EP0268766A2
Expired / Lapsed
EP
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Method of classifying leukocytes by flow cytometry and reagents used in the method
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 Three methods for classifying leukocytes with a flow cytometer by means of optical measurements on fluorochrome-stained blood cells or those which have been so treated as to provide varying intensity in forward scattered light are useful in the practice of clinical testing. Also included are reagents employed in the practice of such methods.
Inventors
- 👤 Syouhei Matsuda
- 👤 Takashi Sakata
- 👤 Tomoyuki Kuroda
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-11-07 00:39
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/5094; G01N15/00; G01N15/10; G01N15/14; G01N15/1456; G01N1/00; G01N1/28; G01N1/30; G01N15/01; G01N2015/011; G01N2015/016; G01N21/00; G01N21/17; G01N21/47; G01N2021/4704; G01N2021/4707; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N2021/6439; Y; Y10; Y10S; Y10S436/00; Y10S436/80; Y10T; Y10T436/00; Y10T436/10; Y10T436/107497; Y10T436/25
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
188 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (a) to (c): (a) a step of preparing a specimen for measurement by adding a sample of anti-coagulated blood to a dye solution composed of a fluorochrome dye for selective staining of eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, a buffer for maintaining a pH of 6.0 - 11.0, and an osmolarity compensating agent for adjusting the osmolarity of the dye solution to a value at which the leukocytes remain unchanged in shape, and of allowing the blood sample to be stained until an equilibrium is reached; (b) a step of permitting the prepared specimen for measurement to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by intensity of fluorescence, and measuring the signals of right-angle (rectangular) scattered light and of fluorescence from leukocytes; and (c) a step of identifying the type of each of the leukocytes based on the right-angle scattered light and fluorescence 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
188 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (a) to (c): (a) a step of preparing a specimen for measurement by adding a sample of anti-coagulated blood to a dye solution composed of a fluorochrome dye for selective staining of eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, a buffer for maintaining a pH of 6.0 - 11.0, and an osmolarity compensating agent for adjusting the osmolarity of the dye solution to a value at which the leukocytes remain unchanged in shape, and of allowing the blood sample to be stained until an equilibrium is reached; (b) a step of permitting the prepared specimen for measurement to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by intensity of fluorescence, and measuring the signals of right-angle (rectangular) scattered light and of fluorescence from leukocytes; and (c) a step of identifying the type of each of the leukocytes based on the right-angle scattered light and fluorescence signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 3
Independent Claim
216 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (a) to (d): (a) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a fluorochrome dye for selective staining of eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, and a buffer for maintaining an acidic pH range; (b) a step of staining the leukocytes in the so-treated blood sample by adding to it a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining a pH suitable for staining, and an osmolarity compensating agent for adjusting the osmolarity of the resulting fluid to a value at which the leukocytes remain unchanged in shape; (c) a step of permitting the stained sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles and ghosts by intensity of fluorescence, and measuring the signals of fluorescence and right-angle (rectangular) scattered light from leukocytes; and (d) a step of identifying the type of each of the leukocytes based on the fluorescence and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 4
Independent Claim
216 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (a) to (d): (a) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a fluorochrome dye for selective staining of eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, and a buffer for maintaining an acidic pH range; (b) a step of staining the leukocytes in the so-treated blood sample by adding to it a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining a pH suitable for staining, and an osmolarity compensating agent for adjusting the osmolarity of the resulting fluid to a value at which the leukocytes remain unchanged in shape; (c) a step of permitting the stained sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles and ghosts by intensity of fluorescence, and measuring the signals of fluorescence and right-angle (rectangular) scattered light from leukocytes; and (d) a step of identifying the type of each of the leukocytes based on the fluorescence and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 5
Dependent Claim
23 words
A method according to Claim 1 or 2 wherein the fluorechrome dye for selective staining of eosinophils and basophils is Astrazon Yellow 3G.
Claim 6
Dependent Claim
87 words
A method according to Claim 1 or 2 wherein the fluorochrome dye for staining the nuclei of leukocytes is at least one member selected from the group consisting of the following dyes: 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-dimethylthiocarbocyanine; 1,1ʹdiethylthiocarbocyanine; 1,1ʹ-diethyl-9-methylthiocarbocyanine bromide; 2-[γ-1ʹethil-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); and Astrazon Orange R.
Claim 7
Independent Claim
203 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (aʹ) to (dʹ): (aʹ) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils, and a buffer for maintaining an acidic pH range; (bʹ) a step of initiating a leukocyte reaction by adding to the lysed blood sample a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining a pH suitable for leukocyte reaction, and an osmolarity compensating agent for adjusting the osmolarity of the resulting reaction solution to a value at which the leukocytes remain unchanged in shape; (cʹ) a step of permitting the sample of flow through a flow cytometer and measuring the signals of forward scattered light and right-angle scattered light from leukocytes; and (dʹ) a step of identifying the type of each of the leukocytes based on the forward scattered light and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 8
Independent Claim
203 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (aʹ) to (dʹ): (aʹ) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils, and a buffer for maintaining an acidic pH range; (bʹ) a step of initiating a leukocyte reaction by adding to the lysed blood sample a second fluid that is composed of a buffer for neutralizing the acid in the buffer in the first fluid and maintaining a pH suitable for leukocyte reaction, and an osmolarity compensating agent for adjusting the osmolarity of the resulting reaction solution to a value at which the leukocytes remain unchanged in shape; (cʹ) a step of permitting the sample of flow through a flow cytometer and measuring the signals of forward scattered light and right-angle scattered light from leukocytes; and (dʹ) a step of identifying the type of each of the leukocytes based on the forward scattered light and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 9
Independent Claim
218 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (aʺ) to (dʺ): (aʺ) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, and a buffer for maintaining an acidic pH range; (bʺ) a step of initiating a leukocyte reaction by adding to the lysed blood sample a second fluid that is composed of a buffer for neutralizing the acid in the first fluid and maintaining a pH suitable for leukocyte reaction, and osmolarity compensating agent for adjusting the osmolarity of the resulting reaction solution to a value at which the leukocytes remain unchanged in shape; (cʺ) a step of permitting the sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by intensity of fluorescence, and measuring the signals of foward scattered light and right-angle scattered light from leukocytes; and (dʺ) a step of identifying the type of each of the leukocytes based on the forward scattered light and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 10
Independent Claim
218 words
A method for classification of leukocytes by flow cytometry which comprises the following steps (aʺ) to (dʺ): (aʺ) a step of lysing the erythrocytes in a sample of anti-coagulated blood by adding it to a hypotonic first fluid composed of a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils, a fluorochrome dye for staining the nuclei of leukocytes, and a buffer for maintaining an acidic pH range; (bʺ) a step of initiating a leukocyte reaction by adding to the lysed blood sample a second fluid that is composed of a buffer for neutralizing the acid in the first fluid and maintaining a pH suitable for leukocyte reaction, and osmolarity compensating agent for adjusting the osmolarity of the resulting reaction solution to a value at which the leukocytes remain unchanged in shape; (cʺ) a step of permitting the sample to flow through a flow cytometer, differentiating leukocytes from all other corpuscles by intensity of fluorescence, and measuring the signals of foward scattered light and right-angle scattered light from leukocytes; and (dʺ) a step of identifying the type of each of the leukocytes based on the forward scattered light and right-angle scattered light signals emitted therefrom, counting the number of detected leukocytes according to their type, and calculating the proportions of individual leukocyte types.
Claim 11
Dependent Claim
28 words
A method according to Claim 5 or 6 wherein the material that selectively decreases the intensity of forward scattered light from eosinophils and basophils is Astrazon Yellow 3G.
Claim 12
Dependent Claim
85 words
A method according to Claim 6 wherein the fluorochrome dye for staining the nuclei of leukocytes is at least one member selected from the group consisting of the following dyes: 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-dimethylthiocarbocyanine; 1,1ʹ-diethylthiocarbocyanine; 1,1ʹ-diethyl-9-methylthiocarbocyanine bromide; 2-[γ-1ʹ-ethil-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); and Astrazon Orange R.
Claim 13
Dependent Claim
28 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a fluorochrome dye for selective staining of eosinophils and basophils.
Claim 14
Dependent Claim
28 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a fluorochrome dye for selective staining of eosinophils and basophils.
Claim 15
Dependent Claim
38 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a fluorochrome dye for selective staining of eosinophils and basophils and a fluorochrome dye for staining the nuclei of leukocytes.
Claim 16
Dependent Claim
38 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a fluorochrome dye for selective staining of eosinophils and basophils and a fluorochrome dye for staining the nuclei of leukocytes.
Claim 17
Dependent Claim
24 words
A reagent system according to Claim 9 or 10 wherein the fluorochrome dye for selective staining of eosinophils and basophils is Astrazon Yellow 3G.
Claim 18
Dependent Claim
33 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils.
Claim 19
Dependent Claim
33 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils.
Claim 20
Dependent Claim
43 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils and a fluorochrome dye for staining the nuclei of leukocytes.
Claim 21
Dependent Claim
43 words
A reagent system for use in the classification of leukocytes into five types by flow cytometry which contains a material that selectively decreases the intensity of forward scattered light from eosinophils and basophils and a fluorochrome dye for staining the nuclei of leukocytes.
Claim 22
Dependent Claim
29 words
A reagent system according to Claim 12 or 13 wherein the material that selectively decreases the intensity of forward scattered light from eosinophils and basophils is Astrazon Yellow 3G.
Claim 23
Dependent Claim
93 words
A reagent system according to any one of Claims 9, 10, 12 and 13 wherein the fluorochrome dye for staining the nuclei of leukocytes is at least one member selected from the group consisting of the following dyes: 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-dimethylthiocarbocyanine; 1,1ʹ-diethylthiocarbocyanine; 1,1ʹ-diethyl-9-methylthiocarbocyanine bromide; 2-[γ-1ʹ-ethil-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); and Astrazon Orange R.
References Cited (Backward Citations)
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