Method for determining the volume of single red blood cells

• US 6,633,368 B2
• Filed: 01/02/2001
• Issued: 10/14/2003
• Est. Priority Date: 01/02/2001
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Berndt, Klaus W.
• Primary Examiner(s): Smith, Zandra V.
• Assistant Examiner(s): Stock, Jr., Gordon J
• Application Number:
  US09/753,205
• Publication Number:
  US 20020126272A1
• Time in Patent Office: 1,015 Days
• Field of Search: 356/39, 356/317, 356/627, 356/335, 422/82.05, 422/82.08, 436/164, 436/165, 250/458.1, 250/459.1, 250/461.2
• US Class Current: 356/39
• 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 2015/012 Red blood cells
• CPC Class Codes: G01N 2015/1497 Particle shape
• CPC Class Codes: G01N 21/03 Cuvette constructions

Abstract

The invention discloses a method for determining the volume of single red blood cells or other particles that are suspended in liquids. The sample is disposed into an optical cuvette suitable for microscopic analysis. A fluorescent dye is added that does not leak into the cells, and that is able to absorb excitation light and emit fluorescence light at wavelengths that are only weakly absorbed by the cells. The cell volume is determined using fluorescence intensity values measured (i) in a first area comprising a single cell, (ii) in a second area close to that cell, and (iii) in said second area, after changing the cuvette thickness by a known amount.

Claims

1. A method for determining the volume of particles in a liquid, the method comprising:

(a) obtaining a suspension of particles in a liquid;

(b) depositing said suspension into a container having at least one transparent window;

(c) adding a fluorescent dye to said suspension in said container that does not substantially leak into said particles, wherein said fluorescent dye is able to absorb excitation light at wavelengths that are only weakly absorbed by said particles and emit fluorescence light at wavelengths that are only weakly absorbed by said particles;

(d) illuminating said suspension with light, whereby the wavelength of said light is selected so that said light is being absorbed by said fluorescent dye but only weakly absorbed by said particles;

(e) measuring a first intensity of fluorescent light that reemerges from said suspension in a first area of said suspension, wherein said first area contains no particles;

(f) changing the thickness of said container in said first area by a known amount by pressing on said container from outside of said container and measuring a second intensity of fluorescent light that reemerges from said suspension in said first area;

(g) measuring a third intensity of fluorescent light that reemerges from said suspension in a second area of said suspension, wherein said second area contains at least one particle;

(h) measuring a fourth intensity of fluorescent light that reemerges from a third area of said suspension close to said at least one particle in said second area; and

(i) determining the volume of said at least one particle based on said fluorescent light intensity values and said known change in thickness of said container.

3. The method of claim 1 wherein the particle is a cell.

5. The method of claim 3 wherein the cell is a human cell.

7. The method of claim 3 wherein the cell is a red blood cell.

9. The method of claim 1 wherein the liquid is a biological or medical sample.

11. The method of claim 9 wherein the biological or medical sample comprises a body fluid.

13. The method of claim 11 wherein the body fluid is selected from the group consisting of blood, urine, sputum, saliva and lymph fluid.

15. The method of claim 9 wherein the biological or medical sample comprises tissue culture cells.

17. The method of claim 9 wherein the biological or medical sample comprises bacterial cells.

19. The method of claim 1 wherein the container is an optical cuvette.

21. The method of claim 19 wherein the optical cuvette comprises at least one transparent window.

23. The method of claim 19 wherein the optical cuvette comprises a microscope slide and a cover slip.

25. The method of claim 1 wherein the dye is selected from the group consisting of TO-PRO-3 and TO-PRO-5.

2. A method for determining the volume of particles in a liquid, the method comprising:

(a) obtaining a suspension of particles in a liquid;

(b) adding a fluorescent dye to said suspension forming a mixture, wherein said fluorescent dye does not substantially leak into said particles and is able to absorb excitation light at wavelengths that are only weakly absorbed by said particles and emit fluorescence light at wavelengths that are only weakly absorbed by said particles;

(c) depositing said mixture into a container having at least one transparent window;

(d) illuminating said mixture with light, whereby the wavelength of said light is selected so that said light is being absorbed by said fluorescent dye but only weakly absorbed by said particles;

(e) measuring a first intensity of fluorescent light that reemerges from said mixture in a first area of said mixture, wherein said first area contains no particles;

(f) changing the thickness of said container in said first area by a known amount by pressing on said container from outside of said container and measuring a second intensity of fluorescent light that reemerges from said mixture in said first area;

(g) measuring a third intensity of fluorescent light that reemerges from said mixture in a second area of said mixture, wherein said second area contains at least one particle;

(h) measuring a fourth intensity of fluorescent light that reemerges from a third area of said mixture close to said at least one particle in said second area; and

determining the volume of said at least one particle based on said fluorescent light intensity values and said known change in thickness of said container.

4. The method of claim 2 wherein the particle is a cell.

6. The method of claim 4 wherein the cell is a human cell.

8. The method of claim 4 wherein the cell is a red blood cell.

10. The method of claim 2 wherein the liquid is a biological or medical sample.

12. The method of claim 10 wherein the biological or medical sample comprises a body fluid.

14. The method of claim 12 wherein the body fluid is selected from the group consisting of blood, urine, sputum, saliva and lymph fluid.

16. The method of claim 10 wherein the biological or medical sample comprises tissue culture cells.

18. The method of claim 10 wherein the biological or medical sample comprises bacterial cells.

20. The method of claim 2 wherein the container is an optical cuvette.

22. The method of claim 20 wherein the optical cuvette comprises at least one transparent window.

24. The method of claim 20 wherein the optical cuvette comprises a microscope slide and a cover slip.

26. The method of claim 2 wherein the dye is selected from the group consisting of TO-PRO-3 and TO-PRO-5.

27. A method for determining the volume of particles in a liquid, the method comprising:

(a) obtaining a suspension of particles in a liquid;

(b) depositing said suspension into a container having at least one transparent window;

(c) adding a fluorescent dye to said suspension in said container that does not substantially leak into said particles, wherein said fluorescent dye is able to absorb excitation light at wavelengths that are only weakly absorbed by said particles and emit fluorescence light at wavelengths that are only weakly absorbed by said particles;

(d) illuminating said suspension with light, whereby the wavelength of said light is selected so that the light is being absorbed by said dye but only weakly absorbed by said particles;

(e) measuring a first intensity of fluorescent light that reemerges from said suspension in a first area of said suspension, wherein said first area is located close to a particle;

(f) changing the thickness of said container in said first area by a known amount by pressing on said container from outside of said container and measuring a second intensity of fluorescent light that reemerges from said suspension in said first area;

(g) measuring a third intensity of fluorescent light that reemerges from said suspension in a second area of said suspension, wherein said second area contains at least one particle; and

(h) determining the volume of said at least one particle based on said fluorescence light intensity values and said known change in thickness of said container.

29. The method of claim 27 wherein the particle is a cell.

31. The method of claim 29 wherein the cell is a human cell.

33. The method of claim 29 wherein the cell is a red blood cell.

35. The method of claim 27 wherein the liquid is a biological or medical sample.

37. The method of claim 35 wherein the biological or medical sample comprises a body fluid.

39. The method of claim 37 wherein the body fluid is selected from the group consisting of blood, urine, sputum, saliva and lymph fluid.

41. The method of claim 35 wherein the biological or medical sample comprises tissue culture cells.

43. The method of claim 35 wherein the biological or medical sample comprises bacterial cells.

45. The method of claim 27 wherein the container is an optical cuvette.

47. The method of claim 45 wherein the optical cuvette comprises at least one transparent window.

49. The method of claim 45 wherein the optical cuvette comprises a microscope slide and a cover slip.

51. The method of claim 27 wherein the dye is selected from the group consisting of TO-PRO-3 and TO-PRO-5.

28. A method for determining the volume of particles in a liquid, the method comprising:

(a) obtaining a suspension of particles in a liquid;

(b) adding a fluorescent dye to said suspension forming a mixture, wherein said fluorescent dye does not substantially leak into said particles and is able to absorb excitation light at wavelengths that are only weakly absorbed by said particles and emit fluorescence light at wavelengths that are only weakly absorbed by said particles;

(c) depositing said mixture into a container having at least one transparent window;

(d) illuminating said mixture with light, whereby the wavelength of said light is selected so that the light is being absorbed by said dye but only weakly absorbed by said particles;

(e) measuring a first intensity of fluorescent light that reemerges from said mixture in a first area of said mixture, wherein said first area is located close to a particle;

(f) changing the thickness of said container in said first area by a known amount by pressing on said container from outside of said container and measuring a second intensity of fluorescent light that reemerges from said mixture in said first area;

(g) measuring a third intensity of fluorescent light that reemerges from said mixture in a second area of said mixture, wherein said second area contains at least one particle; and

(h) determining the volume of said at least one particle based on said fluorescence light intensity values and said known change in thickness of said container.

30. The method of claim 28 wherein the particle is a cell.

32. The method of claim 30 wherein the cell is a human cell.

34. The method of claim 30 wherein the cell is a red blood cell.

36. The method of claim 28 wherein the liquid is a biological or medical sample.

38. The method of claim 36 wherein the biological or medical sample comprises a body fluid.

40. The method of claim 38 wherein the body fluid is selected from the group consisting of blood, urine, sputum, saliva and lymph fluid.

42. The method of claim 36 wherein the biological or medical sample comprises tissue culture cells.

44. The method of claim 36 wherein the biological or medical sample comprises bacterial cells.

46. The method of claim 28 wherein the container is an optical cuvette.

48. The method of claim 46 wherein the optical cuvette comprises at least one transparent window.

50. The method of claim 46 wherein the optical cuvette comprises a microscope slide and a cover slip.

52. The method of claim 28 wherein the dye is selected from the group consisting of TO-PRO-3 and TO-PRO-5.