Measuring volume and constituents of cells

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

preparing a sample by depositing an aliquot of blood to form a monolayer on a microscope slide;

illuminating the sample with incident light at a plurality of illumination wavelengths and obtaining one or more two-dimensional images of the sample, wherein the one or more two-dimensional images comprises at least one image at each of the plurality of illumination wavelengths;

identifying a set of candidate red blood cells in the one or more images;

identifying a set of representative red blood cells from the set of candidate red blood cells;

determining a volume for each cell in the set of representative red blood cells; and

determining a mean cell volume for the sample based on the volumes of each of the cells in the set of representative red blood cells.

2. The method of claim 1 , wherein the one or more two-dimensional images of the sample comprises more than one two-dimensional images, and wherein each of the more than one two-dimensional images corresponds to a different location in the sample.

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

4. The method of claim 1 , wherein at least some of the one or more two-dimensional images of the sample correspond to light emerging from the sample and having a central wavelength in a blue region of the electromagnetic spectrum.

5. The method of claim 1 , wherein the one or more two-dimensional images comprises at least one image at each of four different illumination wavelengths.

6. The method of claim 1 , further comprising, for each member of the set of representative red blood cells:

identifying a set of pixels that correspond to the member;

identifying a subset of the set of pixels that corresponds to a perimeter region of the member; and

removing the subset of pixels from the set of pixels.

7. The method of claim 1 , wherein at least one of the two-dimensional images corresponds to incident light having a central wavelength in a red region of the electromagnetic spectrum, and wherein at least one of the two-dimensional images corresponds to incident light having a central wavelength in a blue region of the electromagnetic spectrum.

8. The method of claim 1 , further comprising, for each cell of the set of representative red blood cells:

for each illumination wavelength, identifying a set of pixels associated with the cell from one of the two-dimensional images of the sample at the illumination wavelength;

for each illumination wavelength, determining mean and maximum optical density values for the identified set of pixels corresponding to the illumination wavelength;

for each illumination wavelength, calculating a wavelength-specific volume of the cell based on the mean and maximum optical density values; and

determining the volume of the cell from the wavelength-specific cell volumes corresponding to the illumination wavelengths.

9. The method of claim 1 , further comprising, for each cell of the set of representative red blood cells:

for each illumination wavelength, identifying a set of pixels associated with the cell from one of the two-dimensional images of the sample at the illumination wavelength;

for each illumination wavelength, determining a total optical density for the cell by calculating a sum of optical densities associated with the identified set of pixels corresponding to the illumination wavelength;

for each illumination wavelength, determining mean and maximum optical density values for the identified set of pixels corresponding to the illumination wavelength;

for each illumination wavelength, calculating a wavelength-specific volume of the cell based on the mean and maximum optical density values; and

determining the volume of the cell from the wavelength-specific cell volumes and the total optical densities corresponding to the illumination wavelengths.

10. The method of claim 1 , wherein identifying the set of representative red blood cells comprises:

for each member of the set of candidate red blood cells, identifying a set of pixels that corresponds to the member;

determining an area and a perimeter of the member based on the set of pixels; and

excluding the member from the set of representative red blood cells if a ratio of a square of the perimeter to the area is larger than a threshold value.

11. The method of claim 1 , wherein determining the volume for each cell in the set of representative red blood cells comprises, for each cell:

determining a total optical density for the cell by calculating a sum of optical densities associated with a set of pixels corresponding to the cell;

determining mean and maximum optical density values among the set of pixels; and

calculating the volume based on the mean and maximum optical density values.

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 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.

13. The method of claim 6 , wherein the subset of pixels corresponds to a perimeter region having a width of three pixels or more.

14. The method of claim 7 , wherein at least one of the two-dimensional images corresponds to incident light having a central wavelength in a yellow region of the electromagnetic spectrum, and wherein at least one of the two-dimensional images corresponds to incident light having a central wavelength in a green region of the electromagnetic spectrum.

15. The method of claim 8 , further comprising, for each cell of the set of representative red blood cells:

determining an area of the cell from one of the two-dimensional images; and

for each illumination wavelength, calculating a wavelength-specific volume of the cell based on the mean and maximum optical density values and the area of the cell.

16. The method of claim 9 , further comprising determining the volume of the cell as a weighted linear combination of the wavelength-specific cell volumes and the total optical densities corresponding to the illumination wavelengths.

17. The method of claim 10 , further comprising, for each member of the set of candidate red blood cells:

determining a convex hull of the member;

determining an area enclosed by the convex hull; and

excluding the member from the set of representative red blood cells if a ratio of the area enclosed by the convex hull to the area of the member exceeds a threshold value.

18. The method of claim 10 , further comprising, for each member of the set of candidate red blood cells, excluding the member from the set of representative red blood cells if the area of the member is outside a selected area range.

19. The method of claim 11 , further comprising calculating the volume based on the mean and maximum optical density values and an offset value.

20. The method of claim 19 , further comprising determining the offset value based on one or more images of reference cells for which volumes of the reference cells are known.

21. The method of claim 12 , 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.

22. The method of claim 21 , 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 volume for each cell in the set of representative red blood cells.