Method of classifying leukocytes by flow cytometry

• US 5,434,081 A
• Filed: 10/01/1993
• Issued: 07/18/1995
• Est. Priority Date: 11/16/1990
• Status: Expired due to Fees

• Current Assignee: Yasunori Maekawa
• Original Assignee: Yasunori Maekawa
• Inventors: Maekawa, Yasunori
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Le, Long V.
• Application Number:
  US08/131,016
• Time in Patent Office: 655 Days
• Field of Search: 436/63, 436/166, 436/172, 436/174, 436/17, 436/800, 436/805, 436/519, 424/11, 435/7.24, 435/29
• US Class Current: 436/17
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/805 Optical property
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]
• CPC Class Codes: Y10T 436/25 including sample preparation

Abstract

A method for leukocyte classification by flow cytometry that uses a first fluid that is hypotonic acidic fluorescent dye solution and a second fluid that is a solution that changes the osmolarity and pH of the first fluid is disclosed. The first fluid to be used in the method incorporates not only the first and second dyes but also the third dye and this insures that even leukocytes with damaged cell membrane can be distributed as entities distinct from other leukocyte types on a two-dimensional plot. And, hence, high precision of leukocyte classification is assured even in the case of assaying blood that contains damaged cells as a result of prolonged standing at room temperature after sampling.

Claims

1. A method of classifying leukocytes by flow cytometry which comprises the steps of:

(a) mixing a sample of blood with hypotonic first fluid comprising a first dye that is a fluorescent dye capable of selectively staining both eosinophils and basophils, said first dye being Astrazon Yellow 3G, a second dye that is a fluorescent dye capable of staining the nuclei of leukocytes, said second dye selected from the group consisting of;

Acridine Red;

Rhodamine S;

Rhodamine 6G

Rhodamine B

Rhodamine 19 perchlorate;

Rhodamine 123

Eosin Y;

Cyanosine;

Cresyl Fast Violet;

Darrow Red;

Acronol Phloxine FFS;

1,1'"'"'-dimethylthiocarbocyanine;

1,1'"'"'-diethylthiocarbocyanine;

1,1-diethyl-9-methylthiocarbocyanine bromide;

2-[γ

-(1'"'"'-ethyl-4'"'"', 5'"'"'-benzothiazolylidene) -propenyl]-1-ethyl-4,5-benzoxazolium iodide;

Astrazon Red 6B;

Basic Violet 16;

2-(p-dimethylaminostyryl-1-ethyl-4,5 benzo-thiazolium iodide;

2,4-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide;

2,6-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; and

Astrazon Orange R;

a third dye that is a fluorescent dye capable of staining the nuclei of leukocytes with damaged cell membrane, said third dye selected from the group consisting of Propidium Iodide, Etidium Bromide and M-264, and a buffer for maintaining a pH in an acidic range to obtain a first sample solution, incubating said first sample solution;

(b) mixing the first sample solution obtained in step (a) with a second fluid comprising a buffer that neutralizes acid in the first fluid for maintaining the pH of the solution at a staining pH and an osmolarity adjusting agent for adjusting the osmolarity of the solution to a value at which the leukocytes remain unchanged in shape to obtain a second sample solution, incubating said second sample solution, said dyes cooperating to enable a combined total incubation period of about one minute;

(c) loading the second sample solution obtained in step (b) in a flow cytometer and obtaining more than one signal for parameters including fluorescence and scattered light in association with individual types of leukocytes;

(d) constructing a first two-dimensional plot of intensities of scattered light and green fluorescence from the signals as emitted from leukocytes, said plot containing distribution zones of each intact leukocyte type, and counting leukocytes within each zone to obtain a number for each leukocyte type located in the distribution zones of intact leukocytes;

(e) further separating and counting leukocytes within distribution zones of individual leukocyte types on said first two-dimensional plot by determining distribution zones for leukocytes that deviate from the distribution zones of intact leukocytes where they would otherwise be located,

(f) Constructing, for the leukocyte groups in those determined distribution zones that deviate from the distribution zones of intact leukocytes, a second two-dimensional plot of the intensities of scattered light and red fluorescence,

(g) separating on said second two-dimensional plot those leukocytes that deviate and counting to obtain a number of such leukocytes in each zone;

(h) combining the number of each leukocyte type located in the distribution zones of intact leukocytes and the number of leukocytes that deviate from said zones to obtain a total number of each type of leukocytes in said blood sample.

2. A method according to claim 1 wherein the individual types of leukocytes are comprised of monocytes and neutrophils, and the distribution zones set in the first two-dimensional plot are the distribution zones of monocytes and neutrophils and, monocytes, intact neutrophils, and neutrophils with damaged cell membranes are separated from one another on the second two-dimensional plot.

7. The method as defined in claims 1, 3, or 4, wherein said step of loading the second sample and obtaining signals includes irradiating said second sample with a single wavelength.

8. The method as defined in claim 7, wherein said single wavelength is 488 nm.

9. The method as defined in claims 1 or 3 comprising the additional step preceding step (b) of:

incubating said first sample for a period of time equal to less than 1 minute.

10. The method as defined in claim 9, comprising the additional step preceding step (c) of:

incubating said second sample for a period of time equal to less than 1 minute.

11. The method as defined in claim 10, wherein said incubation time of said first sample combined with said incubation time of said second sample is less than 1 minute.

3. A method of classifying leukocytes by flow cytometry which comprises the steps of:

(a) mixing a sample of blood with a hypotonic first fluid comprising a first dye that is a fluorescent dye capable of selectively staining both eosinophils and basophils, said first dye being Astrazon Yellow 3G, a second dye that is a fluorescent dye capable of staining the nuclei of leukocytes, said second dye selected from the group consisting of;

Acridine Red;

Rhodamine S;

Rhodamine 6G

Rhodamine B

Rhodamine 19 perchlorate;

Rhodamine 123

Eosin Y;

Cyanosine;

Cresyl Fast Violet;

Darrow Red;

Acronol Phloxine FFS;

1,1'"'"'-dimethylthiocarbocyanine;

1,1'"'"'-diethylthiocarbocyanine;

1,1-diethyl-9-methylthiocarbocyanine bromide;

2-[γ

-(1'"'"'-ethyl-4'"'"', 5'"'"'-benzothiazolylidene) -propenyl]-1-ethyl-4,5-benzoxazolium iodide;

Astrazon Red 6B;

Basic Violet 16;

2-(p-dimethylaminostyryl-1-ethyl-4,5 benzo-thiazolium iodide;

2,4-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide;

2,6-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; and

Astrazon Orange R;

a third dye that is a fluorescent dye capable of staining the nuclei of leukocytes with damaged cell membrane, said third dye selects from the group consisting of Propidium Iodide Etidium Bromide and M-264, and a buffer for maintaining a pH in an acidic range to obtain first sample solution, incubating said first sample solution;

(b) mixing the first sample solution obtained in step (a) with a second fluid comprising a buffer that neutralizes acid in the first fluid for maintaining the pH of the solution at a staining pH and an osmolarity adjusting agent for adjusting the osmolarity of the solution a value at which the leukocytes remain unchanged in shape to obtain a second sample solution, incubating said second sample solution, said dyes cooperating to enable a combined total incubation period of about one minute;

(c) loading the second sample solution obtained in step (b) in a flow cytometer and obtaining more than one signal for parameters including fluorescence and scattered light in association with individual types of leukocytes;

(d) constructing a first two-dimensional plot of intensities of scattered light and green fluorescence from the signals as emitted from leukocytes, said plot containing distribution zones of each leukocyte type, and counting leukocytes within each zone to obtain a number for undamaged leukocytes of each leukocyte type located in the distribution zones of intact leukocytes;

(e) further separating and counting leukocytes within distribution zones of individual leukocyte types on said first two-dimensional plot by determining distribution zones for leukocytes with damaged membranes,

(f) constructing, for the leukocytes with damaged membranes in those determined distribution zones for leukocytes with damaged membranes, a second two-dimensional plot of the intensities of scattered light and red fluorescence,

(g) separating on said second two-dimensional plot those leukocytes with damaged membrane and counting to obtain a number for damaged leukocytes of each leukocyte type located in the zones of leukocytes with damaged membranes;

(h) combining the number of undamaged leukocytes located in the zones of intact leukocytes and the number of damaged leukocytes located in the zones of leukocytes with damaged membranes to obtain a total number of each type of leukocytes in said blood sample.

5. A method according to claim 3 wherein the distribution zones set in the first two-dimensional plot are the distribution zones of monocytes and neutrophils and, monocytes, intact neutrophils and neutrophils with damaged cell membrane are separated from one another on the second two-dimensional plot.

4. A method of classifying leukocytes by flow cytometry which comprises the steps of:

(a) mixing a sample of blood with a hypotonic first fluid comprising a first dye that is a fluorescent dye capable of selectively staining both eosinophils and basophils, said first dye being Astrazon Yellow 3G a second dye that is a fluorescent dye capable of staining the nuclei of leukocytes, said second dye selected from the group consisting of;

Acridine Red;

Rhodamine S;

Rhodamine 6G

Rhodamine B

Rhodamine 19 perchlorate;

Rhodamine 123

Eosin Y;

Cyanosine;

Cresyl Fast Violet;

Darrow Red;

Acronol Phloxine FFS;

1,1'"'"'-dimethylthiocarbocyanine;

1,1'"'"'-diethylthiocarbocyanine;

1,1-diethyl-9-methylthiocarbocyanine bromide;

2-[γ

-(1'"'"'-ethyl-4'"'"', 5'"'"'-benzothiazolylidene) -propenyl]-1-ethyl-4,5-benzoxazolium iodide;

Astrazon Red 6B;

Basic Violet 16;

2-(p-dimethylaminostyryl-1-ethyl-4,5 benzo-thiazolium iodide;

2,4-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide;

2,6-bis(p-dimethylaminostyryl)-1-ethyl-pyridinium iodide; and

Astrazon Orange R;

a third dye that is a fluorescent dye capable of staining the nuclei of leukocytes with damaged cell membrane, said third dye selected from the group consisting of Propidium Iodide, Etidium Bromide and M-264, and a buffer for maintaining a pH in an acidic range to obtain a first sample solution, incubating said first sample solution;

(b) mixing the first sample solution obtained in step (a) with a second fluid comprising a buffer that neutralizes acid in the first fluid for maintaining the pH of the solution at a staining pH and an osmolarity adjusting agent for adjusting the osmolarity of the solution to a value at which the leukocytes remain unchanged in shape to obtain a second sample solution, incubating said second sample solution, said dyes cooperating to enable a combined total incubation period of about one minute;

(c) loading the second sample solution obtained in step (b) in a flow cytometer and obtaining more than one signal for parameters including fluorescence and scattered light in association with individual types of leukocytes;

(d) constructing a first two-dimensional plot of intensity of scattered light plotted against intensity of green fluorescence from the respective signals as emitted from leukocytes in said flow cytometer, said plot defining distribution zones of each intact leukocyte type, and counting to obtain a number for each leukocyte type located in the distribution zones of intact leukocytes;

(e) further separating leukocyte cell groups within each distribution zone, by constructing for each distribution zone a second plot of the intensity of scattered light plotted against intensity of red fluorescence from the respective signals emitted from leukocytes in said flow cytometer, whereby said second plot includes cells having damaged membrane of leukocyte types that would be found in distribution zones for each intact leukocyte type.

(f) distinguishing on said second plot said leukocyte cell groups having damaged membranes, and counting to obtain a number for each type of leukocyte having damaged membranes in each distribution zone; and

(g) combining the number for each leukocyte type located in the distribution zones of intact leukocytes and the number of for each type of leukocyte having damaged membranes to obtain a total number of each type of leukocytes in said blood sample.

6. A method according to claim 4 wherein the distribution zones set in the first two-dimensional plot are the distribution zones of monocytes and neutrophils and, monocytes, intact neutrophils and neutrophils with damaged cell membrane are separated from one another on the second two-dimensional plot.