Coulter Electronics, Inc.

Ted Sena

Harold R Crews

Stephen L Ledis

James H Carter

Timothy J Fischer

Fred L Larsen

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

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.

Multi-purpose blood diluent and lysing agent for differential

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.

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Miami, FL, United States of America

Ted Sena