Measuring volume and constituents of cells

• US 8,345,227 B2
• Filed: 04/13/2012
• Issued: 01/01/2013
• Est. Priority Date: 04/15/2011
• Status: Active Grant

• Current Assignee: Roche Diagnostic Hematology, Inc.
• Original Assignee: Constitution Medical Inc. (Roche Diagnostic Hematology, Inc.)
• Inventors: Zahniser, Michael, Zahniser, Russell
• Primary Examiner(s): STOCK JR, GORDON J
• Application Number:
  US13/446,967
• Publication Number:
  US 20120262705A1
• Time in Patent Office: 263 Days
• Field of Search: None
• US Class Current: 356/40
• CPC Class Codes: G01N 15/0227 using imaging; using holography
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 2015/0084 Platelets
• CPC Class Codes: G01N 2015/012 Red blood cells
• CPC Class Codes: G01N 2015/018 Platelets
• CPC Class Codes: G01N 2015/1472 with colour
• CPC Class Codes: G01N 33/49 Blood chemical methods for determining blood cell populations G01N33/5094; chemical analysis of blood groups or blood types G01N33/80
• CPC Class Codes: G06T 2207/10056 Microscopic image
• CPC Class Codes: G06T 2207/30024 Cell structures in vitro; Tissue sections in vitro
• CPC Class Codes: G06T 7/62 of area, perimeter, diameter or volume
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts
• CPC Class Codes: G06V 20/695 Preprocessing, e.g. image segmentation

Abstract

A method of determining a hemoglobin content value of a red blood cell includes: (a) illuminating the cell with incident light at a plurality of illumination wavelengths; (b) obtaining at least one two-dimensional image of the cell corresponding to each illumination wavelength; (c) for each illumination wavelength, determining a mean optical density and a maximum optical density for the cell; (d) determining an area of the cell; (e) for each illumination wavelength, determining a volume of the cell; (f) for each illumination wavelength, determining an integrated optical density for the cell; and (g) determining the hemoglobin content value of the cell based on the area of the cell, the volumes of the cell corresponding to each of the illumination wavelengths, and the integrated optical densities for the cell corresponding to each of the illumination wavelengths.

Claims

1. A method of determining a hemoglobin content value of a red blood cell, the method comprising:

(a) illuminating the cell with incident light at a plurality of illumination wavelengths;

(b) obtaining at least one two-dimensional image of the cell corresponding to each illumination wavelength;

(c) for each illumination wavelength, determining a mean optical density and a maximum optical density for the cell;

(d) determining an area of the cell;

(e) for each illumination wavelength, determining a volume of the cell based on the area of the cell and the mean optical density and maximum optical density for the cell corresponding to the illumination wavelength;

(f) for each illumination wavelength, determining an integrated optical density for the cell based on the area of the cell and the mean optical density for the cell corresponding to the illumination wavelength; and

(g) determining the hemoglobin content value of the cell based on a weighted combination of the area of the cell, the volumes of the cell corresponding to each of the illumination wavelengths, and the integrated optical densities for the cell corresponding to each of the illumination wavelengths.

2. The method of claim 1, further comprising determining the volume of the cell at each illumination wavelength based on a ratio of the mean optical density to the maximum optical density corresponding to the illumination wavelength.

3. The method of claim 1, wherein the plurality of illumination wavelengths comprises at least three illumination wavelengths.

4. The method of claim 1, wherein the plurality of illumination wavelengths comprises at least four illumination wavelengths.

5. The method of claim 1, further comprising determining the volume of the cell at each illumination wavelength based on a ratio of the mean optical density to a sum of the maximum optical density and a correction factor at the illumination wavelength.

6. The method of claim 1, further comprising:

repeating steps (a) through (g) for a plurality of red blood cells from a sample of blood to determine hemoglobin content values for each one of the plurality of red blood cells; and

determining a mean cell hemoglobin value for the sample from the hemoglobin content values for each one of the plurality of red blood cells.

7. The method of claim 3, wherein the plurality of illumination wavelengths comprises at least one wavelength between 575 nm and 600 nm, at least one wavelength between 525 nm and 570 nm, and at least one wavelength between 400 nm and 475 nm.

8. The method of claim 4, wherein the plurality of illumination wavelengths comprises at least one wavelength between 620 nm and 750 nm, at least one wavelength between 575 nm and 600 nm, at least one wavelength between 525 nm and 570 nm, and at least one wavelength between 400 nm and 475 nm.

9. The method of claim 5, further comprising adding an offset value to the ratio of the mean optical density to the sum of the maximum optical density and the correction factor to determine the volume of the cell at each illumination wavelength.

10. The method of claim 9, further comprising determining values of the correction factor and the offset value from a reference set of blood samples.

11. The method of claim 6, further comprising, for each respective cell of the plurality of red blood cells, excluding the respective cell from the determination of the mean cell hemoglobin value if the area of the respective cell is outside a selected area range.

12. A method of assessing an operating condition of an automated blood analysis device, the method comprising:

operating the device so that the device uses the method of claim 6 to determine a mean cell hemoglobin value for a control composition; and

comparing a reference value of the mean cell hemoglobin for the control composition to the determined value of the mean cell hemoglobin to assess the operating condition of the device.

13. The method of claim 12, further comprising:

determining a difference between the determined and reference values of the mean cell hemoglobin for the control composition; and

re-calibrating the device if the difference exceeds a threshold value.

14. The method of claim 13, wherein re-calibrating the device comprises determining, from a reference set of blood samples, a plurality of weight coefficients that are used to determine the mean cell hemoglobin value for the sample based on the areas of the plurality of cells, the volumes of the plurality of cells corresponding to each of the illumination wavelengths, and the integrated optical densities for the plurality of cells corresponding to each of the illumination wavelengths.

15. The method of claim 6, further comprising, for each respective cell of the plurality of red blood cells, identifying a first set of pixels in at least one of the images, wherein the first set of pixels corresponds to the respective cell.

16. The method of claim 15, further comprising, for each respective cell, identifying a second set of pixels in the at least one of the images, wherein the second set of pixels is identified by removing from the first set of pixels a subset of pixels that correspond to a perimeter region of the respective cell.

17. The method of claim 15, further comprising, for each respective cell, determining the area of the respective cell based on the first set of pixels corresponding to the respective cell.

18. The method of claim 17, further comprising, for each respective cell, determining a perimeter of the respective cell based on the first set of pixels corresponding to the respective cell, and excluding the respective cell from the determination of the mean cell hemoglobin value if a ratio of the perimeter squared to the area for the respective cell exceeds a threshold value.

19. The method of claim 17, further comprising, for each respective cell, determining a convex hull of the respective cell, determining an area enclosed by the convex hull, and excluding the respective cell from the determination of the mean cell hemoglobin value if a ratio of the area enclosed by the convex hull to the area of the respective cell exceeds a threshold value.

20. A system for determining a hemoglobin content value of a red blood cell, the system comprising:

an illumination source configured to illuminate the cell with incident light at a plurality of illumination wavelengths;

a detector configured to obtain at least one two-dimensional image of the cell corresponding to each illumination wavelength; and

an electronic processor configured to;

(a) determine a mean optical density and a maximum optical density for the cell at each illumination wavelength;

(b) determine an area of the cell;

(c) for each illumination wavelength, determine a volume of the cell based on the area of the cell and the mean optical density and maximum optical density for the cell corresponding to the illumination wavelength;

(d) for each illumination wavelength, determine an integrated optical density for the cell based on the area of the cell and the mean optical density for the cell corresponding to the illumination wavelength; and

(e) determine the hemoglobin content value of the cell based on a weighted combination of the area of the cell, the volumes of the cell corresponding to each of the illumination wavelengths, and the integrated optical densities for the cell corresponding to each of the illumination wavelengths.

21. The system of claim 20, wherein the plurality of illumination wavelengths comprises at least one wavelength between 620 nm and 750 nm, at least one wavelength between 575 nm and 600 nm, at least one wavelength between 525 nm and 570 nm, and at least one wavelength between 400 nm and 475 nm.

22. The system of claim 20, further comprising an automated blood sample preparation system.

23. The system of claim 20, wherein the electronic processor is further configured to determine the volume of the cell at each illumination wavelength based on a ratio of the mean optical density to the maximum optical density corresponding to the illumination wavelength.

24. The system of claim 20, wherein the plurality of illumination wavelengths comprises at least four illumination wavelengths.

25. The system of claim 20, wherein the plurality of illumination wavelengths comprises at least three illumination wavelengths.

26. The system of claim 20, wherein the electronic processor is further configured to determine the volume of the cell at each illumination wavelength based on a ratio of the mean optical density to a sum of the maximum optical density and a correction factor at the illumination wavelength.

27. The system of claim 20, wherein the electronic processor is further configured to:

repeat steps (a) through (e) for a plurality of red blood cells from a sample of blood to determine hemoglobin content values for each of the plurality of red blood cells; and

determine a mean cell hemoglobin value for the sample from the hemoglobin content values for each of the plurality of red blood cells.

28. The system of claim 25, wherein the plurality of illumination wavelengths comprises at least one wavelength between 575 nm and 600 nm, at least one wavelength between 525 nm and 570 nm, and at least one wavelength between 400 nm and 475 nm.

29. The system of claim 26, wherein the electronic processor is further configured to add an offset value to the ratio of the mean optical density to the sum of the maximum optical density and the correction factor to determine the volume of the cell at each illumination wavelength.

30. The system of claim 29, wherein the electronic processor is further configured to determine values of the correction factor and the offset value from a reference set of blood samples.

31. The system of claim 27, wherein for each respective cell of the plurality of red blood cells, the electronic processor is further configured to exclude the respective cell from the determination of the mean cell hemoglobin value if the area of the respective cell is outside a selected range.

32. The system of claim 27, wherein the sample is a control composition, and wherein the electronic processor is further configured to:

determine a mean cell hemoglobin value for the control composition; and

compare a reference value of the mean cell hemoglobin for the control composition to the determined value of the mean cell hemoglobin to assess an operating condition of a device that comprises the system.

33. The system of claim 32, wherein the electronic processor is further configured to:

determine a difference between the determined and reference values of the mean cell hemoglobin for the control composition; and

re-calibrate the device if the difference exceeds a threshold value.

34. The system of claim 33, wherein the electronic processor is further configured to re-calibrate the device by determining, from a reference set of blood samples, a plurality of weight coefficients that are used to determine the mean cell hemoglobin value for the sample based on the areas of the plurality of cells, the volumes of the plurality of cells corresponding to each of the illumination wavelengths, and the integrated optical densities for the plurality of cells corresponding to each of the illumination wavelengths.

35. The system of claim 27, wherein the electronic processor is further configured to identify, for each respective cell of the plurality of red blood cells, a first set of pixels in at least one of the images, wherein the first set of pixels corresponds to the respective cell.

36. The system of claim 35, wherein the electronic processor is further configured to identify, for each respective cell, a second set of pixels in the at least one of the images, wherein the second set of pixels is identified by removing from the first set of pixels a subset of pixels that correspond to a perimeter region of the respective cell.

37. The system of claim 35, wherein the electronic processor is further configured to determine, for each respective cell, the area of the respective cell based on the first set of pixels corresponding to the one cell.

38. The system of claim 37, wherein the electronic processor is further configured to:

determine, for each respective cell, a perimeter of the respective cell based on the first set of pixels corresponding to the respective cell; and

exclude the respective cell from the determination of the mean cell hemoglobin value if a ratio of the perimeter squared to the area for the respective cell exceeds a threshold value.

39. The system of claim 37, wherein the electronic processor is further configured to:

determine, for each respective cell, a convex hull of the respective cell, and an area enclosed by the convex hull; and

exclude the respective cell from the determination of the mean cell hemoglobin value if a ratio of the area enclosed by the convex hull to the area of the respective cell exceeds a threshold value.