Method and apparatus for classifying B-lymphocytes

• US 20050069959A1
• Filed: 09/20/2004
• Published: 03/31/2005
• Est. Priority Date: 09/26/2003
• Status: Active Grant

• Current Assignee: Sysmex Corporation
• Original Assignee: Sysmex Corporation
• Inventors: Kamihira, Shimeru, Yoshida, Ayumu
• Granted Patent: US 7,354,727 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10T 436/107497 Preparation composition [e.g., lysing or precipitation, etc.]

Abstract

A B-lymphocyte classifying method includes the steps of (1) preparing a measurement sample by mixing a blood sample with a lysing reagent to lyze erythrocytes and to shrink the B-lymphocytes in the blood sample, (2) letting the measurement sample flow through a flow cell of a flow cytometer, (3) radiating a light beam onto a cell in the measurement sample that is flowing through the flow cell, (4) detecting at least two scattered light emitted from the irradiated cell, (5) specifying a region of a B-lymphocyte cluster in accordance with the detected scattered light, and (6) counting the number of B-lymphocytes in the specified region.

Claims

1. A method for classifying B-lymphocytes, comprising the steps of:

(1) preparing a measurement sample by mixing a blood sample with a lysing reagent to lyze erythrocytes and to shrink B-lymphocytes in said blood sample;

(2) letting said measurement sample flow through a flow cell of a flow cytometer;

(3) radiating a light beam onto a cell in the measurement sample that is flowing through the flow cell;

(4) detecting at least two scattered light emitted from the irradiated cell;

(5) specifying a region of a B-lymphocyte cluster in accordance with the detected scattered light; and

(6) counting the number of B-lymphocytes in the specified region.

2. The method of claim 1, wherein said lysing reagent is a reagent containing ammonium chloride.

3. The method of claim 2, wherein the ammonium chloride is contained at a concentration of 5 to 20 mg/ml.

4. The method of claim 2, wherein the reagent has a pH value of 6.0 to 8.0.

5. The method of claim 1, wherein a mixing ratio of the blood sample to the lysing reagent is 1:

10 to 1;

100 (blood sample;

lysing reagent).

6. The method of claim 1, wherein a reaction time for shrinking the B-lymphocytes is 15 to 60 minutes.

7. The method of claim 1, wherein said scattered light are a forward scattered light and a side scattered light.

8. The method of claim 1, wherein the B-lymphocyte cluster is specified by preparing a two-dimensional scattergram and specifying the region of the B-lymphocyte cluster on the two-dimensional scattergram.

9. An apparatus for classifying B-lymphocytes, comprising:

(1) a measurement sample preparing section for preparing a measurement sample by mixing a blood sample with a lysing reagent to lyze erythrocytes and to shrink B-lymphocytes in said blood sample;

(2) a flow cell for introducing said measurement sample;

(3) a light source for radiating a light beam onto a cell in the measurement sample that is flowing through the flow cell;

(4) a first scattered light detector and a second scattered light detector for detecting scattered light emitted from the irradiated cell;

(5) an analyzing section for specifying a region of a B-lymphocyte cluster in accordance with the detected scattered light and counting the number of B-lymphocytes in the specified region.

10. The apparatus of claim 9, wherein a mixing ratio of the blood sample to the lysing reagent is 1:

10 to 1;

100 (blood sample;

lysing reagent).

11. The apparatus of claim 9, wherein a reaction time for shrinking the B-lymphocytes to such a degree that the B-lymphocytes are distinguishable from other lymphocytes is 15 to 60 minutes.

12. The apparatus of claim 9, wherein said scattered light are a forward scattered light and a side scattered light.

13. The apparatus of claim 9, wherein the analyzing section prepares a two-dimensional scattergram based on the detected first scattered light and second scattered light.

14. The apparatus of claim 13, wherein the analyzing section specifies the region of the B-lymphocyte cluster on the two-dimensional scattergram.