US3883247A
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US
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Method for fluorescence analysis of white blood cells
Assignee: Biophysics Systems Inc
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Filing: 1973-10-30
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Granted: 1975-05-13
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Expires: 1993-10-30
Abstract
Abstract A dye composition for differential analysis of white blood cells comprising a hypotonic aqueous solution of a metachromatic fluorochrome dye, e.g., acridine orange. The solution has a pH factor within normal physiological ranges for human blood. The white cell analysis is made by straining the cells with the fluorescent dye by suspending a sample of fresh blood in the dye solution, subjecting the suspension before dye uptake equilibrium is reached to radiation from a light source (e.g., radiation from a blue laser) having a wave length within the range of absorption of the dye, distinguishing the white cells from other blood particles by detecting fluorescenses (e.g., green fluorescences) emitted from the white cells in response to the radiation, and further distinguishing the various types of white cells by measuring the magnitudes of their respective fluorescence emissions (e.g., red and green fluorescences). Differences in the rates of uptake of fluorochrome dye of the various kinds of white cells are accentuated by altering the aqueous dye solution so that it becomes hypotonic.
Inventors
- 👤 Lawrence R Adams
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-25 14:58
CPC Classifications
G; G01; G01N; G01N21/00; G01N21/62; G01N21/63; G01N21/64; G01N21/6428; A; A61; A61B; A61B5/00; A61B5/41; A61B5/414; A61B5/417; G01N1/00; G01N1/28; G01N1/30; G01N2021/6417; G01N2021/6421; G01N2021/6439; G01N2021/6441
Claims Analysis
Total Claims: 29
Claim 1
Independent Claim
175 words
A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being of hypotonic osmolarity and of physiologically normal pH, and said dye being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said hypotonic diluent solution being further characterized in that it can alter the normal rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.
Claim 2
Independent Claim
175 words
A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being of hypotonic osmolarity and of physiologically normal pH, and said dye being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said hypotonic diluent solution being further characterized in that it can alter the normal rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.
Claim 3
Independent Claim
188 words
A method for distinguishing white blood cells from other particles within a blood sample and for differentiating the white blood cells as between neutrophils, eosinophils, basophils, lymphocytes, monocytes, and immature granulocytes, said method comprising the steps of: a. combining a blood sample with an aqueous solution of acridine orange having a physiologically normal pH factor to form a suspension, said solution having a concentration of acridine orange sufficient to provide a concentration of acridine orange in the suspension within the range of between about 4 X 10 4 and about 4 X 10 2 grams per liter of suspension, and said solution having a salt concentration corresponding to less than the physiologically normal osmolarity; b. subjecting the suspension obtained in step (a) to radiation and distinguishing the white cells from all other blood particles by the detection of green fluorescence emitted from individual white cells in response to the excitation from the radiation; and c. simultaneously detecting the magnitudes of red and green fluorescences emitted from individual white cells in response to excitation from the radiation, and differentially classifying the white cells on the basis of such detection.
Claim 4
Independent Claim
188 words
A method for distinguishing white blood cells from other particles within a blood sample and for differentiating the white blood cells as between neutrophils, eosinophils, basophils, lymphocytes, monocytes, and immature granulocytes, said method comprising the steps of: a. combining a blood sample with an aqueous solution of acridine orange having a physiologically normal pH factor to form a suspension, said solution having a concentration of acridine orange sufficient to provide a concentration of acridine orange in the suspension within the range of between about 4 X 10 4 and about 4 X 10 2 grams per liter of suspension, and said solution having a salt concentration corresponding to less than the physiologically normal osmolarity; b. subjecting the suspension obtained in step (a) to radiation and distinguishing the white cells from all other blood particles by the detection of green fluorescence emitted from individual white cells in response to the excitation from the radiation; and c. simultaneously detecting the magnitudes of red and green fluorescences emitted from individual white cells in response to excitation from the radiation, and differentially classifying the white cells on the basis of such detection.
Claim 5
Dependent Claim
35 words
A method according to claim 2 wherein the blood sample is fresh peripheral blood and wherein said salt concentration within the solution of acridine orange is less than about 7.5 grams per liter of solution.
Claim 6
Dependent Claim
76 words
A mEthod according to claim 2 wherein the suspension obtained in step (a) is directed in step (b) in a fine stream through the flow channel of an optical chamber wherein the suspension is subjected to radiation, the fine stream within the flow channel being so narrow as to be capable of confining the flow to substantially a single cell width such that the cells traverse the radiation beam one at a time in single file.
Claim 7
Dependent Claim
73 words
A method according to claim 4 wherein the radiation employed in step (b) is a blue laser of wavelength in the neighborhood of 488 millimicrons and is directed toward the suspension stream in the form of a narrow beam substantially transverse to the suspension stream and having a beam dimension in the direction of the suspension stream which is on the same order of magnitude as the maximum dimension of an individual cell.
Claim 8
Dependent Claim
36 words
A method according to claim 5 wherein the step of subjecting said suspension to radiation from a blue laser is carried out by energizing an argon ion laser and directing the resulting radiation to said suspension.
Claim 9
Dependent Claim
31 words
A method according to claim 4 wherein the step of subjecting said suspension to radiation is carried out by energizing a helium-cadmium laser and directing the resulting radiation to said suspension.
Claim 10
Dependent Claim
48 words
A method according to claim 2 wherein the red fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 6,500 angstrom units and the green fluorescence radiation is discriminated by an optical filter which passes wavelengths on the order of 5,300 angstrom units.
Claim 11
Dependent Claim
52 words
A method according to claim 2 wherein the differences in the magnitudes of red fluorescence and the differences in the magnitudes of green fluorescence emitted from individual cells are detected by converting the optical signals into electrical signals and displaying the resultant electrical signals upon the face of a cathode ray tube.
Claim 12
Independent Claim
206 words
A method for making a differential blood analysis of white cells, said method comprising the steps of: a. combining undiluted and untreated fresh blood with an aqueous solution of acridine orange in the ratio of up to about 1 part of blood to 5 parts of solution to form a suspension, said solution having a concentration in the neighborhood of 4 X 10 3 grams of acridine orange per liter of solution, said solution containing a buffering agent to maintain the pH factor of the suspension within the normal range for human blood, and said solution having an osmolarity below the normal physiological range for human blood plasma; b. passing the suspension formed in step (a) through a photoanalysis apparatus and subjecting each white cell in the suspension to radiation; c. discriminating the white cells from all blood particles contained in the suspension by detecting fluorescent radiation emissions above a predetermined threshold value from each cell in the green portion of the visible spectrum; d. detecting the magnitudes of the fluorescent radiation emissions from each white cell in the red and green portions of the visible spectrum; and e. classifying cells based upon the differences in magnitudes of red and green fluorescences measured from the cells.
Claim 13
Independent Claim
206 words
A method for making a differential blood analysis of white cells, said method comprising the steps of: a. combining undiluted and untreated fresh blood with an aqueous solution of acridine orange in the ratio of up to about 1 part of blood to 5 parts of solution to form a suspension, said solution having a concentration in the neighborhood of 4 X 10 3 grams of acridine orange per liter of solution, said solution containing a buffering agent to maintain the pH factor of the suspension within the normal range for human blood, and said solution having an osmolarity below the normal physiological range for human blood plasma; b. passing the suspension formed in step (a) through a photoanalysis apparatus and subjecting each white cell in the suspension to radiation; c. discriminating the white cells from all blood particles contained in the suspension by detecting fluorescent radiation emissions above a predetermined threshold value from each cell in the green portion of the visible spectrum; d. detecting the magnitudes of the fluorescent radiation emissions from each white cell in the red and green portions of the visible spectrum; and e. classifying cells based upon the differences in magnitudes of red and green fluorescences measured from the cells.
Claim 14
Dependent Claim
20 words
A method according to claim 10 wherein the osmolarity of the acridine orange solution is between 0.3 and 0.7 isotonic.
Claim 15
Dependent Claim
29 words
A method according to claim 11 wherein the green radiation signals are employed to derive a total count of the white cells from a measured volume of the suspension.
Claim 16
Dependent Claim
130 words
A method according to claim 11 wherein: the magnitudes of the fluorescent radiation emissions from each cell in the red portion of the visible spectrum are detected by converting the red optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the red signals being indicated in terms of the cathode ray tube deflection on one axis; and the magnitudes of the fluOrescent radiation emissions from each cell in the green portion of the visible spectrum are detected by converting the green optical signals to electrical signals and displaying said electrical signals upon the face of a cathode ray tube, the magnitude of the green signals being indicated in terms of the cathode ray tube deflection the other axis.
Claim 17
Dependent Claim
29 words
A method according to claim 13 wherein a camera is placed over the face of the cathode ray tube to record the pattern of spots produced by said method.
Claim 18
Dependent Claim
75 words
A method according to claim 10 wherein: voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and the signals from the overlapping portions of the narrow red and green fields are individually counted for a carefully measured blood sample volume to obtain a quantitative measure of those white cells having the particular characteristics exemplified by overlapping portions of said red and green fields.
Claim 19
Dependent Claim
112 words
A method according to claim 13 wherein: the green fluorescence signals above a predetermined threshold are counted up to a predetermined count value to thereby determine the passage of the number of white cells corresponding to that count value; voltage thresholds are applied to select red fluorescence signals within a narrow field and green fluorescence signals within a narrow field; and the signals from the overlapping portions of the narrow red and green fields are individually counted during the period of the green fluorescence count to thereby obtain a count ratio of those white cells having the particular characteristics examplified by the field common to the selected narrow red and green fields.
Claim 20
Dependent Claim
39 words
A method according to claim 16 wherein the method is repeated with different portions of the same blood sample and with different settings of the voltage thresholds to thereby obtain a series of measurements of cells having different characteristics.
Claim 21
Dependent Claim
52 words
A method according to claim 16 wherein the voltage thresholds are applied by parallel threshold circuits to select green fluorescence signals within a selected narrow field and red fluorescence signals within a selected narrow field simultaneously for each of the lymphocyte, monocyte, neutrophil, basophil, eosinophil and immature granulocyte classes of white cells.
Claim 22
Dependent Claim
121 words
A method according to claim 18 wherein: for each of the said classes of white cells and the green fluorescence signals within a selected narrow field and the red fluorescence signals within a selected narrow field are respectively converted to a digital representation of the fluorescence values on a digital register by an analog-digital converter, each of said registers being connected to the input data buss of a computer; the computer monitors, for each of the said classes of white cells, the green fluorescence values corresponding to the red fluorescence signals within said selected narrow field; and the computer produces a count of the number of cells having fluorescence signals within each selected narrow field of red and green fluorescence signals.
Claim 23
Independent Claim
122 words
A method for making a count of total white cells in a blood sample comprising the steps of: a. combining a fresh blood sample with an aqueous solution of acridine orange to form a suspension, said solution having a physiologically normal pH factor and a concentration of acridine orange in said suspension in the neighborhood of 10 3 grams per liter of suspension, and said solution having an osmolarity below the physiologically normal level of human blood; b. subjecting the suspension obtained from step (a) to radiation from a blue laser and counting the cells from a measured volume of suspension based upon the detection of green fluorescence emitted from each individual cell in response to excitation from the blue laser radiation.
Claim 24
Independent Claim
122 words
A method for making a count of total white cells in a blood sample comprising the steps of: a. combining a fresh blood sample with an aqueous solution of acridine orange to form a suspension, said solution having a physiologically normal pH factor and a concentration of acridine orange in said suspension in the neighborhood of 10 3 grams per liter of suspension, and said solution having an osmolarity below the physiologically normal level of human blood; b. subjecting the suspension obtained from step (a) to radiation from a blue laser and counting the cells from a measured volume of suspension based upon the detection of green fluorescence emitted from each individual cell in response to excitation from the blue laser radiation.
Claim 25
Dependent Claim
33 words
A method according to claim 20 wherein the detection of green fluorescence emitted from the cells Is discriminated by an optical filter which passes radiation in the neighborhood of 5,300 angstrom units wavelength.
Claim 26
Independent Claim
74 words
A METHOD FOR CLASSIFYING VITAL WHITE BLOOD CELLS AMOUNG THE GROUP OF LYMPHOCYTES, MONOCYTES, NEUTROPHILIS, BASOPHILS, EOSINOPHILS AND IMMATURE FORMS OF GRANULOCYTES, SAID METHOD COMPRISING THE STEP OF: A. COMBINING A FRESH BLOOD SAMPLE WITH A HYPOTONIC COLLOIDAL SOLUTION OF A METACHROMATIC FLUOROCHROME DYE AT A PHYSIOLOGICALLY NORMAL PH; AND B. DISTINGUISHING THE GROUPS OF WHITE BLOOD CELLS BY MEASURING THE MAGNITUDES OF METACHROMATIC FLUORESCENCES FROM THE INDIVIDUAL CELLS IN RESPONSE TO OPTICAL RADIATION.
Claim 27
Independent Claim
74 words
A METHOD FOR CLASSIFYING VITAL WHITE BLOOD CELLS AMOUNG THE GROUP OF LYMPHOCYTES, MONOCYTES, NEUTROPHILIS, BASOPHILS, EOSINOPHILS AND IMMATURE FORMS OF GRANULOCYTES, SAID METHOD COMPRISING THE STEP OF: A. COMBINING A FRESH BLOOD SAMPLE WITH A HYPOTONIC COLLOIDAL SOLUTION OF A METACHROMATIC FLUOROCHROME DYE AT A PHYSIOLOGICALLY NORMAL PH; AND B. DISTINGUISHING THE GROUPS OF WHITE BLOOD CELLS BY MEASURING THE MAGNITUDES OF METACHROMATIC FLUORESCENCES FROM THE INDIVIDUAL CELLS IN RESPONSE TO OPTICAL RADIATION.
Claim 28
Independent Claim
163 words
A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said solution being further characterized in that it is capable of altering the differential rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.
Claim 29
Independent Claim
163 words
A method for distinguishing specific types of cells among white blood cells and other particles within a blood sample, said method comprising the steps of: a. forming an aqueous diluent solution of a metachromatic fluorochrome dye, said solution being capable of staining the nuclear material of the white cells to provide fluorescence from the stained nuclear material which is different from the fluorescence of any other blood particle; said solution being further characterized in that it is capable of altering the differential rates of dye uptake by the various components of the white cells; b. staining the vital white cells of a blood sample by suspending the blood sample in the diluent dye solution prepared in step (a); c. subjecting the suspension formed in step (b) before dye uptake equilibrium is reached therein to illumination from a light source having a radiation wavelength absorbable by the stained cellular material; and d. distinguishing the various white cells by the magnitudes of their fluorescence emissions.
References Cited (Backward Citations)
US3684327A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1978-05-12 | 1978-11-11 | 1979-05-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1982-05-12 | 1982-11-11 | 1983-05-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 1986-05-12 | 1986-11-11 | 1987-05-13 | $7700.00 | Window Closed / Lapsed |