A kit and method for analyses of biological specimens

Claim 2

A kit for the quantitation of cell nuclei comprising: a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having predetermined physical characteristics which are detectable after staining; and one or more containers of stain materials; the stain material including stain for simultaneous stain application to the specimen cell objects and the reference means.

Claim 3

A kit as claimed in claim 1 wherein the kit further contains a rinse sulfonating or stain sulfonating agent.

Claim 4

A kit as claimed in claim l or claim 2 wherein the stain material includes Azure A in an amount effective to provide an aqueous solution with a concentration of Azure A in a range from 4.9 to 5.1 mg/ml when mixed with a 0.1 N HCl aqueous solution.

Claim 5

A kit as claimed in any one of the preceding claims wherein the reference means includes a reference material which is trout erythrocytes, chicken erythrocytes, dried DNA, cultured cell lines or a mixture of two or more thereof.

Claim 6

A kit as claimed in any one of the preceding claims wherein the stain is thionin, Azure A, Azure C, acriflavine, pararosanilin, acridine orange, hematoxylin, eosin, methyl green, ethyl green, fast green, diaminobenzidine, alkaline phosphatase, methylene blue, giemsa or a mixture of two or more thereof.

Claim 7

A kit as claimed in any one of the preceding claims for the quantitation of nuclear DNA by light microscopy wherein the stain is thionin and the reference means includes rat liver nuclei.

Claim 8

A kit as claimed in claim 6 wherein the stain material includes thionin in an amount effective to provide an aqueous solution with a concentration of thionin in the range of from l to 5 mg/ml when mixed with 0.1 N HCl.

Claim 9

A kit for use in breast cell analysis by light microscopy, the kit comprising: a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with nuclear material, one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers being diaminobenzidine counterstained with a counterstain which is methylene blue, methyl green, fast green, hematoxylin, or acridine orange in an amount effective to provide an aqueous solution for staining the reference means and specimen cell objects.

Claim 10

A kit for use in breast cell analysis by light microscopy, the kit comprising: a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with nuclear material, one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers being diaminobenzidine counterstained with a counterstain which is methylene blue, methyl green, fast green, hematoxylin, or acridine orange in an amount effective to provide an aqueous solution for staining the reference means and specimen cell objects.

Claim 11

A kit as claimed in claim 8 wherein the kit is for the quantitation of nuclear DNA.

Claim 12

A kit as claimed in claim 8 or claim 9 wherein the counterstain is methyl green or fast green.

Claim 13

A kit for the quantitation of nuclear DNA by light microscopy, the kit comprising: a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA; one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of thionin, Azure A, Azure C, pararosanilin, acriflavine and mixtures thereof in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.

Claim 14

A kit for the quantitation of nuclear DNA by light microscopy, the kit comprising: a microscopic slide, the slide including a reference area and a specimen cell object area for receipt of specimen cell objects, the reference area containing a reference means for staining, the reference means having physical characteristics which permit the reference means to associate with a stain proportionally to an association of the stain with DNA; one or more containers of stain material, the stain material including a stain for simultaneous stain application to the specimen cell objects and the reference means, the stain in the containers selected from the group consisting of thionin, Azure A, Azure C, pararosanilin, acriflavine and mixtures thereof in an effective amount to provide an optical density to the reference means after staining for a predetermined time such that the optical density of the reference means will be substantially a linear function of stain concentration of the reference means after staining.

Claim 15

A kit as claimed in claim 11 wherein the reference means includes a reference material which is selected from the group consisting of rat liver nuclei, trout erythrocytes, chicken erythrocytes, dried DNA, cultured cell lines and mixtures thereof.

Claim 16

A kit for the analysis of human tissue cells for DNA content after staining, the analysis by optical density measuring apparatus, the kit comprising: a microscopic slide; a reference means on the slide having rat liver tissue thereon, the tissue including tetraploid and diploid cells; a specimen cell object area on the slide for receiving thereon human tissue cells; and at least one container of thionin stain material for simultaneous application of stain to the human cells and to the tetraploid and diploid cells of rat liver tissue to provide an effective amount of stained DNA for optical density measurement of DNA in the respective human and rat cells.

Claim 17

A kit for the analysis of human tissue cells for DNA content after staining, the analysis by optical density measuring apparatus, the kit comprising: a microscopic slide; a reference means on the slide having rat liver tissue thereon, the tissue including tetraploid and diploid cells; a specimen cell object area on the slide for receiving thereon human tissue cells; and at least one container of thionin stain material for simultaneous application of stain to the human cells and to the tetraploid and diploid cells of rat liver tissue to provide an effective amount of stained DNA for optical density measurement of DNA in the respective human and rat cells.