# US 5,350,695 A · Methods for the identification and characterization of reticulocytes in whole blood > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,350,695 A - Document ID: US5350695A - Status: Granted - Kind: A - Application number: 07/802,593 - Filed: 1991-12-05 - Granted: 1994-09-27 - Earliest claimed: 1991-12-05 - Assignees: Miles Inc.; Mount Sinai School of Medicine of the City University of New York - Inventors: Gregory M. Colella; Daniel Ben-David; Albert Cupo; Sophie S. Fan; Gena Fischer; Grace E. Martin; Leonard Ornstein - Web page: https://patentgrep.com/patent/US5350695A ## CPC - G01N 33/80: involving blood groups or blood types {or red blood cells (white blood cells G01N33/56972)} - G01N 1/30: Staining; Impregnating {; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis} - G01N 33/5094: {for blood cell populations (red blood cells G01N33/80)} - Y10S 436/80: Fluorescent dyes, e.g. rhodamine - Y10T 436/10: Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing - Y10T 436/101666: Particle count or volume standard or control [e.g., platelet count standards, etc.] - Y10T 436/107497: Preparation composition [e.g., lysing or precipitation, etc.] - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract Methods for characterizing and distinguishing reticulocytes and mature red blood cells use reagent compositions which include organic cationic dyes for staining the reticulocytes in the blood sample and buffer solutions for maintaining a pH of about 6 to about 9. The dyes may be the blue absorption dyes Oxazine 750 or New Methylene Blue. When a zwitterionic surfactant is included in the reagent composition for isovolumetric sphering of the reticulocytes and erythrocytes, and the reagent composition and whole blood sample mixture is passed through the sensing region of a flow cytometer, the light scattered and absorbed by each cell is measured, the erythrocytes can be distinguished from reticulocytes and the volume, hemoglobin concentration and the hemoglobin content of each reticulocyte or erythrocyte, and the mean cell volume, mean corpuscular hemoglobin concentration, and mean cell hemoglobin of the reticulocytes or erythrocytes are calculated from the measured cell-by-cell volume and hemoglobin concentration. One method also includes adjusting the measured absorption signals for pseudo-absorption. ## Claims ### Claim 1 A method for identifying light-absorbing subclasses of cells of interest from other subclasses of cells in a blood sample by flow cytometry which comprises the steps of: (a) mixing an aliquot of said blood sample with an aqueous reagent composition to form a suspension, wherein the reagent composition comprises a solution containing a sphering agent which is a zwitterionic surfactant; (b) passing said suspension of step (a) substantially a cell at a time through an area of focused optical illumination; (c) detecting and measuring the light scattered and light absorbed by each cell; and (d) differentiating said cells of said subclass of interest at least in part on the basis of the measured magnitudes of said scattered and absorbed light. ### Claim 2 (depends on 1) The method of claim 1 wherein step (a) includes adding a dye compound to selectively stain a subclass of interest. ### Claim 3 (depends on 1) The method of claim 1 wherein said detection and measurement step (c) includes collecting the light scattered and absorbed through a collection optics which includes an aperture, and step (d) includes correcting the magnitude of the absorption signal which includes a pseudo-absorption component to remove the pseudo-absorption component due to light scattered outside of the aperture of the collection optics. ### Claim 4 A method for identifying light-absorbing subclasses of cells of interest from other subclasses of cells in a whole blood sample by flow cytometry which comprises the steps of: (a) mixing an aliquot of said blood sample with an aqueous reagent composition to form a suspension, wherein the reagent composition comprises a dye compound which stains the ribonucleic acid of cells in the subclass of interest, a buffer for maintaining a pH of about 6 to about 9, and a sphering agent which is a zwitterionic surfactant and which does not precipitate said dye; (b) passing said suspension of step (a) substantially a cell at a time through an area of focused optical illumination; (c) detecting and measuring the light scattered and light absorbed by each cell; and (d) differentiating said cells of said subclass of interest at least in part on the basis of the measured magnitudes of said scattered said absorbed light. ### Claim 5 (depends on 4) The method of claim 4 wherein the subclass of cells is reticulocytes. ### Claim 6 (depends on 4) The method of claim 4 wherein said optical illumination in step (b) has an excitation wavelength in the red region of the spectrum. ### Claim 7 (depends on 4) The method of claim 4 the total buffer concentration is isotonic to isovolumetrically sphere the cells in the blood sample. ### Claim 8 (depends on 4) The method of claim 4 wherein the dye compound is Oxazine 750 having the formula: ##STR3## or the dye compound is New Methylene Blue having the formula: ##STR4## ### Claim 9 (depends on 7) The method of claim 7 wherein the surfactant is selected from the group consisting of lauramidopropylbetaine, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, cocoamidopropylbetaine and cocoamidosulfobetaine. ### Claim 10 A method for characterizing reticulocytes and erythrocytes in a whole blood sample by flow cytometry which comprises the steps of: (a) mixing an aliquot of said blood sample with a reagent composition comprising an organic cationic dye compound, a sphering agent which is a zwitterionic surfactant and does not precipitate said dye, and a buffer solution to form a suspension, wherein said dye compound stains the ribonucleic acid of the reticulocytes; (b) passing said suspension of step (a) substantially a cell at a time through an area of focused optical illumination; (c) detecting and measuring the light scattered and light absorbed by each cell; (d) identifying stained cells and unstained cells on the basis of the measured magnitudes of said light scattered and light absorbed; and (e) determining the number, the volume, hemoglobin concentration, and RNA concentration of the reticulocytes or erythrocytes on the basis of the measured magnitudes of said scattered and absorbed light. ### Claim 11 (depends on 10) The method of claim 10 wherein the dye compound is Oxazine 750 at a concentration range of from about 2 .mu.g/ml to about 15 .mu.g/ml. ### Claim 12 (depends on 10) The method of claim 10 wherein the pH of the reagent composition is between about 6 and about 9. ### Claim 13 (depends on 10) The method of claim 10 wherein the buffer solution comprises one or more of: K/NaHCO.sub.3 at a concentration from about 5 mM to about 50 mM; Mg Cl.sub.2 at a concentration of from about 0 mM to about 88 mM; KCl at a concentration of from about 4 mM to about 104 mM; Na.sub.3 PO.sub.4 at a concentration of from about 0 mM to about 1.5 mM; and CaCl.sub.2 at a concentration of from about 0 mM to about 0.6 mM; such that the final osmolality of the reagent composition is from about 250 m Osm to about 330 m Osm. ### Claim 14 (depends on 10) The method of claim 10 wherein the buffer solution comprises one or more of: Tris/TEA at a concentration of from about 0 mM to about 150 mM; K.sub.2 Ox/EDTA at a concentration of from about 0 mM to about 121 mM; or KCl/NaCl at a concentration of from about 0 mM to about 155 mM; a such that the final osmolality of the reagent composition is from about 280 m Osm to about 300 m Osm. ### Claim 15 (depends on 10) The method of claim 10 wherein said surfactant is an alkyl betaine or an alkylamidobetaine. ### Claim 16 (depends on 10) The method of claim 10 wherein said surfactant is selected from the group consisting of lauramidopropylbetaine, N-tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, N-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, cocoamidopropylbetaine and cocoamidosulfobetaine. ### Claim 17 (depends on 10) The method of claim 10 wherein said surfactant is present in an amount of from about 3.9 .mu.g/ml to about 148 .mu.g/ml. ### Claim 18 (depends on 10) The method of claim 10 wherein the dye compound is New Methylene Blue at a concentration of from about 10 .mu.g/ml to about 100 .mu.g/ml.