CN112114000A

CN112114000A - Cell analyzer, method for classifying leukocytes based on impedance method and computer-readable storage medium

Info

Publication number: CN112114000A
Application number: CN201910531135.3A
Authority: CN
Inventors: 孔繁钢
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Animal Medical Technology Co Ltd
Priority date: 2019-06-19
Filing date: 2019-06-19
Publication date: 2020-12-22

Abstract

A cell analyzer and a method for classifying leukocytes based on an impedance method, the cell analyzer subjecting a sample to be analyzed to a first treatment including treating the sample with a hemolytic agent; performing a second treatment on the sample, wherein the second treatment comprises treating the sample again by using a hemolytic agent so that the quantity of the red blood cell debris in the sample is less than a preset threshold value; assaying the sample after the first treatment to obtain a first set of parameters of leukocytes; measuring the sample after the second treatment to obtain a second parameter set of leukocytes; and obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells. According to the invention, the sample is processed twice, and at least four classes of the white blood cells are accurately classified according to the results of the two times of measurement.

Description

Cell analyzer, method for classifying leukocytes based on impedance method and computer-readable storage medium

Technical Field

The invention relates to the field of cell analysis, in particular to a cell analyzer and a method for classifying leukocytes based on an impedance method.

Background

Human leukocytes are classified into five types, lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and a clinician can diagnose a patient's condition based on the count and percentage of each type of leukocyte, in combination with the clinical symptoms of the patient.

The white blood cells of mammals such as dogs and cats, which are substantially identical in morphology with human white blood cells, are classified into five types, i.e., lymphocytes, monocytes, neutrophils, eosinophils, and basophils, and similarly, clinicians can diagnose the condition of animals based on the count and percentage of each type of white blood cells in combination with the clinical symptoms of the animals, thereby providing a basis for the diagnosis and the evaluation of the therapeutic effect of the animals.

The blood cell analyzers for testing blood samples that are currently commercially available, for example, the blood cell analyzer for testing blood samples of animals, can be roughly classified into three types.

The type I, three-classification blood cell analyzer realized by impedance method, adds a hemolytic agent into blood sample, counts the leucocyte for one time, can perform three classifications to the leucocyte; such hematology analyzers generally do not separate eosinophils and basophils.

The fourth type of hemocyte analyzer realized by impedance method adds a hemolytic agent into blood sample, counts the leucocytes for one time, and can classify the leucocytes for four times; although this blood cell analyzer can separate eosinophils, its accuracy is not satisfactory.

The flow laser technology realizes the five-classification of the white blood cells, has high classification accuracy, but has higher cost of the whole machine, and is not beneficial to the popularization in small and medium-sized pet hospitals.

Disclosure of Invention

The present application provides a cell analyzer and a method for classifying leukocytes based on the impedance method, which will be described in detail below.

According to a first aspect, an embodiment provides a method of classifying leukocytes based on an impedance method, comprising:

adding a diluent and a blood sample into a white blood cell counting cell;

adding a first dose of a hemolytic agent to the white blood cell count cell;

uniformly mixing the liquid in the leukocyte counting cell;

measuring the liquid in the leucocyte counting pool to obtain a first measuring result, wherein the first measuring result comprises lymphocyte percentage, monocyte percentage and granulocyte percentage;

adding a second dose of the same hemolytic agent to the white blood cell counting cell again, or waiting for a preset time;

uniformly mixing the liquid in the leukocyte counting cell;

measuring the liquid in the leukocyte counting cell to obtain a second measurement result, wherein the second measurement result comprises a leukocyte count value, an eosinophil percentage and an eosinophil count value;

classifying the leukocytes by at least four categories based on the first measurement and the second measurement.

In one embodiment, the second measurement further comprises a basophil percentage and a basophil count.

According to a second aspect, an embodiment provides a method of classifying leukocytes based on an impedance method, comprising:

performing a first treatment of the sample to be analyzed, said first treatment comprising treating the sample with a hemolytic agent;

performing a second treatment on the sample, wherein the second treatment comprises treating the sample again by using a hemolytic agent so that the quantity of the red blood cell debris in the sample is less than a preset threshold value;

assaying the sample after the first treatment to obtain a first set of parameters of leukocytes;

measuring the sample after the second treatment to obtain a second parameter set of leukocytes;

and obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells.

In one embodiment, the first set of parameters includes a lymphocyte percentage, a monocyte percentage, and a granulocyte percentage.

In one embodiment, the first treatment is performed using a dose of hemolysing agent such that red blood cell debris affecting the white blood cell count remains in the sample during the first measurement.

In one embodiment, the second set of parameters includes a white blood cell count value, an eosinophil percentage, and an eosinophil count value.

In one embodiment, the second set of parameters further includes a basophil percentage and a basophil count value.

In one embodiment, the second treatment uses a zero dose of hemolytic agent, and the second treatment further comprises waiting for a predetermined time.

In one embodiment, the second treatment is waited for a predetermined time so that the hemolytic agent used in the first treatment continues to act on the sample to make the sizes of neutrophils and eosinophils in the sample different.

In one embodiment, the second treatment is waited for a predetermined time so that the hemolytic agent used in the first treatment continues to act on the sample to make the sizes of neutrophils, eosinophils, and basophils in the sample different.

In one embodiment, the determining the first processed sample to obtain the first set of parameters of the white blood cells comprises: and measuring the sample after the first treatment to obtain a first white blood cell histogram, performing data processing on the first white blood cell histogram to remove the influence of red blood cell fragments, and acquiring the lymphocyte percentage, the monocyte percentage and the granulocyte percentage according to the first white blood cell histogram after the influence of the red blood cell fragments is removed.

In one embodiment, the obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells includes:

subtracting the eosinophil percentage from the granulocyte percentage to obtain the neutrophil percentage;

and respectively calculating to obtain a lymphocyte count value, a monocyte count value and a neutrophil count value according to the leukocyte count value, the lymphocyte percentage, the monocyte percentage and the neutrophil percentage.

subtracting the eosinophil percentage and the basophil percentage from the granulocyte percentage to obtain the neutrophil percentage;

In one embodiment, the hemolytic agent used in the first treatment and the second treatment is the same hemolytic agent.

In one embodiment, the method further comprises: taking out the part of the sample after the first treatment, and measuring the part of the sample to obtain the parameters of the red blood cells.

According to a third aspect, an embodiment provides a method of classifying leukocytes based on an impedance method, comprising:

obtaining a first set of parameters of white blood cells in a blood sample treated with a first dose of a hemolytic agent and a second set of parameters of white blood cells in the blood sample treated with a third dose of a hemolytic agent, wherein the first dose is less than the third dose;

In one embodiment, the second set of parameters of the white blood cells is obtained by continuing to add the hemolytic agent to the blood sample to the third dose.

According to a fourth aspect, an embodiment provides a cellular analyzer comprising:

the white blood cell counting cell comprises a micropore;

a sampling needle assembly for discharging a sample to be analyzed into the white blood cell counting cell;

the diluent pushing component is used for pushing the diluent to the white blood cell counting cell;

a hemolytic agent pushing component used for pushing hemolytic agent to the white blood cell counting pool;

the blending component is used for blending the liquid in the leucocyte counting cell;

a pressure source component that provides pressure to cause liquid in the white blood cell count cell to pass through the microwells;

a resistance detector for detecting the liquid passing through the micro-hole;

a controller and a processor; wherein:

the controller is used for controlling the diluent pushing component and the sampling needle component to respectively push diluent and discharge blood samples to the white blood cell counting cell, controlling the hemolytic agent pushing component to push a first dose of hemolytic agent to the white blood cell counting cell, then controlling the blending component to blend liquid in the white blood cell counting cell, then controlling the pressure source component to provide pressure so that the liquid in the white blood cell counting cell passes through the micropores, and controlling the resistance-type detector to perform first determination on the liquid passing through the micropores;

the controller then controls the hemolytic agent pushing component to push a second dose of the same hemolytic agent to the white blood cell counting cell, or the controller controls to wait for a preset time;

the controller controls the blending component to blend the liquid in the leucocyte counting cell, then controls the pressure source component to provide pressure so that the liquid in the leucocyte counting cell passes through the micropores, and controls the resistance detector to perform second determination on the liquid passing through the micropores;

the processor acquires a first measurement result according to the data obtained by the first measurement of the resistance detector; the processor obtains a second measurement result according to the data obtained by the second measurement of the resistance detector; wherein the first assay result comprises a lymphocyte percentage, a monocyte percentage, and a granulocyte percentage, and the second assay result comprises a leukocyte count value, an eosinophil percentage, and an eosinophil count value;

the processor classifies the leukocytes at least four times according to the first measurement result and the second measurement result.

According to a fifth aspect, an embodiment provides a cell analyzer comprising:

a leukocyte counting pool;

the resistance detector is used for measuring the sample;

the controller is used for controlling the sampling needle assembly to discharge a sample to be analyzed into the white blood cell counting cell, controlling the hemolytic agent pushing component to push hemolytic agent into the white blood cell counting cell so as to perform first treatment on the sample to be analyzed, and controlling the resistance type detector to determine the sample after the first treatment; the controller then controls the hemolytic agent pushing component to push hemolytic agent to the white blood cell counting cell again so as to perform second processing on the sample, wherein the number of red blood cell fragments in the sample is smaller than a preset threshold value, and the resistance type detector is controlled to determine the sample after the second processing;

and the processor is used for acquiring a first parameter set of the white blood cells according to the data obtained by measuring the sample after the first treatment by the resistance type detector, acquiring a second parameter set of the white blood cells according to the data obtained by measuring the sample after the second treatment by the resistance type detector, and acquiring at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells.

In one embodiment, the controller controls the hemolytic agent pushing component to push the dosage of hemolytic agent used for the first processing to the white blood cell counting cell, so that the sample still has residual red blood cell debris which affects the white blood cell count value when being measured by the resistance type detector for the first time.

In one embodiment, the controller controls the amount of the hemolytic agent used for the second treatment pushed by the hemolytic agent pushing component to the white blood cell counting cell so that the shrinkage rate of the lymphocytes, the monocytes and the neutrophils in the sample is faster than that of the eosinophils.

In one embodiment, the controller controls the amount of the hemolytic agent used for the second treatment pushed by the hemolytic agent pushing component to the white blood cell counting cell so that the shrinkage rate of the lymphocytes, the monocytes and the neutrophils in the sample is faster than that of the eosinophils and the basophils.

In one embodiment, the controller controls the hemolytic agent pushing component to push the leukocyte counting cell for the second treatment with a dosage of hemolytic agent being zero, and controls to wait for a preset time to complete the second treatment.

In one embodiment, the hemolytic agent pushed by the hemolytic agent pushing component for the first treatment and the second treatment is the same hemolytic agent.

According to a sixth aspect, an embodiment provides a cellular analyzer comprising:

a sample preparation component for treating a blood sample with a first dose of a hemolytic agent and treating the blood sample with a third dose of a hemolytic agent;

measuring a blood sample treated with a first dose of a hemolytic agent and a blood sample treated with a third dose of a hemolytic agent to obtain a first parameter set of leukocytes and a second parameter set of leukocytes, respectively;

and the processor is used for obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells.

In one embodiment, the sample preparation component further adds the hemolytic agent to the third dose to obtain the second parameter set of the white blood cells.

According to a seventh aspect, an embodiment provides a computer readable storage medium comprising a program executable by a processor to implement a method as in any of the embodiments herein.

According to the cell analyzer and the method for classifying the white blood cells based on the impedance method of the embodiment, the sample is processed twice, and at least four classifications of the white blood cells are accurately realized according to the results of the two measurements.

Drawings

FIG. 1 is a schematic diagram of the implementation of leukocyte classification based on the impedance method;

FIG. 2 is a schematic view showing the structure of a cell analyzer according to an embodiment;

FIG. 3 is a schematic structural view of a cell analyzer according to another embodiment;

FIG. 4 is a schematic configuration diagram of a cell analyzer according to still another embodiment;

FIG. 5 is a flowchart of a method for classifying leukocytes based on an impedance method according to an embodiment;

FIG. 6 is a flow chart of another embodiment of a method for classifying leukocytes based on an impedance method;

FIG. 7 is a flowchart of a method for classifying leukocytes based on an impedance method according to yet another embodiment;

FIG. 8(a) is a histogram of white blood cells obtained after a first treatment measurement of a blood sample from an example dog; FIG. 8(b) is a histogram of white blood cells obtained after a second treatment measurement of a blood sample from an example dog;

FIG. 9(a) is a histogram of white blood cells obtained after a first treatment measurement of a blood sample from an example cat; FIG. 9(b) is a histogram of white blood cells obtained after a second treatment measurement of a blood sample from an example cat.

Detailed Description

The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. Wherein like elements in different embodiments are numbered with like associated elements. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced with other elements, materials, methods in different instances. In some instances, certain operations related to the present application have not been shown or described in detail in order to avoid obscuring the core of the present application from excessive description, and it is not necessary for those skilled in the art to describe these operations in detail, so that they may be fully understood from the description in the specification and the general knowledge in the art.

Furthermore, the features, operations, or characteristics described in the specification may be combined in any suitable manner to form various embodiments. Also, the various steps or actions in the method descriptions may be transposed or transposed in order, as will be apparent to one of ordinary skill in the art. Thus, the various sequences in the specification and drawings are for the purpose of describing certain embodiments only and are not intended to imply a required sequence unless otherwise indicated where such sequence must be followed.

The numbering of the components as such, e.g., "first", "second", etc., is used herein only to distinguish the objects as described, and does not have any sequential or technical meaning. The term "connected" and "coupled" when used in this application, unless otherwise indicated, includes both direct and indirect connections (couplings).

Referring to fig. 1, a leukocyte classification is realized based on an impedance method, in which a blood sample is first treated with a hemolytic agent to lyse erythrocytes in the blood sample, so as to reduce the influence of erythrocytes on leukocyte classification and counting, and meanwhile, the volume of each type of cells in the leukocytes is inconsistent, and the leukocytes shrink in an inconsistent manner under the action of the hemolytic agent, so that the characteristic of inconsistent volume of each type of cells is further amplified; then under the negative pressure, the blood cells pass through the detection small holes in the leucocyte counting cell one by one, constant current sources are added on two sides of the detection small holes, corresponding pulses can be generated when the cells pass through the detection small holes, the larger the cell volume is, the larger the resistance is increased when the cells pass through the detection small holes, and therefore the generated pulses are larger, namely the amplitude of the pulses is in direct proportion to the volume of the cells, and the frequency of the pulses is in direct proportion to the number of the cells.

Theoretically, the proportion and the number of the five types of leukocytes, namely lymphocytes, monocytes, neutrophils, eosinophils and basophils, can be obtained by five types of leukocyte classification of leukocytes through the method, but in the actual process, only three types of leukocyte classification, namely classification and counting of lymphocytes, monocytes and granulocytes can be generally realized, and eosinophils, basophils and neutrophils can not be further distinguished from granulocytes. In some embodiments, four classifications may be achieved by modifying the structure of the components of the hemolytic agent, i.e., eosinophils may also be separated individually, but accuracy is less than ideal.

The invention realizes at least four classifications of leucocyte more accurately according to the results of two measurements by carrying out two times of treatments and two times of measurements on the sample or the blood sample.

For example, in some embodiments, a hemolytic agent is added twice to the same blood segment of the sample, and after each hemolytic agent addition, a determination is performed once to obtain a histogram of leukocytes, and at least four classifications of leukocytes are finally achieved according to the two histograms of leukocytes; the two added hemolytic agents may be the same hemolytic agent in some embodiments, and may be different hemolytic agents in other embodiments.

For example, in some embodiments, the hemolytic agent is added to the same blood segment of the sample, then the measurement is performed once to obtain the histogram of the white blood cells, then the predetermined time is waited for, so that the hemolytic agent acts on the white blood cells continuously, then the measurement is performed once again to obtain the histogram of the white blood cells, and finally at least four classifications of the white blood cells are realized according to the two histograms of the white blood cells.

For another example, in some embodiments, a hemolytic agent is added to each of two blood segments of the sample, and then the two blood segments are measured, so as to obtain a histogram of leukocytes of the blood segments, and at least four classifications of leukocytes are finally realized according to the histograms of leukocytes of the two blood segments; similarly, the two blood sections are added with the hemolytic agent respectively, and in some embodiments, the two blood sections can be the same hemolytic agent or different hemolytic agents.

The present invention will be described in detail below.

Referring to fig. 2, one embodiment discloses a cell analyzer comprising a white blood cell counting chamber 10 having a micro-well 11, a sampling needle assembly 20, a hemolytic agent pushing component 30, a resistance detector 40, a controller 50 and a processor 60, it being understood that the controller 50 and the processor 60 may be integrated in the processing and controlling components in some examples, or may be separate components in some examples. The sampling needle assembly 20 is used to discharge the sample to be analyzed into the white blood cell 10. The hemolytic agent pushing member 30 is used for pushing hemolytic agent to the white blood cell counting cell 10. The resistance detector 40 is used to measure the sample, and the measurement principle of the resistance detector 40 is based on the above-mentioned impedance method, i.e. measuring the cells passing through the micropores 11 of the leucocyte counting cell 10, and generating corresponding pulses, and outputting the data to the processor 60.

In one embodiment, the controller 50 controls the sampling needle assembly 20 to discharge the sample to be analyzed into the white blood cell counting chamber 10, and controls the hemolytic agent pushing member 30 to push the hemolytic agent into the white blood cell counting chamber to perform a first processing on the sample to be analyzed, and controls the resistive detector 40 to measure the sample after the first processing. In one embodiment, the controller 50 then controls the hemolytic agent pushing component 30 to push the hemolytic agent again to the white blood cell counting chamber 10 to perform the second processing on the sample, and controls the resistance detector 40 to measure the sample after the second processing.

Specifically, the two treatments of the sample may be a first treatment of the sample with a certain amount of hemolytic agent, and a second treatment of the sample with a certain amount of hemolytic agent, where the two treatments of the sample with hemolytic agent may be the same hemolytic agent or different hemolytic agents. The two treatments of the sample may be a first treatment with a certain amount of hemolytic agent and a second treatment with a zero amount of hemolytic agent, that is, the treatment of the sample with hemolytic agent is not performed, but a waiting period is provided, so that the first treatment with hemolytic agent continues to act on the sample. These two cases will be explained separately below.

The first case is to treat the sample twice with a hemolytic agent and to determine chart classification and counting twice, as described in detail below.

In one example, the hemolytic agent used in the first treatment and its dose act to lyse red blood cells in the sample and allow the white blood cells to separate into 3 populations, a lymphocyte population, a monocyte population, and a granulocyte population. In one embodiment, the controller 50 controls the amount of hemolytic agent used for the first processing that the hemolytic agent pushing component 30 pushes to the white blood cell counting cell 10, so that red blood cell debris that affects the white blood cell count value still remains when the sample is measured by the resistance detector 40 for the first time, and thus the difference between the 3 cell groups into which the white blood cells are divided is obvious. The processor 60 obtains a first set of parameters of the white blood cells from the data obtained by the first processing of the sample by the resistance detector 40, wherein the first set of parameters includes a percentage of lymphocytes, a percentage of monocytes, and a percentage of granulocytes. Specifically, the processor 60 may obtain a white blood cell histogram, such as a first white blood cell histogram, based on the data obtained by the resistance detector 40 measuring the first processed sample, and obtain the lymphocyte percentage, the monocyte percentage, and the granulocyte percentage.

In one example, the hemolytic agent and the dosage thereof used in the second treatment are the first action to make the number of erythrocyte fragments in the sample smaller than a predetermined threshold value, so that the erythrocyte fragments do not affect the white blood cell count, the second action to make the shrinkage rate of lymphocytes, monocytes and neutrophils increase, and the shrinkage rate of eosinophils is relatively slow, so that the eosinophil group can be distinguished from the granulocyte group, wherein the actually distinguished eosinophil group comprises the basophil group, but the number of basophil is small relative to the number of eosinophil, so that the distinguished cell group can be approximately regarded as the eosinophil group. In one embodiment, the controller 50 controls the hemolytic agent pushing component 30 to push the dose of hemolytic agent for the second treatment to the white blood cell counting cell 10, so that the shrinkage rate of lymphocytes, monocytes and neutrophils in the sample is faster than that of eosinophils, and the number of red blood cell debris in the sample is smaller than the preset threshold value, i.e. after the sample is treated with the hemolytic agent for the first time and the hemolytic agent for the second time, no red blood cell debris affecting the white blood cell count value exists in the sample in the second measurement, i.e. the number of red blood cell debris in the sample is smaller than the preset threshold value. Processor 60 obtains a second set of parameters for the white blood cells based on data from the second processed sample measured by resistance detector 40, where the second set of parameters includes a white blood cell count value, an eosinophil percentage, and an eosinophil count value in one embodiment. Specifically, processor 60 may derive a leukocyte histogram, such as a second leukocyte histogram, based on data obtained from the second processed sample measured by resistance detector 40, and obtain a leukocyte count, an eosinophil percentage, and an eosinophil count based on the leukocyte histogram. The processor 60 obtains four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells, i.e. four classification and counting of the white blood cells are performed. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the four classification parameters; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the four-classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the four-classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the four-classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the four-classification parameters; subtracting the eosinophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the four-classification parameter, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set to be used as a neutrophil count value in the four-classification parameter; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the four classification parameters; this completes the four sorting and counting of the leukocytes.

The three-classification of the first leukocyte histogram may be performed by any suitable method, for example, as shown in fig. 8(a), first, a first boundary line 1 between a first type of leukocyte and a second type of leukocyte is determined according to a valley point C between two peak points a and B in the first leukocyte histogram, wherein the volume of the first type of leukocyte is smaller than that of the second type of leukocyte, and a region of the first leukocyte histogram having a volume smaller than that of the first boundary line 1 is a first type of leukocyte (e.g., a Lymphocyte (LYM) as shown in fig. 8 (a)).

The two peak points a and B in the first histogram may be determined first, and the valley point C may be determined by any suitable method, for example, the valley point C is the minimum point corresponding to the minimum ordinate value between the peak point a and the peak point B.

Then, continuing as shown in fig. 8(a), a second boundary line 2 between the second type of white blood cell (e.g. Monocyte (MON)) and a third type of white blood cell (e.g. granulocyte (NEU)) is determined according to the first boundary line 1, wherein the second boundary line 2 and the first boundary line 1 are separated by a first predetermined volume, and the volume corresponding to the second boundary line 2 is larger than the volume corresponding to the first boundary line 1;

the first predetermined volume can be reasonably set according to a priori experience, for example, under specific reaction conditions, reaction temperature, reagent (including hemolytic agent and diluent) dosage, the volume of the interval between the valley point and the actual second boundary line 2 under the specific conditions, especially specific hemolytic agent dosage, can be obtained through multiple detections, so as to determine the first predetermined volume, wherein different reaction conditions, reaction temperatures, positions of the valley points of the reagent (including hemolytic agent and diluent) dosage and the first predetermined volume value can be different, and can be reasonably adjusted according to actual conditions.

The second white blood cell histogram classification may be performed by any suitable method, for example, as shown in fig. 8(b), a second critical point D having a slope that is greater than a second threshold slope K for the first time on the curve of the second white blood cell histogram in the direction of volume reduction starting from the maximum volume Vmax (e.g., Vmax ═ 250fL) of the second white blood cell histogram;

wherein the maximum volume Vmax may refer to an end position of a white blood cell in a white blood cell histogram. The slope of a point on the curve of the second white blood cell histogram within a predetermined segment in the volume decreasing direction from the maximum volume Vmax is less than or equal to 0, so the value of the second threshold slope K is set to be less than zero, and specifically the value of the second threshold slope K may be set according to actual circumstances.

Then, a third boundary line 3 between the third type of white blood cells and the fourth type of white blood cells is determined based on the second critical point D, the third boundary line 3 being a straight line passing through the second critical point D and being perpendicular to the abscissa axis of the second histogram of white blood cells, thereby achieving four-classification of white blood cells, the region between the third boundary line 3 and Vmax being a fourth type of white blood cells (e.g., Eosinophils (EOS) — although actually the region between the third boundary line 3 and Vmax is Eosinophils (EOS) and Basophils (BASO), but the number of Basophils (BASO) is small relative to the number of Eosinophils (EOS), and therefore the cells in this region can be considered approximately as both Eosinophils (EOS)), and then the first histogram-derived e.g., granulocyte (NEU) is subtracted from the second histogram-derived E (EOS)), neutrophil NEU) was obtained.

The second histogram classification of leukocytes may be performed by any suitable method, such as determining a fourth borderline 4 between leukocytes of the fourth type and leukocytes of the fifth type on the basis of the third borderline 3, wherein the fourth borderline 4 is distinct from the third borderline 4The three dividing lines 3 are separated by a second predetermined volume S_basoAnd the volume corresponding to the fourth borderline 4 is larger than the volume corresponding to the third borderline 3, the fourth type of white blood cell is the region between the third and fourth borderlines 3 and 4 on the second white blood cell histogram, and the fifth type of white blood cell (e.g. Basophil (BASO)) is the region between the volume corresponding to the second white blood cell histogram and the fourth borderline 4, i.e. the region between the fourth borderline 4 and the maximum volume.

In one example, the hemolytic agent and the dosage thereof used in the second treatment have a first effect of reducing the number of red blood cell debris in the sample to a predetermined threshold value, so that the red blood cell debris does not affect the white blood cell count, and a second effect of increasing the shrinkage rate of lymphocytes, monocytes and neutrophils, and a relatively slower shrinkage rate of eosinophils and basophils, so that the eosinophil population and the basophil population can be distinguished from the granulocytes, respectively. In one embodiment, the controller 50 controls the hemolytic agent pushing component 30 to push the dose of hemolytic agent for the second treatment to the white blood cell counting cell 10, so that the shrinkage rate of lymphocytes, monocytes and neutrophils in the sample is faster than that of eosinophils and basophils, and so that no red blood cell debris affecting the white blood cell count value exists in the sample, i.e. after the sample is treated with the hemolytic agent for the first time and the hemolytic agent for the second time, no red blood cell debris affecting the white blood cell count value exists in the sample in the second measurement, i.e. the number of red blood cell debris in the sample is smaller than the preset threshold value. Processor 60 obtains a second set of parameters for the white blood cells based on data obtained from the second processed sample measured by resistance detector 40, where the second set of parameters includes a white blood cell count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value in one embodiment. Specifically, processor 60 may derive a leukocyte histogram, such as a second leukocyte histogram, based on data obtained from the second processed sample measured by resistance detector 40, and obtain a leukocyte count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value based on the leukocyte histogram. The processor 60 obtains five classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells, namely five classification and counting of the white blood cells are carried out. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the fifth classification parameter; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the fifth classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the fifth classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the five classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the five classification parameters; subtracting the eosinophil percentage and the basophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the five classification parameters, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set and using the neutrophil count value as a neutrophil count value in the five classification parameters; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the fifth categorical parameter; taking the basophil percentage and the basophil count value in the second parameter set as the basophil percentage and the basophil count value in the fifth classification parameter; this completes five classifications and counts of leukocytes.

In some embodiments, the obtaining, by the processor 60, a first set of parameters of the white blood cells according to the data obtained by the measuring the first processed sample by the resistance detector 40 may include: and measuring the sample after the first treatment to obtain a first white blood cell histogram, performing data processing on the first white blood cell histogram to remove the influence of red blood cell fragments, and acquiring the lymphocyte percentage, the monocyte percentage and the granulocyte percentage according to the first white blood cell histogram after the influence of the red blood cell fragments is removed. Specifically, the processor 60 may obtain a white blood cell count value from the first white blood cell histogram, obtain a white blood cell count value from the second white blood cell histogram, and calculate a ratio of the white blood cell count value of the first white blood cell histogram to the white blood cell count value of the second white blood cell histogram; when the ratio is smaller than a preset value, the lymphocyte percentage, the monocyte percentage and the granulocyte percentage are directly obtained from the first white blood cell histogram, when the ratio is larger than or equal to the preset value, a first landmark position between the red blood cell fragments and the white blood cells is determined on the first white blood cell histogram according to the ratio, the first white blood cell histogram after the influence of the red blood cell fragments is removed is obtained, and the lymphocyte percentage, the monocyte percentage and the granulocyte percentage are obtained according to the first white blood cell histogram after the influence of the red blood cell fragments is removed. In one embodiment, the first landmark position between the red blood cell debris and the white blood cell satisfies the following relationship: the ratio of the total area of the first white blood cell histogram to the area of the histogram region to the right of the first landmark is equal to the ratio. In one embodiment, the predetermined value is approximately 1.02.

In the first case, hemolytic agent is added twice, so that the osmotic pressure of the environment where the cells are located is changed twice, the cells are correspondingly contracted, the mapping is determined after the first treatment, the three classification of the white blood cells is realized, the percentages of the lymphocytes, the monocytes and the granulocytes are obtained, the mapping is determined after the second treatment, and the granulocytes are further distinguished; finally, according to the images of the two determinations, the more accurate four-classification and counting or five-classification and counting of the white blood cells are realized.

In the second case, the sample is treated and measured by the hemolytic agent once, and then the sample is waited for the preset time, so that the hemolytic agent continues to act on the sample, and then the measurement is carried out; this will be explained in detail below.

The first processing of the samples in the second case is similar to the first processing of the samples in the first case and is not described in detail here.

The second processing of the sample in the second case is different from the second processing of the sample in the first case.

In one example, the controller 50 controls the hemolytic agent pushing part 30 to push the leukocyte counting cell 10 to zero the dosage of the hemolytic agent for the second treatment, and controls to wait for a preset time to complete the second treatment; that is, in the second case, the sample is processed for the second time and the hemolytic agent is pushed into the leukocyte counting cell 10, but the predetermined time is waited, so that the hemolytic agent used in the first processing continuously acts on the sample, the shrinkage rate of lymphocytes, monocytes and neutrophils is increased, and the shrinkage rate of eosinophils is relatively slow, so that the eosinophil group can be distinguished from the granulocyte group, that is, the actually distinguished eosinophil group includes the basophil group, but since the number of basophils is small relative to the number of eosinophils, the distinguished cell group at this time can be approximately considered as the eosinophil group; the length of the preset time is enough to enable the hemolytic agent used in the first treatment to continuously act on the sample, so that the number of the red blood cell fragments in the sample is less than the preset threshold value, and the counting of the white blood cells cannot be influenced by the red blood cell fragments. Controller 50 then controls resistive detector 40 to measure the second processed sample, and processor 60 obtains a second set of parameters for the white blood cells based on data obtained from the second processed sample measured by resistive detector 40, wherein the second set of parameters includes a white blood cell count value, an eosinophil percentage, and an eosinophil count value in one embodiment. Specifically, processor 60 may derive a leukocyte histogram, such as a second leukocyte histogram, based on data obtained from the second processed sample measured by resistance detector 40, and obtain a leukocyte count, an eosinophil percentage, and an eosinophil count based on the leukocyte histogram. The processor 60 obtains four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells, i.e. four classification and counting of the white blood cells are performed. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the four classification parameters; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the four-classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the four-classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the four-classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the four-classification parameters; subtracting the eosinophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the four-classification parameter, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set to be used as a neutrophil count value in the four-classification parameter; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the four classification parameters; this completes the four sorting and counting of the leukocytes.

In one example, the controller 50 controls the hemolytic agent pushing part 30 to push the leukocyte counting cell 10 to zero the dosage of the hemolytic agent for the second treatment, and controls to wait for a preset time to complete the second treatment; that is, in the second case, the sample is processed for the second time and the hemolytic agent is pushed into the leukocyte counting cell 10, but the predetermined time is waited, so that the hemolytic agent used in the first processing continuously acts on the sample, the shrinking rate of the lymphocytes, the monocytes and the neutrophils is increased, and the shrinking rate of the eosinophils and the basophils is relatively slow, so that the eosinophil group and the basophils can be distinguished from the neutrophil group; the length of the preset time is enough to enable the hemolytic agent used in the first treatment to continuously act on the sample, so that the number of the red blood cell fragments in the sample is less than the preset threshold value, and the counting of the white blood cells cannot be influenced by the red blood cell fragments. The controller 50 then controls the resistive detector 40 to measure the second processed sample, and the processor 60 obtains a second parameter set of the leukocytes according to data obtained by measuring the second processed sample by the resistive detector 40, where the second parameter set includes a leukocyte count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value in one embodiment. Specifically, processor 60 may derive a leukocyte histogram, such as a second leukocyte histogram, based on data obtained from the second processed sample measured by resistance detector 40, and obtain a leukocyte count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value based on the leukocyte histogram. The processor 60 obtains five classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells, namely five classification and counting of the white blood cells are carried out. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the fifth classification parameter; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the fifth classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the fifth classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the five classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the five classification parameters; subtracting the eosinophil percentage and the basophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the five classification parameters, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set and using the neutrophil count value as a neutrophil count value in the five classification parameters; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the fifth categorical parameter; taking the basophil percentage and the basophil count value in the second parameter set as the basophil percentage and the basophil count value in the fifth classification parameter; this completes five classifications and counts of leukocytes.

In the second case, firstly adding hemolytic agent to change the osmotic pressure of the environment where the cells are located, so that the cells shrink correspondingly, and after the treatment, determining the map, realizing the three-classification of the white blood cells, and obtaining the percentages of the lymphocytes, the monocytes and the granulocytes; then waiting for a preset time, on one hand, enabling the hemolytic agent to continuously act for a corresponding time, so that the number of the red blood cell fragments in the sample is smaller than a preset threshold value, the counting of the white blood cells is not influenced, and on the other hand, different contraction rates of different types in the white blood cells are different under the same osmotic pressure, so that the length of a corresponding preset time can be set, and then carrying out second-time image determination, so as to further distinguish the granulocytes; finally, according to the images of the two determinations, the more accurate four-classification and counting or five-classification and counting of the white blood cells are realized.

While the basic description and explanation of the cell analyzer of the present invention are provided above, referring to fig. 3, in some examples, the cell analyzer may further include a diluent pushing component 70, a blending component 80, a pressure source component 90, and the like, wherein the diluent pushing component 70 is used for pushing the diluent to the white blood cell counting cell 10, the blending component 80 is used for blending the liquid in the white blood cell counting cell 10, and the pressure source component 90 provides pressure to make the liquid in the white blood cell counting cell 10 pass through the micropores 11.

Therefore, in an embodiment, the controller 50 is configured to control the diluent pushing component 70 and the sampling needle component 20 to push the diluent and discharge the blood sample to the white blood cell counting chamber 10, and control the hemolytic agent pushing component 30 to push a first dose of hemolytic agent to the white blood cell counting chamber 10, control the blending component 80 to blend the liquid in the white blood cell counting chamber 10, control the pressure source component 90 to provide pressure to make the liquid in the white blood cell counting chamber 10 pass through the micropores 11, and control the resistive detector 40 to perform a first measurement on the liquid passing through the micropores 11, wherein the first dose of hemolytic agent makes the sample still have the red blood cell debris that affects the white blood cell count value when the sample is measured by the resistive detector 40 for the first time, so that the differentiation between the 3 cell groups into which the white blood cells are divided is obvious. The controller 50 then controls the hemolytic agent pushing component 30 to push a second dose of the same hemolytic agent to the white blood cell counting chamber 10, or the controller 50 controls to wait for a preset time; the controller controls the blending component 80 to blend the liquid in the white blood cell counting cell 10, then controls the pressure source component 90 to provide pressure to make the liquid in the white blood cell counting cell 10 pass through the micropores 11, and controls the resistance detector 40 to perform a second measurement on the liquid passing through the micropores 11. Wherein the second amount of the hemolytic agent has a first effect of causing the number of red blood cell debris in the sample to be less than a predetermined threshold such that the red blood cell debris does not affect the white blood cell count, and a second effect of causing the lymphocyte, monocyte and neutrophil to shrink faster and the eosinophil to shrink slower such that the eosinophil population can be distinguished from the neutrophil population; alternatively, the second effect is to increase the rate of shrinkage of lymphocytes, monocytes and neutrophils, while the rate of shrinkage of eosinophils and basophils is relatively slower, thus allowing the differentiation of eosinophil and basophil populations from neutrophils, respectively. Waiting for a preset time, so that the hemolytic agent used in the first treatment continuously acts on the sample to achieve the effect that the number of the erythrocyte fragments in the sample is smaller than a preset threshold value, so that the erythrocyte fragments do not influence the counting of the white blood cells, and in addition, the shrinkage speed of the lymphocytes, the monocytes and the neutrophils is increased, and the shrinkage speed of the eosinophils is relatively slow, so that the eosinophil group can be distinguished from the neutrophil group, or the shrinkage speed of the lymphocytes, the monocytes and the neutrophils is increased, and the shrinkage speed of the eosinophils and the basophils is relatively slow, so that the eosinophil group and the basophils can be distinguished from the neutrophil group.

The processor 60 obtains a first measurement result according to the data obtained by the first measurement of the resistance detector 40, wherein the first measurement result includes a lymphocyte percentage, a monocyte percentage and a granulocyte percentage; processor 60 obtains a second measurement from the data obtained from the second measurement by resistance detector 40, the second measurement including a white blood cell count value, an eosinophil percentage, and an eosinophil count value, and in some embodiments, the second measurement further includes a basophil percentage and a basophil count value. Processor 60 classifies the white blood cells at least four times, such as four times or five times, based on the first and second measurements.

Referring to fig. 4, the cell analyzer in one embodiment may include a sample preparation unit 91, a measurement unit 92, and a processor 93, which will be described in detail below.

The sample preparation unit 91 is used for processing the blood sample with a first amount of hemolytic agent and processing the blood sample with a third amount of hemolytic agent. In some examples, the sample preparation component 91 is configured to process the same blood sample by first processing the blood sample with a first amount of hemolytic agent, and then by continuing to add the hemolytic agent to the blood sample to a third amount, for example, continuing to add a second amount of hemolytic agent to the blood sample, wherein the first amount plus the second amount equals the third amount, thereby achieving processing of the blood sample with the third amount of hemolytic agent; in some examples, the sampling component 91 may also process two fractions of the blood sample, where the first fraction is processed with a first amount of hemolytic agent and the second fraction is processed with a third amount of hemolytic agent.

In some examples, the sample preparation member 91 may include the above-mentioned white blood cell counting cell 10, the sampling needle assembly 20, the hemolytic agent pushing member 30, the diluent pushing member 70, the mixing member 80, and the like.

The measurement unit 92 is configured to measure the blood sample treated with the first amount of hemolytic agent and the blood sample treated with the third amount of hemolytic agent to obtain a first parameter set of white blood cells and a second parameter set of white blood cells, respectively. In one embodiment, the first set of parameters includes lymphocyte percentage, monocyte percentage, and granulocyte percentage, and the second set of parameters includes leukocyte count, eosinophil percentage, and eosinophil count, or the second set of parameters may also include basophil percentage and basophil count.

In some examples, the measurement component 92 may include the resistance detector 40 and the pressure source component 90, etc., described above.

The processor 93 obtains at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells. In some examples, processor 93 may include processor 60 described above, or the like.

Referring to fig. 5, an embodiment of a method for classifying leukocytes based on an impedance method is further disclosed, which may include steps 100 to 140, which are described below.

Step 100: the sample to be analyzed is subjected to a first treatment comprising treating the sample with a hemolytic agent. In one embodiment, the first treatment is performed with a dose of hemolysing agent such that red blood cell debris affecting the white blood cell count remains in the sample during the first measurement. In some embodiments, a portion of the sample after the first treatment may be removed and measured to obtain parameters of the red blood cells.

Step 110: the first processed sample is assayed to obtain a first set of parameters for the white blood cells. In one embodiment, the first set of parameters includes a lymphocyte percentage, a monocyte percentage, and a granulocyte percentage. For example, step 110 may determine that the sample after the first treatment may result in a white blood cell histogram, such as a first white blood cell histogram, based on which the lymphocyte percentage, monocyte percentage, and granulocyte percentage are obtained.

In one embodiment, step 110 includes determining a first set of parameters for the leukocytes from the first processed sample, comprising: and measuring the sample after the first treatment to obtain a first white blood cell histogram, performing data processing on the first white blood cell histogram to remove the influence of red blood cell fragments, and acquiring the lymphocyte percentage, the monocyte percentage and the granulocyte percentage according to the first white blood cell histogram after the influence of the red blood cell fragments is removed. Specifically, the white blood cell count value may be obtained from a first white blood cell histogram, and the white blood cell count value may be obtained from a second white blood cell histogram described below, and the ratio of the white blood cell count value of the first white blood cell histogram to the white blood cell count value of the second white blood cell histogram may be calculated; when the ratio is smaller than a preset value, the lymphocyte percentage, the monocyte percentage and the granulocyte percentage are directly obtained from the first white blood cell histogram, when the ratio is larger than or equal to the preset value, a first landmark position between the red blood cell fragments and the white blood cells is determined on the first white blood cell histogram according to the ratio, the first white blood cell histogram after the influence of the red blood cell fragments is removed is obtained, and the lymphocyte percentage, the monocyte percentage and the granulocyte percentage are obtained according to the first white blood cell histogram after the influence of the red blood cell fragments is removed. In one embodiment, the first landmark position between the red blood cell debris and the white blood cell satisfies the following relationship: the ratio of the total area of the first white blood cell histogram to the area of the histogram region to the right of the first landmark is equal to the ratio. In one embodiment, the predetermined value is approximately 1.02.

Step 120: subjecting the sample to a second treatment comprising treating the sample again with a hemolysing agent such that the amount of red blood cell debris in the sample is less than a predetermined threshold.

In one case, the second treatment is performed with a quantity of hemolytic agent such that the quantity of red blood cell debris in the final sample is less than a predetermined threshold, such that the red blood cell debris does not affect the white blood cell count, and the other effect is such that the shrinkage rate of lymphocytes, monocytes and neutrophils is increased and that of eosinophils is relatively slow, such that the eosinophil population can be distinguished from the neutrophil population, and four classifications and counts of white blood cells can be performed, or such that the shrinkage rate of lymphocytes, monocytes and neutrophils is increased and that of eosinophils and basophils is relatively slow, such that the eosinophil population and the basophils can be distinguished from the neutrophils, and five classifications and counts of white blood cells can be performed. In this case, the hemolytic agent used in the first treatment and the second treatment may be the same hemolytic agent or different hemolytic agents.

In one case, the second treatment uses a zero dose of hemolysis agent, and further comprises waiting a predetermined time. Waiting for a preset time for the second treatment, wherein the hemolytic agent used in the first treatment continuously acts on the sample, so that the quantity of the red blood cell fragments in the sample is smaller than a preset threshold value, and the sizes of the neutrophils and the eosinophils in the sample are different, so that the eosinophil group can be distinguished from the neutrophil group, and four classification and counting of the leukocytes are completed; alternatively, the second treatment is waited for a predetermined time period, so that the hemolytic agent used in the first treatment continuously acts on the sample, the number of red blood cell fragments in the sample is smaller than the predetermined threshold, and the sizes of neutrophils, eosinophils and basophils in the sample are different, so that the eosinophil population and the basophil population can be distinguished from the neutrophil population, and five classifications and counts of leukocytes can be completed.

Step 130: the second processed sample is assayed to obtain a second set of parameters for the white blood cells. For example, the step 130 of determining the second processed sample may result in a white blood cell histogram, such as a second white blood cell histogram, based on which the second set of parameters is obtained. In one embodiment, the second set of parameters includes a white blood cell count value, an eosinophil percentage, and an eosinophil count value. In another embodiment, the second set of parameters comprises a leukocyte count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value.

Step 140: and obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells.

When the second set of parameters includes a leukocyte count value, an eosinophil percentage, and an eosinophil count value, four classifications and counts of leukocytes can be performed based on the first and second sets of parameters of leukocytes. In one embodiment, step 140 subtracts the percentage of eosinophils from the percentage of granulocytes to obtain the percentage of neutrophils; and respectively calculating to obtain a lymphocyte count value, a monocyte count value and a neutrophil count value according to the leukocyte count value, the lymphocyte percentage, the monocyte percentage and the neutrophil percentage. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the four classification parameters; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the four-classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the four-classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the four-classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the four-classification parameters; subtracting the eosinophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the four-classification parameter, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set to be used as a neutrophil count value in the four-classification parameter; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the four classification parameters; this completes the four sorting and counting of the leukocytes.

When the second set of parameters includes a white blood cell count value, an eosinophil percentage, an eosinophil count value, a basophil percentage, and a basophil count value, five classifications and counts of white blood cells can be performed. In one embodiment, step 140 subtracts the percentage of granulocytes from the percentage of eosinophils and the percentage of basophils to obtain the percentage of neutrophils; and respectively calculating to obtain a lymphocyte count value, a monocyte count value and a neutrophil count value according to the leukocyte count value, the lymphocyte percentage, the monocyte percentage and the neutrophil percentage. Specifically, the white blood cell count value in the second parameter set is taken as the white blood cell count value in the fifth classification parameter; taking the lymphocyte percentage in the first parameter set as the lymphocyte percentage in the fifth classification parameter, and obtaining a lymphocyte count value through the lymphocyte percentage in the first parameter set and a leukocyte count value in the second parameter set, wherein the lymphocyte count value is taken as the lymphocyte count value in the fifth classification parameter; taking the percentage of the mononuclear cells in the first parameter set as the percentage of the mononuclear cells in the five classification parameters, and obtaining a mononuclear cell count value through the percentage of the mononuclear cells in the first parameter set and a white blood cell count value in the second parameter set, wherein the mononuclear cell count value is taken as the mononuclear cell count value in the five classification parameters; subtracting the eosinophil percentage and the basophil percentage in the second parameter set from the granulocyte percentage in the first parameter set to obtain a neutrophil percentage which is used as a neutrophil percentage in the five classification parameters, and obtaining a neutrophil count value through the neutrophil percentage and a leukocyte count value in the second parameter set and using the neutrophil count value as a neutrophil count value in the five classification parameters; taking the percentage of eosinophils and the eosinophil count value in the second set of parameters as the percentage of eosinophils and the eosinophil count value in the fifth categorical parameter; taking the basophil percentage and the basophil count value in the second parameter set as the basophil percentage and the basophil count value in the fifth classification parameter; this completes five classifications and counts of leukocytes.

While the method for classifying leukocytes based on the impedance method of the present invention has been described and illustrated, in some cases, the method for classifying leukocytes based on the impedance method may further include steps such as adding a diluent to the leukocyte counting cell, and mixing the liquid in the leukocyte counting cell. For example, referring to fig. 6, an embodiment of a method for classifying leukocytes based on an impedance method may include steps 200 to 270, which are described in detail below.

Step 200: the dilutions and blood samples were added to the white blood cell count cell.

Step 210: adding a first dose of a hemolytic agent to the white blood cell count cell. In one embodiment, the first amount of hemolysis agent is such that the sample remains with red blood cell debris that affects the white blood cell count value when the first assay is performed in step 230, described below.

Step 220: and uniformly mixing the liquid in the leucocyte counting cell.

Step 230: and measuring the liquid in the leucocyte counting pool to obtain a first measuring result, wherein the first measuring result comprises lymphocyte percentage, monocyte percentage and granulocyte percentage.

Step 240: a second dose of the same hemolysing agent is added to the white blood cell count cell again or a preset time is waited for.

In one embodiment, the first effect of the second dose of the hemolytic agent is to reduce the number of red blood cell debris in the sample to a predetermined threshold such that the red blood cell debris does not affect the white blood cell count, and the second effect is to increase the lymphocyte, monocyte and neutrophil shrinkage rate, while the eosinophil shrinkage rate is relatively slow such that the eosinophil population can be distinguished from the neutrophil population; alternatively, the second effect is to increase the rate of shrinkage of lymphocytes, monocytes and neutrophils, while the rate of shrinkage of eosinophils and basophils is relatively slower, thus allowing the differentiation of eosinophil and basophil populations from neutrophils, respectively.

In one embodiment, the predetermined time is waited for, so that the hemolytic agent used in the first treatment continuously acts on the sample, and the effect that the number of the red blood cell debris in the sample is less than the predetermined threshold value is achieved, so that the red blood cell debris does not affect the white blood cell count, and in addition, the shrinkage rate of the lymphocytes, the monocytes and the neutrophils is increased, and the shrinkage rate of the eosinophils is relatively slow, so that the eosinophil population can be distinguished from the neutrophil population, or the shrinkage rate of the lymphocytes, the monocytes and the neutrophils is increased, and the shrinkage rate of the eosinophils and the basophils is relatively slow, so that the eosinophil population and the basophils can be distinguished from the neutrophil population.

Step 250: and uniformly mixing the liquid in the leucocyte counting cell.

Step 260: and measuring the liquid in the leukocyte counting cell to obtain a second measurement result, wherein the second measurement result comprises a leukocyte count value, an eosinophil percentage and an eosinophil count value. In some embodiments, the second measurement further comprises a basophil percentage and a basophil count value.

Step 270: classifying the leukocytes at least four times, for example classifying the leukocytes four times, or classifying the leukocytes five times, based on the first measurement result and the second measurement result.

Referring to fig. 7, an embodiment of a method for classifying leukocytes based on an impedance method may include steps 300 to 310.

Step 300: obtaining a first set of parameters of white blood cells in a blood sample treated with a first dose of a hemolytic agent and a second set of parameters of white blood cells in the blood sample treated with a third dose of a hemolytic agent, wherein the first dose is less than the third dose. In one embodiment, the first set of parameters includes lymphocyte percentage, monocyte percentage, and granulocyte percentage, and the second set of parameters includes leukocyte count, eosinophil percentage, and eosinophil count, or the second set of parameters may also include basophil percentage and basophil count.

In some examples, step 300 obtains the measurement parameters after processing the same blood sample by first processing the blood sample with a first amount of hemolytic agent and then by continuing to add the hemolytic agent to the blood sample to a third amount, e.g., continuing to add a second amount of hemolytic agent to the blood sample, wherein the first amount plus the second amount equals the third amount, thereby achieving processing the blood sample with the third amount of hemolytic agent; in some examples, the measurement parameters obtained in step 300 may be obtained after two fractions of the blood sample are processed, the blood sample is processed with a first amount of hemolytic agent for the first fraction, and the blood sample is processed with a third amount of hemolytic agent for the second fraction.

Step 310: and obtaining at least four classification parameters of the white blood cells according to the first parameter set and the second parameter set of the white blood cells.

The sample or blood sample according to the present invention is a blood sample of an animal, such as a cat or a dog. The following description will be made by way of specific examples.

An example of white blood cell sorting and counting in dogs.

First, 1400uL of a dilution base solution was added to the white blood cell count cell.

Subsequently, 9uL of blood sample was added to the leukocyte count cell, and 700uL of diluent was added to the leukocyte count cell, and the sampling needle was rinsed.

And then, bubbles are formed at the lower end of the leucocyte counting pool to uniformly mix the liquid in the leucocyte counting pool.

The 20uL of sample after mixing in the white blood cell count cell can then be aspirated for red blood cell channel counting.

Next, 0.23mL of hemolytic agent was added to the white blood cell count cell.

And then, bubbles are formed at the lower end of the leucocyte counting pool to uniformly mix the liquid in the leucocyte counting pool. The hemolytic agent is used for dissolving red blood cells, white blood cells are divided into three groups under the action of the hemolytic agent, the lymphocyte group, the monocyte group and the granulocyte group (including neutrophils, eosinophils and basophils) are used, and liquid in the white blood cell counting pool passes through micropores of the white blood cell counting pool under the action of negative pressure. Therefore, the first measurement of the signal of the leukocyte channel results in a leukocyte histogram as shown in fig. 8(a), and a lymphocyte classification value (lymphocyte percentage), a monocyte classification value (monocyte percentage), and a granulocyte classification value (granulocyte percentage) can be obtained.

Then, 0.26mL of the hemolytic agent was added again to the leukocyte counting cell.

The second addition of the hemolytic agent has the effects of accelerating the shrinkage speed of lymphocytes, monocytes and neutrophils, enabling the shrinkage speed of eosinophils and basophils to be relatively low, enabling the eosinophils and basophils to be at the rightmost end of a leukocyte histogram, and enabling liquid in the leukocyte counting cell to pass through micropores of the leukocyte counting cell under the action of negative pressure so as to perform second measurement on signals of the leukocyte channel and obtain the leukocyte histogram shown in figure 8(b), so that the leukocyte count, the eosinophil percentage, the eosinophil count value, the basophil percentage and the basophil count value can be obtained.

And finally, calculating other parameters according to the results of the two images, as follows:

lymphocyte count (white cell count) lymphocyte percentage;

monocyte count value-leukocyte count value-monocyte percentage;

neutrophil percentage-percentage of eosinophils-percentage of basophils;

neutrophil count (white blood cell count) neutrophil percentage;

through the first and second map counting, a plurality of test results of the leukocyte channel can be finally obtained, such as leukocyte count value, lymphocyte count value, monocyte count value, neutrophil count value, eosinophil count value, basophil count value, lymphocyte percentage, monocyte percentage, neutrophil percentage, eosinophil percentage, basophil percentage, and the like.

Yet another example of white blood cell sorting and counting in dogs.

Next, 0.27mL of hemolytic agent was added to the white blood cell count cell.

Then, the reaction was continued for 12 seconds to allow the hemolytic agent to act on the white blood cells and red blood cells.

The 12-second waiting time has the effects that the hemolytic agent further acts on the erythrocytes, so that erythrocyte fragments influencing the white blood cell count do not exist in the sample, and the hemolytic agent further acts on the leukocytes, so that the shrinking speed of lymphocytes, monocytes and neutrophils is increased, the shrinking speed of eosinophils and basophils is relatively low, the eosinophils and basophils are at the rightmost end of the white blood cell histogram, and the liquid in the white blood cell counting cell passes through the micropores of the white blood cell counting cell under the action of negative pressure, so that the signal of the white blood cell channel is measured for the second time, the white blood cell histogram shown in fig. 8(b) is obtained, and therefore, the white blood cell count, the percentage of eosinophils, the count value of eosinophils, the percentage of basophils and the count value of basophils can be obtained.

And finally, calculating other parameters according to the results of the two pictures, wherein the calculation process is similar to that in the example of the dog, and is not repeated herein.

One example of white blood cell sorting and counting for cats.

Next, 0.26mL of hemolytic agent was added to the white blood cell count cell.

And then, bubbles are formed at the lower end of the leucocyte counting pool to uniformly mix the liquid in the leucocyte counting pool. The hemolytic agent is used for dissolving red blood cells, white blood cells are divided into three groups under the action of the hemolytic agent, the lymphocyte group, the monocyte group and the granulocyte group (including neutrophils, eosinophils and basophils) are used, and liquid in the white blood cell counting pool passes through micropores of the white blood cell counting pool under the action of negative pressure. Therefore, the first measurement of the signal of the leukocyte channel results in a leukocyte histogram as shown in fig. 9(a), and a lymphocyte classification value (lymphocyte percentage), a monocyte classification value (monocyte percentage), and a granulocyte classification value (granulocyte percentage) can be obtained.

Then, 0.23mL of the hemolytic agent was added again to the leukocyte counting cell.

The second addition of the hemolytic agent has the effects of accelerating the shrinkage speed of lymphocytes, monocytes and neutrophils, enabling the shrinkage speed of eosinophils and basophils to be relatively low, enabling the eosinophils and basophils to be at the rightmost end of a leukocyte histogram, and enabling liquid in the leukocyte counting cell to pass through micropores of the leukocyte counting cell under the action of negative pressure so as to perform second measurement on signals of the leukocyte channel and obtain the leukocyte histogram shown in figure 9(a), so that the leukocyte count, the eosinophil percentage, the eosinophil count value, the basophil percentage and the basophil count value can be obtained.

Yet another example of white blood cell sorting and counting for cats.

Next, 0.26mL of hemolytic agent was added to the white blood cell count cell.

The 12-second waiting time has the effects that the hemolytic agent further acts on the erythrocytes, so that erythrocyte fragments influencing the white blood cell count do not exist in the sample, and the hemolytic agent further acts on the leukocytes, so that the shrinking speed of lymphocytes, monocytes and neutrophils is increased, the shrinking speed of eosinophils and basophils is relatively low, the eosinophils and basophils are at the rightmost end of the white blood cell histogram, and the liquid in the white blood cell counting cell passes through the micropores of the white blood cell counting cell under the action of negative pressure, so that the signal of the white blood cell channel is measured for the second time, the white blood cell histogram shown in fig. 9(b) is obtained, and therefore, the white blood cell count, the percentage of eosinophils, the count value of eosinophils, the percentage of basophils and the count value of basophils can be obtained.

Those skilled in the art will appreciate that all or part of the functions of the various methods in the above embodiments may be implemented by hardware, or may be implemented by computer programs. When all or part of the functions of the above embodiments are implemented by a computer program, the program may be stored in a computer-readable storage medium, and the storage medium may include: a read only memory, a random access memory, a magnetic disk, an optical disk, a hard disk, etc., and the program is executed by a computer to realize the above functions. For example, the program may be stored in a memory of the device, and when the program in the memory is executed by the processor, all or part of the functions described above may be implemented. In addition, when all or part of the functions in the above embodiments are implemented by a computer program, the program may be stored in a storage medium such as a server, another computer, a magnetic disk, an optical disk, a flash disk, or a removable hard disk, and may be downloaded or copied to a memory of a local device, or may be version-updated in a system of the local device, and when the program in the memory is executed by a processor, all or part of the functions in the above embodiments may be implemented.

The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.

Claims

1. A method for classifying leukocytes based on an impedance method, comprising:

adding a diluent and a blood sample into a white blood cell counting cell;

adding a first dose of a hemolytic agent to the white blood cell count cell;

uniformly mixing the liquid in the leukocyte counting cell;

2. The method of claim 1, wherein said second measurement further comprises a basophil percentage and a basophil count value.

3. A method for classifying leukocytes based on an impedance method, comprising:

4. The method of claim 3, wherein the first set of parameters comprises a lymphocyte percentage, a monocyte percentage, and a granulocyte percentage.

5. The method of claim 4, wherein the first treatment is performed using a quantity of hemolysing agent such that red blood cell debris affecting the white blood cell count remains in the sample at the time of the first measurement.

6. The method of claim 4, wherein said second set of parameters comprises a white blood cell count value, a percentage of eosinophils, and an eosinophil count value.

7. The method of claim 6, wherein the second set of parameters further comprises a basophil percentage and a basophil count value.

8. The method of claim 3, 6 or 7, wherein the second treatment uses a zero dose of hemolytic agent and further comprises waiting a predetermined time.

9. The method of claim 8, wherein the second treatment is performed for a predetermined period of time such that the hemolysis agent used in the first treatment continues to act on the sample to differentiate the size of neutrophils and eosinophils in the sample.

10. The method of claim 8, wherein the second treatment is performed for a predetermined period of time such that the hemolytic agent used in the first treatment continues to act on the sample to cause the size of neutrophils, eosinophils, and basophils in the sample to vary.

11. The method of claim 4, wherein determining the first processed sample to obtain the first set of parameters for the white blood cells comprises: and measuring the sample after the first treatment to obtain a first white blood cell histogram, performing data processing on the first white blood cell histogram to remove the influence of red blood cell fragments, and acquiring the lymphocyte percentage, the monocyte percentage and the granulocyte percentage according to the first white blood cell histogram after the influence of the red blood cell fragments is removed.

12. The method of claim 6, wherein obtaining at least four classification parameters of the white blood cells from the first and second sets of parameters of the white blood cells comprises:

13. The method of claim 7, wherein obtaining at least four classification parameters of the white blood cells from the first and second sets of parameters of the white blood cells comprises:

14. The method of claim 3, wherein the hemolysing agent used in the first and second treatments is the same hemolysing agent.

15. The method of claim 3, further comprising: taking out the part of the sample after the first treatment, and measuring the part of the sample to obtain the parameters of the red blood cells.

16. A method for classifying leukocytes based on an impedance method, comprising: obtaining a first set of parameters of white blood cells in a blood sample treated with a first dose of a hemolytic agent and a second set of parameters of white blood cells in the blood sample treated with a third dose of a hemolytic agent, wherein the first dose is less than the third dose;

17. The method of claim 16, wherein the second set of parameters for the white blood cells is obtained by continuing to add the hemolytic agent to the blood sample to the third dose.

18. A cellular analyzer, comprising:

the white blood cell counting cell comprises a micropore;

a resistance detector for detecting the liquid passing through the micro-hole;

a controller and a processor; wherein:

19. The cellular analyzer of claim 18, wherein the second measurement further comprises a basophil percentage and a basophil count value.

20. A cellular analyzer, comprising:

a leukocyte counting pool;

the resistance detector is used for measuring the sample;

21. The cell analyzer of claim 20, wherein the controller controls the amount of hemolytic agent used for the first treatment pushed by the hemolytic agent pushing component to the white blood cell counting cell such that red blood cell debris affecting the white blood cell count value remains in the sample when the sample is first measured by the resistance type detector.

22. The cell analyzer of claim 21, wherein the controller controls the amount of the hemolytic agent used for the second treatment that the hemolytic agent pushing component pushes to the white blood cell counting cell so that the lymphocyte, monocyte and neutrophil in the sample shrink faster than the eosinophil shrink rate.

23. The cell analyzer of claim 21, wherein the controller controls the amount of the hemolytic agent used for the second treatment that the hemolytic agent pushing component pushes to the white blood cell counting cell so that the lymphocyte, monocyte and neutrophil in the sample shrink faster than the eosinophil and basophil shrink.

24. The cell analyzer of claim 21, wherein the controller controls the hemolytic agent pushing component to push the leukocyte counter for the second treatment to zero dosage and controls to wait for a predetermined time to complete the second treatment.

25. The cell analyzer of any one of claims 20 to 24, wherein the hemolytic agent pushed by the hemolytic agent pushing component for the first treatment and the second treatment is the same hemolytic agent.

26. A cellular analyzer, comprising:

27. The cell analyzer of claim 29, wherein the sample preparation component obtains the second set of parameters for the white blood cells by continuing to add the hemolytic agent to the blood sample to the third dose.

28. A computer-readable storage medium, characterized by comprising a program executable by a processor to implement the method of any one of claims 1 to 19.