Method for detecting platelet aggregation
The problem of pseudo-thrombocytopenia and fluorescent dye dependence is solved by detecting platelet aggregation using light at specific wavelengths, combined with dilution reagents and flow cytometry, achieving more accurate platelet counts.
Patent Information
- Application Number
- CN202410906598.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2023-07-10
- Filing Date
- 2024-07-08
- Publication Date
- 2025-01-10
AI Technical Summary
The existing platelet agglutination detection methods have pseudo-thrombocytopenia, which leads to misdiagnosis. Hemolytic reagents and fluorescent dyes are often used, which affects the accuracy of the detection.
Using light at the first wavelength of 315nm or above 600nm and the second wavelength of 750nm or above 610nm or above 750nm, platelet aggregation is detected by side and front scattering light information to avoid hemolytic reagents and fluorescent dyes. The sample is prepared using dilution reagents and combined with flow method Cytometer for analysis.
It improves the accuracy of platelet agglutination detection, reduces pseudo-thrombocytopenia, provides more reliable platelet count results, avoids misdiagnosis, and does not rely on fluorescent dyes, simplifies the detection process.
Smart Images
Figure CN119290715A_ABST
Abstract
Description
[Technical field] The present invention relates to a method for detecting platelet aggregation. [Background technology] Blood contains various blood cell components such as red blood cells, white blood cells, and platelets. Among these, platelets are anucleated cells with a diameter of 2 to 3 μm, and there are 150,000 to 350,000 / μL in normal blood. However, it is known that the number of platelets decreases in the blood of patients with idiopathic thrombocytopenic purpura or acute leukemia. Generally speaking, when the number of platelets in the blood becomes less than 10,000 / μL, it is judged that blood transfusion is required. Therefore, it is important to correctly classify and count platelets in clinical sites. Platelet aggregation occurs in blood collection tubes due to the mixing of tissue fluid during blood collection, insufficient mixing, or the effect of EDTA. Since the size of platelet aggregation is different from that of monomeric platelets, platelet aggregation is not counted as platelet count when blood is analyzed by an automatic blood cell analyzer. Therefore, in blood with platelet aggregation, the number of platelets decreases apparently. This phenomenon is called pseudo-thrombocytopenia and can lead to misdiagnosis. When pseudo-thrombocytopenia is suspected, it is necessary to collect blood and analyze blood cells again. Patent Document 1 describes a method for measuring platelet aggregation. In Patent Document 1, red blood cells in a blood sample are lysed with a hemolytic reagent, the remaining blood cells are stained with a fluorescent dye, and analyzed using an automatic blood cell analyzer. Furthermore, platelet aggregation is determined and counted from a scattergram based on scattered light intensity and fluorescence intensity. [Prior art literature] [Patent Literature] [Patent Document 1] U.S. Patent Application Publication No. 2020 / 0132589 [Summary of the Invention] [Problems to be solved by the invention] The present invention aims to provide a new means for enabling detection of platelet aggregation.
Methods for solving the problem
[13] .
[0001] A method for detecting platelet aggregation, comprising: a step of acquiring first scattered light information and second scattered light information generated by irradiating a measurement sample containing particles with light of a first wavelength and light of a second wavelength, and a step of acquiring information indicating the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information; The first wavelength is greater than or equal to 315 nm and less than or equal to 600 nm, and the second wavelength is greater than or equal to 610 nm and less than or equal to 750 nm.
[0002] The method described in [1] above, wherein The first scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength, The second scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light having the second wavelength.
[0003] The method described in [1] or [2] above, wherein the measurement sample is a sample prepared by mixing whole blood and a diluent reagent.
[0004] The method according to any one of [1] to [3] above, wherein the measurement sample does not contain a hemolytic reagent and a fluorescent dye.
[0005] The method according to any one of [1] to [4] above, wherein Before the step of acquiring the first scattered light information and the second scattered light information, the method further includes the step of mixing whole blood and a diluent to prepare the measurement sample. The preparation step does not include a step of hemolyzing red blood cells contained in the measurement sample, and does not include a step of staining the blood cells contained in the measurement sample with a fluorescent dye.
[0006] The method according to any one of [2] to [5] above, wherein The above-mentioned side scattered light related information is the side scattered light intensity, and the above-mentioned front scattered light related information is the front scattered light intensity. In the step of obtaining the information indicating the presence or absence of platelet aggregation, particles in the measurement sample are classified into a group containing platelets and a group containing blood cells other than platelets based on the side scattered light intensity and the forward scattered light intensity. When the platelet-containing group includes particles showing a forward scattered light intensity equal to or higher than a threshold value, information indicating the presence of platelet aggregation is obtained.
[0007] The method described in [6] above further includes the step of classifying the group containing blood cells other than platelets into a group containing red blood cells and a group containing white blood cells based on the side scattered light intensity and / or the forward scattered light intensity.
[0008] The method described in [6] or [7] above, wherein in the process of obtaining the information indicating the presence or absence of platelet aggregation, a scatter plot based on the side scattered light intensity and the forward scattered light intensity is prepared, and in the scatter plot, a group containing the platelets is determined.
[0009] The method according to any one of [2] to [5] above, wherein The above-mentioned side scattered light related information is the side scattered light intensity, and the above-mentioned front scattered light related information is the front scattered light intensity. The step of obtaining the information indicating the presence or absence of platelet aggregation comprises: a step of classifying particles in the measurement sample into a group including leukocytes and a group including blood cells other than leukocytes based on the side scattered light intensity and the forward scattered light intensity; and a step of classifying the group containing blood cells other than white blood cells into a group containing platelets and a group containing red blood cells based on the forward scattered light intensity; When the platelet-containing group includes particles showing a forward scattered light intensity equal to or higher than a threshold value, information indicating the presence of platelet aggregation is obtained.
[0010] The method described in [9] above, wherein in the step of obtaining the information indicating the presence or absence of platelet aggregation, a scattergram based on the side scattered light intensity and the forward scattered light intensity is prepared, and in the scattergram, a group containing the platelets is determined.
[0011] The method according to any one of [2] to [5] above, wherein The above-mentioned side scattered light related information is the side scattered light intensity, and the above-mentioned front scattered light related information is the front scattered light intensity. In the step of acquiring the information indicating the presence or absence of platelet aggregation, a scattergram based on the forward scattered light intensity and the side scattered light intensity is prepared, and a group of particles appearing in a designated area in the scattergram is detected as platelet aggregation.
[0012] The method described in
[11] above, wherein in the step of obtaining the information indicating the presence or absence of platelet aggregation, a scatter plot based on the forward scattered light intensity and the side scattered light intensity is prepared, and in the scatter plot, at least one selected from white blood cells, platelets and red blood cells is classified.
[0013] The method described in any one of [1] to
[12] above, wherein the first wavelength is greater than 315 nm and less than 490 nm. [Effects of the invention] The present invention provides a method for detecting platelet aggregation and a blood cell analysis reagent used in the method. [Brief description of the attached drawings]
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Fig.10
【 Fig.11 (A) is a schematic diagram showing the positions of the clusters containing PLT and the clusters containing blood cells other than PLT in a scatter plot with the first side scattered light (SSC-1) intensity on the horizontal axis and the second forward scattered light (FSC-2) intensity on the vertical axis. (B) is a schematic diagram showing the positions of platelet aggregation in the scatter plot of (A). "PLTc?" is a particle suspected of platelet aggregation.
Fig.12
【 Fig.13 (A) is a schematic diagram illustrating the positions of the clusters containing WBCs and the clusters containing blood cells other than WBCs in a scatter plot with SSC-1 on the horizontal axis and FSC-2 intensity on the vertical axis. (B) is a schematic diagram illustrating the positions of platelet aggregation in the scatter plot of (A).
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Explanation of symbols
Claims
1. A method for detecting platelet aggregation, comprising: a step of acquiring first scattered light information and second scattered light information generated by irradiating a measurement sample containing particles with light of a first wavelength and light of a second wavelength, and a step of acquiring information indicating the presence or absence of platelet aggregation based on the first scattered light information and the second scattered light information, The first wavelength is between 315 nm and 600 nm, and The second wavelength is greater than 610 nm and less than 750 nm.
2. The method of claim 1, wherein The first scattered light information is information related to side scattered light generated from the particles by irradiating the measurement sample with light of the first wavelength, and The second scattered light information is information related to forward scattered light generated from the particles by irradiating the measurement sample with light of the second wavelength.
3. The method according to claim 1, wherein the measurement sample is a sample prepared by mixing whole blood and a diluent. The method according to claim 1 , wherein the measurement sample does not contain a hemolytic reagent and a fluorescent dye.
5. The method according to claim 1, Before the step of acquiring the first scattered light information and the second scattered light information, the method further includes the step of mixing whole blood and a diluent reagent to prepare the measurement sample. The preparation step does not include a step of hemolyzing red blood cells contained in the measurement sample, and does not include a step of staining the blood cells contained in the measurement sample with a fluorescent dye.
6. The method of claim 2, wherein The side scattered light related information is the side scattered light intensity, and The forward scattered light related information is the forward scattered light intensity, In the step of obtaining information indicating the presence or absence of platelet aggregation, particles in the measurement sample are classified into a group containing platelets and a group containing blood cells other than platelets based on the side scattered light intensity and the forward scattered light intensity. When the platelet-containing group includes particles showing a forward scattered light intensity equal to or greater than a threshold value, information indicating the presence of platelet aggregation is acquired. 7 . The method according to claim 6 , further comprising the step of classifying the population containing blood cells other than platelets into a population containing red blood cells and a population containing white blood cells based on the side scattered light intensity and / or the forward scattered light intensity.
8. The method of claim 6, wherein In the step of obtaining information indicating the presence or absence of platelet aggregation, a scattergram based on the side scattered light intensity and the forward scattered light intensity is prepared, In the scatter plot, the platelet-containing population was identified.
9. The method of claim 2, wherein The side scattered light related information is the side scattered light intensity, and The forward scattered light related information is the forward scattered light intensity, The step of obtaining information indicating the presence or absence of platelet aggregation comprises: a step of classifying particles in the measurement sample into a group including leukocytes and a group including blood cells other than leukocytes based on the side scattered light intensity and the forward scattered light intensity; and a step of classifying the group containing blood cells other than white blood cells into a group containing platelets and a group containing red blood cells based on the forward scattered light intensity; When the platelet-containing group includes particles showing a forward scattered light intensity equal to or greater than a threshold value, information indicating the presence of platelet aggregation is acquired.
10. The method of claim 9, wherein In the step of obtaining information indicating the presence or absence of platelet aggregation, a scattergram based on the side scattered light intensity and the forward scattered light intensity is prepared, In the scatter plot, the platelet-containing population was identified.
11. The method of claim 2, wherein The side scattered light related information is the side scattered light intensity, and The forward scattered light related information is the forward scattered light intensity, In the step of obtaining information indicating the presence or absence of platelet aggregation, a scattergram based on the forward scattered light intensity and the side scattered light intensity is prepared, In the scattergram, a group of particles appearing in a designated area is detected as platelet aggregation.
12. The method of claim 11, wherein In the step of obtaining information indicating the presence or absence of platelet aggregation, a scattergram based on the forward scattered light intensity and the side scattered light intensity is prepared, In the scattergram, at least one selected from the group consisting of leukocytes, platelets, and erythrocytes is classified.
13. The method of claim 1, wherein the first wavelength is greater than 315 nm and less than 490 nm.
Citation Information
Patent Citations
Method and device for identifying platelet aggregation and cell analyzer
US20200132589A1