EP0485945A2
Expired / Lapsed
EP
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Method of classifying leukocytes by flow cytometry
Assignee: Sysmex Corp
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Filing: 1991-11-12
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Granted: —
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Expires: 2011-11-12
Abstract
Abstract A method for leukocyte classification which relies basically upon flow cytometry that uses a first fluid that is hypotonic acidic fluorescent dye solution and a second fluid that is a solution that changes the osmolarity and pH of the first fluid is disclosed. In accordance with the method, a fluorescent dye that specifically stains the nuclei of damaged leukocyte cells is added to the first fluid and the resulting fluid is used in measurements. The first fluid to be used in the method incorporates not only the first and second dyes but also the third dye and this insures that even leukocytes with damaged cell membrane can be distributed as entities distinct from other leukocyte types on a two-dimensional plot. And, hence, high precision of leukocyte classification is assured even in the case of assaying blood that contains damaged cells as a result of prolonged standing at room temperature after sampling.
Inventors
- 👤 Yasunori Maekawa
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 04:53
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/1456; G01N15/01; G01N2015/016; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N2021/6439; G01N2021/6441; Y; Y10; Y10S; Y10S436/00; Y10S436/80; Y10S436/805; Y10T; Y10T436/00; Y10T436/10; Y10T436/107497; Y10T436/25
Claims Analysis
Total Claims: 6
Claim 1
Independent Claim
284 words
A method of classifying leukocytes by flow cytometry which comprises the following steps (a) to (e): (a) mixing blood with a hypotonic first fluid comprising a first dye that is a fluorescent dye capable of selectively staining both eosinophils and basophils, a second dye that is a fluorescent dye capable of staining the nuclei of leukocytes, a third dye that is a fluorescent dye capable of staining the nuclei of leukocytes with damaged cell membrane, and a buffer for maintaining a pH in the acidic range; (b) mixing the sample solution obtained in step (a) with a second fluid comprising a buffer that neutralizes the acid in the first fluid for maintaining the pH of the solution at a staining pH and an osmolarity adjusting agent for adjusting the osmolarity of the solution to a value at which the leukocytes remain unchanged in shape; (c) loading the thus prepared sample solution in a flow cytometer and obtaining more than one signal for parameters including fluorescence and scattered light in association with individual types of leukocytes; (d) constructing a first two-dimensional plot of the intensities of two of the signals as emitted from leukocytes; and (e) separating the distribution zones of individual leukocyte types on said first two-dimensional plot by setting the distribution zones of cell groups that, on account of a certain factor such as the lapse of time after blood sampling, deviate from the zones where they would otherwise be located, constructing, for the cell groups in those set zones, a second two-dimensional plot of the intensities of two other signals, separating on said second two-dimensional plot those cell groups of the deviated distribution and counting the number of cells in each zone.
Claim 2
Dependent Claim
27 words
A method according to Claim 1 wherein the third dye in the first fluid is selected from the group consisting of Propidium iodide, Etidium bromide and M-264.
Claim 3
Dependent Claim
18 words
A method according to Claim 1 wherein the first dye in the first fluid is Astrazon Yellow 3G.
Claim 4
Dependent Claim
70 words
A method according to Claim 1 wherein the second dye in the first fluid is selected from the group consisting of the following members: Acridine Red; Rhodamine S; Rhodamine 6G; Rhodamine B; Rhodamine 19 perchlorate; Rhodamine 123; Eosin Y; Cyanosine; Cresyl Fast Violet; Darrow Red; Acronol Phloxine FFS; 1,1'-dimethylthiocarbocyanine; 1,1'-diethylthiocarbocyanine; 1,1'-diethyl-9-methylthiocarbocyanine bromide; 2-[γ-(1'-ethyl-4',5'-benzothiazolylidene)-propenyl]-1-ethyl-4,5-benzoxazolium iodide; Astrazon Red 6B; Basic Violet 16; 2-(p-dimethylaminostyryl)-1-ethyl-4,5-benzothiazolium iodide; 2,4-bis(p-dimethylaminostyryl)-1-ethylpyridinium iodide; 2,6-bis(p-dimethylaminostyryl)-1-ethylpyridinium iodide; and Astrazon Orange R.
Claim 5
Independent Claim
47 words
A method according to any one of the preceding claims wherein the first two-dimensional plot is that of the intensity of green fluorescence vs the intensity of side-scattered light, and the second two-dimensional plot is that of the intensity of red fluorescence vs that of side-scattered light.
Claim 6
Dependent Claim
43 words
A method according to Claim 5 wherein the zones set in the first two-dimensional plot are the distribution zones of monocytes and neutrophils and, monocytes, intact neutrophils and neutrophils with damaged cell membrane are separated from one another on the second two-dimensional plot.
References Cited (Backward Citations)
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