Coulter Electronics, Inc.

Coulter International Corp.

Harold R Crews

Stephen L Ledis

James H Carter

Ted Sena

David L Chastain, Jr

Timothy J Fischer

Dave Chastain, Jr

Frank J Lucas

Gerald E Jaffe

Eileen Landrum

Nazle Kuylen

Fred L Larsen

Francis J Lucas

Adry Galiounghi

Ravindra S Shukla

Nancy M Garcia

Maria Barcelon

The invention provides a cell control product for use with a cytoenzymology assay to confirm enzymatic activity in devices employing electronic and/or optical means. The cell control product comprises a lyophilized mammalian cell which is capable of being rehydrated in water to exhibit cellular structure and cellular enzymatic activity so that said lyophilized cells can function effectively as a cytoenzymology cell control in an enzymatic analysis. The cellular structure of the cell control is capable of being authenticated by light scatter or microscopy analysis and the cellular enzymatic activity is capable of being authenticated by fluorescence analysis. The cell control exhibits a real time stability when stored at between 2 and 8.degree. C. for at least a two month period of time. Preferably the mammalian cell is an abnormal cell selected from Molt 4, CCRF-CEM and HL-60 cell lines.

Cell control used to confirm enzymatic activity

The necessity to detect blast cells in a patient sample to determine a treatment regime for leukemia has long been desired. The present invention relates to a method and assay reagents for identification of blast cells in a fluid sample. The method further enables the determination of the lineage of the blast cells. The new method provides a determination of blast cells in less time than prior art methods.

Identification of blast cells in a leukocyte cell preparation

A lytic reagent composition and a kit of a lytic reagent system are disclosed for the rapid isolation, identification and/or analysis of at least five subpopulations of leukocytes from a whole blood sample. The composition and system have application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. The lytic reagent composition of this invention comprises saponin and a carboxylic acid of the formula RCOOH wherein R is H or a C.sub.1-3 aliphatic hydrocarbon radial optionally substituted by one or more carbonyl and/or hydroxy groups. The kit of a lytic reagent system comprises the lytic reagent composition and a quench reagent.

Method and reagent system for isolation, identification and/or analysis

A method and reagent system are disclosed for the rapid isolation, identification and/or analysis of leukocytes from a whole blood sample. The method and reagent system of this invention has application to any environment in which the study and/or analysis of the leukocyte fraction of whole blood requires their isolation in their native or near native state. One of the environments in which this invention can be used to advantage is in the performance of white cell differentiation on automated instrumentation designed for that purpose. In one of the preferred embodiments of this invention, the lytic reagent can contain a mixture of both formic and acetic acid, with the formic acid comprising the major component thereof and the acetic acid being present in only minor quantities, (if at all). This reagent system is used to selectively effect stromatolysis of red blood cells and create subtle modifications to the leukocyte population to enable their automated differentiation into five (5) sub-populations. The advantage and uniqueness of this reagent system is the surprising speed at which it is able to effect the foregoing objectives (generally less than 10 seconds at room temperature) and the ability to further differentiate the leukocyte population (notably, the granulocyte population). Following stromatolysis, a quenching agent is added to retard the reactivity of the lytic reagent system and, thus, inhibit any further dramatic changes to the leukocyte population. The treatment of the whole blood sample in the foregoing manner is further unique in that the leukocyte fraction of the sample has retained its characteristic immunochemical response.

Lysing reagent system for isolation, identification and/or analysis of

An isotonic multipurpose blood diluent, and a method for use of this diluent with a weak lysing reagent system which is especially suitable for routine enumeration of traditional hemogram values, and also the determination of lymphoid-myeloid populations of leukocytes, particularly in automatic particle counting systems. This blood diluent is capable of affording accurate, reproducible test results. It is an osmotically balanced aqueous solution of preselected pH containing Procaine hydrochloride for maintaining erythrocyte morphology during operation, N-(2-acetamido)iminodiacetic acid (ADA) as a blood cell stabilizing agent, and bacteriostatic agents including sodium 1-hydroxypyridine-2-thione, and dimethylolurea which, together with the ADA, allow preferential determination of myeloid-lymphoid leukocytes, and other hematological values. The lysing agent is a mixture of an aqueous solution of at least one quaternary ammonium salt having surface active properties, and an alkali metal cyanide.

Multi-purpose blood diluent and lysing agent for differential

This invention describes a reagent system, including saponin in a lysing reagent and a rapidly active cross-linking agent such as glutaraldehyde as a fixing reagent, which reproducably affects whole blood to cause the red blood cells to stromatolyze and modifies the leukocytes to generate data to define four distinct clusters for detection and classification by flow analysis instrumentation. The clusters represent the four major leukocyte types found in blood: lymphocytes, monocytes, neutrophils and eosinophils, thus providing a method of leukocyte differential analysis. The parameters used for the leukocyte classification include combinations of two or more of DC (Coulter) volume, high frequency (RF) size, opacity (RF size/DC volume), light scatter at various angular ranges, and fluorescence at various wavelengths of illumination.

Method and reagents for differential determination of four populations

A method and reagent system is described for differential determination of more that one class of leukocyte populations, using an automatic blood cell analyzer. The reagent system includes a blood diluent and stromatolyzing reagent. The blood diluent is an osmotically balanced aqueous solution of ingredients for maintaining erythrocyte morphology, blood cell stabilizing, buffering and bacteriostatic action. The stromatolyzing reagent is a mixture of an aqueous solution of quaternary ammonium salts, a chromagen forming agent and an additive which is a non-cationic surfactant. The quaternary ammonium salts, with the additive, serve the purpose of positioning, relative to one another and a volume reference point, the lymphoid and myeloid populations. The additive also acts to prevent or to reduce the amount of protein deposit in the sensing orifices of the analyzer, which deposit tends to accumulate from the cell debris resulting from blood cell stromatolyzation.

A method and reagent system is described for differential determination of three-populations of leukocytes (lymphocyte, monocyte and granulocyte), using an automatic counting system. The reagent system includes a blood diluent and lysing reagent. The blood diluent is an osmotically balanced aqueous solution of ingredients at a preselected pH for maintaining erythrocyte morphology, blood cell stabilizing, buffering and bacteriostatic action. The lysing reagent is a mixture of an aqueous solution of quaternary ammonium salts, which lysing reagent is added to the diluted blood under more mild conditions of concentration and at a slower rate than is current practice in order to obtain an unexpected volumetric modification of at least one of the three-populations of leukocytes, whereby volumetric differential analysis can be accomplished.

Method for three-volume differential determination of lymphocyte,

This invention utilizes for stabilization of platelets a combination of iodoacetamide and an iminodiacetic acid or salt thereof, together with a compatible bacteriostatic agent, in an aqueous electrolytic solution which is maintained at a preselected range of pH and osmolality. In a preferred formulation, the stabilizing solution contains iodoacetamide, N-(2-acetamido)iminodiacetic acid and sodium penicillin. Ethylenediaminetetraacetic acid, or salts thereof, are optionally present as an additional ingredient.

Method of stabilizing platelets for determining multiple platelet

Stabilized reference control and calibrator compositions for determining multiple platelet parameters in stand alone platelet controls and whole blood reference controls are prepared from platelets stabilized with a fixative-stabilizing composition containing glutaraldehyde and a non-ionic surfactant which is a mixture of ethoxylates of certain isomeric linear alcohols.

An isotonic cleansing agent and method for its use in electronic instrumentation systems for determining hematological components of blood samples are described. The cleansing agent comprises an aqueous solution of a non-hemolytic polyoxyethylated alkylphenol detergent, dimethylolurea, and an isotonic and isoconductive saline base which is free of phosphates. The dimethylolurea serves as a cell stabilizer, and also acts as a germicidal agent, together with other added germicidal agents, particularly sodium 1-hydroxypyridine-2-thione.

Cleansing composition for electronic particle counting apparatus; and

A process for preparing whole blood reference controls having long term stability up to six months for devices using electronic means for whole blood determinations including platelet count and mean cell volume and red blood distribution width. The preconditioning diluent for the red blood cells consists essentially of an aqueous solution of lactose, sodium azide, and a non-ionic surfactant and is pH buffered and osmolality adjusted. The media of the whole blood control includes lactose, fungicides and antibiotics, and supplementary agents including purine nucleosides. It also includes additional components which alter red blood cell membranes including bile salts and cholic acid derivatives, phenothiazine compounds and the salts thereof having antihistamine properties, and 4-aminobenzoic acid ester derivatives and their salts having local anesthetic properties. In the process, the red blood cells are separated from the whole blood and preconditioned by the diluent to attain desired size distribution width and volume. The preconditioned red blood cells then are mixed in the media in suitable number for yielding the desired cell count for the whole blood reference control.

Process for preparing whole blood reference controls having long term

An isotonic multipurpose blood diluent, and a method for use of this diluent with a weak lysing reagent system which is especially suitable for routine enumeration of traditional hemogram values, and also the determination of lymphoid-myeloid populations of leukocytes, particularly in automatic particle counting systems. This blood diluent is capable of affording accurate, reproducible test results. It is an osmotically balanced aqueous solution of preselected pH containing Procaine hydrochloride for maintaining erythrocyte morphology during operation, N-(2-acetamido)iminodiacetic acid (ADA) as a blood cell stabilizing agent, and bacteriostatic agents including sodium 1-hydroxypyridine-2-thione, dimethylolurea and chlorhexidene diacetate which, together with the ADA, allow preferential determination of myeloid-lymphoid leukocytes, and other hematological values. The lysing agent is a mixture of an aqueous solution of at least one quaternary ammonium salt having surface active properties, and an alkali metal cyanide.

Growing community of inventors

Miami, FL, United States of America

Harold R Crews