Patent EP0259834A2 successfully refreshed from Google Patents.
EP0259834A2
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EP
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Method of classifying leucocytes by flow cytometry
Assignee: Sysmex Corp
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Filing: 1987-09-08
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Granted: —
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Expires: 2007-09-08
Abstract
Abstract A method for classifying leukocytes with a flow cytometer by means of optical measurements on fluorochrome-stained blood cells is useful in the practice of clinical testing.
Inventors
- 👤 Tomoyuki Kuroda
- 👤 Takashi Sakata
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-09 03:57
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/1456; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/5094; G01N1/00; G01N1/28; G01N1/30; G01N15/02; G01N15/0205; G01N2015/0238; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N2021/6439
Claims Analysis
Total Claims: 8
Claim 1
Independent Claim
183 words
A method for classifying leukocytes by flow cytometry which comprises the following steps (a) to (c): (a) preparing a specimen for measuring by adding a fresh sample of anti-coagulated blood to a dye solution and allowing it to stand until equilibrium is reached, said dye solution consisting of a buffer for maintaining a pH in the range of 3.5 - 10.0, an inorganic salt for maintaining the osmolarity of the dye solution at one half to twice the physiological osmolarity so that the leukocites in the blood will not deform excessively, and a fluorochrome dye for differentially staining the leukocytes according to their cytochemical characters; (b) permitting the prepared specimen for measurement to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by the intensity of fluorescence, and measuring the signal of right-angle (rectangular) scattered light and at least one fluorescence signal emitted from the leukocytes; and (c) identifying the type of each of the leukocytes based on the two signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 2
Independent Claim
183 words
A method for classifying leukocytes by flow cytometry which comprises the following steps (a) to (c): (a) preparing a specimen for measuring by adding a fresh sample of anti-coagulated blood to a dye solution and allowing it to stand until equilibrium is reached, said dye solution consisting of a buffer for maintaining a pH in the range of 3.5 - 10.0, an inorganic salt for maintaining the osmolarity of the dye solution at one half to twice the physiological osmolarity so that the leukocites in the blood will not deform excessively, and a fluorochrome dye for differentially staining the leukocytes according to their cytochemical characters; (b) permitting the prepared specimen for measurement to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by the intensity of fluorescence, and measuring the signal of right-angle (rectangular) scattered light and at least one fluorescence signal emitted from the leukocytes; and (c) identifying the type of each of the leukocytes based on the two signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 3
Dependent Claim
80 words
A method according to Claim 1 for classifying leukocytes into at least four types by using one of the following dyes in step (a): Group I: Xanthene dyes Pyronine Y Rhodamine 3GO Fluorescein Group II: Oxacarbocyanine dyes DiOC1(3) DiOC2(3) DiOC3(S) DiOC5(3) DiOC6(3) Group III: Acridine dyes Acridine Orange Brilliant Phosphine Rhoduline Orange Euchrysin, 3RX Flavophosphine R Coriphosphine O Group IV : Azine dyes Neutral Red Group V : Diphenylmethane dyes Auramine O Group VI : Methine dyes Astrazon Orange G
Claim 4
Dependent Claim
94 words
A method according to Claim 1 for classifying leukocytes into at least three types by using one of the following dyes in step (a): Group I : Xanthene dyes Acridine Red; Rhodamine S; Rhodamine 6G; Rhodamine B; Rhodamine 19 perchlorate; Rhodamine 123; Eosin Y; Cyanosine; Group VII: Oxazine dyes Cresyl Fast Violet; Darrow Red; Group VIII: Cyanine dyes Acronol Phloxine FFS; 1,1ʹ-dimethylthiocarbocyanine; 1-1ʹ-diethylthiocarbocyanine; 1-1ʹ-diethyl-9-methylthiocarbocyanine bromide; 2-[γ-(1ʹ-ethyl-4ʹ,5ʹ-benzothiazolylidene) propenyl]-1-ethyl-4,5-benzoxazolium iodide; Group IX : Styryl dyes Astrazon Red 6B; C.I.Basic Violet 16; 2-(p-dimethylaminostyryl) -1-ethyl-4,5-benzothiazolium iodide; 2,4-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; 2,6,-bis(p-dimethylaminostyryl)-1-ethyl-pyridinum iodide; TA-2 (Nippon Kankoh-Shikiso Kenkyusho Co., Ltd., Okayama, Japan
Claim 5
Dependent Claim
84 words
A method according to Claim 1 for classifying leukocytes into at least five types by using two or more of the following dyes in combination in step (a): Group I: Xanthene dyes Pyronine Y Rhodamine 3GO Fluorescein Group II: Oxacarbocyanine dyes DiOC1(3) DiOC2(3) DiOC3(3) DiOC5(3) DiOC6(3) Group III: Acridine dyes Acridine Orange Brilliant Phosphine Rhoduline Orange Euchrysin, 3RX Flavophosphine R Coriphosphine O Group IV : Azine dyes Neutral Red Group V : Diphenylmethane dyes Auramine O Group VI : Methine dyes Astrazon Orange G
Claim 6
Dependent Claim
48 words
A method according to Claim 4 for classifying leukocytes into at least five types by additionally using one of the following dyes in step (a): Rhodamine S; Rhodamine 19 perchlorate; Acronol Phloxine FFS; 2-[γ-(1ʹ-ethyl-4ʹ,5ʹ-benzothiazolylidene)propenyl]-1-ethyl-4,5-benzoxazolium iodide; 1,1ʹ-diethyl-9-methylthiocarbocyanine bromide; Astrazon Red 6B; and TA-2(Nippon Kankoh-Shikiso Kenkyusho Co., Ltd., Okayama, Japan).
Claim 7
Dependent Claim
44 words
A method according to Claim 1 wherein one of an Ar ion laser, a He-Ne laser, Krypton ion laser, a He-Cd laser, a Hg arc lamp and a Xe arc lamp is used as a light source for the flow cytometer in step (b).
Claim 8
Dependent Claim
99 words
A method according to Claim 6 which employs one or more of the dyes listed below: Acridine Red; Rhodamine S; Rhodamine 6G; Rhodamine B; Rhodamine 19 perchlorate; Rhodamine 123; Eosin Y; Cyanosine; Cresyl Fast Violet; Darrow Red; Acronol Phloxine FFS (1,1ʹ,3,3,3ʹ,3ʹ,hexamethylindocarbocyanine); 1,1ʹ-dimethylthiocarbocyanine; 1,1ʹ-diethylthiocarbocyanine; 1,1ʹ-diethyl-9-methylthiocarbocyanine bromide [9-Me-DiSC2(3)]; 2-[γ-(1ʹ-ethyl-4ʹ,5ʹ-benzothiazolylidene)propenyl]-1-ethyl-4,5-benzoxazolium iodide; Astrazon Red 6B; C.I. Basic Violet 16; 2-(p-dimethylaminostyryl)-1-ethyl-4,5-benzothiazolium iodide 2-4-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; 2-6-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; TA-2 (Nippon Kankoh-Shikiso Kenkyusho Co., Ltd. Okayama, Japan; Pyronine Y; Rhodamine 3GO; Fluorescein; DiOC1(3); DiOC2(3); DiOC3(3); DiOC5(3); DiOC6(3); Acridine Orange; Brilliant Phosphine; Rhoduline Orange; Euchrysin 3RX; Flavophosphine R; Coriphosphine O; Neutral Red; Auramine O; and Astrazon Orange G.
References Cited (Backward Citations)
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