# US 8,541,227 B2 · Cell counting > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,227 B2 - Document ID: US8541227B2 - Status: Granted - Kind: B2 - Application number: 11/884,247 - Filed: 2006-02-06 - Published: 2008-08-14 - Granted: 2013-09-24 - Earliest claimed: 2005-02-25 - Assignees: Dako Denmark A/S - Applicants: Nanna K Christensen; Jesper Laursen; Lars Winther - Inventors: Nanna K Christensen; Jesper Laursen; Lars Winther - Web page: https://patentgrep.com/patent/US8541227B2 ## CPC - G01N 15/1459: {the analysis being performed on a sample stream} - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - G01N 33/543: with an insoluble carrier for immobilising immunochemicals - G01N 33/54366: {Apparatus specially adapted for solid-phase testing} - G01N 33/54393: {Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding} - G01N 2015/1486: {Counting the particles} - Y10T 436/101666: Particle count or volume standard or control [e.g., platelet count standards, 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The inventions relates to compositions and method for determining the absolute counts of cells per unit volume of a sample. Such a method comprises: (a) providing a container containing (i) a predetermined quantity of microparticles; and (ii) a cell-binding agent; in which the microparticles are disposed in or on a matrix which adheres to at least one wall of the container such that substantially all the microparticles are thereby attached to the container; (b) adding a known volume of sample to the container; (c) determining the ratio of microparticles to cells by counting microparticles and cells in a volume of the sample; and (d) determining the absolute count of cells by multiplying the number of cells per microparticle by the concentration of microparticles in the sample. Preferably, the matrix retains substantially all the microparticles in or on the container during routine handling, including inversion, of the container, in the absence of mechanical retaining means such as a retainer grid in the container. ## Claims ### Claim 1 A method for determining the absolute counts of cells of interest per unit volume of a sample, said method comprising: (a) providing a container containing: (i) a matrix adhered to at least one wall of said container, said matrix being semi-solid, of gel-like consistency, or in dry form; (ii) a predetermined quantity of reference microparticles, distinguishable from a labeled cell-binding agent, disposed in or on said matrix such that substantially all the reference microparticles are thereby attached to said container; and (iii) the labeled cell-binding agent in or on the matrix, wherein the labeled cell-binding agent is capable of binding to and identifying said cells of interest in the sample; (b) adding a known sample volume of said sample to said container, whereby: (i) said known sample volume, labeled cell binding agent, and matrix are mixed; (ii) said labeled cell-binding agent binds to cells of interest are in said known sample volume; and (iii) said reference microparticles are released into the added known sample volume; (c) counting, in a portion of the sample volume obtained in (b), both the number of said cells of interest identified as cells bound to said labeled cell-binding agent and the number of reference microparticles, and from this determining the ratio of said cells of interest per reference microparticles in said portion of the sample volume; and (d) determining an absolute count of said cells of interest in said known sample volume by multiplying the ratio of cells of interest per reference microparticle obtained in (c) by the predetermined quantity of reference microparticles in said known sample volume. ### Claim 2 (depends on 1) The method according to claim 1, in which said matrix retains substantially all said reference microparticles in or on said container during routine handling of said container. ### Claim 3 (depends on 1) The method according to claim 1, in which said matrix retains substantially all said reference microparticles in or on said container when said container is inverted. ### Claim 4 (depends on 1) The method according to claim 1 in which said matrix is the only means for retaining said reference microparticles in or on said container. ### Claim 5 (depends on 1) The method according to claim 1, in which said matrix comprises a carbohydrate. ### Claim 6 (depends on 1) The method according to claim 1, in which said matrix comprises a sugar, a mixture of sugars, a polymer, or a protein. ### Claim 7 (depends on 1) The method according to claim 1, in which said matrix comprises a 1:2, 1:1, or 2:1 mixture of any two components chosen from the group consisting of fructose, trehalose, and raffinose. ### Claim 8 (depends on 1) The method according to claim 1, in which said matrix is present at a quantity of less than 5 mg. ### Claim 9 (depends on 1) The method according to claim 1, in which said matrix comprises an antioxidant. ### Claim 10 (depends on 1) The method according to claim 1, in which substantially all said reference microparticles are released from said at least one wall of said container into said quantity known sample volume of said sample on addition of said known sample volume of said sample to said container. ### Claim 11 (depends on 1) The method according to claim 1, in which substantially all said reference microparticles are discrete when counted. ### Claim 12 (depends on 1) The method according to claim 1, in which said counting in (c) comprises the step of counting said cells of interest and said reference microparticles in a flow cytometer. ### Claim 13 (depends on 1) The method according to claim 1, in which said cells of interest comprise microorganisms. ### Claim 14 (depends on 13) The method according to claim 13, in which said microorganisms comprise yeast or bacteria. ### Claim 15 (depends on 1) The method according to claim 1, in which said cells of interest comprise lymphocytes and said sample comprises unlysed whole blood. ### Claim 16 (depends on 1) The method according to claim 1, in which said labeled cell-binding agent comprises an antibody capable of binding to an antigen selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD33, CD34, CD38, CD45, CD56, CD57, CD64, CDw65, CD117, and CD133. ### Claim 17 (depends on 1) The method according to claim 1, in which said reference microparticles are chosen from the group consisting of polystyrene beads, latex beads, agarose beads, and acrylamide beads. ### Claim 18 (depends on 1) The method according to claim 1, in which (c) comprises the step of individually detecting said reference microparticles and said labeled cell-binding agent by virtue of a signal-generating means. ### Claim 19 (depends on 18) The method according to claim 18, in which said signal generating means comprises a fluorochrome selected from the group consisting of: fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Cy5, PE-Cy5.5, PE-Cy7, PE-A680, PE-TR (TEXAS RED), allophycocyanin (APC), APC-Cy7, PACIFIC BLUE, CASCADE YELLOW, ALEXA dyes, coumarins, and Q-DOTS. ### Claim 20 (depends on 19) The method according to claim 19, in which said reference microparticles and said labeled cell-binding agent are labeled with different fluorochromes. ### Claim 21 (depends on 1) The method according to claim 1, in which said container contains more than one labeled cell-binding agent. ### Claim 22 (depends on 1) The method according to claim 1, in which said labeled cell-binding agent comprises an anti-CD3 antibody coupled to phycoerythrin and in which the container contains a second labeled cell-binding agent comprising an anti-CD4 antibody coupled to APC and a third labeled cell-binding agent comprising an anti-CD45 antibody coupled to FITC. ### Claim 23 (depends on 1) The method according to claim 1, in which said cell-binding agent comprises an anti-CD34 antibody coupled to phycoerythrin and in which the container contains a second labeled cell-binding agent comprising an anti-CD45 antibody coupled to FITC or APC. ### Claim 24 (depends on 1) The method according to claim 1, in which the container contains propidium iodide and TWEEN. ### Claim 25 (depends on 1) The method according to claim 1, in which the container is disposable. ### Claim 26 An apparatus for preparing a sample for absolute counting of cells of interest in said sample, said apparatus comprising: (a) a container; (b) a matrix adhered to at least one wall of said container, said matrix being semi-solid, of gel-like consistency, or in dry form; (c) a predetermined quantity of reference distinguishably distinct microparticles disposed on or in said matrix such that substantially all said reference microparticles are thereby attached to said container; and (d) a labeled cell-binding agent in or on the matrix, in which said matrix is configured to release said reference microparticles into said sample upon addition of a known volume of said sample to said container. ### Claim 27 A kit for preparing a sample for determining the absolute counts of cells of interest in said sample comprising: (a) an apparatus comprising: (i) a container; (ii) a matrix adhered to at least one wall of said container, said matrix being semi-solid, of gel-like consistency, or in dry form; (iii) a predetermined quantity of reference distinguishably distinct microparticles disposed on or in said matrix such that substantially all said reference microparticles are thereby attached to said container; and (iv) a labeled cell-binding agent; in or on said matrix, in which said matrix is configured to release said reference microparticles into said sample upon addition of said sample to said container; and (b) instructions for adding incubating, and mixing said sample in said apparatus. ### Claim 28 (depends on 27) The kit according to claim 27, in which said cells of interest comprise CD3+ and CD4+ cells. ### Claim 29 (depends on 27) The kit according to claim 27, wherein the labeled cell-binding agent is of a type that binds to and enables determining of said absolute counts of cells of interest present in a sample.