CN112014196A - Dilution method for classifying and counting white blood cells - Google Patents
Dilution method for classifying and counting white blood cells Download PDFInfo
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- CN112014196A CN112014196A CN202010821561.3A CN202010821561A CN112014196A CN 112014196 A CN112014196 A CN 112014196A CN 202010821561 A CN202010821561 A CN 202010821561A CN 112014196 A CN112014196 A CN 112014196A
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- 210000000265 leukocyte Anatomy 0.000 title claims abstract description 42
- 238000003113 dilution method Methods 0.000 title claims abstract description 9
- 239000003219 hemolytic agent Substances 0.000 claims abstract description 34
- 210000004369 blood Anatomy 0.000 claims abstract description 27
- 239000008280 blood Substances 0.000 claims abstract description 27
- 238000002156 mixing Methods 0.000 claims abstract description 26
- 238000000034 method Methods 0.000 claims abstract description 17
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 6
- RBTBFTRPCNLSDE-UHFFFAOYSA-N 3,7-bis(dimethylamino)phenothiazin-5-ium Chemical compound C1=CC(N(C)C)=CC2=[S+]C3=CC(N(C)C)=CC=C3N=C21 RBTBFTRPCNLSDE-UHFFFAOYSA-N 0.000 claims description 3
- 229960000907 methylthioninium chloride Drugs 0.000 claims description 3
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 230000002949 hemolytic effect Effects 0.000 claims description 2
- 239000008213 purified water Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 15
- 239000012535 impurity Substances 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 7
- 230000000007 visual effect Effects 0.000 description 9
- 210000000601 blood cell Anatomy 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- 239000012560 cell impurity Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000004820 blood count Methods 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
Images
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a dilution method for classifying and counting white blood cells, which is applied to the process of detecting and analyzing the white blood cells by using an image recognition technology, and comprises the following steps of mixing an acquired blood sample with a hemolytic agent in a volume ratio of 1: (7-11) mixing to obtain a mixed sample. The dilution method of the invention comprises the following steps of mixing a blood sample with a hemolytic agent according to the volume ratio of 1: (7-11), the obtained mixed sample is appropriate in the number of leucocytes, rapid focusing in the image acquisition process is facilitated, and the detection efficiency is improved; meanwhile, the impurities of other cells in the mixed sample are less, so that the recognition and counting of the white blood cells are not influenced, and the accuracy of a classification result is ensured.
Description
Technical Field
The invention relates to the technical field of blood cell detection, in particular to a dilution method for classifying and counting leucocytes.
Background
In the cell counting analysis process using image recognition technology as an analysis method, a general operation flow is to place a sample to be detected under an electron microscope for image acquisition, and obtain a counting result through image analysis processing. Before this, it was necessary to mix and dilute the blood sample with a hemolytic agent for two purposes: firstly, other blood cells except the white blood cells in the blood sample are dissolved and removed; one is to control the white blood cells in the sample to a suitable density for observation and counting.
The above dilution process is performed by using hemolytic agent, and the dilution ratio of several conventional leukocyte hemolytic agents is 20 times. However, in practice, there are problems as follows:
firstly, the dilution multiple is higher, which results in the number of the white blood cells in the unit visual field being too small, the accuracy and the reliability of the counting result can be reduced by too few white blood cells, the common solution is to make up for the deficiency by increasing the counting area, namely, the accuracy and the reliability of the counting result can be ensured by increasing the observation of a plurality of visual fields on the original basis, and the operation is time-consuming.
Secondly, the density of white blood cells in the visual field is small, the difficulty of focusing is increased, the operation steps are increased, and the efficiency is reduced.
On the other hand, if the dilution factor is too low, other cells are not completely dissolved, and the foreign objects in the visual field are too many, which causes interference to the recognition and counting of the white blood cells; it also results in too dark a background color and also adds difficulty to cell identification.
In view of the above, a new technical solution is needed to solve the above technical problems.
Disclosure of Invention
The invention aims to provide a method for diluting leukocyte differential counting, which can solve the problems of small number of leukocytes and impurity interference in visual field caused by improper dilution ratio in the prior art.
In order to achieve the purpose, the invention adopts the following technical means:
a method for diluting a differential count of white blood cells, which is applied to a process of detecting and analyzing white blood cells by using an image recognition technology, wherein the diluting method comprises the following steps of mixing a collected blood sample with a hemolytic agent in a volume ratio of 1: (7-11) mixing to obtain a mixed sample.
As a further improvement, the hemolytic agent comprises hydrochloric acid, methylene blue and pure water.
As a further improvement, the hemolytic agent comprises a quaternary ammonium salt and a buffering agent.
As a further improvement, the blood sample is quantitatively sampled by a sampler, and the hemolytic agent is quantitatively filled in a mixing bottle; the volume ratio of the blood collection amount of the sampler to the filling amount of the mixing bottle is 1: (7-11).
As a further improvement, the blood sampling amount of the sampler is 20 microliters, and the filling amount of the mixing bottle is 140-220 microliters.
Compared with the prior art, the invention has the following technical effects:
the dilution method of the invention comprises the following steps of mixing a blood sample with a hemolytic agent according to the volume ratio of 1: (7-11), the obtained mixed sample is appropriate in the number of leucocytes, rapid focusing in the image acquisition process is facilitated, and the detection efficiency is improved; meanwhile, the impurities of other cells in the mixed sample are less, so that the recognition and counting of the white blood cells are not influenced, and the accuracy of a classification result is ensured.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows a flow chart of a differential white blood cell count detection assay of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
In the description of the present invention, it is to be understood that the terms "central," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," "circumferential," and the like are used in the orientations and positional relationships indicated in the drawings for convenience in describing the invention and to simplify the description, and are not intended to indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and are not to be considered limiting of the invention.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In the present invention, unless otherwise expressly stated or limited, the first feature "on" or "under" the second feature may be directly contacting the first and second features or indirectly contacting the first and second features through an intermediate. Also, a first feature "on," "over," and "above" a second feature may be directly or diagonally above the second feature, or may simply indicate that the first feature is at a higher level than the second feature. A first feature being "under," "below," and "beneath" a second feature may be directly under or obliquely under the first feature, or may simply mean that the first feature is at a lesser elevation than the second feature.
Referring to fig. 1, the present invention provides a method for diluting white blood cell differential count, which is applied to a process using an image recognition technology as a counting method, such as three-differential counting and five-differential counting of blood. The process flow of such cell analysis generally comprises the following main steps:
s1, consumable preparation: mainly comprises a sampler and a mixing bottle, wherein the sampler is used for collecting blood samples, and hemolytic agents are filled in the mixing bottle and are used for dissolving blood cells except leucocytes and simultaneously maintaining the stable shape of the leucocytes;
s2, blood sample collection: namely, collecting fresh blood by using a sampler;
s3, uniformly mixing: dripping the collected blood sample into a mixing bottle, and reversing and uniformly mixing;
s4, filling a counting plate into a pool, and performing detection on a machine after sedimentation: and adding the mixed sample into a counting cell of a counting plate, and moving to a microscope for observation to obtain a counting result.
In step S3, the mixing volume ratio of the blood sample and the hemolytic agent is 1: (7-11).
In step S2, the blood sample may be collected quantitatively, for example, 5 microliters, 10 microliters, 20 microliters or other quantities, and accordingly, the volume of the hemolytic agent in the mixing bottle is adjusted according to the above ratio.
If the mixing bottle is filled with a fixed amount of hemolytic agent in the step S1 and a fixed amount of blood sampling is adopted in the step S2, the volume ratio of the blood sampling amount to the filling amount is 1: (7-11).
The invention is explained in more detail below with reference to a few specific examples.
Example one
The hemolytic agent used in this example mainly contains a quaternary ammonium salt and a buffer.
Quantitatively collecting blood with a hemostix, wherein the blood collection amount is 20 microliters; the hemolytic agent filled in the mixing bottle is 140 microliter, 180 microliter and 220 microliter; two comparative examples were set up, corresponding to 100 microliters, 300 microliters of hemolytic agent in the mixing vial.
The following experimental indexes are measured:
| amount of hemolysis | 100μL | 140μL | 180μL | 220μL | 300μL |
| Density of white blood cells | 6 to 9 | 5 to 7 | 4 to 6 | 4 to 5 | 0 to 1 |
| Impurities | Much more | Hardly any | Hardly any | Hardly any | Hardly any |
| Time of focus | 10-20 seconds | 10-20 seconds | 10-20 seconds | 10-20 seconds | More than 30 seconds |
Description of the drawings: the white blood cell density in the above table refers to the statistics of the number of effective white blood cells in an observation cell; impurities refer to the number of other cells within an observation cell; the in-focus time refers to the average elapsed time to find a clear white blood cell image.
It should be noted that the amount of leukocytes in blood is the least, and conventionally, statistics is performed on the observation results of a plurality of samples, and most of the observation results fall into more than 4 interval ranges of each observation cell, which indicates that the density of the batch of samples is appropriate; if most of the observations are less than 4, i.e., deviate from the range, the density of the batch of samples is low, which is not favorable for observation.
As can be seen from the above table, the hemolytic agent is exemplified by 140 microliters, 180 microliters, and 220 microliters, and all of the above three assessment aspects perform well: the leukocyte density is suitable, the impurities are almost avoided, the focusing time is short, and accurate and reliable counting results and high-level detection efficiency are ensured.
In the example of 100 microliters of hemolytic agent, although the density of white blood cells is sufficient, the amount of hemolytic agent is too small, so that other cell impurities in the mixed sample are too much, which interferes with the identification of individual white blood cells in the final image, and results in inaccurate counting result.
In the case of 300. mu.l of hemolytic agent, although the impurity rate is low, the leukocyte density in the visual field is too low, the focusing time is too long, and the efficiency is affected; and the density of the white blood cells in the visual field is uneven, so that the reliability of the final counting result is greatly reduced.
Example two
The hemolytic agent used in this embodiment is a hemolytic agent conventionally matched with three-kind blood, and is prepared from hydrochloric acid, methylene blue and pure water.
Quantitatively collecting blood with a hemostix, wherein the blood collection amount is 20 microliters; the hemolytic agent filled in the mixing bottle is 160 microliter, 200 microliter and 240 microliter; two comparative examples were set up with 100 microliters, 300 microliters of hemolysing agent in the mixing vial.
The following experimental indexes are measured:
| amount of hemolysis | 100μL | 140μL | 180μL | 220μL | 300μL |
| Density of white blood cells | 6 to 9 | 4 to 7 | 4 to 6 | 4 to 5 | 0 to 1 |
| Impurities | Much more | Hardly any | Hardly any | Hardly any | Hardly any |
| Time of focus | 10-20 seconds | 10-20 seconds | 10-20 seconds | 10-20 seconds | More than 30 seconds |
Description of the drawings: the white blood cell density in the above table refers to the statistics of the number of effective white blood cells in an observation cell; impurities refer to the number of other cells within an observation cell; the in-focus time refers to the average elapsed time to find a clear white blood cell image.
It should be noted that the white blood cells in the blood are the least blood cells, conventionally, the observed results of a plurality of samples are counted, and most observed results fall into more than 4 interval ranges of each observed cell, which indicates that the density of the batch of samples is proper; if most of the observations are less than 4, i.e., deviate from the range, the density of the batch of samples is low, which is not favorable for observation.
As can be seen from the above table, the hemolytic agent is exemplified by 140 microliters, 180 microliters, and 220 microliters, and all of the above three assessment aspects perform well: the leukocyte density is suitable, the impurities are almost avoided, the focusing time is short, and accurate and reliable counting results and high-level detection efficiency are ensured.
In the example of 100 microliters of hemolytic agent, although the density of white blood cells is sufficient, the amount of hemolytic agent is too small, so that other cell impurities in the mixed sample are too much, which interferes with the identification of individual white blood cells in the final image, and results in inaccurate counting result.
In the case of 300. mu.l of hemolytic agent, although the impurity rate is low, the leukocyte density in the visual field is too low, the focusing time is too long, and the efficiency is affected; and the density of the white blood cells in the visual field is uneven, so that the reliability of the final counting result is greatly reduced.
It should be noted that the hemolytic agents in the above embodiments are only two preferred embodiments of the present invention, and in other embodiments, other leukocyte hemolytic agents, such as glacial acetic acid, may be substituted for the hemolytic agents, so as to achieve the object of the present invention.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (5)
1. A method for diluting differential counting of white blood cells, which is applied to the process of detecting and analyzing white blood cells by using an image recognition technology, and is characterized in that the diluting method comprises the following steps of mixing a collected blood sample with a hemolytic agent in a volume ratio of 1: (7-11) mixing to obtain a mixed sample.
2. The method of claim 1, wherein the hemolytic agent comprises hydrochloric acid, methylene blue, and purified water.
3. The method of claim 1, wherein the hemolysing agent comprises a quaternary ammonium salt and a buffer.
4. The dilution method according to claim 1, wherein the blood sample is quantitatively sampled by a sampler, and the hemolytic agent is quantitatively filled by a mixing bottle; the volume ratio of the blood collection amount of the sampler to the filling amount of the mixing bottle is 1: (7-11).
5. The dilution method according to claim 4, wherein the sampler takes up 20. mu.l of blood and the mixing flask takes up 140 to 220. mu.l of blood.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4581223A (en) * | 1980-03-12 | 1986-04-08 | Lawrence Kass | Individual leukocyte determination by means of differential metachromatic dye sorption |
| CN101078720A (en) * | 2006-05-22 | 2007-11-28 | 深圳迈瑞生物医疗电子股份有限公司 | Improved reagent and method for classifying leucocyte |
| CN101470109A (en) * | 2007-12-25 | 2009-07-01 | 深圳迈瑞生物医疗电子股份有限公司 | Method for improving accuracy of blood sample leukocyte classifying result |
| US20120288889A1 (en) * | 2011-05-10 | 2012-11-15 | Horiba, Ltd. | Reagent for blood cell counting and blood analysis method |
| CN104297134A (en) * | 2014-11-05 | 2015-01-21 | 深圳市开立科技有限公司 | Hemolytic agent and application thereof as well as classifying and counting method for white blood cells |
| CN109270281A (en) * | 2017-07-18 | 2019-01-25 | 深圳市帝迈生物技术有限公司 | Improve the method and apparatus of leukocyte differential count result accuracy and count results repeatability |
| CN111189762A (en) * | 2020-03-14 | 2020-05-22 | 深圳联开生物医疗科技有限公司 | Four-classification white blood cell analyzer and analysis method |
| CN211043062U (en) * | 2019-11-12 | 2020-07-17 | 湖南伊鸿健康科技有限公司 | Blood cell counting plate and blood cell analysis system |
-
2020
- 2020-08-15 CN CN202010821561.3A patent/CN112014196A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4581223A (en) * | 1980-03-12 | 1986-04-08 | Lawrence Kass | Individual leukocyte determination by means of differential metachromatic dye sorption |
| CN101078720A (en) * | 2006-05-22 | 2007-11-28 | 深圳迈瑞生物医疗电子股份有限公司 | Improved reagent and method for classifying leucocyte |
| CN101470109A (en) * | 2007-12-25 | 2009-07-01 | 深圳迈瑞生物医疗电子股份有限公司 | Method for improving accuracy of blood sample leukocyte classifying result |
| US20120288889A1 (en) * | 2011-05-10 | 2012-11-15 | Horiba, Ltd. | Reagent for blood cell counting and blood analysis method |
| CN104297134A (en) * | 2014-11-05 | 2015-01-21 | 深圳市开立科技有限公司 | Hemolytic agent and application thereof as well as classifying and counting method for white blood cells |
| CN109270281A (en) * | 2017-07-18 | 2019-01-25 | 深圳市帝迈生物技术有限公司 | Improve the method and apparatus of leukocyte differential count result accuracy and count results repeatability |
| CN211043062U (en) * | 2019-11-12 | 2020-07-17 | 湖南伊鸿健康科技有限公司 | Blood cell counting plate and blood cell analysis system |
| CN111189762A (en) * | 2020-03-14 | 2020-05-22 | 深圳联开生物医疗科技有限公司 | Four-classification white blood cell analyzer and analysis method |
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Application publication date: 20201201 |