Reticulocyte quantitating reagent for flow cytometry

Claim 1

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample for a period of about ten minutes with a composition of auramine O, said composition containing 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes, and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or relative RNA content in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.

Claim 2

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample for a period of about ten minutes with a composition of auramine O, said composition containing 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes, and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or relative RNA content in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.

Claim 6

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample for a period of about thirty seconds with a composition of auramine O, said composition containing 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or the numbers of reticulocytes in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.

Claim 7

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample for a period of about thirty seconds with a composition of auramine O, said composition containing 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or the numbers of reticulocytes in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.

Claim 8

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry without the use of a chelating reagent or an amino-group reacting reagent, comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample with a composition consisting essentially of 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, water, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or the numbers of reticulocytes in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.

Claim 9

A method for quantitating reticulocytes in a fresh blood sample stained with auramine O by flow cytometry without the use of a chelating reagent or an amino-group reacting reagent, comprising the steps of: (a) fluorescent staining RNA in blood cells of the fresh blood sample with a composition consisting essentially of 20 to 2500 μg/ml of auramine O, alkaline metal salt in an amount sufficient to stabilize the shape of the blood cells, water, and buffer to maintain the pH of the composition at a value in the range of 6 to 10; (b) flowing said stained cells through a detecting area of a flow cytometer having a light source with a suitable excitation wavelength; (c) detecting scattered light and fluorescence above 520 nm emitted from cells; (d) distinguishing platelets from cells by said detected scattered light; (e) discriminating mature erythrocytes, reticulocytes and leucocytes by RNA content using auramine O fluorescence; and (f) calculating the ratio of reticulocytes to the total erythrocytes and/or the numbers of reticulocytes in a unit volume of fresh blood and/or relative RNA content in a unit volume of fresh blood.