Reagent and method for classification and counting of leukocytes

• US 6,004,816 A
• Filed: 05/15/1998
• Issued: 12/21/1999
• Est. Priority Date: 05/19/1997
• Status: Expired due to Term

• Current Assignee: Sysmex Corporation
• Original Assignee: Sysmex Corporation
• Inventors: Mizukami, Toshihiro, Sakata, Takashi, Ishiyama, Munetaka
• Primary Examiner(s): WALLENHORST, MAUREEN
• Application Number:
  US09/080,016
• Time in Patent Office: 585 Days
• Field of Search: 436/8, 436/10, 436/17, 436/18, 436/63, 436/164, 436/172, 436/175, 436/800, 435/2, 252/408.1
• US Class Current: 436/10
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: G01N 33/52 Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements immunological elements G01N33/54386; involving labelled immunochemicals G01N33/58; for haemoglobin or occult blood G01N33/72
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/10 Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

A reagent for classification and counting of leukocytes, the reagent containing at least one dye which has the following structural formula ##STR1## where R₁ is a hydrogen atom or an alkyl group, R₂ and R₃ each represent a hydrogen atom, a lower alkyl group or a lower alkoxy group, R₄ represents a hydrogen atom, an acyl group or an alkyl group, Z represents a sulfur atom, an oxygen atom, or a carbon atom having a lower alkyl group, n denotes 0, 1 or 2, and X^- represents an anion,

and specifically binds to RNA to increase in fluorescence intensity, and a method for classification and counting of leukocytes which uses the reagent, whereby abnormal cells such as immature leukocytes and abnormal leukocytes can be classified and counted easily and highly accurately, and at the same time, classification and counting of normal leukocytes as well as the counting of leukocytes can be performed.

Claims

1. A reagent for classification and counting of leukocytes, said reagent containing at least one dye which has the following structural formula ##STR8## where R₁ is a hydrogen atom or an alkyl group, R₂ and R₃ each represent a hydrogen atom, a lower alkyl group or a lower alkoxy group, R₄ represents a hydrogen atom, an acyl group or an alkyl group, at least one of R₁ and R4 is an alkyl group having 8 to 18 carbon atoms, Z represents a sulfur atom, an oxygen atom, or a carbon atom having a lower alkyl group, n denotes 0, 1 or 2, and X represents an anion,

and specifically binds to RNA to increase in fluorescence intensity.

2. A method for classification and counting 2 of leukocytes, comprising the steps of:

1) mixing a blood sample with a hemolytic agent which lyses erythrocytes in the blood sample to such a degree as not to impede measurement, thereby bringing normal or abnormal blood cells to a state suitable for staining;

2) mixing a sample prepared in the step

     1) with a dye which has the following structural formula ##STR9## where R₁ is a hydrogen atom or an alkyl group, R₂ and R₃ each represent a hydrogen atom, a lower alkyl group or a lower alkoxy group, R₄ represents a hydrogen atom, an acyl group or an alkyl group, at least one of R₁ and R₄ is an alkyl group having 8 to 18 carbon atoms, Z represents a sulfur atom, an oxygen atom or a carbon atom having a lower alkyl group, n denotes 0, 1 or 2, and X represents an anion, and specifically binds to cellular RNA to increase in fluorescence intensity, thereby fluorescence-staining nucleated cells in the blood sample;

3) measuring an assay sample prepared in the step

     2) with a flow cytometer to measure at least one scattered light and at least one fluorescence; and

4) classifying normal leukocytes into at least 5 populations, and counting them, by the use of differences in the intensities of the scattered light and the fluorescence measured in step

     3).

3. The method for classification and counting of leukocytes as claimed in claim 2, wherein the hemolytic agent contains at least one nonionic surfactant and at least one cationic surfactant and has a pH of 4.5 to 11.0.

4. The method for classification and counting of leukocytes as claimed in claim 3, wherein the hemolytic agent further contains an organic acid having at least one aromatic ring or a salt thereof.

5. The method for classification and counting of leukocytes as claimed in claim 2, wherein the scattered light is at least one member selected from the group consisting of forward low-angle scattered light, forward high-angle scattered light, and lateral scattered light.

6. The method for classification and counting of leukocytes as claimed in claim 2, which can simultaneously perform the classification and counting of the abnormal blood cells.

7. The method for classification and counting of leukocytes as claimed in any one of claim 2 to 6, wherein the abnormal cells are cells with RNA content increased over the normal cells.

8. The method for classification and counting of leukocytes as claimed in claim 7, wherein the abnormal cells are one or more members selected from the group consisting of immature granulocytes, myeloblasts, erythroblasts and atypical lymphocytes.

9. A reagent kit for classification and counting of leukocytes, comprising:

1) a hemolytic agent which lyses erythrocytes in a blood sample to such a degree as not to impede measurement, thereby bringing normal or abnormal blood cells to a state suitable for staining; and

2) a staining solution containing at least one dye which has the following structural formula ##STR10## where R₁ is a hydrogen atom or an alkyl group, R₂ and R₃ each represent a hydrogen atom, a lower alkyl group or a lower alkoxy group, R₄ represents a hydrogen atom, an acyl group or an alkyl group, at least one of R₁ and R₄ is an alkyl group having 8 to 18 carbon atoms, Z represents a sulfur atom, an oxygen atom or a carbon atom having a lower alkyl group, n denotes 0, 1 or 2, and X represents an anion,

and specifically binds to RNA to increase in fluorescence intensity.