Metachromatic dye sorption and fluorescent light emmisive means for differential determination of developmental stages of neutrophilic granulocytic cells and other leukocytes

Claim 1

In analysis of human blood cells present in a donor specimen in a fixative-free aqueous environment under influence of a fluorescent light emissive energy source, the improvement in identification, enumeration and study of monocytes present in said specimen which comprises staining said specimen with an aqueous solution of a basic, cationic, quaternary organic dyestuff selected from the group consisting of basic orange #21, basic red #13, basic red #36, basic red #49, basic violet #7, basic violet #15, basic violet #16 basic violet #36, basic violet #39 basic violet #40 and carboeyanine; K-5 subjecting said dye stained specimen to emissive wave energy to thereby stimulate fluorescence of said dye exposed cells, emitted fluorescence light providing means for differentiation of monocytes present in the specimen from all other blood cells.

Claim 7

A method of supervital analysis of human normal or pathologic blood cells present in a donor specimen in an aqueous fixative free environment which comprises exposing said specimen (a) to the dyestuff basic orange #21, (b) stimulating fluorescence of each of the stained species present in said specimen by impact of wave energy and differentiating each of the said blood cells one from the other by the presence or absence of characteristic fluorescent color and pattern of the nucleus and cytoplasm and by the number, size, arrangement, pattern and fluorescent color or quanta of light energy intensities emitted by the granules present, their size, number and locus of the granules in the cytoplasm, if present.

Claim 8

A method of supervital analysis of human normal or pathologic blood cells present in a donor specimen in an aqueous fixative free environment which comprises exposing said specimen (a) to the dyestuff basic orange #21, (b) stimulating fluorescence of each of the stained species present in said specimen by impact of wave energy and differentiating each of the said blood cells one from the other by the presence or absence of characteristic fluorescent color and pattern of the nucleus and cytoplasm and by the number, size, arrangement, pattern and fluorescent color or quanta of light energy intensities emitted by the granules present, their size, number and locus of the granules in the cytoplasm, if present.

Claim 12

A method of fixative-free, supravital human blood cell analysis whereby optical differentiation, comparison and enumeration are made possible of T-lymphocytes and B-lymphocytes present in a donor specimen which comprises metachromatic staining of said specimen in a fixative-free aqueous environment with basic orange #21 dye, subjecting said dyed specimen to a fluorescent light stimulating energy source and thereby differentiating T-cells present from B-cells by a cluster of bright yellow fluorescent granules in the cytoplasm of the T-cells, which may show some fluorescent dull green coloration of the nucleus, and B-cells present whose cytoplasm is essentially free from any fluorescent granules and/or fluorescent color but the nucleus of which does exhibit dull green fluorescence thereby providing required identification.

Claim 13

A method of fixative-free, supravital human blood cell analysis whereby optical differentiation, comparison and enumeration are made possible of T-lymphocytes and B-lymphocytes present in a donor specimen which comprises metachromatic staining of said specimen in a fixative-free aqueous environment with basic orange #21 dye, subjecting said dyed specimen to a fluorescent light stimulating energy source and thereby differentiating T-cells present from B-cells by a cluster of bright yellow fluorescent granules in the cytoplasm of the T-cells, which may show some fluorescent dull green coloration of the nucleus, and B-cells present whose cytoplasm is essentially free from any fluorescent granules and/or fluorescent color but the nucleus of which does exhibit dull green fluorescence thereby providing required identification.

Claim 14

A method of identifying, differentiating and enumerating dye responsive cells in a human blood specimen which comprises staining the specimen in a fixative-free aqueous environment which basic orange #21 dye, subjecting the dyed specimen to fluorescent light emissive wave energy to stimulate fluorescent light emissions from said dye responsive cells, each of said cells emitting quanta of fluorescent light energy of hue, value, chroma and intensity which provides identifying means for differentiation and enumeration of each one of said dye responsive cells.

Claim 15

A method of identifying, differentiating and enumerating dye responsive cells in a human blood specimen which comprises staining the specimen in a fixative-free aqueous environment which basic orange #21 dye, subjecting the dyed specimen to fluorescent light emissive wave energy to stimulate fluorescent light emissions from said dye responsive cells, each of said cells emitting quanta of fluorescent light energy of hue, value, chroma and intensity which provides identifying means for differentiation and enumeration of each one of said dye responsive cells.

Claim 17

A method of supravital human blood analysis whereby optical differentiation, enumeration and comparison of each of the individuals in a granulocytic myeloid series including, when present; promyelocytes, myelocytes, metamyelocytes, bands or neutrophils is achieved by metachromatically staining at least one supravital, fixative free human blood specimen fraction by contact in an aqueous environment with a basic orange #21 dye solution, thereby differentiating the promyelocytes by fluorescent bright yellow primary granules on one side area of the cytoplasm; myelocytes by a relatively even distribution of bright yellow primary granules throughout the cytoplasm; metamyelocytes by a smaller mass of fluorescent bright yellow primary granules in the smaller crescent opposite an unstained clear protrusion of cytoplasm inwardly into the nucleus; the bands by a relatively small cluster of primary bright yellow fluorescent granules in a dominant field of cytoplasm and generally bifurcating the mass of the nucleus; and the neutrophils by a less distinct cytoplasm exhibiting a deep green fluorescence in which the rare granules present fluoresce bright yellow, each member of the foregoing series having an apparently decreasing proportionate cell volume of the nucleus remaining substantially unstained, the first three members of the series having a generally larger over all cell volume cell size than the last two members of the series.

Claim 18

A method of supravital human blood analysis whereby optical differentiation, enumeration and comparison of each of the individuals in a granulocytic myeloid series including, when present; promyelocytes, myelocytes, metamyelocytes, bands or neutrophils is achieved by metachromatically staining at least one supravital, fixative free human blood specimen fraction by contact in an aqueous environment with a basic orange #21 dye solution, thereby differentiating the promyelocytes by fluorescent bright yellow primary granules on one side area of the cytoplasm; myelocytes by a relatively even distribution of bright yellow primary granules throughout the cytoplasm; metamyelocytes by a smaller mass of fluorescent bright yellow primary granules in the smaller crescent opposite an unstained clear protrusion of cytoplasm inwardly into the nucleus; the bands by a relatively small cluster of primary bright yellow fluorescent granules in a dominant field of cytoplasm and generally bifurcating the mass of the nucleus; and the neutrophils by a less distinct cytoplasm exhibiting a deep green fluorescence in which the rare granules present fluoresce bright yellow, each member of the foregoing series having an apparently decreasing proportionate cell volume of the nucleus remaining substantially unstained, the first three members of the series having a generally larger over all cell volume cell size than the last two members of the series.