CN118225654A - Blood cell counting kit and classification counting method - Google Patents

Abstract

The invention discloses a blood cell counting kit and a classified counting method, comprising a first reagent and a second reagent; the first reagent comprises: buffering agents, cationic surfactants, zwitterionic surfactants, nonionic surfactants, organic alcohols, preservatives; the second reagent includes a fluorescent dye. The invention greatly reduces the dosage of the cationic surfactant by adding the milder zwitterionic surfactant, and uses the cationic surfactant, the zwitterionic surfactant and the nonionic surfactant to compound the system, thereby achieving the purposes of further mildly controlling the treatment degree of the white blood cells, simultaneously taking a small amount of organic alcohol as the fixation effect, and finally, being capable of correctly classifying and counting the nucleated red blood cells, the basophils and other white blood cells in the fresh whole blood sample and the aged blood sample which is placed for 24-48 hours at normal temperature.

Description

Blood cell counting kit and classification counting method

Technical Field

The invention relates to the field of in-vitro diagnosis, in particular to a blood cell counting kit and a classification counting method.

Background

In blood cell analysis, the proper classification of basophils in a blood sample is usually achieved after a specific treatment. In addition, during the measurement of white blood cells, erythrocytes are usually destroyed by lysis, preventing interference with the measurement. However, when the blood sample contains nucleated red blood cells, the nuclei of the nucleated red blood cells are not dissolved by the hemolytic agent, resulting in interference with the measurement of white blood cells.

Aged blood samples refer to blood samples that are placed for a period of time after collection; after the blood sample is isolated, the blood cells still continue to perform metabolic activities, so that oxygen in the sample is continuously consumed and metabolites are continuously produced; however, since the cells have no division regenerating ability after leaving the living tissue, metabolism of proteins, nucleic acids and the like in blood cells is gradually unbalanced and the cells are gradually aged with the time of leaving. Therefore, compared with fresh blood, the aged blood in the same blood sample has the morphology and internal structure of nucleated red blood cells and white blood cells changed, so that the classification effect of nucleated red blood cells and white blood cells of the aged blood is greatly different from that of fresh blood, but in clinical rechecking, the aged blood is still required to accurately area and count nucleated red blood cells, other white blood cells except basophils and basophils.

However, the existing kit is difficult to simultaneously test a fresh sample and an aged sample, so that the classification count is inaccurate when the reagent detects the fresh sample or the aged sample.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a blood cell counting kit and a classification counting method, so as to solve the problem that the kit in the prior art cannot simultaneously meet the measurement of fresh samples and aged samples.

In order to solve the problems, the invention adopts the following solutions:

A blood cell count kit comprising a first reagent and a second reagent; the first reagent comprises: buffering agents, cationic surfactants, zwitterionic surfactants, nonionic surfactants, organic alcohols, preservatives; the second reagent includes a fluorescent dye.

Further, the amount of the buffer is 500-5000mg/L; the dosage of the cationic surfactant is 50-5000mg/L; the consumption of the zwitterionic surfactant is 100-6000mg/L; the dosage of the nonionic surfactant is 500-7000mg/L; the dosage of the organic alcohol in the first reagent is 100-8000 mg/L; the dosage of the preservative is 0.1-2g/L; the solvent of the first reagent is water; the concentration of the fluorescent dye is 1-50mg/100mL, and the solvent is organic alcohol.

Further, the organic alcohol in the first reagent is selected from methanol, ethanol, ethylene glycol, propylene glycol, isopropanol or phenoxyethanol; the solvent organic alcohol of the fluorescent dye is selected from methanol, ethanol or glycol.

Further, the buffer consists of an organic acid and an organic base; the organic acid is selected from citric acid, salicylic acid, malic acid, benzoic acid or phthalic acid; the organic base is selected from sodium citrate, sodium salicylate, potassium benzoate or potassium hydrogen benzoate; the pH of the buffer is 2.0-5.0.

Further, the cationic surfactant is selected from one or a combination of two or more of tetradecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide, dodecyl trimethyl ammonium chloride, sunflower trimethyl ammonium bromide, sunflower trimethyl ammonium chloride, octyl trimethyl ammonium bromide and octyl trimethyl ammonium chloride.

Further, the zwitterionic surfactant is selected from one or a combination of more of betaine type zwitterionic surfactant, amino acid zwitterionic surfactant or imidazoline amphoteric surfactant.

Further, the nonionic surfactant is selected from one or a combination of a plurality of polyoxyethylene nonionic surfactants or polyoxyethylene polyol ester nonionic surfactants.

Further, the preservative is selected from isothiazolinone, phenoxyethanol or sodium benzoate.

A blood cell sorting and counting method comprising the steps of:

Mixing a blood sample with a first reagent to prepare a cell suspension;

Adding a second reagent to the cell suspension;

detecting forward scattered light and lateral fluorescence signals of the cell suspension;

Sorting and counting nucleated erythrocytes, basophils and other leukocytes in blood cells based on forward scattered light and lateral fluorescence signals;

The first reagent comprises: buffering agents, cationic surfactants, zwitterionic surfactants, nonionic surfactants, organic alcohols, preservatives; the second reagent includes a fluorescent dye.

Compared with the prior art, the invention has the following beneficial effects:

Compared with the prior art, the invention utilizes the characteristic that the basophils and the nucleated erythrocytes can keep the form stably under the acidic condition, and can simultaneously determine the nucleated erythrocytes, the basophils and other leukocytes for correct classification and counting through one-time treatment; the use amount of the cationic surfactant is greatly reduced by adding the milder zwitterionic surfactant, and the cationic surfactant, the zwitterionic surfactant and the nonionic surfactant are compounded, so that the treatment degree of the white blood cells is further milder and controllable, and meanwhile, a small amount of organic alcohol is used as a fixing effect, and finally, the nucleated red blood cells, the basophils and other white blood cells in the fresh whole blood sample and the aged blood sample which are placed for 24-48 hours at normal temperature can be correctly classified and counted.

Drawings

FIG. 1 is a scatter plot of the reagent of example 1 of the present invention for basophils and other leukocyte classification in a normal human fresh whole blood sample 1;

FIG. 2 is a scatter plot of the class of basophils and other leukocytes in whole blood sample 1 of example 1 of the present invention for 48 hours of normal human aging;

FIG. 3 is a scatter plot of the reagent of example 2 of the present invention for nucleated red blood cells, basophils, and other white blood cell classification in a fresh whole blood sample 2 of a patient;

FIG. 4 is a scatter plot of the nucleated red blood cells, basophils, and other white blood cell classifications of the agent of example 2 of the present invention used in a whole blood sample 2 aged for 48 hours in a patient;

FIG. 5 is a scatter plot of the reagent of example 3 of the present invention for basophils and other leukocyte classification in normal human whole blood sample 3;

FIG. 6 is a scatter plot of the reagent of example 4 of the present invention for sorting nucleated red blood cells, basophils, and other white blood cells in a fresh whole blood sample 4 of a patient;

FIG. 7 is a scatter plot of the reagent of example 5 of the present invention for classifying nucleated red blood cells, basophils, and other white blood cells in a patient aged whole blood sample 5;

FIG. 8 is a scatter plot of the reagent of example 6 of the present invention for classifying nucleated red blood cells, basophils, and other white blood cells in a patient aged whole blood sample.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art without the exercise of inventive faculty, are intended to be within the scope of the invention.

A blood cell counting kit, comprising a first reagent and a second reagent;

The first reagent comprises:

(a) Buffering agent: 500-5000mg/L, preferably 500-4000mg/L;

(b) Cationic surfactant: 50-5000mg/L, preferably 50-2500mg/L;

(c) Zwitterionic surfactants: 100-6000mg/L, preferably 200-5000mg/L;

(d) Nonionic surfactant: 500-7000mg/L, preferably 500-6000mg/L;

(e) Organic alcohol: 100-8000 mg/L, preferably 600-5000mg/L;

(f) Preservative: 0.1-2g/L, preferably 0.3-1 g/L;

the pH of the reagent is adjusted to 2.0-5.0 by 1mol/L hydrochloric acid, and the first reagent solvent is water.

The second reagent comprises:

(a) Fluorescent dye, the concentration is: 1-50mg/100mL, preferably 1-20mg/100mL; the solvent is an organic alcohol.

The ratio of the first reagent to the second reagent is conventional and common in the art, and preferably the ratio of the first reagent to the second reagent is: 50:1.

The buffer in the first reagent consists of an aromatic organic acid and an organic base at a pH of 2.0-5.0; the organic acid is selected from citric acid, salicylic acid, malic acid, benzoic acid or phthalic acid; the organic base is selected from sodium citrate, sodium salicylate, potassium benzoate or potassium hydrogen benzoate.

Buffers are used to stabilize the pH of the hemolysis agent under acidic conditions to act as components of the reagent. Under acidic conditions, both basophils and nucleated erythrocytes are stable, and when the blood sample is treated with the hemolytic agent, nucleated erythrocytes, basophils and other leukocytes can be measured simultaneously, and correctly classified and counted.

The buffer pH is controlled between 2.0 and 5.0, preferably between 2.0 and 4.0.

The cationic surfactant in the first reagent is quaternary ammonium salt cationic surfactant with chain length of 8-14, and the cationic surfactant can be one cationic surfactant or a combination of two or more cationic surfactants.

The magnitude of the hemolytic ability of the cationic surfactant is positively correlated with the chain growth of the main chain thereof, and the more the C atoms of the main chain are, the stronger the hemolytic ability is, and the required amount is correspondingly reduced. The optimal amount is that red blood cells and platelets are fully dissolved into cell fragments, and simultaneously, cell membranes of white blood cells and nucleated red blood cells are partially damaged to the extent that dyes can fully enter the inside of the cells, and the optimal amount is smaller than the amount when all white blood cells are naked.

The cationic surfactant is selected from one or more of tetradecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium chloride, dodecyl trimethyl ammonium bromide, dodecyl trimethyl ammonium chloride, sunflower trimethyl ammonium bromide, sunflower trimethyl ammonium chloride, octyl trimethyl ammonium bromide and octyl trimethyl ammonium chloride.

The cationic surfactant is used for hemolyzing erythrocytes in the whole blood sample, so that the cell membranes of leukocytes and nucleated erythrocytes except basophils are greatly damaged, and each cell is shrunken, namely nuggeted; and simultaneously, the cell membranes of the white blood cells and the nucleated red blood cells are treated to cause damage which allows the passing degree of the fluorescent dye to be satisfied.

The zwitterionic surfactant in the first reagent may be one zwitterionic surfactant or a combination of two or more zwitterionic surfactants. The zwitterionic surfactant is selected from betaine type zwitterionic surfactants: dodecyl dimethyl betaine, cocamidopropyl betaine, lauramidopropyl betaine, lauryl dihydroxyethyl betaine, and the like; or from amino acid zwitterionic surfactants: sodium dodecylaminopropionate, 3-sulfopropyl cetyl dimethyl ammonium, and the like, or imidazoline amphoteric surfactant: sodium cocoyl amphoacetate, 3-sulfopropyl dodecyl dimethyl betaine, and the like.

The zwitterionic surfactant has good compatibility with the cationic surfactant and the nonionic surfactant; the amphoteric ion surfactant can carry positive and negative ions at the same time, so that the amphoteric ion surfactant has a good stabilizing effect on leucocyte membranes, particularly on aged samples, and can relieve excessive damage of the cationic surfactant on leucocytes.

The nonionic surfactant in the first reagent may be one nonionic surfactant or a combination of two or more nonionic surfactants. The nonionic surfactant is selected from polyoxyethylene nonionic surfactants, polyoxyethylene ether type: polyoxyethylene lauryl ether, polyoxyethylene (20) cetyl ether, polyoxyethylene nonylphenol ether and the like; polyoxyethylene polyol ester type: polyoxyethylene sorbitan laurate (Tween-20), polyoxyethylene sorbitan stearate (Tween-40), and polyoxyethylene sorbitan oleate (Tween-60).

Nonionic surfactants can treat leukocytes more gently to some extent and lyse cell debris; can shrink the internal substances of cells and can amplify the difference of scattered light intensities of different internal structures of leukocyte subclasses to a certain extent.

The organic alcohol in the first reagent is selected from methanol, ethanol, ethylene glycol, propylene glycol, isopropanol, phenoxyethanol, and the like. The micromolecular organic alcohol can help the shrinkage of substances such as proteins in cells, prevents excessive loss of cytoplasm in nucleated red blood cells and white blood cells, thereby protecting cell membranes of the nucleated red blood cells and the white blood cells from being excessively damaged by cationic surfactant and maintaining the stability of certain forms and internal structures of the nucleated red blood cells and the white blood cells; especially for aged blood, the cells themselves become extremely fragile, and the small molecular organic alcohol helps to maintain the fixation of proteins inside nucleated red blood cells and white blood cells in the aged blood, maintain the cellular characteristics thereof, and maintain the possibility of classifying nucleated red blood cells and white blood cells in the aged blood.

The preservative in the first reagent is selected from isothiazolinone, phenoxyethanol, sodium benzoate and the like; can inhibit the growth of microorganisms during long-term storage of the hemolytic agent in the effective period, thereby preventing deterioration of the reagent and ensuring more stable long-term storage and use of the reagent.

The fluorescent dye in the second reagent is selected from the polymethine dye/cyanine dye excited in the wavelength range of 630-650 nm; the dye can be specifically combined with nucleic acid substances (such as DNA, RNA and organelles with similar properties) in cells, and can excite fluorescent dye under the irradiation of red laser in the wavelength range of 630-650nm, and different types of cells generate difference in fluorescence intensity due to different amounts of nucleic acid substances in the cells, and are further amplified and identified through fluorescence signal intensity. And the affinity of the fluorescent dye to white blood cells is stronger than that of nucleated red blood cells, so that the white blood cells can be more obviously dyed. Based on the difference in fluorescent signals from cells stained with the fluorescent dye, nucleated erythrocytes, leukocytes other than basophils, and basophils can be recognized more efficiently. Fluorescent dyes are capable of staining nucleic acids.

The organic alcohol in the second reagent is selected from methanol, ethanol or glycol; the long-term stability of the fluorochrome in aqueous solutions is poor, whereas the stability in non-aqueous solutions is greatly increased, so that a suitable solvent, preferably an organic solvent, needs to be selected for dissolving the fluorochrome.

The invention also discloses a blood cell classifying and counting method, which comprises the following steps:

(a) Mixing a blood sample with a first reagent to prepare a cell suspension;

(b) Adding a second reagent to the cell suspension, and detecting forward scattered light and lateral fluorescence signals of the cell suspension;

(c) Nucleated erythrocytes, basophils and other leukocytes in blood cells were classified and counted based on forward scattered light and lateral fluorescence signals.

The method comprises the following specific steps: 1mL of the first reagent, 20uL of the second reagent and 20uL of the whole blood sample to be detected are repeatedly mixed, after reaction is carried out for 10 seconds at 40 ℃, the volume information of the cells is measured by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees and the nucleic acid information in the cells is measured by adopting a laser detection method of an excitation light source with 633 nm.

According to the invention, for aged blood which is placed for 24-48 hours at normal temperature after blood sample collection, at the moment, white blood cells, nucleated red blood cells and the like in the sample are more easily damaged, and the existing hemolytic agents on the market often use excessive cationic surfactants to increase hemolysis and difference among cells, at the moment, the aged blood is excessively damaged, and nucleated red blood cells, other white blood cells except basophils and basophils cannot be accurately distinguished. The reagent for blood cell analysis provided by the invention can effectively distinguish nucleated red blood cells, basophils and other white blood cells of a fresh whole blood sample and an aged blood sample which is placed for 24-48 hours at normal temperature at the same time, and accurately count.

Example 1

A blood cell count kit comprising the following components:

A first reagent: 1.5g of salicylic acid, 2.5g of sodium salicylate, 5.0g of octyl trimethyl ammonium bromide, 0.1g of lauramidopropyl betaine, 5.0g of polyoxyethylene (20) cetyl ether, 4.0g of propylene glycol and 0.1g of isothiazolinone,

The pure water is fixed to 1L,1mol/L hydrochloric acid is used for adjusting the pH of the reagent to about 3.0;

and (2) a second reagent: 6.5mg of polymethine dye and 100mL of ethylene glycol.

1ML of the first reagent, 20uL of the second reagent and 20uL of fresh whole blood sample 1 of a normal person to be detected (the normal person sample does not contain nuclear red blood cells) are repeatedly mixed, after reaction is carried out for 10 seconds at 40 ℃, the volume information of the cells is measured by adopting a laser detection method of an excitation light source of 633nm by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescence signal. After the completion of the test, the whole blood sample 1 was left at room temperature for 48 hours, and then retested in the above-described manner.

FIG. 1 is a scatter plot of the reagent of example 1 of the present invention for basophils and other leukocyte classification in a normal human fresh whole blood sample 1. WBC means white blood cells other than basophils, and BASO means basophils.

FIG. 2 is a scatter plot of the class of basophils and other leukocytes in whole blood sample 1 of example 1 of the present invention used for 48 hours of normal human aging. WBC means white blood cells other than basophils, and BASO means basophils.

As can be seen from fig. 1 and 2, the reagent of example 1 is well suited for achieving basophil and other leukocyte classification with a clear demarcation between them for fresh normal human whole blood sample 1. For the normal human whole blood sample 1 aged for 48 hours, the reagent of the embodiment 1 can still well realize the classification of the nucleated red blood cells, the basophils and other white blood cells, has obvious demarcation, and can meet the clinical requirement on the accuracy of the classification of the whole blood cells.

Example 2

A blood cell count kit comprising the following components:

A first reagent: 3.5g of citric acid, 1.0g of potassium hydrogen benzoate, 4.0g of sunflower-base trimethyl ammonium chloride, 5.0g of dodecyl dimethyl betaine, 1.5g of polyoxyethylene sorbitan oleate, 0.5g of phenoxyethanol and 0.6g of isothiazolinone, wherein the pH of the reagent is adjusted to about 3.5 by using 1mol/L hydrochloric acid after the volume is fixed to 1L by pure water;

and (2) a second reagent: 5mg of polymethine dye and 100mL of ethylene glycol;

1mL of the first reagent, 20uL of the second reagent and 20uL of whole blood sample 2 (nucleated red blood sample, 2 nucleated red blood cells in every 100 white blood cells are detected by a mirror), and after reacting for 10 seconds at 40 ℃, the volume information of the cells is measured by using a laser detection method of an excitation light source of 633nm by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescent signal. After the completion of the test, the whole blood sample 2 was left at room temperature for 48 hours, and then retested in the above-described manner.

FIG. 3 is a scatter plot of the reagent of example 2 of the present invention for nucleated red blood cells, basophils, and other white blood cell classification in a fresh whole blood sample 2 of a patient. WBC means white blood cells other than basophils, BASO means basophils, and NRBC means nucleated red blood cells.

FIG. 4 is a scatter plot of the nucleated red blood cells, basophils, and other white blood cell classifications of the agent of example 2 of the present invention used in a whole blood sample 2 aged for 48 hours in a patient. WBC means white blood cells other than basophils, BASO means basophils, and NRBC means nucleated red blood cells.

As can be seen from fig. 3 and 4, the reagent of example 2 is well suited for achieving the classification of nucleated erythrocytes, basophils and other leukocytes with a clear demarcation between them for a fresh patient whole blood sample 2. For the whole blood sample 2 of the patient aged for 48 hours, the reagent of the embodiment 2 can still well realize the classification of the nucleated red blood cells, the basophils and other white blood cells, has obvious demarcation, and can meet the clinical requirement on the accuracy of the classification of the whole blood cells.

Example 3

A blood cell count kit comprising the following components:

A first reagent: malic acid 0.2 g g, potassium benzoate 0.3g, tetradecyl trimethyl ammonium bromide 0.05g, cocamidopropyl betaine 5.5g, polyoxyethylene lauryl ether 7.0g, methanol 0.1g, isothiazolinone 2.0g,

The pure water is fixed to 1L,1mol/L hydrochloric acid is used for adjusting the pH value of the reagent to about 2.0;

And (2) a second reagent: 1mg of polymethine dye and 100mL of ethylene glycol.

1ML of the first reagent, 20uL of the second reagent and 20uL of fresh whole blood sample 3 (normal sample) to be detected are repeatedly mixed, after reaction is carried out for 10 seconds at 40 ℃, the volume information of cells is measured by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees by using a laser detection method of an excitation light source of 633nm, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescence signal.

FIG. 5 is a scatter plot of the reagent of example 3 of the present invention for basophils and other leukocyte classification in normal human whole blood sample 3. WBC means white blood cells other than basophils, and BASO means basophils.

As can be seen from fig. 5, the reagent of example 3 is well suited for achieving basophil and other leukocyte classification with a clear demarcation between them for fresh normal human whole blood samples.

Example 4

A blood cell count kit comprising the following components:

a first reagent: 2.5g of benzoic acid, 2.5g of potassium benzoate, 1.0g of tetradecyl trimethyl ammonium chloride, 0.1g of sodium dodecyl aminopropionate, 0.5g of polyoxyethylene sorbitan laurate, 8.0g of isopropanol and 0.1g of phenoxyethanol, wherein the volume of pure water is fixed to 1L, and 1mol/L hydrochloric acid is used for regulating the pH of the reagent to about 5.0;

and (2) a second reagent: 45mg of cyanine dye and 100mL of ethylene glycol.

1ML of the first reagent, 20uL of the second reagent and 20uL of fresh whole blood sample 4 (patient whole blood sample, nucleated red blood sample) to be detected are repeatedly mixed, 1 nucleated red blood cell in every 100 white blood cells is subjected to microscopic examination, after reaction is carried out for 10 seconds at 40 ℃, the volume information of the cells is measured by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees by using a laser detection method of 633nm, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescent signal.

FIG. 6 is a scatter plot of the reagent of example 4 of the present invention for nucleated red blood cells, basophils, and other white blood cell classification in a fresh whole blood sample 4 of a patient. WBC means white blood cells other than basophils, BASO means basophils, and NRBC means nucleated red blood cells.

As can be seen from FIG. 6, the reagent of example 4 is capable of achieving well-defined classification of nucleated red blood cells, basophils and other white blood cells with a clear demarcation between them for fresh patient whole blood samples containing nucleated red blood cells.

Example 5

A blood cell count kit comprising the following components:

A first reagent: 1g of citric acid, 3.5g of potassium hydrogen benzoate, 4.0g of sunflower-base trimethyl ammonium chloride, 5.0g of dodecyl dimethyl betaine, 1.5g of polyoxyethylene sorbitan oleate, 0.5g of phenoxyethanol and 0.6g of isothiazolinone, wherein the pH of the reagent is adjusted to about 3.5 by 1mol/L hydrochloric acid after the volume of pure water is fixed to 1L;

and (2) a second reagent: 5mg of polymethine dye and 100mL of ethylene glycol;

1mL of the first reagent, 20uL of the second reagent and 20uL of the whole blood sample 5 (nucleated red blood sample, 2 nucleated red blood cells in every 100 white blood cells are detected by a mirror) to be placed for 24 hours at normal temperature are repeatedly mixed, after reaction is carried out for 10 seconds at 40 ℃, the volume information of the cells is measured by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees by using a laser detection method of an excitation light source with 633nm, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescent signal.

FIG. 7 is a scatter plot of the reagent of example 5 of the present invention for classifying nucleated red blood cells, basophils, and other white blood cells in a patient aged whole blood sample 5. The results of the test are shown in FIG. 7, in which WBC represents white blood cells other than basophils, BASO represents basophils, and NRBC represents nucleated red blood cells.

As can be seen from FIG. 7, for the aged whole blood sample 5 of the patient containing nucleated red blood cells, the reagent of example 5 can well realize the classification of nucleated red blood cells, basophils and other white blood cells, and the boundaries between them are obvious, so that the clinical requirement of the classification accuracy of the whole blood cells can be satisfied.

Example 6

A blood cell count kit comprising the following components:

A first reagent: salicylic acid 2.0g, sodium citrate 0.5g, dodecyl trimethyl ammonium chloride 1.0g, 3-sulfopropyl dodecyl dimethyl betaine 1.5g, nonylphenol polyoxyethylene ether 2.0g, ethylene glycol 0.5g, isothiazolinone 0.6g, pure water to 1L,1mol/L hydrochloric acid to adjust the pH of the reagent to about 2.8;

And (2) a second reagent: 8.5mg of polymethine dye and 100mL of ethylene glycol;

1mL of the first reagent, 20uL of the second reagent and 20uL of the whole blood sample 6 (a nucleated red blood sample, 1 nucleated red blood cell in every 100 white blood cells is detected by a mirror) to be placed for 48 hours at normal temperature are repeatedly mixed, after reaction for 10 seconds at 40 ℃, the volume information of the cells is measured by adopting a forward low-angle laser signal with a measuring angle of 2-6 degrees by using a laser detection method with an excitation light source of 633nm, and the nucleic acid information in the cells is measured by adopting a 90-degree lateral fluorescent signal.

FIG. 8 is a scatter plot of the reagent of example 6 of the present invention for nucleated red blood cells, basophils, and other white blood cell classifications in a patient aged whole blood sample 6. As a result of the measurement, WBC represents white blood cells other than basophils, BASO represents basophils, and NRBC represents nucleated red blood cells, as shown in FIG. 8.

As can be seen from FIG. 8, the reagent of example 6 can well achieve classification of nucleated red blood cells, basophils and other white blood cells, and the separation between them is obvious, so as to meet the clinical requirement of the classification accuracy of the whole blood cells.

Compared with the prior art, the invention can simultaneously determine and count the nucleated erythrocytes, the basophils and other leukocytes by one treatment by utilizing the characteristic that the basophils and the nucleated erythrocytes can stably maintain the form under the acidic condition. The method has the advantages that the method is simple in structure, convenient to use, and easy to operate, the use amount of the cationic surfactant is greatly reduced, and the cationic surfactant, the zwitterionic surfactant and the nonionic surfactant are compounded, so that the treatment degree of the white blood cells is further gentle and controllable, a small amount of organic alcohol is added as fixation, and the nucleated red blood cells, the basophils and other white blood cells in a fresh whole blood sample and an aged blood sample which is placed at normal temperature for 24-48 hours can be accurately classified and counted.

The above examples merely represent a few embodiments of the present invention, which are described in more detail and are not to be construed as limiting the scope of the patent. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (9)

1. A blood cell count kit comprising a first reagent and a second reagent; the first reagent comprises: buffering agents, cationic surfactants, zwitterionic surfactants, nonionic surfactants, organic alcohols, preservatives; the second reagent includes a fluorescent dye.

2. A blood cell count kit according to claim 1 wherein said buffer is present in an amount of 500-5000mg/L; the dosage of the cationic surfactant is 50-5000mg/L; the consumption of the zwitterionic surfactant is 100-6000mg/L; the dosage of the nonionic surfactant is 500-7000mg/L; the dosage of the organic alcohol in the first reagent is 100-8000 mg/L; the dosage of the preservative is 0.1-2g/L; the solvent of the first reagent is water; the concentration of the fluorescent dye is 1-50mg/100mL, and the solvent is organic alcohol.

3. A blood cell count kit according to claim 2 wherein the organic alcohol in said first reagent is selected from the group consisting of methanol, ethanol, ethylene glycol, propylene glycol, isopropanol and phenoxyethanol; the solvent organic alcohol of the fluorescent dye is selected from methanol, ethanol or glycol.

4. A blood cell count kit according to claim 1 wherein said buffer is comprised of an organic acid and an organic base; the organic acid is selected from citric acid, salicylic acid, malic acid, benzoic acid or phthalic acid; the organic base is selected from sodium citrate, sodium salicylate, potassium benzoate or potassium hydrogen benzoate; the pH of the buffer is 2.0-5.0.

5. A blood cell counting kit according to claim 1, wherein the cationic surfactant is selected from one or a combination of two or more of tetradecyltrimethylammonium bromide, tetradecyltrimethylammonium chloride, dodecyl trimethylammonium bromide, dodecyl trimethylammonium chloride, sunflower trimethylammonium bromide, sunflower trimethylammonium chloride, octyl trimethylammonium bromide, octyl trimethylammonium chloride.

6. A blood count kit according to claim 1 wherein said zwitterionic surfactant is selected from the group consisting of betaine-type zwitterionic surfactants, amino acid zwitterionic surfactants or imidazoline amphoteric surfactants.

7. A blood cell count kit according to claim 1 wherein said nonionic surfactant is selected from the group consisting of one or more of polyoxyethylene-type nonionic surfactants or polyoxyethylene polyol ester-type nonionic surfactants.

8. A blood count kit according to claim 1 wherein said preservative is selected from isothiazolinone, phenoxyethanol or sodium benzoate.

9. A blood cell sorting and counting method comprising the steps of:

Mixing a blood sample with a first reagent to prepare a cell suspension;

Adding a second reagent to the cell suspension;

detecting forward scattered light and lateral fluorescence signals of the cell suspension;

Sorting and counting nucleated erythrocytes, basophils and other leukocytes in blood cells based on forward scattered light and lateral fluorescence signals;

The first reagent comprises: buffering agents, cationic surfactants, zwitterionic surfactants, nonionic surfactants, organic alcohols, preservatives; the second reagent includes a fluorescent dye.