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Detection method and apparatus of activated neutrophils

A technology for neutrophils and eosinophils, which is applied in the measurement device, measurement of scattering characteristics, fluorescence/phosphorescence, etc., can solve the problems of undisclosed activated neutrophil detection, etc., and achieve the effect of good accuracy

Active Publication Date: 2013-05-01
SYSMEX CORP
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Problems solved by technology

However, none of these patent documents disclose the detection of activated neutrophils

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  • Detection method and apparatus of activated neutrophils
  • Detection method and apparatus of activated neutrophils
  • Detection method and apparatus of activated neutrophils

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preparation example Construction

[0102] [Preparation of measurement samples]

[0103] In the above preparation steps, the order of mixing the living body sample, the fluorescent dye, and the hemolytic agent is not particularly limited. For example, a fluorescent dye and a hemolytic agent are first mixed, and this mixed solution may be mixed with a living body sample. Alternatively, the hemolytic agent and the living body sample are first mixed, and this mixed solution is also mixed with a fluorescent dye. In embodiments of the invention, mixing in any order yields equivalent results.

[0104] In an embodiment of the present invention, the mixing of the living body sample, the fluorescent dye and the hemolytic agent is that the volume ratio of the living body sample: the mixture of the fluorescent dye and the hemolytic agent is 1:5 to 1:1000, preferably mixed to 1:10 to 1 :100 is preferred. In this case, it is preferable that the ratio of the fluorescent dye to the hemolytic agent in the mixture is 1:1 to 1...

Embodiment 1

[0158]Peripheral blood was collected from a healthy person using a heparin blood collection tube for blood tests. The obtained blood was centrifuged at room temperature using a monomer separation solution (Dainippon Sumitomo Pharmaceutical Co., Ltd.) to obtain a multinucleated cell sample with a purity of 90% or higher. In addition, this operation was carried out according to the instructions of the manufacturer (Sumitomo Dainippon Pharmaceutical Co., Ltd.). Then, the obtained multinucleated cells (2×10 5 each) suspended in 1 mL of 0.1% bovine serum albumin (phosphate-buffered saline). To stain neutrophils among multinucleated cells, CD16 (anti-human CD16 Fcγ receptor III (clone: ​​DJ130c) mouse monoclonal antibody / RPE-labeled neutrophil-specific antibody for flow cytometry; Dako Company) (final concentration 750 μg / L), incubated at room temperature for 10 minutes. Next, APF (aminophenylfluorescein; Sekisui Medical) (final concentration: 10 μM) was added as a reagent for R...

reference example 1

[0167] In order to verify the results of Example 1 above, it was examined whether stimulated and activated neutrophils also showed increased ROS production ability in microscopic observation.

[0168] In the same manner as in Example 1, samples containing activated neutrophils were prepared from peripheral blood of healthy individuals. However, only PMA was used as a neutrophil-stimulating substance. In addition, as control samples, a sample not treated with APF and a stimulating substance (-APF), and a sample not treated with a stimulating substance (+APF) were prepared.

[0169] Each prepared sample was transferred to a glass bottom dish coated with poly-L-lysine, and the cells in the sample were examined with a confocal laser microscope (I×81 (manufactured by Olympus), CSU-X1 (Yokogawa Corporation) mechanism), ImagEM (manufactured by Hamamatsu Photonics)) to observe neutrophils.

[0170] The results are shown in Figure 11 . exist Figure 11 In the PMA-added sample, ne...

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Abstract

A detection method and an apparatus of activated neutrophils are provided, to provide a detection method of activated neutrophils enabling the classification of normal leukocytes and the detection of activated neutrophils, a measurement sample in which erythrocytes in a biotic sample are hemolyzed and nucleic acid of leukocytes is stained by a nucleic acid staining fluorochrome is prepared, the sample is irradiated with light, and leukocytes in the biotic sample are classified into at least a cluster containing neutrophils and a cluster containing eosinophils based on scattered light intensity and fluorescence intensity obtained by measuring the scattered light intensity and fluorescence intensity generated from particles in the sample to detect particles present between the cluster containing neutrophils and the cluster containing eosinophils as activated neutrophils.

Description

【Technical field】 [0001] The invention relates to a method and a device for detecting activated neutrophils in living samples. 【Background technique】 [0002] Normal white blood cells are usually classified into: granulocytes including neutrophils, eosinophils and basophils, and lymphocytes and monocytes. Among them, neutrophils account for 50 to 60% of white blood cells, and play an important role in living body defense against pathogenic microorganisms such as bacteria and fungi. For example, when a foreign body invades a living body due to bacterial infection or the like, neutrophils are activated by the inflammatory reaction of the living body, migrate to the infected site, and swallow the foreign matter. Foreign substances swallowed by neutrophils are sterilized as reactive oxygen species (ROS) of various decomposing enzymes and powerful toxic substances produced in neutrophils. [0003] On the other hand, neutrophils activated by stimulation (hereinafter also referre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N21/47
CPCG01N33/5094G01N15/1468
Inventor 河野麻理高木由里
Owner SYSMEX CORP
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