CN114813522A - Blood cell analysis method and system based on microscopic amplification digital image - Google Patents
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Abstract
The blood cell analysis method and system based on the microscopic magnification digital image are used for calculating the concentration and the volume of target cells in blood, identifying the target cells in the selected X microscopic magnification digital images and obtaining the number NTC of the target cells in the selected X microscopic magnification digital images; calculating a single target cell volume VTC = target cell area STC × first volume correction factor CVC1 in the microscopic magnified digital image; calculating a single target cell volume VTC = a first absorption parameter α 1 × each target cell area STC × a second volume correction factor CVC 2; calculate single target cell volume VTC = target cell area STC × first cell height coefficient TH 1. The concentration and the volume of the target cells are calculated, the method is simple, and the accuracy of counting and volume calculation is high; the method has the advantages that the method has very accurate foundation for the statistical analysis of the volume of the single target cell, and the statistical analysis aiming at the volume of the single target cell and other combined parameters can be expanded, so that deeper valuable information can be obtained for clinic.
Description
Technical Field
The application belongs to the technical field of acquiring characteristics and parameters of each component in a target sample based on a cell suspension microscopic magnification digital image, and particularly relates to the technical field of calculating the concentration and volume of various cells in a blood sample based on a digital image.
Background
In the prior art, there are different methods for analyzing the concentration and volume of various cells in blood cells. One of the methods is to dilute the blood sample and then convert the blood sample into a sample by counting the blood sample under a microscope by adopting a bovine and abalone blood cell counting plate. The counting process needs manual work, is time-consuming and labor-consuming, and is very easy to make mistakes. The second is the detection of the concentration, volume and hemoglobin of blood cells by a blood analyzer.
In the prior art, flow cytometry is generally used for cell type identification and cell volume and quantity calculation. Some flow cytometers use electrical principles, such as electrical impedance principle, i.e., coulter principle, to design counting channels corresponding to different types of cells, so as to detect the volume and quantity of different types of cells. Or the radio frequency conductance method is used for identifying and counting cell types of different types of cells due to different conductances of cell characteristics. As shown in figure 1, in the electrical impedance method flow cytometer, a blood cell passes through a slit, and electrolyte environments with direct current are arranged inside and outside the slit, so that when the cell passes through, instantaneous potential change can be caused, electric pulses are formed, and the number of the electric pulses reflects the number of the cells, and the size of the cells reflects the volume of the cells. The cell volume is classified as a target cell within a certain range. This method is one of the common detection principles of three-class blood analyzers.
The other part of the flow cytometry adopts an optical or photochemical principle, such as a laser scattering method shown in fig. 2 and 3, the diluted and dyed cells are injected into a sheath flow mechanism, laser irradiates the cells flowing into the sheath flow mechanism, optical characteristics corresponding to the characteristics of the cells are generated due to different characteristics of the cells, for example, corresponding scattering is generated, and volume and quantity information of the corresponding cells can be obtained by detecting corresponding optical signals. Flow cytometry also uses laser light scattering, which means that when different cells are irradiated with a laser beam, scattered light is generated at various angles according to the cell characteristics. The scattered light information obtained by the signal detector is comprehensively analyzed, and cells of normal types can be accurately distinguished. The low angle scattered light (forward scattered light) reflects the number and surface area size of the cells, and the high angle scattered light (side scattered light) reflects the complexity of the cells' internal particles, nuclei, etc. The method is one of the main detection principles of the modern five-classification blood analyzer. The method can obtain the size parameter of a single cell, so the volume parameter of the single cell can be obtained.
No matter the cell analyzer of electricity principle or optics or photochemistry principle, it analyzes for artifical ox abalone board, and its result accuracy reliability and efficiency have all had great promotion. However, the flow cytometry requires that single cells of a small sample be sequentially identified; therefore, precise fluid channels are required to be designed for the flow of the single cells, and the design of a corresponding complex optical system is matched so as to adapt to the capture of the photoelectric parameters of the single cells. Complex runner design and optical system design are needed, the system hardware design is complex and high in cost, faults are very easy to occur, and regular maintenance is usually needed to ensure that the runner and the optical system maintain normal working states; in addition, the single cell flow channel has low cell identification efficiency; when the shape characteristics of the cells change, the accuracy of recognition may decrease.
The application designs a blood cell analysis method based on a bright field microscopic magnification digital image, which can identify and count different cell types based on the bright field microscopic magnification digital image and calculate the volumes of different types of cells; the blood cell analysis method in the present application performs a whole blood cell analysis with extremely simple hardware costs. Moreover, the method is very visual and has better accuracy because of being based on bright field images; the design of a sheath flow mechanism in the flow cytometry is not provided, and the design of a complex spectrophotometer is also not provided; the whole technical scheme is extremely simple, the system efficiency from research and development to use and maintenance is very high, and the cost is extremely low.
In practical applications, such as the identification and counting of blood cell types, a plurality of cell types are involved in a digital image, the actual size of different cell types is greatly different, the WBC diameter is larger than the RBC diameter; the diameter of the red blood cell RBC is equivalent to that of the reticulocyte, the diameter of the red blood cell RBC and the reticulocyte is larger than that of the platelet, and the size difference is large; and the concentration difference of different cell types in the same amount of body fluid sample is larger; when a cell suspension is imaged by using a microscopic digital image, the number of different types of cells in the same image is often not in an order of magnitude, for example, in a microscopic digital image with a magnification of 40, the number of red blood cells may be hundreds or thousands, but the number of white blood cells is only one digit. These features all present difficulties in efficiently using microscopic digital images for cell analysis.
WBC is an abbreviation for "white blood cell" in english, meaning white blood cells in chinese; WBC in a hematology analyzer means the concentration of white blood cells in units of "counts/L";
RBC is an abbreviation for "red blood cell" in English, meaning red blood cell in Chinese; in hematology analyzers, RBC means red blood cell concentration in units of "counts/L";
HCT is an abbreviation for "hematocrit", also known as hematocrit (PCV), which means hematocrit in Chinese; in hematology analyzers, HCT means the volume ratio of erythrocytes to whole blood after anticoagulation; the unit is%;
CV is an abbreviation of "corpuscular volume" in english, and chinese means volume of red blood cells; the unit is "fL";
MCV is an abbreviation of "mean corpuscular volume" in english, and chinese means mean red blood cell volume; MCV in a hematology analyzer means the average volume of all red blood cells, i.e., the mean red blood cell volume, in "fL" femtoliters;
HGB is an abbreviation for english "hemoglobin", which means hemoglobin in chinese; the meaning of HGB in a blood analyzer is the hemoglobin content per volume of blood, i.e. the hemoglobin concentration, in "g/L";
CH is an abbreviation of English "corpuscular hemoglobin", and Chinese means hemoglobin of red blood cells; the meaning of CH in a hematology analyzer is the hemoglobin content of a single red blood cell in units of "pg";
MCH is an abbreviation of "mean corpuscular hemoglobin" in English, and Chinese means mean red blood cell hemoglobin content; MCH in a hematology analyzer means the mean corpuscular hemoglobin content of individual erythrocytes in units of "pg" picograms;
MCHC is an abbreviation of "mean corpuscle hemoglobin concentration" in english, and chinese means mean corpuscular hemoglobin concentration; MCHC in a hematology analyzer means the mean corpuscular hemoglobin content per unit volume of red blood cells, in "g/L";
in the calculation process of the traditional blood analyzer, MCHC = HGB/RBC/MCV, MCHC = MCH/MCV = HGB/RBC/MCV, and MCH = HGB/RBC.
Disclosure of Invention
The technical problem to be solved by the application is to avoid the defects in the prior art and provide a blood cell analysis method based on a micro-magnification digital image; and can perform computational analysis of the concentration and volume of various blood cells based on the microscopic magnification digital image. The technical scheme for solving the technical problems is that the blood cell analysis method based on the micro-magnification digital image is used for calculating the concentration of target cells in blood, and the micro-magnification digital image is a group of micro-magnification digital images obtained by tiling a blood cell monolayer in a suspension liquid; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; comprises the following steps of 2A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic magnification digital image, and obtaining the number NTC of the target cells in the selected X-piece microscopic magnification digital image; and step 2B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the corresponding view field area S of all the microscopic amplified digital images participating in the operation; and step 2C: acquiring the height H of a cell suspension in a known imaging target area; calculating target cell concentration in blood = target cell number NTC ÷ (field area S × H); the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes. The scheme is used for calculating the concentration of red blood cells, white blood cells, platelets and reticulocytes.
The technical scheme for solving the technical problems is that the blood cell analysis method based on the micro-magnification digital image is used for calculating the concentration of target cells in blood, and the micro-magnification digital image is a group of micro-magnification digital images obtained by tiling a blood cell monolayer in a suspension liquid; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; comprises the following steps of 3A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic magnification digital image, and obtaining the number NTC of the target cells in the selected X-piece microscopic magnification digital image; identifying the reference particles in the selected X-piece microscopic magnification digital image, and obtaining the number of the reference particles in the selected X-piece microscopic magnification digital image; and step 3B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the number RRC of reference particles in all the microscopic amplification digital images participating in operation; and step 3C: calculating target cell concentration = target cell number NTC ÷ reference particle number RRC × reference particle concentration C in blood; the suspension contains reference particles of known reference particle concentration C; the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes.
The technical scheme for solving the technical problems is that the blood cell analysis method based on the micro-magnification digital image is used for calculating the volume of target cells in blood, and the micro-magnification digital image is a group of micro-magnification digital images obtained by tiling a blood cell monolayer in a suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; the method comprises the following steps of 4A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic amplification digital images, and obtaining the area STC of each target cell in the selected X-piece microscopic amplification digital images; and step 4B: obtaining a known first volume correction factor CVC 1; calculating a single target cell volume VTC = target cell area STC × first volume correction factor CVC1 in the microscopic magnified digital image; the target cells in the blood comprise any one or more of red blood cells and reticulocytes.
The blood cell analysis method based on the microscopic magnification digital image further comprises a step 4AA of calculating a first volume correction coefficient CVC 1; step 4AA includes: step 4AA 1: taking the same blood cell sample to be analyzed, and obtaining the average cell volume ZSC of target cells by external equipment; step 4AA 2: taking the same blood cell sample to be analyzed as in the step 4AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 4AA 3: identifying target cells in the micro-amplification digital image, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells; step 4AA 4: the first volume correction factor CVC1= mean cell volume ZSC ÷ target cell area mean STCA.
In the blood cell analysis method based on the microscopic magnification digital image, the first volume correction coefficient CVC1 is a fixed first cell height coefficient TH 1.
The technical scheme for solving the technical problems is that the blood cell analysis method based on the micro-magnification digital image is used for calculating the volume of target cells in blood, and the micro-magnification digital image is a group of micro-magnification digital images obtained by tiling a blood cell monolayer in a suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; comprises the following steps of 5A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic amplification digital images, and obtaining the area STC of each target cell in the selected X-piece microscopic amplification digital images; and step 5B: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image; obtaining a known second volume correction factor CVC 2; calculating a single target cell volume VTC = a first absorption parameter α 1 × each target cell area STC × a second volume correction factor CVC 2; the target cells in the blood are red blood cells or reticulocytes.
The method for analyzing blood cells based on the micro-magnified digital image further comprises a step 5AA of calculating a second volume correction factor CVC 2; step 5AA includes: step 5AA 1: taking the same blood cell sample to be analyzed, and obtaining the average cell volume ZSC of target cells by external equipment;
step 5AA 2: taking the same blood cell sample to be analyzed as the blood cell sample to be analyzed in the step 5AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 5AA 3: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image; step 5AA 4: the second volume correction factor CVC2= mean cell volume ZSC ÷ first absorption parameter α 1.
The blood cell analysis method based on the micro-magnification digital image comprises the following steps before each target cell area STC in the micro-magnification digital image is obtained, and the steps are as follows, step 4A 1: acquiring data of a long axis AX =2a and a short axis BY of each target cell; minor axis BY =2 b; step 4a 2: according to whether the ratio between the major axis AX and the minor axis BY falls within a set threshold range, the threshold range is a threshold range having a center value of 1; projecting the morphology of the target cell in the microscopic magnification digital image if the ratio between the major axis AX and the minor axis BY falls within a set threshold rangeRecognized as a circle; target cell area STC = π × a 2 Or target cell area STC = π × b 2 If the ratio of the long axis AX to the short axis BY is larger than a set threshold range, identifying the form of the target cell projection in the microscopic magnification digital image as an ellipse; target cell area STC = π × a × b; where a is the radius of the major axis AX and b is the radius of the minor axis BY.
The blood cell analysis method based on the microscopic magnification digital image further comprises a step 4C or a step 5C or a step 14C: summing each individual target cell volume VTC in the microscopic magnified digital image to obtain all target cell volumes AVC, obtaining all target cell numbers NTC in the microscopic magnified digital image, calculating the target cell mean volume MCV = all target cell volumes AVC ÷ all target cell numbers NTC.
The technical scheme for solving the technical problems is that the blood cell analysis method based on the micro-magnification digital image is used for calculating the volume of target cells in blood, and the micro-magnification digital image is a group of micro-magnification digital images obtained by tiling a blood cell monolayer in a suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; comprising the steps of 15A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-number of microscopic amplification digital images, and obtaining the area STC of each target cell in the microscopic amplification digital images; the target cells in the blood are platelets or leukocytes; step 15B: obtaining a known third volume correction factor CVW 3; calculating individual target cell volume VTC = target cell area STC in the microscopic magnification digital image 1.5 X third volume correction factor CVW 3; the third volume correction factor CVW3 includes a platelet volume correction factor CPLT and a first leukocyte volume correction factor CVW 1.
The method for analyzing blood cells based on the micro-magnified digital image further comprises a step 15AA of calculating a third volume correction factor CVW 3; step 15AA includes: step 15AA 1: taking the same blood cell sample to be analyzed, and obtainingTaking the average cell volume ZSC of the target cells; step 15AA 2: taking the same blood cell sample to be analyzed as in the step 15AA1, carrying out pretreatment to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 15AA 3: identifying target cells in the micro-amplification digital image, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells; step 13AA 4: third volume correction factor CVW3= mean cell volume ZSC ÷ mean target cell area STCA 1.5 。
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M: and adding the individual target cell volumes VTC to average according to the individual target cell volumes VTC, and calculating to obtain the target cell average volume.
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M 2: outputting a VTC histogram of the individual target cell volumes based on the individual target cell volumes VTC; the histogram is used for counting the distribution rule of different target cell volumes.
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M 3: acquiring the content CH of each target hemoglobin, and outputting a CH-CV combined scatter diagram according to the volume VTC of each target cell and the content CH of each target hemoglobin; and the CH-CV combined scatter diagram is used for counting the distribution rule of the hemoglobin of target cells with different volumes.
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M 4: the step of displaying at least one CH range indicator and at least one CV range indicator on a CH-CV joint scatter plot.
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M 5: obtaining hemoglobin concentration CHC of each target cell; or obtaining the content CH of each target hemoglobin, and calculating CHC = the content CH of the target hemoglobin ÷ the volume VTC of each target cell; outputting a CHC-CV combined scatter diagram according to the volume of each target cell and the hemoglobin concentration CHC of the target cell; and the CHC-CV combined scatter diagram is used for counting the distribution rule of hemoglobin of target cells with different volumes.
The blood cell analysis method based on the microscopic magnification digital image comprises the following steps of 4M 6: the step of displaying at least one CHC range indicator and at least one CV range indicator on a CHC-CV joint scattergram.
A blood cell analysis system for blood cell analysis comprising a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor executes the program to implement a method of blood cell analysis based on micro-magnified digital images as described above. Method for producing a composite material
A readable storage medium on which a computer program is stored, characterized in that the program, when executed by a processor, implements a method for blood cell analysis based on micro-magnified digital images as described above.
Compared with the prior art, one of the beneficial effects of the application is that the target cell concentration and volume are calculated based on the number of the target cells in the micro-magnification digital image, the method is simple, the accuracy of counting and volume calculation is high, and therefore the calculation accuracy of the cell concentration is also high. A cell suspension containing blood cells is placed in the imaging target area, wherein the height of the cavity for containing the cell suspension can be used as the height H of the cell suspension; the cell suspension holding cavity is a specially designed cavity, and the cell suspension is basically in a monolayer cell flat state and is used for cell suspension imaging.
Compared with the prior art, the method has the second beneficial effect that the statistical analysis of the volume of the single target cell has a very accurate basis, the statistical analysis aiming at the volume of the single target cell can be developed, and deeper valuable information is obtained for clinic. Statistical analysis of various target cell volumes has significant clinical value in the typing of a variety of anemias.
Compared with the prior art, the cell suspension system has the advantages that the cell morphology is kept complete, the volume measurement of the cell is more accurate, the single cell volume and the single cell hemoglobin content are combined, the single hemoglobin content and the single hemoglobin volume are combined to perform statistical analysis, and deeper multi-dimensional valuable information is obtained for clinic. In particular, the single cell hemoglobin content in combination with the statistical analysis of the volume of individual hemoglobins has an extremely important clinical value in the typing of various anemias.
Compared with the prior art, the method has the beneficial effects that on the basis of the information in the micro-amplification digital image, the concentration and volume calculation of the red blood cells and the reticulocytes are mutually verified by a plurality of calculation methods, so that the reliability of calculation is ensured; the volume calculation of the red blood cells adopts a cylindrical model for calculation, and the volume calculation of the platelets and the white blood cells adopts a spherical model for calculation, which is more close to clinical practice.
Compare with prior art, the beneficial effect of this application is five, based on the information in the micro-magnification digital image, can carry out concentration, the volume calculation of multiple cell simultaneously, and low cost need not to carry out centrifugal step in advance, also need not complicated flow cytometry and spectrophotometer's measurement process. All the calculations are based on the fact that a microscopic amplification digital image with enough amplification factor and enough information is obtained, the working idea of the original cell analyzer is overturned, and the method is based on the application of an AI big data image processing technology in the field of cell analysis and is the true digitization of the cell analyzer.
Drawings
FIG. 1 is one of the schematic diagrams of a prior art split-flow cytometer;
FIG. 2 is a second schematic diagram of a prior art split-flow cytometer;
FIG. 3 is a third schematic diagram of a prior art split-flow cytometer;
FIG. 4 is a schematic block diagram of a microscopic image acquisition apparatus;
FIG. 5 is a schematic view of the composition of an optical portion in the microscopic image obtaining apparatus;
FIG. 6 is a schematic view of an imaging target area; the reference number 100 in the figure is an imaging target area, and comprises a chip base for bearing cell suspension, namely a kit chip; reference numeral 200 is a cell suspension tiling area, and reference numeral 300 is a selected primary field of view target imaging area;
FIG. 7 is a schematic view of a primary field of view of an imaging target area; it can be seen that the primary field of view target imaging area 300 in FIG. 6 is divided into 16 primary fields of view 310;
FIG. 8 is a schematic illustration of a graded field of view of an imaging target region; the primary field of view target imaging area 300 is divided into 16 primary fields of view 310; one of the primary views 310 is further divided into 25 secondary views 510;
FIG. 9 is a partial magnified view of one of the primary fields of view of FIG. 8, divided into 25 secondary fields of view 510;
FIG. 10 is a second schematic view of the hierarchical view of the imaging target region in the hierarchical view digital image acquisition method; the primary field of view target imaging area 300 is divided into 9 primary fields of view 310; one of the primary views 310 is further divided into 16 secondary views 510;
FIG. 11 is a portion of a photomicrographic image showing the different states of one of the red blood cells of the target cell; partial cells in the cell suspension are in a completely flattened state, and a central plane where a cell main body is located is orthogonal to an imaging light beam, so that a complete orthographic projection image of the cell main body can be obtained; part of cells in the cell suspension liquid are in a lateral suspension state, and a central plane where a cell main body is located is not orthogonal to an imaging light beam, so that only partial projection images of the cells can be obtained; the target cell, as shown at 800 in FIG. 9, is in a state where the projection is a incomplete circle or ellipse; whereas the upper left portion of fig. 11 has several target cells closer to the complete orthographic projection image;
fig. 12 is a schematic view of an elliptical projection of fig. 11, with the major axis AX =2a and the minor axis BY =2 b;
FIG. 13 is a single cell CV histogram of healthy dogs; the statistical distribution characteristics of CV are embodied very intuitively;
FIG. 14 is a CV histogram of individual cells from another dog showing a relatively small CV value, which is a common clinical symptom of iron deficiency anemia, iron juvenile cell anemia, thalassemia;
FIG. 15 is a single cell CV histogram of healthy cats; the statistical distribution characteristics of CV are embodied very intuitively;
FIG. 16 is a single cell CV histogram of a diseased cat; the CV value of single cells in the graph is large, and the single cells are clinically common in megaloblastic anemia;
FIG. 17 is a CH-CV scattergram of healthy dogs;
FIG. 18 is a CHC-CV scattergram for healthy dogs;
FIG. 19 is a CH-CV scatter plot for healthy cats;
FIG. 20 is a CHC-CV scatter plot for healthy cats; the CHC-CV scattergram is more aggregated relative to the CH-CV scattergram, and can more intuitively reflect some pathological conditions;
FIG. 21 is a dot-plot of CH-CV for a patient, showing that the CH/CV is small and that clinical symptoms are common in iron deficiency anemia;
FIG. 22 is a scatter plot of CH-CV values in a diseased cat, showing that both CH/CV values are small and that clinical symptoms are common in iron deficiency anemia.
Detailed Description
Embodiments of the present application will be described in further detail below with reference to the drawings.
As shown in fig. 4 to 6, a microscope imaging system based on the method for acquiring a graded-field digital image includes a main controller, a microscope imaging assembly, a driving assembly and an illumination light source assembly; the microscopic imaging assembly comprises a lens assembly and a camera assembly, the lens assembly and the camera assembly are combined together to move together, and the microscopic imaging assembly is used for acquiring a digitalized image after microscopic magnification in an imaging target area range; the microscopic imaging assembly is connected with the driving assembly, and the driving assembly controls the distance of the microscopic imaging assembly relative to an imaging target area; the driving assembly is electrically connected with the main controller, receives the instruction of the main controller, can drive the microscopic imaging assembly to move along the imaging optical axis, and adjusts the distance between the microscopic imaging assembly and an imaging target area so as to acquire a clear microscopic magnification digital image; the imaging target area is arranged between the illumination light source assembly and the microscopic imaging assembly; the illumination light source assembly is used for imaging illumination.
In fig. 5, reference numeral 600 is a microscopic imaging assembly, reference numeral 620 is a camera assembly, and reference numeral 610 is a lens assembly; reference numeral 100 is a target imaging region; reference numeral 700 is an illumination light source assembly; a lens assembly 610 disposed over the imaging target area for forming a microscopic magnified digital image of the imaging target area; the camera assembly 620 is used to acquire a digitized image of the micro-magnified digital image.
As shown in fig. 6, a portion 300 of the imaging target area 200 is selected, and the imaging target area of the portion is divided into N1 primary fields of view; the sizes of the primary visual field ranges are set to be different or the same, and N1 is a natural number more than or equal to 2; dividing each primary view corresponding imaging target area into M2 secondary views; the sizes of the secondary visual field ranges are set to be different or the same, and M2 is a natural number more than or equal to 2; optionally selecting one primary view, taking any one secondary view as a focusing target, and adjusting the distance between an imaging target area and the microscopic imaging assembly to enable the microscopic imaging assembly to acquire a clear microscopic magnification digital image of the secondary view; and moving the microscopic imaging assembly in the horizontal direction under the state that the focal length is maintained, so that the microscopic imaging assembly sequentially acquires M2 clear microscopic magnification digital images corresponding to the secondary visual field.
As shown in fig. 6, in an embodiment of the hierarchical view digital image acquisition method, the size of the imaging target area is 12mm × 14 mm; i.e. the area marked 200 in fig. 6. Selecting and dividing not less than one quarter of the imaging target area, namely the area marked with the reference numeral 300 in FIG. 6, into N1 primary fields of view; the microscopic magnification ranges from 20 times to 100 times. In addition to selecting a quarter of the imaged target area, the size of a third or fifth of the imaged target area or other area may be selected. The specific area size of the imaging target area can be adjusted along with the dilution degree between the body fluid and the diluent in the cell suspension. The blood cells in the cell suspension may include stained cells or unstained cells; in the case of only partial cell analysis, such as only red blood cell analysis, a non-staining cell suspension may be used.
In the embodiment shown in fig. 7-9, N1 is equal to 16; m2 equals 25; n1 has a value in the range of 10 to 20. In the embodiment shown in FIG. 10, N1 is equal to 9; m2 equals 16. The number of regions actually divided can be adjusted according to the size and magnification of the divided regions to obtain the best combination. The blood cells in the cell suspension include stained blood cells and unstained blood cells; the ratio of the volume of blood in the cell suspension to the volume of staining reagent ranges from 1:200 to 1: 260.
since the cell concentration in the present application refers to the number of cells per unit volume, the essence of concentration calculation is cell counting. In the present application, a microscopic digital image is a set of microscopic digital images obtained based on a monolayer of blood cells laid flat in a suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1; selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration or volume of the target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic magnification digital image, and obtaining the number NTC of the target cells in the selected X-piece microscopic magnification digital image; x may be 1, but of course may be other values; the specific values can be chosen in balance according to the microscopic magnification, the size of the camera field of view for obtaining the digital image and the dilution concentration of the suspension, as long as the number can meet the basic requirements on statistics.
In an embodiment of a blood cell analysis method for calculating a concentration of target cells in blood, in particular for calculating a concentration of red blood cells, white blood cells, platelets, reticulocytes, the method for calculating a concentration of red blood cells, platelets, reticulocytes comprises the steps of 2A: identifying target cells in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the target cells in each microscopic amplification digital image; and step 2B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the corresponding view field area S of all the microscopic amplified digital images participating in the operation; and step 2C: acquiring the height H of a cell suspension in a known imaging target area; calculating target cell concentration in blood = target cell number NTC ÷ (field area S × H); the micro-magnified digital image in the above step comprises at least one micro-magnified digital image acquired based on a graded-field digital image acquisition method; the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes.
A cell suspension containing blood cells is placed in the imaging target area, wherein the height of the cavity for containing the cell suspension can be used as the height H of the cell suspension; the cell suspension holding cavity is a specially designed cavity, and a monolayer of cells in the cell suspension is laid flat for imaging the cell suspension. For the process of imaging cell suspension, please refer to patent application No. CN2020112669182 entitled "method and system for imaging cell suspension sample and kit".
In an embodiment of a blood cell analysis method for calculating a concentration of target cells in blood, particularly for calculating a concentration of red blood cells, white blood cells, platelets, and reticulocytes, the method for calculating a concentration of red blood cells, platelets, and reticulocytes includes the following steps 3A: identifying target cells in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the target cells in each microscopic amplification digital image; identifying the reference particles in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the reference particles in each microscopic amplification digital image; and step 3B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the number RRC of reference particles in all the microscopic amplification digital images participating in operation; and step 3C: calculating target cell concentration = target cell number NTC ÷ reference particle number RRC × reference particle concentration C in blood; the micro-magnified digital image in the above step comprises at least one micro-magnified digital image acquired based on a graded-field digital image acquisition method; the cell suspension comprises reference particles with a known reference particle concentration C; the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes.
In an embodiment of the blood cell analysis method for calculating the volume of target cells of blood, in particular for calculating the volume of red blood cells and reticulocytes, one of the red blood cell and reticulocyte volume calculation methods, the method comprises the steps of 4A: identifying target cells in the micro-amplification digital image by using an AI algorithm, and obtaining the area STC of each target cell in the micro-amplification digital image; and step 4B: obtaining a known first volume correction factor CVC 1; calculating a single target cell volume VTC = target cell area STC × first volume correction factor CVC1 in the microscopic magnified digital image; the micro-magnified digital image in the above step comprises at least one micro-magnified digital image acquired based on a graded-field digital image acquisition method; the target cells in the blood include any one or more of erythrocytes and reticulocytes.
In the above embodiment, the method further comprises a step 4AA of calculating a first volume correction coefficient CVC 1; step 4AA includes: step 4AA 1: taking the same blood cell sample to be analyzed, and acquiring the average cell volume ZSC of target cells by using external equipment; step 4AA 2: taking the same blood cell sample to be analyzed as in the step 4AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 4AA 3: identifying target cells in the micro-amplification digital image, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells; step 4AA 4: the first volume correction factor CVC1= mean cell volume ZSC ÷ target cell area mean STCA.
The method for obtaining the average cell volume ZSC of the target cells by using an external device can be various. The method of obtaining the same may be different for different types of cells among them. The corresponding mean cell volume ZSC can be obtained for platelets, reticulocytes and leukocytes using any standardized measuring device known in the art. The standardized measuring device may be a state of the art blood cell analysis system or other device of higher measurement accuracy.
For red blood cells, the process of one of the methods for obtaining the mean cell volume ZSC thereof comprises: firstly, carrying out cell stratification by a centrifugal method to obtain the volume ratio of a red blood cell layer to a whole blood sample, namely the red blood cell specific volume HCT; calculating the volume of red blood cells in the corresponding whole blood sample according to the obtained specific volume of red blood cells HCT and the volume of the corresponding whole blood sample, i.e. the volume of red blood cells in the corresponding whole blood sample = the volume of the whole blood sample x specific volume of red blood cells HCT; using the red blood cell concentration obtained from an external standardized measuring device, and calculating the number of red blood cells in the corresponding whole blood sample = volume of the whole blood sample × red blood cell concentration according to the red blood cell concentration; mean cell volume ZSC = corresponding to the volume of red blood cells in the whole blood sample ÷ corresponding to the number of red blood cells in the whole blood sample. The external standardized measuring device for obtaining the concentration of the red blood cells can be any one of the prior art. The specific volume of red blood cells HCT can also be obtained using standardized measuring devices known in the art. It should be noted that, in the present application, in the process of calculating various correction coefficients by using an external device, a necessary unit conversion process, that is, a quantitative conversion process of the same sample, is included by default, and the above conversion processes are all prior art and are not described again.
Another embodiment of the blood cell analysis method for calculating the target cell volume of blood, particularly for calculating the volume of red blood cells and reticulocytes, i.e. the second method for calculating the volume of red blood cells and reticulocytes, comprises the steps of 5A: identifying target cells in the micro-amplification digital image by using an AI algorithm, and obtaining the area STC of each target cell in the micro-amplification digital image; and step 5B: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnification digital image; obtaining a known second volume correction factor CVC 2; calculating a single target cell volume VTC = a first absorption parameter α 1 × each target cell area STC × a second volume correction factor CVC 2; the micro-magnified digital image in the above step comprises at least one micro-magnified digital image acquired based on a graded-field digital image acquisition method; the target cells in the blood are red blood cells or reticulocytes. lg is the logarithmic operator.
Note that the first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image was calculated. The area where a single cell is located is selected to be subjected to rectangular screen capture, that is, a rectangular area with the cell as the center is selected to calculate the first absorption parameter α 1. The blank mean gray scale value means the mean gray scale value of the area not occupied by the cells in the rectangular area. The mean gray value of the cell pixels means the mean gray value of the area occupied by the cells in the rectangular area.
In the above embodiment, the method further comprises a step 5AA of calculating a second volume correction coefficient CVC 2; step 5AA includes: step 5AA 1: taking the same blood cell sample to be analyzed, and acquiring the average cell volume ZSC of target cells by using external equipment; step 5AA 2: taking the same blood cell sample to be analyzed as the blood cell sample to be analyzed in the step 5AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 5AA 3: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image; step 5AA 4: the second volume correction factor CVC2= mean cell volume ZSC ÷ first absorption parameter α 1. Since the first absorption parameter α 1 is a parameter that is strongly correlated with cell size; according to the lambertian law, the size of the first absorption parameter α 1 and the cell size have a linear relationship, and thus the volume of the target cell can be obtained by a volume correction coefficient using the absorption parameter. The procedure for obtaining the cell volume ZSC and the number of target cells using an external device was the same as in the previous example.
Another embodiment of the blood cell analysis method for calculating the target cell volume of blood, particularly for calculating the volume of red blood cells and reticulocytes, i.e. the third method for calculating the volume of red blood cells and reticulocytes, comprises the steps of 14A: identifying target cells in the micro-amplification digital image by using an AI algorithm, and obtaining the area STC of each target cell in the micro-amplification digital image; step 14B: obtaining a known first cell height coefficient TH 1; calculating a single target cell volume VTC = target cell area STC x first cell height coefficient TH1 in the microscopic magnified digital image; the micro-magnified digital image in the above step comprises at least one micro-magnified digital image acquired based on a graded-field digital image acquisition method; the target cells in the blood include red blood cells. The first cell height coefficient TH1 is a reference coefficient given according to the parameter ranges of various target cells in the clinical study database.
In practical clinical application, especially in veterinary equipment, some animal blood samples which cannot be obtained by testing and comparing by using the conventional blood analyzer can also be used for obtaining corresponding calculation parameters by using the method. In many of the above embodiments, before obtaining each target cell area STC in the microscopic magnification digital image, the following steps, step 4a1, are included: acquiring data of a long axis AX and a short axis BY of each target cell; major axis AX =2 a; minor axis BY =2 b; step 4a 2: depending on whether the ratio between the major axis AX and the minor axis BY falls within a set threshold range, which is a threshold range with a center value of 1, such as 0.99 to 1.01; if the ratio between the long axis AX and the short axis BY falls within a set threshold range, identifying the form of the target cell projection in the microscopic magnification digital image as a circle; target cell area STC = π x (a + b) 2 /4, or target cell area STC = π × a 2 Or target cell area STC = π × b 2 If the ratio between the major axis AX and the minor axis BY does not fall within a set threshold range, identifying the form of the target cell projection in the microscopic magnification digital image as an ellipse; target cell area STC = π × a × b.
As shown in fig. 9 and 10, fig. 9 is a partial portion of a photomicrographic image showing the different states of one of the target cells, red blood cells; partial cells in the cell suspension are in a completely flattened state, and a central plane where a cell main body is located is orthogonal to an imaging light beam, so that a complete orthographic projection image of the cell main body can be obtained; part of cells in the cell suspension liquid are in a lateral suspension state, and a central plane where a cell main body is located is not orthogonal to an imaging light beam, so that only partial projection images of the cells can be obtained; the target cell, as shown at 800 in FIG. 9, is in a state where the projection is a incomplete circle or ellipse; whereas the upper left portion of fig. 9 has several target cells that are closer to the full orthographic projection image. Fig. 10 is a schematic diagram of an elliptical projection of fig. 9, with the major axis AX =2a and the minor axis BY =2 b.
When the projection area of the red blood cell is calculated, judging the projection approximate circle degree according to whether the ratio of the long axis AX to the short axis BY falls into a set threshold range; if the partial image falls within the set threshold range, identifying the partial image as the orthographic projection of the red blood cell; the volume of the red blood cells can be calculated by using a cylinder model and directly multiplying the area of the orthographic projection by the height parameter to obtain the volume of the red blood cells. If the partial projection does not fall into the set threshold range, the partial projection is considered to be an ellipse, and the partial projection is identified as a non-orthographic projection of the red blood cell; when calculating the volume of the red blood cells, the area of the ellipse may be converted into the area of the circular orthographic projection, and then the volume of the red blood cells may be calculated. That is, the area is multiplied by a/b on the basis of the elliptical area, or the area is directly calculated by taking the major axis as the radius and is used as the cylindrical bottom area when the volume of the red blood cells is calculated.
In the above embodiment of the blood cell analysis method for calculating the volume of red blood cells and reticulocytes, the method further comprises step 4C or step 5C or step 14C: summing the individual target cell volumes VTC in the microscopic magnified digital image to obtain the volume AVC of all target cells, obtaining the number NTC of all target cells in the microscopic magnified digital image, calculating the mean target cell volume MCV = the volume AVC ÷ the number NTC of all target cells.
In another embodiment of the blood cell analysis method for blood target cell volume calculation, in particular for platelet volume calculation, i.e. one of the platelet volume calculation methods, at least one microscopic magnification digital image is acquired based on the above-mentioned graded-field digital image acquisition method; the target cells in the blood are platelets; comprising the steps of 15A: identification in a micro-magnified digital image using an AI algorithmAnd obtaining an area STC of each target cell in the micro-magnified digital image; step 15B: obtaining a known platelet volume correction coefficient CPLT; calculating individual target cell volume VTC = target cell area STC in the microscopic magnification digital image 1.5 X platelet volume correction coefficient CPLT.
In the above embodiment, the method further includes a step 15AA of calculating a platelet volume correction coefficient CPLT; step 15AA includes: step 15AA 1: taking the same blood cell sample to be analyzed, and acquiring the average cell volume ZSC of target cells by using external equipment; step 15AA 2: taking the same blood cell sample to be analyzed as in the step 15AA1, carrying out pretreatment to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension; step 15AA 3: identifying target cells in the micro-amplification digital image by using an AI algorithm, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells; step 13AA 4: platelet volume correction factor CPLT = mean cell volume ZSC ÷ mean target cell area STCA 1.5 。
In one embodiment of a blood cell analysis method for calculating a concentration of target cells in blood, particularly for calculating a concentration of white blood cells, i.e., a white blood cell concentration calculation method, N2 × M2 microscopic magnification digital images acquired based on a graded-field digital image acquisition method; the target cells in the blood are leukocytes; comprises the following steps of 11A: identifying target cells in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the target cells in each microscopic amplification digital image; step 11B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the corresponding view field area S of all the microscopic amplified digital images participating in the operation; and step 11C: acquiring the height H of a cell suspension in a known imaging target area; the target cell concentration in blood = target cell number NTC ÷ (field area S × H) was calculated.
In another embodiment of the blood cell analysis method for calculating the concentration of target cells in blood, particularly for calculating the concentration of leukocytes, i.e., the leukocyte concentration calculation method, N2 × M2 microscopic magnification digital images are acquired based on the graded-field digital image acquisition method; the target cells in the blood are leukocytes; comprises the following steps of 12A: identifying target cells in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the target cells in each microscopic amplification digital image; identifying the reference particles in each microscopic amplification digital image by using an AI algorithm, and obtaining the number of the reference particles in each microscopic amplification digital image; and step 12B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the number RRC of reference particles in all the microscopic amplification digital images participating in operation; and step 12C: calculating target cell concentration = target cell number NTC ÷ reference particle number RRC × reference particle concentration C in blood; the reference particle concentration C is known.
In another embodiment of the blood cell analysis method for blood target cell volume calculation, particularly for leukocyte volume calculation, i.e., one of the leukocyte volume calculation methods, N2 × M2 microscopic magnification digital images acquired based on the graded-field digital image acquisition method; the target cells in the blood are leukocytes; the microscopic magnification digital image is used for calculating the volume of the red blood cells; comprises the following steps of 13A: identifying target cells in the micro-amplification digital image by using an AI algorithm, and obtaining the area STC of each target cell in the micro-amplification digital image; step 13B: obtaining a known first leukocyte volume correction factor CVW 1; calculating individual target cell volume VTC = target cell area STC in the microscopic magnification digital image 1.5 X first leukocyte volume correction factor CVW 1.
In the above embodiment, the method further comprises a step 13AA of calculating a first leukocyte volume correction factor CVW 1; step 13AA includes: step 13AA 1: taking the same blood cell sample to be analyzed, and acquiring the average cell volume ZSC of target cells by using external equipment; step 13AA 2: taking the same blood cell sample to be analyzed as in the step 13AA1, carrying out pretreatment to prepare a cell suspension, and injecting the cell suspension into an imaging target area; making blood cell monolayerFlatly paving the suspension liquid, and acquiring a microscopic magnification digital image of the blood cell monolayer flatly paving the suspension liquid; step 13AA 3: identifying target cells in the micro-amplification digital image by using an AI algorithm, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells; step 13AA 4: first leukocyte volume correction factor CVW1= mean cell volume ZSC ÷ mean target cell area average STCA 1.5 。
An embodiment of the method for analyzing blood cells based on a microscopic magnification digital image comprises the steps of 4M: and adding the individual target cell volumes VTC to average according to the individual target cell volumes VTC, and calculating to obtain the target cell average volume. Step 4M 2: outputting a VTC histogram of the individual target cell volumes based on the individual target cell volumes VTC; the histogram is used for counting the distribution rule of different target cell volumes. FIG. 13 is a single cell CV histogram of healthy dogs; FIG. 14 is a CV histogram of individual cells from another dog showing a relatively small CV value, which is a common clinical symptom of iron deficiency anemia, iron juvenile cell anemia, thalassemia; FIG. 15 is a single cell CV histogram of healthy cats; FIG. 16 is a single cell CV histogram of a diseased cat; the CV value of single cells in the graph is large, and the clinical application is common in megaloblastic anemia. As can be seen in fig. 13-15, the histogram very intuitively characterizes the statistical distribution of CVs. In a specific embodiment, the number of red blood cells is about 25000, but in fig. 13 to 22, only a small part of about 500 to 1000 data are extracted and displayed in a histogram; in practical applications, the number of presentations may be set according to clinical needs.
An embodiment of the method for analyzing blood cells based on microscopic magnification digital images comprises the steps of 4M 3: acquiring the content CH of each target hemoglobin, and outputting a CH-CV combined scatter diagram according to the volume VTC of each target cell and the content CH of each target hemoglobin; and the CH-CV combined scatter diagram is used for counting the distribution rule of the hemoglobin of target cells with different volumes. FIG. 17 is a CH-CV scattergram of healthy dogs; FIG. 19 is a CH-CV scatter plot for healthy cats; FIG. 21 is a dot-plot of CH-CV for a patient, showing that the CH/CV is small and that clinical symptoms are common in iron deficiency anemia; FIG. 22 is a scatter plot of CH-CV values in a diseased cat, showing that both CH/CV values are small and that clinical symptoms are common in iron deficiency anemia. The CH-CV combined scatter diagram can simultaneously and intuitively see two pieces of maintained cell information, namely the volume and the hemoglobin distribution rule.
An embodiment of the method for analyzing blood cells based on microscopic magnification digital images comprises the steps of 4M 4: the step of displaying at least one CH range indicator and at least one CV range indicator on a CH-CV joint scatter plot. Step 4M 5: obtaining hemoglobin concentration CHC of each target cell; or obtaining the content CH of each target hemoglobin, and calculating CHC = the content CH of the target hemoglobin ÷ the volume VTC of each target cell; outputting a CHC-CV combined scatter diagram according to the volume of each target cell and the hemoglobin concentration CHC of the target cell; and the CHC-CV combined scatter diagram is used for counting the distribution rule of hemoglobin of target cells with different volumes. Step 4M 6: the step of displaying at least one CHC range indicator and at least one CV range indicator on a CHC-CV joint scattergram. FIG. 18 is a CHC-CV scattergram for healthy dogs; FIG. 20 is a CHC-CV scatter plot for healthy cats; compared with a CH-CV scattergram, the CHC-CV scattergram is more aggregated and can more intuitively reflect some pathological conditions.
In an embodiment of the blood cell analysis system, the blood cell analysis system is used for blood cell analysis, and comprises a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor executes the program to implement the blood cell analysis method based on the micro-magnified digital image. In an embodiment of the readable storage medium, a computer program is stored thereon, which when executed by a processor implements the method for blood cell analysis based on micro-magnified digital images as described above.
The first and second numbers in this application are for convenience of expression and do not necessarily indicate the order of magnitude and timing. The letter numbers in step 1 are also for convenience of expression, and do not necessarily indicate the sequential relationship in time sequence.
The above descriptions of fig. 1 to 22 are only examples of the present application, and are not intended to limit the scope of the present application, and all equivalent structures or equivalent flow transformations that are made by using the contents of the specification and the drawings, or are directly or indirectly applied to other related technical fields are also included in the scope of the present application.
Claims (19)
1. A blood cell analysis method based on a microscopic magnification digital image is used for calculating the concentration of target cells in blood and is characterized in that,
the microscopic magnification digital image is a set of microscopic magnification digital images obtained based on a monolayer of blood cells plated in suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1;
comprises the following steps of 2A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic magnification digital image, and obtaining the number NTC of the target cells in the selected X-piece microscopic magnification digital image;
and step 2B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the corresponding view area S of all the micro-amplified digital images participating in the operation;
and step 2C: acquiring the height H of a cell suspension in a known imaging target area; calculating target cell concentration in blood = target cell number NTC ÷ (field area S × H);
the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes.
2. A blood cell analysis method based on a microscopic magnification digital image is used for calculating the concentration of target cells in blood and is characterized in that,
the microscopic magnification digital image is a set of microscopic magnification digital images obtained based on a monolayer of blood cells plated in suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1;
comprises the following steps of 3A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic magnification digital image, and obtaining the number NTC of the target cells in the selected X-piece microscopic magnification digital image; identifying the reference particles in the selected X-piece microscopic magnification digital image, and obtaining the number of the reference particles in the selected X-piece microscopic magnification digital image;
and step 3B: calculating the number NTC of target cells in all the microscopic amplification digital images participating in operation; calculating the number RRC of reference particles in all the microscopic amplification digital images participating in operation;
and step 3C: calculating target cell concentration = target cell number NTC ÷ reference particle number RRC × reference particle concentration C in blood;
the suspension contains reference particles of known reference particle concentration C;
the target cells in the blood comprise any one or more of red blood cells, white blood cells, platelets and reticulocytes.
3. A blood cell analysis method based on a microscopic magnification digital image is used for calculating the volume of target cells in blood,
the microscopic magnification digital image is a set of microscopic magnification digital images obtained based on a monolayer of blood cells plated in suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1;
the method comprises the following steps of 4A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic amplification digital images, and obtaining the area STC of each target cell in the selected X-piece microscopic amplification digital images;
and step 4B: obtaining a known first volume correction factor CVC 1; calculating a single target cell volume VTC = target cell area STC × first volume correction factor CVC1 in the microscopic magnified digital image;
the target cells in the blood comprise any one or more of red blood cells and reticulocytes.
4. The method for analyzing blood cells based on micro-magnified digital images according to claim 3,
further comprising a step 4AA of calculating a first volume correction factor CVC 1;
step 4AA includes:
step 4AA 1: taking the same blood cell sample to be analyzed, and obtaining the average cell volume ZSC of target cells by external equipment;
step 4AA 2: taking the same blood cell sample to be analyzed as in the step 4AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension;
step 4AA 3: identifying target cells in the micro-amplification digital image, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells;
step 4AA 4: the first volume correction factor CVC1= mean cell volume ZSC ÷ target cell area mean STCA.
5. The method for analyzing blood cells based on micro-magnified digital images according to claim 3,
the first volume correction factor CVC1 is a fixed first cell height factor TH 1.
6. A blood cell analysis method based on a microscopic magnification digital image is used for calculating the volume of target cells in blood,
the microscopic magnification digital image is a set of microscopic magnification digital images obtained based on a monolayer of blood cells plated in suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1;
comprises the following steps of 5A: selecting X pieces of microscopic amplification digital images from the N multiplied by M pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-piece microscopic amplification digital images, and obtaining the area STC of each target cell in the selected X-piece microscopic amplification digital images;
and step 5B: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image; obtaining a known second volume correction factor CVC 2; calculating a single target cell volume VTC = a first absorption parameter α 1 × each target cell area STC × a second volume correction factor CVC 2;
the target cells in the blood are red blood cells or reticulocytes.
7. The method for analyzing blood cells based on micro-magnified digital images according to claim 6,
further comprising a step 5AA of calculating a second volume correction factor CVC 2;
step 5AA includes:
step 5AA 1: taking the same blood cell sample to be analyzed, and obtaining the average cell volume ZSC of target cells by external equipment;
step 5AA 2: taking the same blood cell sample to be analyzed as the blood cell sample to be analyzed in the step 5AA1, pretreating to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension;
step 5AA 3: calculating a first absorption parameter α 1= lg (blank mean gray value/cell pixel mean gray value) in the microscopic magnified digital image;
step 5AA 4: the second volume correction factor CVC2= mean cell volume ZSC ÷ first absorption parameter α 1.
8. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3 or 6,
before each target cell area STC in a microscopic-magnified digital image is obtained, the following steps are included,
step 4a 1: acquiring data of a long axis AX =2a and a short axis BY of each target cell; minor axis BY =2 b;
step 4a 2: according to whether the ratio between the major axis AX and the minor axis BY falls within a set threshold range, the threshold range is a threshold range having a center value of 1; if the ratio between the long axis AX and the short axis BY falls within a set threshold range, identifying the form of the target cell projection in the microscopic magnification digital image as a circle; target cell area STC = π × a 2 Or target cell area STC = π × b 2 ;;
If the ratio of the long axis AX to the short axis BY is larger than a set threshold range, identifying the form of the target cell projection in the microscopic magnification digital image as an ellipse; target cell area STC = π × a × b;
where a is the radius of the major axis AX and b is the radius of the minor axis BY.
9. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3 or 6,
further comprising step 4C or step 5C or 14C: summing each individual target cell volume VTC in the microscopic magnified digital image to obtain all target cell volumes AVC, obtaining all target cell numbers NTC in the microscopic magnified digital image, calculating the target cell mean volume MCV = all target cell volumes AVC ÷ all target cell numbers NTC.
10. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3 or 6, comprising,
step 4M 3: acquiring the content CH of each target hemoglobin, and outputting a CH-CV combined scatter diagram according to the volume VTC of each target cell and the content CH of each target hemoglobin; and the CH-CV combined scatter diagram is used for counting the distribution rule of the hemoglobin of target cells with different volumes.
11. The method for analyzing blood cells based on micro-magnified digital images according to claim 10, comprising,
step 4M 4: the step of displaying at least one CH range indicator and at least one CV range indicator on a CH-CV joint scatter plot.
12. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3 or 6, comprising,
step 4M 5: obtaining hemoglobin concentration CHC of each target cell; or obtaining the content CH of each target hemoglobin, and calculating CHC = the content CH of the target hemoglobin ÷ the volume VTC of each target cell;
outputting a CHC-CV combined scatter diagram according to the volume of each target cell and the hemoglobin concentration CHC of the target cell; and the CHC-CV combined scatter diagram is used for counting the distribution rule of hemoglobin of target cells with different volumes.
13. The method for analyzing blood cells based on micro-magnified digital images according to claim 12, comprising,
step 4M 6: the step of displaying at least one CHC range indicator and at least one CV range indicator on a CHC-CV joint scattergram.
14. A blood cell analysis method based on a microscopic magnification digital image is used for calculating the volume of target cells in blood,
the microscopic magnification digital image is a set of microscopic magnification digital images obtained based on a monolayer of blood cells plated in suspension; the group of the microscopic magnification digital images comprises N multiplied by M microscopic magnification digital images; n and M are both natural numbers more than or equal to 1;
comprising the steps of 15A: selecting X pieces of microscopic amplification digital images from the NxM pieces of microscopic amplification digital images for calculating the concentration of target cells, wherein X is a natural number more than or equal to 1; identifying target cells in the selected X-number of microscopic amplification digital images, and obtaining the area STC of each target cell in the microscopic amplification digital images; the target cells in the blood are platelets or leukocytes;
step 15B: obtaining a known third volume correction factor CVW 3; calculating individual target cell volume VTC = target cell area STC in the microscopic magnification digital image 1.5 X third volume correction factor CVW 3;
the third volume correction factor CVW3 includes a platelet volume correction factor CPLT and a first leukocyte volume correction factor CVW 1.
15. The method for analyzing blood cells based on micro-magnified digital images according to claim 14,
further comprising a step 15AA of calculating a third volume correction factor CVW 3;
step 15AA includes:
step 15AA 1: taking the same blood cell sample to be analyzed, and facilitating the acquisition of the average cell volume ZSC of target cells;
step 15AA 2: taking the same blood cell sample to be analyzed as in the step 15AA1, carrying out pretreatment to prepare a cell suspension, and injecting the cell suspension into an imaging target area; plating the blood cell monolayer in the suspension and obtaining a microscopic digital image of the blood cell monolayer plated in the suspension;
step 15AA 3: identifying target cells in the micro-amplification digital image, obtaining the area STC of each target cell in the micro-amplification digital image, and calculating the average value STCA of the area of the target cells;
step 13AA 4: third volume correction factor CVW3= mean cell volume ZSC ÷ mean target cell area STCA 1.5 。
16. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3, 6 or 14, comprising,
step 4M: and adding the individual target cell volumes VTC to average according to the individual target cell volumes VTC, and calculating to obtain the target cell average volume.
17. The method for analyzing blood cells based on micro-magnified digital images according to any one of claims 3, 6 or 14, comprising,
step 4M 2: outputting a VTC histogram of the individual target cell volumes based on the individual target cell volumes VTC; the histogram is used for counting the distribution rule of different target cell volumes.
18. A blood cell analysis system for blood cell analysis comprising a memory, a processor and a computer program stored in the memory and executable on the processor, wherein the processor when executing the program implements a method for blood cell analysis based on micro-magnified digital images according to any one of claims 1 to 17.
19. A readable storage medium on which a computer program is stored, which program, when being executed by a processor, is adapted to carry out a method of blood cell analysis based on micro-magnified digital images according to any one of claims 1 to 17.
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