Measuring volume and constituents of cells

• US 8,488,111 B2
• Filed: 04/13/2012
• Issued: 07/16/2013
• 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:
  US13/447,045
• Publication Number:
  US 20120262704A1
• Time in Patent Office: 459 Days
• Field of Search: None
• US Class Current: 356/39
• CPC Class Codes: G01N 15/0227 using imaging; using holography
• CPC Class Codes: G01N 15/1433 using image recognition
• 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 for determining a mean cell volume for a blood sample includes: illuminating the sample with incident light at a plurality of illumination wavelengths and obtaining a two-dimensional image of the sample at each of the plurality of illumination wavelengths; identifying a plurality of cells that appear in each of the images; for each one of the plurality of cells, determining an integrated optical density corresponding to each of the plurality of illumination wavelengths; for each one of the plurality of cells, determining a cell volume based on the integrated optical densities corresponding to each of the plurality of illumination wavelengths; and determining the mean cell volume for the blood sample from the cell volumes for each one of the plurality of cells.

Claims

1. A method for determining a mean cell volume for a blood sample, the method comprising:

illuminating the sample with incident light at a plurality of illumination wavelengths and obtaining a two-dimensional image of the sample at each of the plurality of illumination wavelengths;

identifying a plurality of cells that appear in each of the images;

for each one of the plurality of cells, determining an integrated optical density corresponding to each of the plurality of illumination wavelengths;

for each one of the plurality of cells, determining a cell volume based on the integrated optical densities corresponding to each of the plurality of illumination wavelengths; and

determining the mean cell volume for the blood sample from the cell volumes for each one of the plurality of cells.

2. The method of claim 1, further comprising:

for each one of the plurality of cells, determining an area of the cell; and

for each one of the plurality of cells, determining the cell volume based on the area of the cell and the integrated optical densities corresponding to each of the plurality of illumination wavelengths.

3. The method of claim 1, wherein the two-dimensional image corresponds to incident light that is reflected from, or transmitted by, the sample.

4. The method of claim 1, wherein for each one of the plurality of cells, determining the integrated optical density corresponding to each of the plurality of illumination wavelengths comprises:

determining an area of the cell;

for each of the plurality of illumination wavelengths, determining a mean optical density for the cell based a corresponding one of the images; and

determining the integrated optical density corresponding to each wavelength based on the area of the cell and the mean optical density for the cell corresponding to the wavelength.

5. The method of claim 1, wherein for each one of the plurality of cells, the cell volume is determined based on a weighted linear combination of the integrated optical densities corresponding to each of the plurality of illumination wavelengths.

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

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

8. 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 1 to determine a mean cell volume for a control composition; and

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

9. The method of claim 1, wherein identifying the plurality of cells comprises, for each one of the plurality of cells, identifying a first set of pixels in each image that corresponds to the cell.

10. The method of claim 1, further comprising:

for each one of the plurality of cells, determining a volume corresponding to each of the plurality of illumination wavelengths; and

for each one of the plurality of cells, determining the cell volume based on the integrated optical densities and the volumes corresponding to each of the plurality of illumination wavelengths.

11. The method of claim 5, further comprising determining weight coefficients for the weighted linear combination from a reference set of blood samples.

12. The method of claim 6, 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.

13. The method of claim 7, 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.

14. The method of claim 8, further comprising:

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

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

15. The method of claim 14, 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 cell volume based on the integrated optical densities corresponding to each of the plurality of illumination wavelengths.

16. The method of claim 9, wherein identifying the plurality of cells comprises, for each one of the plurality of cells, removing pixels from the first set of pixels in each image that correspond to a perimeter region of the cell.

17. The method of claim 9, further comprising, for each one of the plurality of cells, determining an area of the cell based on the first set of pixels.

18. The method of claim 9, further comprising, for each one of the plurality of cells, determining a convex hull of the cell, determining an area enclosed by the convex hull, and excluding the cell from the determination of the mean cell volume if a ratio of the area enclosed by the convex hull to the area of the cell exceeds a threshold value.

19. The method of claim 16, wherein the perimeter region of the cell has a thickness of 0.5 microns or more.

20. The method of claim 17, further comprising, for each one of the plurality of cells, determining a perimeter of the cell based on the first set of pixels, and excluding the cell from the determination of the mean cell volume if a ratio of the perimeter squared to the area exceeds a threshold value.

21. The method of claim 17, further comprising, for each one of the plurality of cells, excluding the cell from the determination of the mean cell volume if the area of the cell is outside a selected area range.

22. The method of claim 10, wherein, for each one of the plurality of cells, determining the volume corresponding to each of the plurality of illumination wavelengths comprises:

for each of the plurality of illumination wavelengths, determining a mean optical density and a maximum optical density for the cell; and

for each of the plurality of illumination wavelengths, determining the volume of the cell based on a ratio of the mean optical density to the maximum optical density corresponding to the wavelength.

23. The method of claim 10, wherein for each one of the plurality of cells, the cell volume is determined based on a weighted linear combination of the integrated optical densities and the volumes corresponding to each of the plurality of illumination wavelengths.

24. The method of claim 10, wherein, for each one of the plurality of cells, determining the volume corresponding to each of the plurality of illumination wavelengths comprises:

for each of the plurality of illumination wavelengths, determining a mean optical density and a maximum optical density for the cell; and

for each of the plurality of illumination wavelengths, determining the volume of the cell based on a ratio of the mean optical density to the sum of the maximum optical density corresponding to the wavelength and a correction factor at the wavelength.

25. The method of claim 10, further comprising:

for each one of the plurality of cells, determining an area of the cell; and

for each one of the plurality of cells, determining the cell volume based on the area of the cell and the integrated optical densities and volumes corresponding to each of the plurality of illumination wavelengths.

26. The method of claim 23, further comprising determining weight coefficients for the weighted linear combination from a reference set of blood samples.

27. The method of claim 24, 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.

28. The method of claim 27, further comprising determining values of the correction factor and the offset value from a reference set of blood samples.

29. The method of claim 25, wherein for each one of the plurality of cells, the cell volume is determined based on a weighted linear combination of the area and the integrated optical densities and volumes corresponding to each of the plurality of illumination wavelengths.

30. The method of claim 29, further comprising determining weight coefficients for the weighted linear combination from a reference set of blood samples.

31. A system for determining a mean cell volume for a blood sample, the system comprising:

an illumination source configured to direct incident light at a plurality of illumination wavelengths to the sample;

a detector configured to obtain two-dimensional images of the sample at each of the plurality of illumination wavelengths; and

an electronic processor configured to;

identify a plurality of cells that appear in each of the images;

for each one of the plurality of cells, determine an integrated optical density corresponding to each of the plurality of illumination wavelengths;

for each one of the plurality of cells, determine a cell volume based on the integrated optical densities corresponding to each of the plurality of illumination wavelengths; and

determine the mean cell volume for the blood sample from the cell volumes for each one of the plurality of cells.

32. The system of claim 31, wherein the system further comprises an automated blood sample preparation system.

33. The system of claim 31, 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 400nm and 475 nm.

34. The system of claim 31, wherein the electronic processor is further configured to:

for each one of the plurality of cells, determine an area of the cell; and

for each one of the plurality of cells, determine the cell volume based on the area and the integrated optical densities corresponding to each of the plurality of illumination wavelengths.

35. The system of claim 31, wherein the electronic processor is further configured to:

for each one of the plurality of cells, determine a volume corresponding to each of the plurality of illumination wavelengths; and

for each one of the plurality of cells, determine the cell volume based on the integrated optical densities and the volumes corresponding to each of the plurality of illumination wavelengths.

36. The system of claim 35, wherein the electronic processor is further configured to:

for each one of the plurality of cells, determine an area of the cell; and

for each one of the plurality of cells, determine the cell volume based on the area and the integrated optical densities and volumes corresponding to each of the plurality of illumination wavelengths.

37. A computer readable storage device, having encoded thereon computer readable instructions that, when executed by a processor, cause the processor to:

receive a plurality of images of a blood sample, each of the plurality of images corresponding to a different wavelength of illumination light incident on the sample;

identify a plurality of cells that appear in each of the images;

for each one of the plurality of cells, determine an integrated optical density corresponding to each of the plurality of illumination wavelengths;

for each one of the plurality of cells, determine a cell volume based on the integrated optical densities corresponding to each of the plurality of illumination wavelengths; and

determine a mean cell volume for the blood sample from the cell volumes for each one of the plurality of cells.