US4528274A — Multi-purpose blood diluent and lysing agent for …

Claim 1

Independent Claim 122 words

A stromatolysing reagent for use in the determination of at least two leukocyte populations in blood, two such populations being lymphoid and myeloid, the blood being processed through a blood cell analyzer using the Coulter Principle of operation, which employs a leukocyte sensing zone, the blood having been first diluted with an electrically conductive isotonically balanced diluent, wherein said stromatolysing reagent comprises an aqueous solution of at least two quaternary ammonium salts having surface active properties and an additive which is at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for positioning said leukocyte populations relative to one another and relative to a blood cell volume reference point, within the time constraints of said blood cell analyzer.

Claim 2

Dependent Claim 122 words

A stromatolysing reagent for use in the determination of at least two leukocyte populations in blood, two such populations being lymphoid and myeloid, the blood being processed through a blood cell analyzer using the Coulter Principle of operation, which employs a leukocyte sensing zone, the blood having been first diluted with an electrically conductive isotonically balanced diluent, wherein said stromatolysing reagent comprises an aqueous solution of at least two quaternary ammonium salts having surface active properties and an additive which is at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for positioning said leukocyte populations relative to one another and relative to a blood cell volume reference point, within the time constraints of said blood cell analyzer.

Claim 3

Dependent Claim 16 words

The stromatolysing reagent of claim 1 wherein said non-cationic surfactant additive is a nonionic polyoxyethylated alkylphenol.

Claim 4

Dependent Claim 54 words

The stromatolysing reagent of claim 2 wherein said additive has the following product characteristics: ______________________________________ Appearance clear, almost water-white liquid 1% solution clear and colorless solubility in water readily soluble, even in cold water pH of 1% solution neutral stability stable to acids, alkalis and metallic ions cloud point clear at 212° C. ______________________________________

Claim 5

Dependent Claim 84 words

The stromatolysing reagent of claim 1 wherein the quaternary ammonium salts are those with C 12 and C 14 , alkyl radicals which have useful ranges of approximately 10 to 55 g/L and 9 to 20 g/L, respectively, with said additive having a useful range of approximately 4 to 20 mL/L; such that the selection of any two of these three components anywhere within their ranges will provide a useful reagent when the amount of the third component is properly selected within its range.

Claim 6

Dependent Claim 36 words

The stromatolysing reagent of claim 4 wherein said salts are dodecyltrimethylammonium chloride and tetradecyltrimethylammonium bromide, and said additive is selected from the group consisting of: 1. Polyoxyethylated alkylphenols, 2. Poly(oxypropylene)poly(oxyethylene)condensates, and 3. Polyethylene glycol p-isoalkylphenyl ethers.

Claim 7

Dependent Claim 98 words

The stromatolysing reagent of claim 1 wherein the quaternary ammonium salts are those with C 12 and C 14 , alkyl radicals which have useful ranges of approximately 10 to 55 g/L and 9 to 20 g/L, respectively, with said additive having a useful range of approximately 0.5 to 5 ml/L and being an alkali metal salt of a C 10 -C 18 alkyl sulfate; such that the selection of any two of these three components anywhere within their ranges will provide a useful reagent when the amount of the third component is properly selected within its range.

Claim 8

Dependent Claim 15 words

The stromatolysing reagent of claim 6 where said salts are dodecyltrimethylammonium chloride and tetradecyltrimethylammonium bromide.

Claim 9

Dependent Claim 14 words

The stromatolysing reagent of claim 1 wherein said non-cationic surfactant additive is a poly(oxypropylene)polyoxyethylene)condensate.

Claim 10

Dependent Claim 17 words

The stromatolysing reagent of claim 1 wherein said non-cationic surfactant additive is a polyethylene glycol p-isoalkylphenyl ether.

Claim 11

Dependent Claim 24 words

The stromatolysing reagent of claim 1 wherein said non-cationic surfactant additive is an alkali metal salt of a C 10 -C 18 alkyl sulfate.

Claim 12

Dependent Claim 32 words

The stromatolysing reagent of claim 1 which includes the following three ingredients in approximately the concentrations indicated: 1. Tetradecyltrimethylammonium bromide: 9 g/L 2. Dodecyltrimethylammonium chloride: 55 g/L 3. Polyoxyethylated alkylphenol: 12 mL/L.

Claim 13

Dependent Claim 32 words

The stromatolysing reagent of claim 1 which includes the following three ingredients in approximately the concentrations indicated: 1. Tetradecyltrimethylammonium bromide: 20 g/L 2. Dodecyltrimethylammonium chloride: 10 g/L 3. Polyoxyethylated alkylphenol: 8 mL/L.

Claim 14

Dependent Claim 37 words

The stromatolysing reagent of claim 1 which includes the following three ingredients in approximately the concentrations indicated: 1. Tetradecyltrimethylammonium bromide: 13 g/L 2. Dodecyltrimethylammonium chloride: 70 g/L 3. Polyethylene glycol p-isoalkylphenyl ether or polyoxyethylated alkylphenol: 8 mL/L.

Claim 15

Dependent Claim 33 words

The stromatolysing reagent of claim 1 which includes the following three ingredients in approximately the concentrations indicated: 1. Tetradecyltrimethylammonium bromide: 13 g/L 2. Dodecyltrimethylammonium chloride: 35 g/L 3. Sodium lauryl sulfate: 1.25 g/L.

Claim 16

Dependent Claim 17 words

A stromatolysing reagent according to claim 1 for use in the measurements of two populations of leukocytes.

Claim 17

Dependent Claim 17 words

A stromatolysing reagent according to claim 1 for use in the measurements of three populations of leukocytes.

Claim 18

Dependent Claim 41 words

The stromatolysing reagent of claim 1 for the measurements of three populations of leukocytes, stromatolysing reagent including the following three ingredients in approximately the concentrations indicated: 1. Tetradecyltrimethylammonium bromide: 6.5 g/L 2. Dodecyltrimethylammonium chloride: 18.8 g/L 3. Polyoxyethylated alkylphenol: 3 mL/L.

Claim 19

Dependent Claim 128 words

In a method for determination of at least two leukocyte populations in blood which is being processed through a blood cell analyzer using the Coulter Principle of operation which includes the step of: I. diluting a blood sample with an electronically conductive isotonically balanced diluent, the improvement which comprises the step of: II. stromatolysing with a reagent comprising an aqueous solution of at least two quaternary ammonium salts having surface active properties, and an additive which contains at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for affecting the normal leukocyte volume distributions during said stromatolysation so as to cause the volume distribution measurements of said blood cell analyzer to lie within predetermined limits, with reference to at least one volume reference point.

Claim 20

Dependent Claim 128 words

In a method for determination of at least two leukocyte populations in blood which is being processed through a blood cell analyzer using the Coulter Principle of operation which includes the step of: I. diluting a blood sample with an electronically conductive isotonically balanced diluent, the improvement which comprises the step of: II. stromatolysing with a reagent comprising an aqueous solution of at least two quaternary ammonium salts having surface active properties, and an additive which contains at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for affecting the normal leukocyte volume distributions during said stromatolysation so as to cause the volume distribution measurements of said blood cell analyzer to lie within predetermined limits, with reference to at least one volume reference point.

Claim 21

Dependent Claim 38 words

The method of claim 18 in which the diluent comprises an aqueous solution of: 1. buffering means, 2. cell membrane stabilizing means, 3. germicidal means, and 4. means for producing an osmotically balanced and substantially neutral pH solution.

Claim 22

Dependent Claim 76 words

The method of claim 18 in which the diluent comprises an aqueous solution of: 1. Procaine hydrochloride, 2. N-(2-acetamido)iminodiacetic acid (ADA), 3. Dimethylolurea, and 4. Alkali metal sulfate and alkali metal halide, said diluent being an osmotically balanced and substantially neutral pH solution; and said stromatolysing reagent further comprises an alkali metal cyanide; and said ingredients are present in a concentration range which is effective to enable the differential determination of leukocyte populations and hemogram values.

Claim 23

Dependent Claim 27 words

The method of claim 18 wherein said quaternary ammonium salts are selected from the group consisting of: 1. Dodecyltrimethylammonium chloride, 2. Tetradecyltrimethylammonium bromide, and 3. Hexadecyltrimethylammonium bromide.

Claim 24

Dependent Claim 41 words

The method of claim 18 wherein said non-cationic surfactant additive is selected from the group consisting of: 1. Polyoxyethylated alkylphenols, 2. Poly(oxypropylene)poly(oxyethylene)condensates, 3. Polyethylene glycol p-isoalkylphenyl ethers, and 4. An alkali metal salt of a C 10 -C 18 alkyl sulfate.

Claim 25

Dependent Claim 117 words

In a method for the size differentiation of at least two populations of leukocytes in blood, the blood being processed through a blood cell analyzer using the Coulter Principle of operation which employs a leukocyte sensing zone, the blood having been first diluted with an electrically conductive isotonically balanced diluent, the improvement wherein said stromatolysing reagent comprises an aqueous solution of at least two quaternary ammonium salts having surface active properties and an additive which is at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for positioning said leukocyte populations relative to one another and relative to a blood cell volume reference point, within the time constraints of said blood cell analyzer.

Claim 26

Dependent Claim 117 words

In a method for the size differentiation of at least two populations of leukocytes in blood, the blood being processed through a blood cell analyzer using the Coulter Principle of operation which employs a leukocyte sensing zone, the blood having been first diluted with an electrically conductive isotonically balanced diluent, the improvement wherein said stromatolysing reagent comprises an aqueous solution of at least two quaternary ammonium salts having surface active properties and an additive which is at least one non-cationic surfactant, said salts and additive being present in sufficient amounts for positioning said leukocyte populations relative to one another and relative to a blood cell volume reference point, within the time constraints of said blood cell analyzer.

Claim 27

Dependent Claim 76 words

In the method of claim 23 the improvement wherein said quaternary ammonium salts have the formula ##STR2## where R 1 is a long chain alkyl radical having 10 to 18 carbon atoms, R 2 , R 3 , R 4 are short chain alkyl radicals having 1 to 6 carbon atoms and X - is a salt forming radical such as Cl - , Br - , PO 4 3- and CH 3 PO 4 .

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	1988-07-08	1989-01-07	1989-07-09	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	1992-07-08	1993-01-07	1993-07-09	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	1996-07-08	1997-01-07	1997-07-09	$7700.00	Window Closed / Lapsed

Multi-purpose blood diluent and lysing agent for differential determination of lymphoid-myeloid population of leukocytes

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