US 6,664,110 B1Granted

Erythroblast diagnostic flow-cytometry method and reagents

AssigneeSysmex Corporation

InventorsTomohiro Tsuji, Takashi Sakata, Yoshiro Ikeuchi, Shin'ichiro Oguni

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Abstract

Reagents and a method for simple and rapid discrimination and counting of erythroblasts in peripheral blood or circulatory system-related samples accurately with high precision is disclosed. The reagents include a hemolytic agent for dissolving erythrocytes in a body fluid sample and for conditioning leukocytes and erythroblasts in the sample to be suitable for staining, and including at least one fluorescent dye selected to stain leukocytes and erythroblasts differentially. When the selected fluorescent dye is mixed with the sample, a detectable difference in fluorescence intensity at least between leukocytes and erythroblasts arises under laser illumination in flow cytometric analysis. The reagents further include surfactant added to the hemolytic agent, selected to enable flow cytometric discrimination of erythroblasts in the body fluid sample by their maturation stages.

Claims25

Dependent claims

Reagents for discriminating and counting erythroblasts in body fluid samples by flow cytometry, consisting essentially of one fluorescent dye selected to stain leukocytes strongly and crythroblasts weakly in the body fluid samples to produce a flow-cytometric detectable intense fluorescence in the leukocytes relative to the erytlroblasts, the one fluorescent dye being selected from the group consisting of the following:

wherein R1, R2 are either a hydrogen meolecule, an alkyl group, an alkynyl group or an alkyl group substituted with a hydroxyl, Y, Z are either sulfur, oxygen, nitrogen or carbon having a lower alkyl groups, n is 0, 1 or 2, and X− is an anion;
wherein R1 is a hydrogen molecule or a dimethylamino group, R2 is an alkyl group, R3 is a hydrogen molecule or a dimethylamino group, n is 1 or 2, and X− is an anion;
wherein R1 is a hydrogen molecule or an alkyl group, R2 is a dimethylamino group, R3 is a hydrogen molecule or an amino group, R4 is a hydrogen molecule, an alkyl group or an amino group, R5 is a hydrogen molecule or a dimethylamino group, X− is an anion, and Y is sulfur or oxygen;
wherein R1 is a hydrogen molecule or a hydroxyl, R2, R3 are a hydrogen molecule or a sulfonic group, and Y+ is an alkali metal ion;
and
a hemolytic agent containing intra-molecularly at least one of an organic acid having at least one aromatic ring, and a salt thereof, in an aqueous solution having a pH of approximately 2.0-5.0 and an osmotic pressure of approximately 100 mOsm/kg or less, for dissolving erythrocytes in a body fluid sample to an extent such that said hemolytic agent does not interfere with discrimination and counting by flow cytometry of erythroblasts in the body fluid sample, and such that said hemolytic agent conditions leukocytes and erythroblasts in the body fluid sample to be suitable for staining;
wherein said reagent has a pH of approximately 2.0 to 5.0.

4 more elements

A method for discriminating and counting erythroblasts from body fluid samples by flow cytometry, comprising the preparatory steps of:

(a) mixing a body fluid sample with a hemolytic agent containing intra-molecularly at least one of an organic acid having at least one aromatic ring and a salt thereof, in an aqueous solution having a pH of approximately 2.0-5.0 and an osmotic pressure of approximately 100 mOsm/kg or less, the hemolytic agent therein selected for dissolving erythrocytes within body fluid samples to an extent that does not interfere with flow-cytometric assaying, and for conditioning leukocytes and erythroblasts to be suitable for staining;
and
(b) staining leukocytes strongly and erythroblasts weakly in the body fluid samples to produce a flow-cytometric detectable intense fluorescence in the leukocytes relative to the erythroblasts by mixing the sample as prepared in said step (a) with one fluorescent dye selected from the group consisting of the following:
wherein R1, R2 are either a hydrogen molecule, an alkyl group, an alkynyl group or an alkyl group substituted with a hydroxyl, Y, Z are either sulfur, oxygen, nitrogen or carbon having lower alkyl groups, n is 0, 1 or 2, and X− is an anion;
wherein R1 is a hydrogen molecule or a dimethylamino group, R2 is an alkyl group, R3 is a hydrogen molecule or a dimethylamino group, n is 1 or 2, and X− is anion;
wherein R1 is a hydrogen molecule or an alkyl group, R2 is a dimethylamino group, R3 is a hydrogen molecule or an amino group, R4 is a hydrogen molecule, an alkyl group or an amino group, R5 is a hydrogen molecule or a dimethylamino group, X− is an anion, and Y is either sulfur or oxygen;
wherein R1 is a hydrogen molecule or a hydroxyl, R2, R3 are a hydrogen molecule or a sulfonic group, and Y+ is an alkali metal ion;
said hemolytic agent/dye mixture having a pH of approximately 2.0 to 5.0, said method further comprising the steps of:
(c) flow cytometrically assaying the sample as prepared in said step (b) by measuring at least one scattered light parameter and at least one fluorescence parameter;
and
(d) discriminating and counting erythroblasts utilizing intensity differences in scattered light and in fluorescence as measured in said step (c).

8 more elements

Reagents for discriminating and counting erythroblasts in body fluid samples by flow cytometry, consisting essentially of one fluorescent dye selected to stain leukocytes strongly and erythroblasts weakly in the body fluid samples to produce a flow-cytometric detectable intense fluorescence in the leukocytes relative to the erythroblasts, the one fluorescent dye being selected from the group consisting of the following:

wherein R1 is a hydrogen molecule or a dimethylamino group, R2 is an alkyl group, R3 is a hydrogen molecule or a dimethylamino group, n is 1 or 2, and X− is an anion;
wherein R1 is a hydrogen molecule or an alkyl group, R2 is a dimethylamino group, R3 is a hydrogen molecule or an amino group, R4 is a hydrogen molecule, an alkyl group or an amino group, R5 is a hydrogen molecule or a dimethylamino group, X− is an anion and Y is sulfur or oxygen;
wherein R1 is a hydrogen molecule or a hydroxyl, R2, R3 are a hydrogen molecule or a sulfonic group, and Y+ is an alkali metal ion;
and
a hemolytic agent in an aqueous solution having a pH of approximately 2.0-5.0 and an osmotic pressure of approximately 100 mOsm/kg or less for dissolving erythrocytes in a body fluid sample to an extent such that said hemolytic agent does not interfere with discrimination and counting by flow cytometry of erythroblasts in the body fluid sample, and such that said hemolytic agent conditions leukocytes and erythroblasts in the body fluid sample to be suitable for staining;
wherein said reagent has a pH of approximately 2.0 to 5.0.

3 more elements

Reagents for discriminating and counting erythroblasts in body fluid samples by flow cytometry, consisting essentially of one fluorescent dye selected to stain leukocytes strongly and erythroblasts weakly in the body fluid samples to produce a flow-cytometric detectable intense fluorescence in the leukocytes relative to the erythroblasts, the one fluorescent dye being selected from the group consisting of the following:

wherein R1 is a hydrogen molecule or a dimethylamino group, R2 is an alkyl group, R3 is a hydrogen molecule or a dimethylamino group, n is 1 or 2, and X− is an anion;
wherein R1 is a hydrogen molecule or an alkyl group, R2 is a dimethylamino group, R3 is a hydrogen molecule or an amino group, R4 is a hydrogen molecule, an alkyl group or an amino group, R5 is a hydrogen molecule or a dimethylamino group, X− is an anion, and Y is sulfur or oxygen;
wherein R1 is a hydrogen molecule or a hydroxyl, R2, R3 are a hydrogen molecule or a sulfonic group, and Y+ is an alkali metal ion;
and
a hemolytic agent containing intra-molecularly at least one of an organic acid having at least one aromatic ring, and a salt thereof, in an aqueous solution having a pH of approximately 2.0-5.0 and an osmotic pressure of approximately 100 mOsm/kg or less, for dissolving erythrocytes in a body fluid sample to an extent such that said hemolytic agent does not interfere with discrimination and counting by flow cytometry of erythroblasts in the body fluid sample, and such that said hemolytic agent conditions leukocytes and erythroblasts in the body fluid sample to be suitable for staining;
wherein said reagent has a pH of approximately 2.0 to 5.0.

3 more elements

A method for discriminating and counting erythroblasts from body fluid samples by flow cytometry, comprising the preparatory steps of:

(a) mixing a body fluid sample with a hemolytic agent containing intra-molecularly at least one of an organic acid having at least one aromatic ring, and a salt thereof, in an aqueous solution having a pH of approximately 2.0-5.0 and an osmotic pressure of approximately 100 mOsm/kg or less, the hemolytic agent therein selected for dissolving erythrocytes within body fluid samples to an extent that does not interfere with flow-cytometric assaying, and for conditioning leukocytes and erythroblasts to be suitable for staining;
and
(b) staining leukocytes strongly and erythroblasts weakly in the body fluid samples to produce a flow-cytometric detectable intense fluorescence in the leukocytes relative to the erythroblasts by mixing the sample as prepared in said step (a) with one fluorescent dye selected from the group consisting of the following:
wherein R1 is a hydrogen molecule or a dimethylamino group, R2 is an alkyl group, R3 is a hydrogen molecule or a dimethylamino group, n is 1 or 2, and X− is anion;
wherein R1 is a hydrogen molecule or an alkyl group, R2 is a dimethylamino group, R3 is a hydrogen molecule or an amino group, R4 is a hydrogen molecule, an alkyl group or an amino group, R5 is a hydrogen molecule or a dimethylamino group, X− is an anion, and Y is either sulfur or oxygen;
wherein R1 is a hydrogen molecule or a hydroxyl, R2, R3 are a hydrogen molecule or a sulfonic group, and Y+ is an alkali metal ion;
said haemolytic agent/dye mixture having a pH of approximately 2.0 to 5.0, said method further comprising the steps of:
(c) flow cytometrically assaying the sample as prepared in said step (b) by measuring at least one scattered light parameter and at least one fluorescence parameter;
and
(d) discriminating and counting erythroblasts utilizing intensity differences in scattered light and in fluorescence as measured in said step (c).

7 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/207,995Filed 9 Dec 1998
Priority: JP 10-33691627 Nov 1998
Granted: 16 Dec 2003
Term ends: Not estimatedThe source records don’t support an estimate.

Classification9 codes

G01N 33/5094
Involving human or animal cells: for blood cell populations
G01N 15/1459
Without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals: the analysis being performed on a…
G01N 33/80
Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing:…
G01N 2015/1006
Investigating individual particles: for cytology