Patent US3916205A successfully refreshed from Google Patents.
US3916205A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Differential counting of leukocytes and other cells
Assignee: Bio Rad Laboratories Inc
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Filing: 1973-05-31
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Granted: 1975-10-28
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Expires: 1993-05-31
Abstract
Abstract A mixture of dyes for differentially staining biological cells. A system for the automatic absolute counting of leukocytes, erythrocytes and reticulocytes and differential counting of leukocytes utilizes a composition characterized by specific fluorescent dyes for staining a blood specimen and includes means for irradiating, counting and classifying leukocyte types. When the stained sample is irradiated, characteristic fluorescent intensities in specific spectral ranges uniquely identify particular leukocyte types.
Inventors
- 👤 Marcos Kleinerman
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-23 23:10
CPC Classifications
G; G01; G01N; G01N33/00; G01N33/48; G01N33/50; G01N33/5005; G01N33/5094; G01N15/00; G01N15/10; G01N15/14; G01N15/1468; G01N15/147; G01N15/01; G01N2015/016; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; G01N2021/6439; G01N2021/6441
Claims Analysis
Total Claims: 23
Claim 1
Independent Claim
121 words
Method of identifying a blood cell, and comprising the steps of: staining said blood cell with a plurality of dyes, at least one of which is a fluoresecent dye; illuminating the dyed cell with a sequence of exposures to radiation, each of said exposures being at a limited wavelength band corresponding to the wavelength absorption band of a respective one of said dyes; observing during each such exposure the characteristic wavelength band of emission or transmission by a selected one of said dyes in said dyed cell in response to each of said exposures; and comparing with one another the relative intensities of the emission or transmission observed for each sequential exposure, whereby said cell can be characterized by such comparison.
Claim 2
Dependent Claim
121 words
Method of identifying a blood cell, and comprising the steps of: staining said blood cell with a plurality of dyes, at least one of which is a fluoresecent dye; illuminating the dyed cell with a sequence of exposures to radiation, each of said exposures being at a limited wavelength band corresponding to the wavelength absorption band of a respective one of said dyes; observing during each such exposure the characteristic wavelength band of emission or transmission by a selected one of said dyes in said dyed cell in response to each of said exposures; and comparing with one another the relative intensities of the emission or transmission observed for each sequential exposure, whereby said cell can be characterized by such comparison.
Claim 3
Dependent Claim
83 words
Method as defined in claim 1 wherein another dye of said plurality of dyes is a sensitizer capable of transferring energy absorbed in its characteristic absorption wavelength band to molecules of said fluorescent dye in proximity to said sensitizer; and further including the step of observing the fluorescent emission from said dyed cell in the characteristic wavelength emission band of said fluorescent dye in response to an exposure of said dyed cell to radiation in the characteristic wavelength absorption band of said sensitizer.
Claim 4
Dependent Claim
50 words
A method as defined in claim 1 wherein said plurality is at least three, and one of said dyes differentially dyes nucleic acids of said cell, one of said dyes stains the basic portions of said cell and the third of said dyes dyes the acidic portions of said cell.
Claim 5
Dependent Claim
38 words
A method as defined in claim 1 wherein at least two of said dyes are fluorescent dyes which dye different types of blood cells so that each said type fluoresces under each said illumination with a different intensity.
Claim 6
Dependent Claim
16 words
A method as defined in claim 4 wherein said cell types are leukocytes, erythrocytes and reticulocytes.
Claim 7
Dependent Claim
16 words
A method as defined in claim 4 wherein said cell types are different types of leukocytes.
Claim 8
Dependent Claim
40 words
The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes sulfonated triazinyl derivatives of 4,4'' diamino stilbene for imparting a blue fluorescence to the protein of leukocytes and to neutrophil and eosinophil granules.
Claim 9
Dependent Claim
29 words
The method for identifying leukocytes types as claimed in claim 6 wherein said plurality of dyes includes 8-p-toluidino 1-naphthalene sulfonic acid for imparting a blue-green fluorescence to eosinophil granules.
Claim 10
Dependent Claim
27 words
The method for identifying leukocytes distributed as claimed in claim 6 wherein said plurality of dyes includes brilliant sulfalfavine for imparting a blue-green fluorescence to eoSinophil granules.
Claim 11
Dependent Claim
30 words
The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes ethidium bromide for imparting a red fluorescence to the nuclei of all leukocytes.
Claim 12
Independent Claim
96 words
A system for identifying leukocytes, erythrocytes and reticulocytes randomly distributed in a medium, said system comprising: a. means for staining said leukocytes, erythrocytes and reticulocytes with a dye composition including sulfonated triazinyl derivatives of 4,4'' diamino stilbene, sulfonated fluorescent derivatives of 1,8 napthalimide, and a phenanthridinium dye; b. source means for sequentially illuminating the stained leukocytes, erythrocytes and reticulocytes with optical radiation in the characteristic absorption wavelength band of each said dye and c. detector means optically coupled to said irradiated leukocytes, erythrocytes and reticulocytes for identifying each said type cell according to its fluorescent intensity.
Claim 13
Independent Claim
96 words
A system for identifying leukocytes, erythrocytes and reticulocytes randomly distributed in a medium, said system comprising: a. means for staining said leukocytes, erythrocytes and reticulocytes with a dye composition including sulfonated triazinyl derivatives of 4,4'' diamino stilbene, sulfonated fluorescent derivatives of 1,8 napthalimide, and a phenanthridinium dye; b. source means for sequentially illuminating the stained leukocytes, erythrocytes and reticulocytes with optical radiation in the characteristic absorption wavelength band of each said dye and c. detector means optically coupled to said irradiated leukocytes, erythrocytes and reticulocytes for identifying each said type cell according to its fluorescent intensity.
Claim 14
Dependent Claim
18 words
The system as claimed in claim 11 wherein said sulfonated fluorescent derivatives of 1,8 naphthalimide is brilliant sulfaflavine.
Claim 15
Dependent Claim
14 words
The system as claimed in claim 11 wherein said phenanthridinium dye is ethidium bromide.
Claim 16
Dependent Claim
29 words
The system as claimed in claim 11 wherein said detector means includes photodetector means for measuring light scattered by said leukocytes for differentiating said leukocytes according to their size.
Claim 17
Independent Claim
159 words
A system for differentially classifying and counting leukocyte types, particularly eosinophils, neutrophils, monocytes and lymphocytes stained with at least a pair of compatible fluorescent dyes and fixed on slide means, said system comprising: means for illuminating said slide means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said slide means for detecting, during each of said exposures, the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposuresbut not the characteristic wavelength band emitted by any of the other dyes responsively to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
Claim 18
Independent Claim
159 words
A system for differentially classifying and counting leukocyte types, particularly eosinophils, neutrophils, monocytes and lymphocytes stained with at least a pair of compatible fluorescent dyes and fixed on slide means, said system comprising: means for illuminating said slide means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said slide means for detecting, during each of said exposures, the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposuresbut not the characteristic wavelength band emitted by any of the other dyes responsively to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
Claim 19
Independent Claim
223 words
A system for differentially classifying and counting leukocyte types randomly distributed in a blood sample, said system comprising: means for staining the leukocyte types in said sample with a dye composition comprising a plurality of fluorescent dyes, at least one of said fluorescent dyes imparting a first characteristic fluorescence to the nuclei of all said leukocytes, said other fluorescent dyes imparting a second characteristic fluorescence to the cytoplasm of eosinophils, neutrophils, monocytes and lymphocytes; means for flowing the stained leukocytes in said sample in single file; source means for illuminating said stained leukocytes passing in single file through said flow means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said flow means for detecting, during each of said exposures, only the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposures but not any wavelength band emitted by any other of said dyes in response to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
Claim 20
Independent Claim
223 words
A system for differentially classifying and counting leukocyte types randomly distributed in a blood sample, said system comprising: means for staining the leukocyte types in said sample with a dye composition comprising a plurality of fluorescent dyes, at least one of said fluorescent dyes imparting a first characteristic fluorescence to the nuclei of all said leukocytes, said other fluorescent dyes imparting a second characteristic fluorescence to the cytoplasm of eosinophils, neutrophils, monocytes and lymphocytes; means for flowing the stained leukocytes in said sample in single file; source means for illuminating said stained leukocytes passing in single file through said flow means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said flow means for detecting, during each of said exposures, only the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposures but not any wavelength band emitted by any other of said dyes in response to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
Claim 21
Dependent Claim
131 words
System for identifying a blood cell stained with a plurality of compatible dyes, at least one of which is a fluorescent dye, said system comprising, in combination: means for illuminating said cell with a sequence of exposures to radiation, each exposure of said sequence being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; means for observing during each of said exposures only the characteristic wavelength band emitted or transmitted by one of said dyes in said cell in response to a corresponding one of said exposures; means for generating a signal in response to each emission or transmission from said cell resulting from each of said exposures; and means for comparing said signals to obtain a characteristic value for said cell.
Claim 22
Dependent Claim
131 words
System for identifying a blood cell stained with a plurality of compatible dyes, at least one of which is a fluorescent dye, said system comprising, in combination: means for illuminating said cell with a sequence of exposures to radiation, each exposure of said sequence being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; means for observing during each of said exposures only the characteristic wavelength band emitted or transmitted by one of said dyes in said cell in response to a corresponding one of said exposures; means for generating a signal in response to each emission or transmission from said cell resulting from each of said exposures; and means for comparing said signals to obtain a characteristic value for said cell.
Claim 23
Dependent Claim
110 words
System as defined in claim 17 wherein said means for illuminating includes means for providing at least two of said exposures at different wavelength bands corresponding to the absorption bands of respective ones of said dyes; and said means for observing includes means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye due only to exposure of said cell to radiation in the wavelength absorption band of said fluorescent dye, and means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye due only to exposure of said cell to radiation in the wavelength absorption band of another of said dyes.
References Cited (Backward Citations)
US3315229A
Cited
US3497690A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1978-10-27 | 1979-04-28 | 1979-10-28 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-10-27 | 1983-04-28 | 1983-10-28 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-10-27 | 1987-04-28 | 1987-10-28 | $7700.00 | Window Closed / Lapsed |