. The invention also relates to a corresponding blood analyzer. The invention can accurately, sensitively, low-cost and rapidly identify parasites in blood.

Description

Blood analysis method and blood analyzer

Technical Field

The present invention relates to the field of in vitro diagnostics, in particular to a blood analysis method and a blood analyzer.

Background

Blood cell parasitic micro-organisms refer to parasites, such as malaria or toxoplasma, that are parasitic in the blood or blood cells of animals. The period of onset of parasites in the blood is short and hazardous, so that early and accurate diagnosis of parasites in blood cells is necessary for effective disease management and monitoring.

Currently, three methods are mainly available for detecting blood cell parasitic micro-organisms. The first method is the accurate detection and quantification of blood cell parasitic micro-organisms by microscopic examination of blood smears, which however is highly dependent on the training and skill of the operator. The second method is a Rapid Diagnostic Test (RDT) based on antigen-antibody reactions, which does not require too high an operating skill, but is generally expensive and lacks sufficient sensitivity for low levels of blood cell parasites. The third method is to realize the detection of parasites in blood cells when blood routine screening is carried out by using a blood cell analyzer, and the method is simple and easy to operate, has low cost and does not depend on the training and skills of operators.

However, the accuracy of detecting parasites in blood with a blood cell analyzer is still not high at present.

Disclosure of Invention

The object of the present invention is therefore to provide a blood analysis method and a blood analyzer which enable a simple, sensitive, rapid and accurate detection of parasites in blood on the basis of flow cytometry, using fluorescent labeling techniques, in a low-cost routine blood detection process.

In order to achieve the above object, a first aspect of the present invention provides a blood analysis method, comprising the steps of:

treating the same blood sample to be tested with a dye reagent comprising a first dye and a hemolytic agent for lysing erythrocytes to obtain a sample fluid to be tested, wherein the first dye is capable of staining parasites in blood and the first dye comprises a compound having the structure of formula i:

wherein R is ₁ And R is ₂ Identical or different, and independently selected from C _1-18 Straight-chain or branched alkyl, C _1-18 Straight-chain or branched alkylene-M, M is selected from the group consisting of sulfonic acid, phenyl, carboxyl, mercapto, amino, R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 Alkyl, Y is absent or a counter anion;

passing particles in the sample liquid to be detected through an optical detection area one by one and irradiating the particles flowing through the optical detection area with light to obtain scattered light information and fluorescence information generated by the particles in the sample liquid to be detected after the particles are irradiated with the light, wherein the fluorescence information comprises first fluorescence information from the first dye; and

identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

The second aspect of the present invention also proposes a blood analyzer comprising:

the sampling device is used for quantitatively sucking a blood sample to be detected;

a sample preparation device having a reaction tank for receiving the blood sample to be tested sucked by the sampling device and receiving a dye reagent containing a first dye and a hemolyzing agent for lysing red blood cells supplied from the reagent supply, and a reagent supply section, the blood sample to be tested sucked by the sampling device being mixed with the dye reagent and the hemolyzing agent supplied from the reagent supply section in the reaction tank to prepare a sample liquid to be tested, wherein the first dye is capable of staining parasites and includes a compound having a structure of formula i:

an optical detection device including a light source for emitting a light beam to irradiate the flow cell, a flow cell which communicates with the reaction cell and through which particles in the sample liquid to be detected can pass one by one, a scattered light detector for detecting scattered light information generated by the particles passing through the flow cell after being irradiated with light, and a fluorescence detector for detecting fluorescence information generated by the particles passing through the flow cell after being irradiated with light, the fluorescence information including first fluorescence information from the first dye; and

A processor configured to acquire the scattered light information and the fluorescence information from the optical detection device and identify parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information.

In the technical schemes provided by the invention, firstly, a cyanine dye capable of dyeing parasites, especially benzothiazole cyanine dye and a hemolytic agent for dissolving red blood cells are adopted to treat a blood sample to be tested so as to obtain a sample liquid to be tested, then, flow cytometry is adopted to obtain scattered light information and fluorescence information of the sample liquid to be tested, and whether the parasites exist in the sample liquid to be tested can be identified based on the scattered light information and the fluorescence information. Thus, parasites in blood can be detected accurately, sensitively, at low cost and rapidly.

Drawings

FIG. 1 is a schematic flow chart of one embodiment of a blood analysis method according to the present invention;

fig. 2 is a first scatter plot for identifying parasites according to one embodiment of the present invention;

FIG. 3 is a schematic flow chart of another embodiment of a blood analysis method according to the present invention;

FIG. 4 is a scatter plot of parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample, in accordance with the present invention;

FIG. 5 is a schematic flow chart diagram of yet another embodiment of a blood analysis method in accordance with the present invention;

FIG. 6 is a scatter plot of parasite identification results and leukocyte classification results obtained simultaneously in one test of the same blood sample in accordance with the present invention;

FIG. 7 is a schematic representation of the emission spectra of two dyes according to an embodiment of the present invention;

FIG. 8 is a schematic diagram of the emission spectrum and excitation spectrum of a large Stokes shift dye according to an embodiment of the present invention;

FIG. 9 is a schematic diagram of an embodiment of a blood analyzer in accordance with the present invention;

FIGS. 10 to 12 are schematic structural views of different embodiments of an optical detection device according to the present invention;

fig. 13 is a scatter diagram of the parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample, according to example 1 of the present invention;

fig. 14 is a scatter diagram of the parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample, according to example 2 of the present invention;

fig. 15 is a scatter diagram of the parasite identification results and nucleated red blood cell identification results obtained simultaneously in one test of the same blood sample, according to example 3 of the present invention;

Fig. 16 is a scatter diagram of the results of parasite identification and leukocyte classification obtained simultaneously in one test of the same blood sample in accordance with example 4 of the present invention;

fig. 17 is a scatter diagram of the results of parasite identification and leukocyte classification obtained simultaneously in one test of the same blood sample in accordance with example 5 of the present invention;

fig. 18 is a scatter diagram of the results of parasite identification and leukocyte classification obtained simultaneously in one test of the same blood sample in accordance with example 6 of the present invention;

FIG. 19 shows the change in fluorescence spectrum with increasing DNA concentration of the dye of example 9;

FIG. 20 shows the change in fluorescence spectrum with increasing concentration of RNA for the dye of example 9;

FIG. 21 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations in example 9;

FIG. 22 shows the result of observing the staining of HepG2 living cells by the compound II under the laser microscope in example 10;

FIG. 23 shows the result of observing the staining of HepG2 living cells by the compound III under the laser microscope in example 11;

FIG. 24 shows the evaluation results of the dye stability in example 12;

FIG. 25 shows the evaluation results of dye cell penetrating power in example 13.

Detailed Description

Embodiments of the present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which it is shown, however, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

It should be noted that, the term "first\second\third" related to the embodiment of the present invention is merely to distinguish similar objects, and does not represent a specific order for the objects, it is to be understood that "first\second\third" may interchange a specific order or sequence where allowed.

As a necessary instrument for routine analysis of blood in modern clinical blood department, a blood cell analyzer (also called a blood analyzer and a hemocytometer) taking flow cytometry as a basic principle has the advantages of rapidness, accuracy, extremely simple operation and the like for detecting blood cells, and is an indispensable full-automatic blood cell rapid detection instrument in modern clinical examination.

Based on this, the invention proposes a technical solution for rapid detection of parasites in blood based on flow cytometry and fluorescent labelling techniques.

In an embodiment of the invention, the parasite is selected from: roundworm, hookworm, tapeworm, trichomonas vaginalis, liver fluke, paragonium wegener, toxoplasma gondii, cysticercus suis, trichina, amoeba, leishmania donovani, plasmodium, schistosome, filarial, artemia, scabies, follicular mites, lice, fleas.

As used herein, the term "C _1-18 By straight or branched alkyl "is meant a radical obtained by removing one hydrogen atom from a straight or branched alkane containing from 1 to 18 carbon atoms, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, t-butyl, isobutyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl.

As used herein, the term "C _1-6 Alkyl "refers to a group obtained by removing one hydrogen atom from a straight or branched alkane containing 1 to 6 carbon atoms, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, t-butyl, isobutyl, and the like.

As used herein, the term "C _1-6 By straight chain alkyl "is meant a group derived from a straight chain alkane containing 1 to 6 carbon atoms with one hydrogen atom removed, specific examples of which include, but are not limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl.

As used herein, the term "C _1-18 The straight-chain or branched alkylene group means a group obtained by removing two hydrogen atoms from a straight-chain or branched alkane having 1 to 18 carbon atoms, and specific examples thereof include, but are not limited to, methylene, ethylene, propylene, butylene and the like.

As used herein, the term "C _1-6 The straight chain alkylene group means a group obtained by removing two hydrogen atoms from a straight chain alkane having 1 to 6 carbon atoms.

As used herein, the term "halogen" includes fluorine, chlorine, bromine and iodine.

As used herein, the term "halo" refers to the substitution of a hydrogen on a group or compound with one or more halogen atoms, including perhalo and partially halo.

As shown in fig. 1, an embodiment of the present invention first provides a blood analysis method 100 for rapidly detecting parasites in blood based on flow cytometry and fluorescent labeling techniques, and the blood analysis method 100 includes the following steps S110, S120 and S130.

In step S110, the same blood sample to be measured is treated with a dye reagent containing a first dye and a hemolyzing agent for lysing erythrocytes to obtain a sample solution to be measured. The first dye is capable of staining parasites in the blood. That is, when parasites are present in the blood sample to be tested, treating the blood sample to be tested with the first dye can cause the parasites therein to be stained. The first dye is a cyanine dye, in particular a benzothiazole-based cyanine dye, and comprises a compound having the structure of the general formula i:

wherein R is ₁ And R is ₂ Identical or different, and is independentAt the site selected from C _1-18 Straight-chain or branched alkyl, C _1-18 Straight-chain or branched alkylene-M, M is selected from the group consisting of sulfonic acid, phenyl, carboxyl, mercapto, amino, R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 Alkyl, Y is absent or a counter anion.

For example, in step S110, a hemolytic agent and a dye reagent may be sequentially added to the same blood sample to be tested to obtain a sample solution to be tested, and then the sample solution to be tested is incubated, so that the dye reagent can sufficiently dye the substance to be tested in the sample solution to be tested. In step S110, for example, the dye reagent may be mixed with the hemolysis agent in advance to obtain a mixed reagent, and then the mixed reagent and the blood sample to be tested may be mixed at 250:1-1000: mixing in a volume ratio of 1, and after mixing uniformly, incubating the mixed sample liquid to be tested at a temperature of 25-50 ℃ for 10 seconds-1 minute, preferably for 20-40 seconds.

It will be appreciated that in embodiments of the invention, the haemolytic agent is used to lyse the red blood cells of the blood, breaking them into fragments, but is able to maintain the morphology of the white blood cells substantially unchanged.

In some embodiments, the hemolysis agent may comprise any one or a combination of several of cationic surfactants, nonionic surfactants, anionic surfactants, amphiphilic surfactants, buffer pairs. The cationic surfactant is, for example, at least one or a combination of several selected from dodecyl trimethyl ammonium chloride, octyl trimethyl ammonium bromide and tetradecyl trimethyl ammonium chloride. The nonionic surfactant is, for example, at least one or a combination of several selected from long-chain fatty alcohol polyoxyethylene, alkylphenol polyoxyethylene, fatty acid polyoxyethylene and fatty amine polyoxyethylene. The buffer pair is selected from at least one or a combination of a plurality of phosphates, citrates and Tris-HCl. The anionic surfactant is selected from at least one or a combination of several of dodecylbenzene sulfonic acid, sodium fatty alcohol acyl sulfate, sodium ethoxylated fatty acid methyl ester sulfonate, sodium secondary alkyl sulfonate and alcohol ether carboxylate.

In other embodiments, the hemolytic agent may include at least one of an alkyl glycoside, a triterpenoid saponin, a steroid saponin.

In step S120, particles in the sample liquid to be measured are made to pass through the optical detection area one by one and the particles flowing through the optical detection area are irradiated with light, so as to obtain scattered light information and fluorescence information generated by the particles in the sample liquid to be measured after being irradiated with light. The fluorescence information includes at least first fluorescence information from the first dye, that is, the first fluorescence information includes a fluorescence signal generated by the particles in the sample liquid to be measured after being combined with the first dye under light excitation.

That is, in step S120, scattered light information and fluorescence information of the sample liquid to be measured are obtained based on the flow cytometry principle. When light, such as a laser beam, irradiates the particles flowing through the optical detection zone, the characteristics of the particles themselves (e.g., volume, degree of staining, cell content size and content, nucleus density, etc.) may block or redirect the laser beam, thereby producing scattered light at various angles corresponding to its characteristics, which, upon receipt by the signal detector, may yield optical information related to the structure and composition of the particles, i.e., scattered light information and fluorescence information of the present invention. Here, the scattered light information includes, for example, at least one of forward scattered light information and side scattered light information. Among them, the number and volume of forward scattering photoreactive particles, the complexity of Side Scatter (SS) reaction cell internal structures (such as intracellular particles or nuclei), and the content of nucleic acid substances in Fluorescence (FL) reaction cells. By using the optical information, various particles in the sample liquid to be detected can be identified.

The first dyes proposed according to the invention comprising compounds having the structure of the formula I have one or more of the following advantages: the thermal stability is good; has high biological (parasites, cells) penetrability; can be specifically combined with DNA, and is favorable for specific recognition and accurate measurement of the DNA; the fluorescent dye has good living cell permeability, can enter cells to dye nucleic acid under the condition of not damaging cell membranes, and has low toxicity and low carcinogenicity; blue-green light with smaller wavelength can be used for excitation, so that tiny particles can be identified, and the detection capability of the tiny particles is improved; the common green semiconductor laser can be used as a light source, so that the use cost is greatly reduced; simple structure, easy obtaining of raw materials for preparing the catalyst, high synthesis yield and easy realization of industrialization.

The compounds of the present invention may be synthesized by methods common in the art. Illustratively, the benzothiazole compounds of the present invention may be synthesized by the following methods: starting first with unsubstituted or substituted methylbenzothiazole, this is reacted with R ₂ X (X is F, cl, br or I) is heated for reflux reaction, and the intermediate I in the form of quaternary ammonium salt is obtained. And then, carrying out heating reflux reaction on the connecting molecule 4-hydroxy isophthalaldehyde and the intermediate I (the molar ratio of the intermediate I to the 4-hydroxy isophthalaldehyde is more than or equal to 2) so as to enable the intermediate I to be condensed with the connecting molecule, thereby obtaining the benzothiazole compound.

When R is ₂ Is C _1-18 In the case of straight-chain or branched alkylene-sulphonic acid groups, intermediate I may also be synthesised by the following method: starting from unsubstituted or substituted methylbenzothiazole or the like, intermediate I is obtained by ring opening reaction with the appropriate sultone.

The above method is suitable for R ₁ And R is ₂ The same is true.

When R is ₁ And R is ₂ When not identical, the benzothiazole compounds of the present invention may be synthesized by the following exemplary methods: r is R ₁ Or R is ₂ The substituted methylbenzothiazole reacts with the connecting molecule 4-hydroxy isophthalaldehyde by heating and refluxing (4-hydroxy isophthalaldehyde and R) ₁ Or R is ₂ The molar ratio of substituted methylbenzothiazole is about 2), giving the formyl-containing intermediate I'; allowing the resulting intermediate I' to react with R ₂ Or R is ₁ The substituted methylbenzothiazole is subjected to condensation reaction to obtain the benzothiazole compound.

In the above process, each intermediate or product may be recovered by isolation and purification techniques well known in the art to achieve the desired purity.

The various starting materials used in the above-described process are commercially available or may be prepared from the starting materials known in the art by methods known to those skilled in the art or methods disclosed in the prior art.

In an embodiment of the invention, the scattered light information comprises scattered light signal intensity and the fluorescence information comprises fluorescence signal intensity.

In step S130, parasites in the sample liquid to be tested are identified based on the scattered light information and the first fluorescence information.

In some embodiments, step S130, i.e. identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information, may comprise: generating a first scatter plot based on the scattered light information and the first fluorescence information; and identifying parasites in the sample fluid to be tested based on the first scattergram.

In the embodiment of the invention, the scatter diagram can be a two-dimensional scatter diagram or a three-dimensional scatter diagram, and two-dimensional or three-dimensional characteristic information of a plurality of particles is distributed on the scatter diagram, wherein an X coordinate axis, a Y coordinate axis and a Z coordinate axis of the scatter diagram all represent one characteristic of each particle. For example, in a scatter plot, the X-axis represents forward scattered light signal intensity, the Y-axis represents fluorescence signal intensity, and the Z-axis represents side scattered light signal intensity. It should be noted that the scatter plot herein is not limited to graphical form, but may be in a data form, such as a digital form of a table or list having an equivalent or similar resolution to the scatter plot, or in any other suitable manner known in the art.

Further, the scattered light information may comprise forward scattered light information FSC, in particular forward scattered light signal strength. Accordingly, generating the first scatter plot based on the scattered light information and the first fluorescence information may include: a first scatter plot is generated based on the forward scattered light information FSC, in particular the forward scattered light signal intensity, and the first fluorescence information FL1, in particular the first fluorescence signal intensity, as shown in fig. 2.

Further, as shown in fig. 2, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information may include:

acquiring a parasite characteristic region P1 and a white blood cell region P2 from the first scattergram, wherein the intensity of first fluorescent information of the parasite characteristic region is larger than that of the white blood cell region; and is also provided with

Parasites in the sample liquid to be tested are identified based on the parasite characterization area P1.

Here, through the repeated studies of the inventors, in a scatter diagram having forward scattered light information as an abscissa and first fluorescent information as an ordinate, for example, a two-dimensional scatter diagram shown in fig. 2, a certain specific region exists above a white blood cell group (i.e., white blood cell region) (i.e., the first fluorescent signal intensity of the specific region is not less than the first fluorescent signal intensity of the white blood cell region), when parasites are present in a blood sample, there are always scattered spots of clusters in the specific region, and when parasites are not present in the blood sample, there are no scattered spots of clusters in the specific region. Thus, the scattered dots of clusters in this characteristic region, which may also be referred to as parasite characteristic region, may be used to characterize the parasite particle clusters. When the number of scattered points in the characteristic region exceeds a predetermined threshold value, then the presence of parasites in the blood sample to be tested is considered.

In some embodiments, identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information may include: the number of parasites in the sample liquid to be tested is determined on the basis of the scattered light information, in particular the forward scattered light information, and the first fluorescence information. For example, in the embodiment shown in fig. 2, the number of scattered points in the parasite characterization area P1 can be used to characterize the number of parasites in the sample fluid to be tested.

In some possible specific examples, the scatter data of a large number of blood samples containing parasites (e.g. the number of scatter points in the parasite characterization area P1) and the actual number of parasites in these blood samples may be collected in advance, and a correlation curve of the scatter data of the parasites and the actual number may be obtained by fitting, so as to obtain a corresponding calculation model, e.g. a linear calculation model. In the actual test of the blood sample to be tested, the method of the invention acquires the parasite scattered point data of the blood sample to be tested, and based on the parasite scattered point data of the blood sample to be tested and the predetermined calculation model, the number of parasites in the blood sample to be tested can be estimated, thereby realizing quantitative analysis of the parasites.

In some embodiments, the blood analysis method 100 may further include: and identifying microorganisms in the sample liquid to be detected based on the scattered light information and the first fluorescence information, in particular identifying microorganisms in the sample liquid to be detected based on the first scatter diagram. Therefore, the detection of microorganisms and parasites in blood can be realized simultaneously by one-time detection of the same blood sample to be detected, the blood detection efficiency is improved and the reagent cost is saved under the condition of not increasing the blood consumption.

In the embodiments of the present invention, the microorganism means a microorganism which is difficult to be seen by naked eyes and which can be observed by means of an optical microscope or an electron microscope. Microorganisms include bacteria, viruses, fungi, and a few algae, among others.

In some embodiments, the microorganism is selected from the group consisting of:

bacteria (e.g., staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, bacillus dysenteriae, bacillus pertussis, bacillus diphtheriae, neisseria meningitidis, mycobacterium tuberculosis, clostridium tetani, bacillus leptospiriformis, group A hemolytic streptococcus, brucella, bacillus cholerae, typhoid bacillus, bacillus anthracis, neisseria gonorrhoeae, vibrio cholerae, pseudomonas klebsiella, and paratyphoid A, B or C.paratyphi),

Viruses (e.g., influenza virus, mumps virus, rubella virus, japanese encephalitis virus, dengue virus, epidemic hemorrhagic fever virus, rabies virus, human papilloma virus, polio virus, measles virus, varicella zoster virus, hepatitis virus, novel enterovirus type 70, coxsackie virus type A24 variant, human immunodeficiency virus, poxvirus (e.g., monkey poxvirus)),

fungi (e.g., candida albicans, trichophyton rubrum, and epizoon floccosum),

mycoplasma (such as mycoplasma pneumoniae, ureaplasma urealyticum, mycoplasma hominis, and mycoplasma genitalium),

chlamydia (e.g., chlamydia trachomatis, chlamydia pneumoniae, chlamydia psittaci, and chlamydia of livestock),

rickettsia (e.g., rickettsia praecox, rickettsia moellendorffimbriae, rickettsia tsutsugamushi) and,

actinomycetes (e.g., actinomycetes israeli),

spirochetes (e.g., leptospira, treponema pallidum).

In some embodiments, R ₁ And R is ₂ Independently selected from C _1-6 Straight chain alkyl, C _1-6 The linear alkylene group M is selected from sulfonic acid group, phenyl group, carboxyl group, mercapto group and amino group.

In some embodiments, R ₁ And R is ₂ At least one of which is C _1-18 Straight-chain or branched alkylene-sulfonic acid groups, C _1-18 Straight-chain or branched alkylene-carboxyl groups.

In some embodiments, R ₁ And R is ₂ Is different and independently selected from C _1-6 Straight chain alkyl, benzyl, C _1-6 Straight chain alkylene-carboxyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Straight chain alkylene-mercapto, C _1-6 Linear alkylene-amino groups. In other embodiments, R ₁ And R is ₂ Identical and selected from C _1-6 Straight chain alkyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Linear alkylene-carboxyl groups.

In some embodiments, R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 An alkyl group.

In some embodiments, when R ₃ When hydrogen, R ₁ And R is ₂ Not both methyl and R ₁ And R is ₂ Not both benzyl.

In some embodiments, Y in formula I is a counter anion, in which case Y may be selected from the group consisting of halide (e.g., F ^- 、Cl ^- 、Br ^- 、I ^- )、ClO ₄ ^- 、PF ₆ ^- 、CF ₃ SO ₃ ^- 、BF ₄ ^- Acetate, methanesulfonate or p-toluenesulfonate. In other embodiments, Y in formula I is absent, thisIn this case, the compound may be an inner salt. The "inner salt" is also referred to in the art as a "zwitterionic". For example, the compounds of the present invention may contain both an acidic group (e.g., a sulfonic acid group or a carboxyl group) and a basic group (e.g., an amino group or a thiazole ring) within the molecule, which are neutralized with each other to form an internal salt.

It will be appreciated that when a compound contains a plurality of acidic groups and/or a plurality of basic groups in the molecule, each acidic group and/or each basic group may act as a salt-forming group. Salts of the compounds formed by various salt-forming means are included within the scope of the present invention.

In some embodiments, the compounds of the present invention may have any of the structures shown in table 1 below:

structural formula of the compound of Table 1

Preferably, the compound of the present invention is a compound represented by the above structural formula 5, 6 or 9.

In some embodiments, the dye reagents of the present invention may be stored in a water-soluble organic phase such as glycerol, glycol, ethylene glycol, and the like.

In some embodiments, the dye reagents of the invention may be stored alone or in combination with a hemolysis agent.

In some embodiments, in step S110, the dye reagent may further include a second dye different from the first dye, the second dye being capable of staining cells in blood. At this time, the fluorescence information obtained in step S120 further includes second fluorescence information FL2 from the second dye, particularly second fluorescence signal intensity, that is, the second fluorescence information includes a fluorescence signal generated under light excitation after the particles in the sample liquid to be measured are combined with the second dye. Thus, the cell parameters of the sample liquid to be measured, such as white blood cell parameters, nucleated red blood cell parameters and the like, can be further obtained according to the scattered light information and the second fluorescence information.

As some implementations, a second dye capable of staining nucleated cells, such as white blood cells and nucleated red blood cells, in particular capable of identifying nucleated red blood cells, is used in step S110. Accordingly, as shown in fig. 3, the blood analysis method 100 may further include step S140: at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be measured is obtained based on the scattered light information and the second fluorescent information, and in particular, the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample liquid to be measured are obtained based on the scattered light information and the second fluorescent information. Therefore, the parasite detection and the leucocyte and/or nucleated red blood cell detection in the blood can be simultaneously realized by one-time detection of the same blood sample to be detected, the blood detection efficiency is improved and the reagent cost is saved under the condition of not increasing the blood consumption.

In some embodiments, the second dye used to stain nucleated cells may be a nucleic acid dye capable of binding to a nucleic acid substance within the cells, or may be a protein dye capable of binding to a protein substance within the cells. In one example, the second dye for staining nucleated cells includes, for example, a compound having the following chemical formula iii.

Further, the scattered light information may comprise forward scattered light information FSC. Accordingly, identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information, step S130, may comprise: identifying parasites in the sample liquid to be tested based on forward scattered light information FSC and first fluorescence information FL1, as shown in fig. 4A; and obtaining at least one of the white blood cell count, the basophil count, and the nucleated red blood cell count of the sample liquid to be measured based on the scattered light information and the second fluorescence information, that is, step S140 may include: at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample liquid to be measured is acquired based on the forward scattered light information FSC and the second fluorescence information FL2, as shown in fig. 4B.

As a further implementation, a second dye capable of staining leukocytes in blood, in particular a second dye for leukocyte classification, is used in step S110. Accordingly, as shown in fig. 5, the blood analysis method 100 may further include step S150: and acquiring a leukocyte classification result of the sample liquid to be detected based on the scattered light information and the second fluorescence information. In a specific example, the white blood cells in the sample liquid to be tested can be classified into a lymphocyte population, a monocyte population, a neutrophil population, and an eosinophil population based on the scattered light information and the second fluorescence information, and each type of white blood cell can be counted to obtain a lymphocyte count and/or a proportion of the lymphocyte count to the white blood cell count, a proportion of the monocyte count to the white blood cell count, a proportion of the neutrophil count and/or the neutrophil count to the white blood cell count, and a proportion of the eosinophil count to the white blood cell count. In another example, white blood cells in the sample liquid to be measured may be classified into a lymphocyte group, a monocyte group, a neutrophil group, an eosinophil group and a basophil group based on the scattered light information and the second fluorescence information, and the white blood cells of each type may be counted. Therefore, the parasite detection and the leucocyte detection in the blood can be realized simultaneously by one-time detection of the same blood sample to be detected, the blood detection efficiency is improved and the reagent cost is saved under the condition of not increasing the blood consumption.

In some embodiments, the second dye for leukocyte classification may also be a nucleic acid dye capable of binding to intracellular nucleic acid species, including for example cyanine-based cationic compounds, for further details reference to chinese patent application CN101750274a filed by applicant at 2019, 9, 28, the entire disclosure of which is incorporated herein by reference.

In some embodiments, the second dye for leukocyte classification is a non-DNA or RNA specific nucleic acid dye, e.g., comprising a compound having formula ii below.

Further, the scattered light information may include side scattered light information SSC and forward scattered light information FSC. Accordingly, identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information, step S130, may comprise: identifying parasites in the sample liquid to be tested based on the forward scattered light information FSC and the first fluorescence information FL1, as shown in fig. 6A; and acquiring the white blood cell classification result of the sample liquid to be measured based on the scattered light information and the second fluorescence information, that is, step S150 may include: a white blood cell classification result of the sample liquid to be measured, for example, a white blood cell four classification result as described above, is obtained based on the side scattered light information SSC and the second fluorescent information FL2, as shown in fig. 6B. For example, a second scattergram is generated based on the side scattered light information and the second fluorescence information, and white blood cells in the sample liquid to be measured are classified into a neutrophil group, a lymphocyte group, a monocyte group, and an eosinophil group on the second scattergram according to the gating technique and these cell groups are counted.

Further, step S150 may also include obtaining a white blood cell classification result of the sample liquid to be measured based on the forward scattered light information FSC, the side scattered light information SSC, and the second fluorescence information FL 2.

In some embodiments, where the blood sample to be measured is treated with a dye reagent comprising a first dye and a second dye, in step S120, illuminating the particles flowing through the optical detection zone with light comprises: particles flowing through the optical detection zone are irradiated with light of a single wavelength, in particular blue light, for example blue light having a wavelength of around 450 nm.

Here, in the case where two dyes, i.e., a first dye and a second dye, are excited simultaneously with one excitation light, in order to better distinguish and collect first fluorescent information corresponding to the first dye and second fluorescent information corresponding to the second dye, thereby more accurately distinguishing white cells and parasites in blood by the two dyes under hemolysis conditions, the first dye and the second dye are selected such that the absolute value of the difference in wavelengths corresponding to the peaks of emission spectra of the first dye and the second dye is greater than 30 nm and less than 80 nm. Alternatively or additionally, the first dye and the second dye are selected such that the amount of overlap of the emission spectra of the first dye and the second dye is not more than 50%. By selecting such a first dye and a second dye, the detection interference of the first fluorescent signal and the second fluorescent signal with each other can be greatly reduced, i.e. the discrimination of the first fluorescent signal and the second fluorescent signal can be greatly increased.

Fig. 7 shows a schematic diagram of the emission spectra of the first dye and the second dye, the curve indicated by the dotted line being the emission spectrum 210 of the first dye, and the curve indicated by the solid line being the emission spectrum 220 of the second dye. Wherein, the peak point of the emission spectrum 210 of the first dye is a, and the peak point of the emission spectrum 220 of the second dye is D. Here, the absolute difference (i.e., the difference in wavelength corresponding to the peak value) between the abscissa of the peak point a and the peak point D is greater than 30 nm and less than 80 nm. Further, the amount of overlap of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye may be a ratio of a first polygonal area equal to a curved polygonal area surrounded by three points E, G, and C, and a second polygonal area equal to a curved polygonal area surrounded by the emission spectrum 210 of the first dye (or the emission spectrum 220 of the second dye) and the reference line 230, wherein the reference line 230 is a dashed line transverse to the horizontal axis as shown in fig. 7, which is at 5% of the normalized peak of the emission spectrum 210 of the first dye and the emission spectrum 220 of the second dye. Points E and F are the left and right intersection points of the emission spectrum 220 of the second dye with the reference line 230, respectively, and points B and C are the left and right intersection points of the emission spectrum 210 of the first dye with the reference line 230, respectively. Here, the amount of overlap of the emission spectrum 210 of the first dye with the emission spectrum 220 of the second dye is no more than 50%.

It is further advantageous, especially in the case of irradiation with light of a single wavelength, that the absolute value of the difference between the wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than 40 and less than 80 nm, preferably greater than 50 nm and less than 80 nm, more preferably greater than 50 nm and less than 70 nm, so that the mutual detection interference of the first fluorescent signal and the second fluorescent signal can be further reduced.

Furthermore, it is advantageous if the emission spectra of the first dye and the second dye overlap by no more than 35%, preferably no more than 15%, so that the detection interference of the first fluorescent signal and the second fluorescent signal with each other can also be further reduced. Wherein, the smaller the overlapping amount of the emission spectra of the first dye and the second dye, the more favorable it is to distinguish the first fluorescent signal from the second fluorescent signal.

Next, some embodiments of the first dye of the present invention are described.

In some embodiments, the first dye may be a large stokes shift dye. Here, the large stokes shift dye means a dye in which a difference between wavelengths corresponding to respective peaks of an emission spectrum and an excitation spectrum is larger than a predetermined threshold.

Fig. 8 is a schematic spectrum diagram of a large stokes shift dye whose excitation spectrum (also referred to as absorption spectrum) 310 is shown by a dotted line and emission spectrum 320 is shown by a solid line. Wherein, the peak point of the excitation spectrum 310 is A1, and the peak point of the emission spectrum 320 is A2. The difference between the abscissa of each of the peak points A2 and A1 (i.e., the difference between the wavelengths corresponding to the peaks of each of the emission spectrum and the excitation spectrum) is greater than a predetermined threshold. The predetermined threshold may be, for example, greater than 30 nanometers and less than 150 nanometers, preferably greater than 50 nanometers and less than 100 nanometers.

The use of large stokes shift dyes in particular reduces the detection interference of the first fluorescent signal and the second fluorescent signal with each other.

The present invention also provides a blood analyzer 400 based on flow cytometry and fluorescent labeling techniques. As shown in fig. 9, the blood analyzer 400 includes a sampling device 410, a sample preparation device 420, an optical detection device 430, and a processor 440. The blood analyzer 400 also includes a fluid path system (not shown) for communicating the sampling device 410, the sample preparation device 420, and the optical detection device 430 for fluid communication between these devices.

The sampling device 410 is used for quantitatively sucking a blood sample to be measured. For example, the sampling device 410 has a pipette with a pipette nozzle and has a drive device for driving the pipette to quantitatively aspirate a blood sample to be measured through the pipette nozzle. The sampling device may deliver the collected blood sample to be tested to the sample preparation device 420.

The sample preparation device 420 has a reaction cell and a reagent supply. The reaction tank is used for receiving the blood sample to be measured sucked by the sampling device 410, and receiving the dye reagent containing the first dye and the hemolysis agent for dissolving the red blood cells provided by the reagent supply part, and the blood sample to be measured sucked by the sampling device 410 is mixed with the dye reagent and the hemolysis agent provided by the reagent supply part in the reaction tank to prepare a sample liquid to be measured. The first dye is capable of staining parasites and comprises a compound having the structure of formula i:

Wherein R is ₁ And R is ₂ Identical or different, and independently selected from C _1-18 Straight-chain or branched alkyl, C _1-18 Straight-chain or branched alkylene-M, M is selected from the group consisting of sulfonic acid, phenyl, carboxyl, mercapto, amino, R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 Alkyl, Y is absent or a counter anion.

The optical detection device 430 includes a light source for emitting a light beam to illuminate the flow cell, the flow cell being in communication with the reaction cell and particles in the sample liquid to be measured can pass through the flow cell one by one, a scatter detector for detecting scattered light information generated by the particles passing through the flow cell after being illuminated by the light, and a fluorescence detector for detecting fluorescence information generated by the particles passing through the flow cell after being illuminated by the light, the fluorescence information including first fluorescence information from the first dye.

In this context, a flow cell refers to a chamber adapted to detect focused liquid flow of light scattering signals and fluorescent signals. When a particle, such as a blood cell, passes through the detection aperture of the flow cell, the particle scatters the incident light beam from the light source directed toward the detection aperture in various directions. The light detector may be arranged at one or more different angles relative to the incident light beam to detect light scattered by the particles to obtain a light scattering signal. Since different particles have different light scattering properties, the light scattering signal can be used to distinguish between different populations of particles. In particular, the light scattering signal detected in the vicinity of the incident light beam is generally referred to as a forward light scattering signal or a small angle light scattering signal. In some embodiments, the forward light scatter signal may be detected from an angle of about 1 ° to about 10 ° from the incident light beam. In other embodiments, the forward light scatter signal may be detected from an angle of about 2 ° to about 6 ° from the incident light beam. The light scattering signal detected in a direction at about 90 ° to the incident light beam is generally referred to as a side light scattering signal. In some embodiments, the side scatter signal may be detected from an angle of about 65 ° to about 115 ° from the incident light beam. Typically, fluorescent signals from blood cells stained with a fluorescent dye are also typically detected in a direction that is about 90 ° from the incident light beam.

In some embodiments, the optical detection device 430 includes a forward scatter detector for detecting forward scattered light or a side scatter detector for detecting side scattered light. The optical detection device 430 preferably includes a forward scatter detector and a side scatter detector.

Fig. 10 shows a specific example of the optical detection device 430. The optical detection device 430 has a light source 401, a beam shaping assembly 402, a flow cell 403, and a forward scatter detector 404 arranged in that order. On one side of the flow chamber 403, a dichroic mirror 406 is arranged at an angle of 45 ° to the straight line. Lateral light emitted by the particles in flow chamber 403, a portion of which passes through dichroic mirror 406, is captured by fluorescence detector 405 disposed behind dichroic mirror 106 at a 45 ° angle to dichroic mirror 406; another portion of the side light is reflected by the dichroic mirror 406 and captured by a side scatter detector 407 arranged in front of the dichroic mirror 406 at an angle of 45 ° to the dichroic mirror 406.

The processor 440 is configured to process the optical signals collected by the optical detection device 430 to obtain a desired result, for example, a two-dimensional scattergram or a three-dimensional scattergram may be generated according to various collected optical signals, and a particle analysis may be performed on the scattergram according to a gating (gating) method. The processor 440 may also perform a visualization process on the intermediate or final operation results, which are then displayed by the display device 450.

In some embodiments, the processor 440 includes, but is not limited to, a central processing unit (Central Processing Unit, CPU), a micro control unit (Micro Controller Unit, MCU), a Field programmable gate array (Field-Programmable Gate Array, FPGA), a Digital Signal Processor (DSP), etc., for interpreting computer instructions and processing data in computer software. For example, the processor 440 is configured to execute each computer application program in the computer-readable storage medium, thereby enabling the blood analyzer 400 to perform a corresponding detection procedure and analyze the optical signal detected by the optical detection device 430 in real time.

In addition, blood analyzer 400 includes a first housing 460 and a second housing 470. The display device 450 may be, for example, a user interface. The optical detection device 130 and the processor 140 are disposed inside the second housing 170. The sample preparation device 420 is disposed inside the first housing 460, for example, and the display device 450 is disposed on an outer surface of the first housing 460 and is used for displaying a detection result of the blood cell analyzer, for example. In other embodiments, a computer with a display may be communicatively coupled to the blood analyzer 400, for example, where the computer is installed remotely from the laboratory where the blood cell analyzer is located, such as in a doctor's office.

In an embodiment of the invention, the processor 440 is configured to obtain scattered light information and fluorescence information from the optical detection device 430 and to identify parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information.

In some embodiments, processor 440 may be further configured to perform the following steps in identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

generating a first scatter plot based on the scattered light information and the first fluorescence information; and is also provided with

Identifying parasites in the sample liquid to be tested based on the first scatter diagram.

Further, the scattered light information comprises forward scattered light information, in which case the processor 440 may be further configured to generate a first scatter plot based on the forward scattered light information and the first fluorescence information.

Further, the processor 440 may be further configured to:

acquiring a parasite characteristic region and a leukocyte region from the first scattergram, wherein the intensity of the first fluorescence information of the parasite characteristic region is greater than that of the first fluorescence information of the leukocyte region; and is also provided with

Identifying parasites in the sample liquid to be tested based on the parasite characteristic regions.

In some embodiments, processor 440 may be further configured to perform the following steps in identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information: and acquiring the number of parasites in the sample liquid to be detected based on the scattered light information and the first fluorescence information.

In some embodiments, the processor 440 may be further configured to: and identifying microorganisms in the sample liquid to be detected based on the scattered light information and the first fluorescence information.

In some embodiments, Y in formula I is a counter anion, in which case Y may be selected from the group consisting of halide (e.g., F ^- 、Cl-、Br-、I-)、ClO ₄ -、PF ₆ -、CF ₃ SO ₃ -、BF ₄ -, acetate, methanesulfonate or p-toluenesulfonate. In other embodiments, Y in formula I is absent, in which case the compound may be an internal salt.

In some embodiments, the dye reagent may further include a second dye, different from the first dye, that is capable of staining cells in blood. At this time, the fluorescence information obtained by the optical detection device 430 further includes second fluorescence information from the second dye. Thus, the cell parameters of the sample liquid to be measured, such as white blood cell parameters, nucleated red blood cell parameters and the like, can be further obtained according to the scattered light information and the second fluorescence information.

Fig. 11 shows another specific example of the optical detection device 430. The optical detection device 430 has a laser 431, a front light assembly 432, a flow cell 433, a forward scatter detector 434, a first dichroic mirror 435, a side scatter detector 436, a second dichroic mirror 437, a first fluorescence detector 438, and a second fluorescence detector 439. The first fluorescence detector 438 is configured to detect a first fluorescence signal corresponding to a first dye generated by the particles passing through the flow cell 433 after being irradiated with light, and the second fluorescence detector 439 is configured to detect a second fluorescence signal corresponding to a second dye generated by the particles passing through the flow cell 433 after being irradiated with light. Here, the laser 431, the front light module 432, the flow cell 433, and the forward scattered light detector 434 are sequentially arranged on the optical axis in the optical axis direction, and the front light module is configured to concentrate the excitation light emitted by the laser 431 to the detection region of the flow cell 433 in the particle flow direction so that the particles flowing through the detection region of the flow cell 433 can generate scattered light. On one side of flow cell 433, first dichroic mirror 435 is disposed at a 45 ° angle to the optical axis. A portion of the lateral light generated by the particles as they flow through the detection zone of the flow chamber 433 is reflected by the first dichroic mirror 435 and captured by the lateral scatter detector 436, while another portion of the lateral light passes through the first dichroic mirror 435 to the second dichroic mirror 437, the second dichroic mirror 437 also being arranged downstream of the first dichroic mirror 435 at an angle of 45 ° to the optical axis. A portion of the lateral light transmitted through first dichroic mirror 435 is reflected by second dichroic mirror 437 and captured by first fluorescence detector 438, while another portion is transmitted through second dichroic mirror 437 and captured by second fluorescence detector 439.

In other embodiments, as shown in fig. 12, the forward scatter detector 434 may also be arranged oblique to the optical axis, unlike the optical detection apparatus shown in fig. 11. On the optical axis, a mirror 4341 is arranged downstream of the flow cell in the optical axis direction, which reflects forward scattered light of the particles into a forward scattered light detector 434 arranged obliquely to the optical axis.

As some implementations, the second dye is a dye that is capable of staining leukocytes and nucleated erythrocytes in blood, particularly a dye for identifying nucleated erythrocytes. Accordingly, the processor 440 may be further configured to: and acquiring at least one of a white blood cell count, a basophil count and a nucleated red blood cell count of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes forward scattered light information. Accordingly, the processor 440 may be further configured to: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescent information, and obtaining at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample fluid to be tested based on the forward scattered light information and the second fluorescent information.

As a further implementation, the second dye is a dye that is capable of staining leukocytes in blood, in particular for leukocyte classification. Accordingly, the processor 440 may be further configured to: and acquiring a leukocyte classification result of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

Further, the scattered light information includes side scattered light information and forward scattered light information. Accordingly, the processor 440 may be further configured to: identifying parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescent information, and obtaining a white blood cell classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescent information.

Preferably, the light source may be configured to illuminate the flow cell with light of a single wavelength. For example, the optical detection device 430 has only one laser source that emits blue-green light, in particular a laser source that emits blue light.

Further embodiments and advantages of the blood analyzer 400 provided by the present invention are referred to above in the description of the blood analysis method 100 of the present invention and are not described in detail herein.

The invention also proposes the use of DNA-specific dyes (i.e. dyes that bind specifically to deoxyribonucleic acid) for identifying parasites in blood samples to be tested using flow cytometry. Wherein the DNA specific dye comprises a compound having the structure of formula I:

Wherein R is ₁ And R is ₂ Identical or different, and independently selected from C _1-18 Straight-chain or branched alkyl, C _1-18 A linear or branched alkylene group-M selected from the group consisting of sulfonic acid groups, phenyl groups, carboxyl groups, mercapto groups, and amino groups; r is R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 Alkyl, hydroxy, C _1-6 Alkoxy, halo C _1-6 An alkyl group; and Y is absent or a counter anion.

For further embodiments and advantages of the DNA specific dyes proposed in the present invention, reference is made to the above description of the blood analysis method 100 of the present invention, which is not repeated here.

Example 1

Dye reagent A1 and hemolytic agent B1 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 1 in the above table 1 and the second dye comprises a compound having the chemical formula iii.

Then, 20 microliter of dye reagent A1, 1 milliliter of hemolytic agent B1 and 20 microliter of anticoagulated malaria patient blood sample to be tested are mixed, and the mixture is incubated for 30 seconds at 42 ℃ to form a test sample liquid for measurement; next, a sample liquid to be tested is tested using the sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 13A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 13B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 13A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 13B.

Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the nucleated red blood cell detection in blood by one-time detection of the same blood sample to be detected.

Example 2

Dye reagent A2 and hemolytic agent B2 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 2 in the above table 1 and the second dye comprises a compound having the chemical formula iii.

Then, 20 microliter of dye reagent A2 and 1 milliliter of hemolytic agent B2 are mixed with 20 microliter of anticoagulated malaria patient blood sample to be tested, and the mixture is incubated for 30 seconds at 42 ℃ to form a test sample liquid for measurement; next, a sample liquid to be tested is tested using the sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 14A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 14B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 14A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 14B.

Example 3

Dye reagent A3 and hemolytic agent B3 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 3 in table 1 above and the second dye comprises a compound having the formula:

then, 20 microliter of dye reagent A3 and 1 milliliter of hemolytic agent B3 are mixed with 20 microliter of anticoagulated malaria-sensitive patient blood sample to be detected, and the mixture is incubated for 30 seconds at 42 ℃ to form a detection sample liquid for detection; next, a sample liquid to be tested is tested using the sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention to obtain forward scattered light intensity FSC, first fluorescence intensity FL1, and second fluorescence intensity FL2; generating a first scatter plot as shown in fig. 15A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 15B from the forward scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 15A, and white blood cells, nucleated red blood cells, and basophils in the blood sample to be tested can be identified and counted based on the second scattergram shown in fig. 15B.

Example 4

Dye reagent A4 and hemolytic agent B4 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 2 in the above table 1 and the second dye comprises a compound having the formula ii.

Then, 20 microliter of dye reagent A4 and 1 milliliter of hemolytic agent B4 are mixed with 20 microliter of anticoagulated malaria patient blood sample to be tested, and the mixture is incubated for 30 seconds at 42 ℃ to form a test sample liquid for measurement; next, a sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention is used to test a sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescent light intensity FL1 and second fluorescent light intensity FL2; generating a first scatter plot as shown in fig. 16A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 16B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 16A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 16B, resulting in the white blood cell classification results shown in table 2.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk model BC-6800) using a DIFF channel (using michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 2.

TABLE 2 results of leukocyte classification

As can be seen from Table 2, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

Example 5

Dye reagent A5 and hemolytic agent B5 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 3 in the above table 1 and the second dye comprises a compound having the formula ii.

Then, 20 microliter of dye reagent A5 and 1 milliliter of hemolytic agent B5 are mixed with 20 microliter of anticoagulated malaria patient blood sample to be tested, and the mixture is incubated for 30 seconds at 42 ℃ to form a test sample liquid for measurement; next, a sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention is used to test a sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescent light intensity FL1 and second fluorescent light intensity FL2; generating a first scatter plot as shown in fig. 17A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 17B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 17A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 17B, resulting in the white blood cell classification results shown in table 3.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk model BC-6800) using a DIFF channel (using michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 3.

TABLE 3 results of leukocyte classification

Parameters (parameters)	BC-6800	The method according to the invention
			Percent lymphocyte (%)	30.6	30.3
Percentage of monocytes (%)	5.3	5.4
			Percent neutrophil (%)	59.1	59.2
Percentage of eosinophils (%)	2.3	2.2

As can be seen from Table 3, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

Example 6

Dye reagent A6 and hemolytic agent B6 were first prepared according to the following formulation.

Wherein the first dye comprises a compound having the structural formula 4 in table 1 above and the second dye comprises a compound having the formula:

then, 20 microliter of dye reagent A6 and 1 milliliter of hemolytic agent B6 are mixed with 20 microliter of anticoagulated malaria patient blood sample to be tested, and the mixture is incubated for 30 seconds at 42 ℃ to form a test sample liquid for measurement; next, a sample analyzer having a blue laser with an excitation wavelength of about 450nm according to the present invention is used to test a sample liquid to be tested to obtain forward scattered light intensity FSC, side scattered light intensity SSC, first fluorescent light intensity FL1 and second fluorescent light intensity FL2; generating a first scatter plot as shown in fig. 18A from the forward scattered light intensity information and the first fluorescence intensity, and generating a second scatter plot as shown in fig. 18B from the side scattered light intensity information and the second fluorescence intensity; the presence of malaria parasites in the blood sample to be tested can be identified based on the first scattergram shown in fig. 18A, and the white blood cells in the blood sample to be tested can be four-classified based on the second scattergram shown in fig. 18B, resulting in the white blood cell classification results shown in table 4.

The same blood sample to be tested was tested on an existing blood analyzer (michaelk, model BC-6800) using a DIFF channel (using the michaelk BC-6800 DIFF kit) to obtain the white blood cell classification results shown in table 4.

TABLE 4 results of leukocyte classification

Parameters (parameters)	BC-6800	The method according to the invention
			Percent lymphocyte (%)	30.5	30.3
Percentage of monocytes (%)	10.0	10.2
			Percent neutrophil (%)	54.5	54.2
Percentage of eosinophils (%)	0.9	1.0

As can be seen from Table 4, the results of the classification of white blood cells obtained according to the present invention are substantially identical to those obtained according to the prior BC-6800. Therefore, the embodiment of the invention can simultaneously realize the parasite detection and the leucocyte detection in blood by one-time detection of the same blood sample to be detected.

EXAMPLE 7 Synthesis of Compound I

The structure of compound I can be represented by structural formula 1.

In the first step, the compound (E) -4-2- (5-formyl-2-hydroxystyryl) benzothiazole-3-butyl-1-sulfonic acid inner salt (structure shown on the right side of formula I) is prepared according to formula I below.

Into the reaction flask were added 5mL of methanol, and 1.33mmol of 4-hydroxy-isophthalaldehyde (Shanghai Bi Co., ltd.) and 0.67mmol of (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonate and 2.66mmol of pyridine (Shanghai Bi Co., ltd.) were sequentially added. The reaction was stirred at 80℃for 43 hours.

The reaction mixture was suction-filtered, the filter cake was washed 3 times with 10mL of ethanol, and the filter cake was collected and dried under reduced pressure to give 0.44mmol of a yellow solid powder, which was (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonic acid inner salt. The yield of this reaction of formula I was about 33%.

The compounds were subjected to nuclear magnetic resonance testing and the results are as follows.

1H NMR(400MHz,DMSO-d6)δ＝12.04(s,1H),9.95(s,1H),8.78(d,J＝2.0Hz,1H),8.42-8.40(m,2H),8.34-8.24(m,2H),7.94-7.86(m,2H),7.80(t,J＝7.6Hz,1H),7.18-7.12(m,1H),5.00-4.88(m,2H),2.59–2.56(m,2H),2.08-2.10(m,2H),1.89-1.79(m,2H)。

The second step was to prepare an inner salt of 4- (2- ((E) -3- ((Z)) -2- (3-methylbenzothiazole-2 (3H) -enyl) vinyl) -6-oxo-1, 4-cyclohexadien-1-yl) vinyl) benzothiazol-3-yl) butanesulfonic acid according to the following reaction formula II (compound I).

To the reaction flask was added 5mL of acetic acid, 0.44mmol of the inner salt of (E) -4- (2- (5-formyl-2-hydroxystyryl) benzothiazol-3-yl) -butyl-1-sulfonic acid (structure shown in the left side of reaction formula II) obtained in the second step, 0.72mmol of 2, 3-dimethylbenzothiazole salt (Shanghai Haihong biomedical science and technology Co., ltd.) and 1.58mmol of sodium acetate were sequentially added to the reaction flask, and reacted at 110℃for 12 hours.

The reaction mixture was filtered and the filter cake was purified with 5mL acetonitrile/water=1: 1 for 3 times to obtain a crude product.

The above crude product was purified by preparative HPLC to give about 0.01mmol of a red solid powder which was 4- (2- ((E) -3- ((Z)) -2- (3-methylbenzothiazole-2 (3H) -enyl) vinyl) -6-oxo-1, 4-cyclohexadien-1-yl) vinyl) benzothiazol-3-yl) butanesulfonic acid inner salt (compound I). The yield of reaction formula II was about 20%.

The product was subjected to nuclear magnetic resonance testing and the results were as follows:

1H NMR(400MHz,DMSO-d6)δ＝8.35-8.14(m,4H),8.09-7.94(m,3H),7.91-7.82(m,2H),7.77-7.74(m,1H),7.70-7.63(m,2H),7.59-7.55(m,1H),7.44-7.29(m,1H),6.39(d,J＝9.2Hz,1H),4.68-4.66(m,2H),4.15-4.13(m,3H),2.62-2.58(m,2H),2.01-1.92(m,2H),1.85-1.81(m,2H)。

the test result is verified to be in accordance with the structure of the structural formula 1.

EXAMPLE 8 Synthesis of Compound II

The structure of compound II can be represented by structural formula 2.

In the first step, 2-methyl-3- (butylsulphonic acid) benzothiazole salt is prepared according to the following reaction scheme I (structure is shown on the right side of reaction scheme I).

50mL of toluene was weighed into a vessel, and 34mmol of benzothiazole (left side of the reaction formula I) and 40mmol of 1, 4-butansultone (Shanghai Honghai Biomedicine Co., ltd.) were charged into the vessel. Reflux and stir under nitrogen for 24 hours before stopping the reaction.

The mixture after the reaction was suction filtered, and then the filter cake was washed 3 times with 50mL of ethyl acetate to obtain a yellow solid product, namely 2-methyl-3- (butylsulfonic acid) benzothiazole salt. The yield of this reaction of formula I was about 62%.

In a second step, compound II (4- (2- ((E) -6-oxo-3- ((Z) -2- (3- (4-sulfobutyl) benzo [ d ] thiazol-2-ylidene) ethylidene) cyclohexyl-1, 4-dien-1-yl) vinyl) benzo [ d ] thiazol-3-yl) butanesulfonic acid is prepared according to reaction formula II below.

30mL of acetic acid was weighed into a vessel, and 1.7mmol of the 2-methyl-3- (butylsulfonic acid) benzothiazole salt obtained in the first reaction (structure shown above arrow of reaction formula II), 0.7mmol of 4-hydroxy isophthalaldehyde, and 2.2mmol of sodium acetate were added into the vessel. The reaction was stirred under nitrogen at 80℃for 24 hours.

The reacted mixture was poured into 50mL of a petroleum ether:ethyl acetate=5:1 solution prepared in advance, and then the supernatant was poured out to obtain a crude product. The crude product was slurried with 10mL acetonitrile/water 1:1 to give about 0.34mmol of a brown solid powder, compound II, in about 52% yield.

The product was subjected to nuclear magnetic resonance testing and the results are as follows.

¹ H NMR(400MHz,DMSO-d6，TMS)δ＝8.87(bs,1H),8.42-8.34(m,4H),8.30-8.22(m,2H),8.21-8.07(m,3H),7.88-7.73(m,4H),7.09(d,J＝8.0Hz,1H),5.00-4.95(m,4H),2.68-2.65(m,4H),2.11-2.03(m,4H),1.90-1.84(m,4H).

The test result is verified to be in accordance with the structure of the structural formula 2.

Example 9 evaluation of the specificity of the dye

And (3) measuring a fluorescent intensity change graph of the dye compound II along with the increase of calf thymus DNA and RNA concentration and a linear relation graph of the maximum fluorescent emission peak intensity and calf thymus DNA and RNA concentration to evaluate the specificity of the dye.

An aqueous solution of calf thymus DNA having a certain concentration was prepared, and the absorbance at 260nm was measured by an ultraviolet absorption spectrophotometer to give a concentration of 1.8mM. 100. Mu.L of calf thymus DNA, which had been designated as 1.8mM, was taken and added with 290. Mu.L of water to dilute the aqueous solution of calf thymus DNA at 0.5 mM. 1.5. Mu.L of DMSO (dimethyl sulfoxide) solution of Compound B was prepared, and M-60LN hemolytic agent (Michael) was added to 3mL, and the mixture was placed in a cuvette to measure the fluorescence intensity. Subsequently, 0.6. Mu.L of 0.5mM calf thymus DNA aqueous solution was placed in a cuvette each time, and after the buffer was stirred uniformly, the cuvette was left to stand in an environment of 37℃for 3 minutes, and then the fluorescence intensity was measured. Finally, the calf thymus DNA concentration in the cuvette was 1. Mu.M. Taking the intensity of the maximum fluorescence emission peak of each calf thymus DNA concentration as a linear relation graph of the fluorescence intensity and the calf thymus DNA concentration. Experiments on the linear relationship between the concentration of RNA and the fluorescence intensity were also performed according to the above procedure. The used instrument is an ultraviolet-visible spectrophotometer, and the model is as follows: hp8453; fluorescence spectrophotometer, model: FP-6500.

FIG. 19 shows the change in fluorescence spectrum of Compound II with increasing DNA concentration. FIG. 20 shows the change in fluorescence spectrum of Compound II with increasing RNA concentration. FIG. 21 is a graph showing the linear relationship between fluorescence intensity and calf thymus DNA and RNA concentrations.

As can be seen from the figure, compound II has a concentration dependence on DNA, but not on RNA, indicating that compound II can specifically bind to DNA.

Example 10 staining of HepG2 living cells by Compound II under a laser microscope

10. Mu.L of PBS buffer at 1mM concentration, which was added to a six-well plate in which HepG2 cells were cultured, was added at 37℃with 5% CO ₂ Is incubated for 30min in a cell incubator. Then PBS was washed 3 times with shaking, and then cell culture medium was added thereto, followed by confocal laser scanning microscopy to observe cell morphology. The type of the used instrument is as follows: FV1000IX81, japan.

Fig. 22 shows the observation results. The middle panel is a white field micrograph of compound II staining HepG2 living cells, the left panel is a fluorescent micrograph of compound II staining HepG2 living cells, and the right panel is a superposition of the bright field and fluorescent images. As can be seen from the figure, the compound II can be used for clearly staining HepG2 cell nuclei, which shows that the dye has good permeability and strong nucleic acid staining capability.

Example 11 staining of HepG2 living cells by Compound III of formula 3 under laser microscopy

10. Mu.L of PBS buffer at a concentration of 1mM in compound III was added to a six-well plate in which HepG2 cells were cultured, and incubated at 37℃in a 5% CO2 cell incubator for 30min. Then PBS was washed 3 times with shaking, and then cell culture medium was added thereto, followed by confocal laser scanning microscopy to observe cell morphology. The type of the used instrument is as follows: FV1000IX81, japan.

Fig. 23 shows the observation results. The middle panel is a white field micrograph of compound III staining HepG2 living cells, the left panel is a fluorescent micrograph of compound III staining HepG2 living cells, and the right panel is a superposition of the bright field and fluorescent images. As can be seen from the figure, the compound III can be used for clearly staining HepG2 cell nuclei, which shows that the dye has good permeability and strong nucleic acid staining capability.

Example 12 evaluation of dye stability

The dye is used for commercialized cell dye agents, needs to have certain high-temperature stability, and the existing dye is limited in practical commercialized application due to poor high-temperature stability. In order to improve the situation, the invention designs from the aspect of molecular structure, and develops various novel dyes so as to improve the stability of the dyes.

To evaluate the high temperature stability, 50mg/L of different dye/glycol solutions were placed in a 50℃incubator, 0.5ml of the solutions were measured for dye concentration at different times, and degradation curves were drawn according to the dye concentration at different time points. The dyes tested were: dyes QCy-DT, dye 5 (compound V of formula 5), dye 6 (compound VI of formula 6), dye 9 (compound IX of formula 9) reported in literature (Nucleic Acids Research,2015,Vol.43,No.18 8651-8663). The used instrument is a UV-VIS ultraviolet visible spectrophotometer, model: TCC-240A, SHIMADZU, japan.

FIG. 24 shows degradation curves, four curves corresponding to dye 5, dye 6, dye 9, dye QCY-DT in order from top to bottom. The graph shows that the degradation degree of the dye QCY-DT is the most serious, and the degradation conditions of the dye 5, the dye 6 and the dye 9 are slight, which indicates that the introduction of the electron withdrawing group at the compound R3 can improve the stability of the compound to a certain extent, and is beneficial to the commercial application of the compound such as blood cell staining.

Example 13 evaluation of dye cell penetration

The dye is used for commercialized cell dye agents, and has better cell penetration, and the existing dye has weaker penetration capability on living cells due to smaller molecular structure log P value (logarithmic value of distribution coefficient ratio of a compound in n-octanol and water) and is limited in practical commercialized application. In order to improve the situation, the invention designs from the angle of molecular structure, introduces various chemical groups, and improves the penetration of dye molecules to cells while improving the stability of the dye.

To verify cell penetration, different dye compounds were prepared as 1mM PBS buffer, 10. Mu.L, respectively, and added to 12-well plates of cultured HepG2 cells at 37℃and 5% CO ₂ Is incubated in a cell incubator. Then, at different time points, PBS was used for washing 3 times with shaking, then cell culture medium was added, and fluorescence of each channel was measured by a multifunctional enzyme-labeled instrument (Thermo, USA).

Fig. 25 shows the test results, four curves corresponding to dye 6, dye 9, dye 5, dye QCy-DT in order from top to bottom. As shown in the figure, the dye 5, 6, 9 has better penetration to cells than the dye QCY-DT.

The features or combinations of features mentioned above in the description, in the drawings and in the claims may be used in any combination with one another or individually, as long as they are significant and do not contradict one another within the scope of the invention. The advantages and features described for the blood analysis method provided by the invention are applicable in a corresponding manner to the application of the blood analyzer and the DNA specific dye provided by the invention and vice versa.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, and all equivalent modifications made by the present invention and the accompanying drawings, or direct/indirect application in other related technical fields are included in the scope of the present invention.

Claims

1. A blood analysis method, characterized in that the blood analysis method comprises the steps of:

2. The method of claim 1, wherein identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information comprises:

3. The blood analysis method of claim 2, wherein the scattered light information comprises forward scattered light information; and is also provided with

Generating a first scatter plot based on the scattered light information and the first fluorescence information includes: the first scatter plot is generated based on the forward scattered light information and the first fluorescence information.

4. A method of blood analysis according to claim 3 wherein identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information comprises:

obtaining a parasite characteristic region and a leukocyte region from the first scattergram, the parasite characteristic region having a first fluorescent information intensity greater than a first fluorescent information intensity of the leukocyte region; and is also provided with

5. The method of blood analysis according to any one of claims 1 to 4, wherein identifying parasites in the sample liquid to be tested based on the scattered light information and the first fluorescence information comprises:

and acquiring the number of parasites in the sample liquid to be detected based on the scattered light information and the first fluorescence information.

6. The blood analysis method according to any one of claims 1 to 5, further comprising:

and identifying microorganisms in the sample liquid to be detected based on the scattered light information and the first fluorescence information.

7. The method for analyzing blood according to any one of claims 1 to 6, wherein R ₁ And R is ₂ Independently selected from C _1-6 Straight chain alkyl, C _1-6 A linear alkylene group-M, M being selected from the group consisting of sulfonic acid groups, phenyl groups, carboxyl groups, mercapto groups, amino groups; or alternatively

R ₁ And R is ₂ At least one of which is C _1-18 Linear or branched alkylene-sulfonic acid groups; or alternatively

R ₁ And R is ₂ Is different and independently selected from C _1-6 Straight chain alkyl, benzyl, C _1-6 Straight chain alkylene-carboxyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Straight chain alkylene-mercapto, C _1-6 A linear alkylene-amino group; or alternatively

R ₁ And R is ₂ Identical and selected from C _1-6 Straight chain alkyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Linear alkylene-carboxyl groups.

8. The method of blood analysis according to any one of claims 1 to 7, wherein R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 An alkyl group; and/or

When R is ₃ When hydrogen, R ₁ And R is ₂ Not both methyl and R ₁ And R is ₂ Not both benzyl.

9. The method according to any one of claims 1 to 8, wherein when Y is a counter anion, Y is selected from the group consisting of halide, clO ₄ -、PF ₆ -、CF ₃ SO ₃ -、BF ₄ -, acetate, methanesulfonate or p-toluenesulfonate; when Y is absent, the compound is an inner salt.

10. The method of any one of claims 1 to 9, wherein the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells and nucleated red blood cells in blood, the fluorescent information further comprising second fluorescent information from the second dye; and is also provided with

The blood analysis method further comprises: and acquiring at least one of a white blood cell count, a basophil count and a nucleated red blood cell count of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

11. The blood analysis method of claim 10 wherein the scattered light information comprises forward scattered light information;

Identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information comprises: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescence information; and is also provided with

Obtaining at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample fluid to be tested based on the scattered light information and the second fluorescence information includes: and acquiring at least one of a white blood cell count, a basophil count and a nucleated red blood cell count of the sample liquid to be detected based on the forward scattered light information and the second fluorescence information.

12. The method of any one of claims 1 to 9, wherein the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells in blood, the fluorescent information further comprising second fluorescent information from the second dye; and is also provided with

The blood analysis method further comprises: and acquiring a white blood cell classification result of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

13. The blood analysis method according to claim 12, wherein the scattered light information includes side scattered light information and forward scattered light information;

The obtaining the white blood cell classification result of the sample liquid to be detected based on the scattered light information and the second fluorescence information comprises the following steps: and acquiring a white blood cell classification result of the sample liquid to be detected based on the side scattered light information and the second fluorescence information.

14. The method of any one of claims 10 to 13, wherein illuminating particles flowing through the optical detection zone with light comprises: the particles flowing through the optical detection zone are illuminated with light of a single wavelength.

15. A blood analyzer, the blood analyzer comprising:

16. The blood analyzer of claim 15, wherein the processor is further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

17. The blood analyzer of claim 16, wherein the scattered light information comprises forward scattered light information; and is also provided with

The processor is further configured to generate the first scatter plot based on the forward scattered light information and the first fluorescence information.

18. The blood analyzer of claim 17, wherein the processor is further configured to:

19. The blood analyzer according to any one of claims 15-18, wherein the processor is further configured to perform the following steps when identifying parasites in the sample fluid to be tested based on the scattered light information and the first fluorescence information:

20. The blood analyzer of any one of claims 15-19, wherein the processor is further configured to:

21. The blood analyzer of any one of claims 15 to 20, wherein R ₁ And R is ₂ Independently selected from C _1-6 Straight chain alkyl, C _1-6 A linear alkylene group-M, M being selected from the group consisting of sulfonic acid groups, phenyl groups, carboxyl groups, mercapto groups, amino groups; or alternatively

R ₁ And R is ₂ Identical and selected from C _1-6 Straight chain alkyl, C _1-6 Straight chain alkylene-sulfonic acid group, C _1-6 Linear alkylene-carboxyl groups; and/or

R ₃ Selected from hydrogen, sulfonic acid group, halogen, cyano group, C _1-6 An alkyl group; and/or

When R is ₃ When hydrogen, R ₁ And R is ₂ Not both methyl and R ₁ And R is ₂ Not both benzyl; and/or

When Y is a counter anion, Y is selected from the group consisting of halide and ClO ₄ ^- 、PF ₆ ^- 、CF ₃ SO ₃ ^- 、BF ₄ ^- Acetate, methanesulfonate or p-toluenesulfonate; when Y is absent, the compound is an inner salt.

22. The blood analyzer of any one of claims 15 to 21, wherein the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells and nucleated red blood cells in blood, the fluorescence information further comprising second fluorescence information from the second dye; and is also provided with

The processor is further configured to: and acquiring at least one of a white blood cell count, a basophil count and a nucleated red blood cell count of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

23. The blood analyzer of claim 22, wherein the scattered light information comprises forward scattered light information; and is also provided with

The processor is further configured to: identifying parasites in the sample fluid to be tested based on the forward scattered light information and the first fluorescent information, and obtaining at least one of a white blood cell count, a basophil count, and a nucleated red blood cell count of the sample fluid to be tested based on the forward scattered light information and the second fluorescent information.

24. The blood analyzer of any one of claims 15 to 21, wherein the dye reagent further comprises a second dye different from the first dye, the second dye being capable of staining white blood cells in blood, the fluorescence information further comprising second fluorescence information from the second dye; and is also provided with

The processor is further configured to: and acquiring a white blood cell classification result of the sample liquid to be detected based on the scattered light information and the second fluorescence information.

25. The hematology analyzer of claim 24, wherein the scattered light information includes side scattered light information and forward scattered light information; and is also provided with

The processor is further configured to: identifying parasites in the sample liquid to be tested based on the forward scattered light information and the first fluorescent information, and obtaining a white blood cell classification result of the sample liquid to be tested based on the side scattered light information and the second fluorescent information.

26. The blood analyzer of any one of claims 22-25, wherein the light source is configured to illuminate the flow cell with a single wavelength of light.