Identifying and measuring reticulocytes

Abstract

Methods and systems for identifying reticulocytes in a blood sample deposited on a substrate include: illuminating the sample with incident light at two different wavelengths, obtaining a two-dimensional image of the sample corresponding to a first one of the wavelengths, and obtaining a two-dimensional image of the sample corresponding to a second one of the wavelengths; analyzing the images to identify a set of representative red blood cells; determining an area of each of the red blood cells in the set; determining a color value of each of the red blood cells in the set; and, for each one of the red blood cells in the set, identifying the red blood cell as a reticulocyte if the area of the red blood cell exceeds an area cutoff value and the color value of the red blood cell is less than a color cutoff value.

1 . A method for identifying reticulocytes in a blood sample deposited on a substrate, the method comprising:
 illuminating the sample with incident light at two different wavelengths, obtaining a two-dimensional image of the sample corresponding to a first one of the wavelengths, and obtaining a two-dimensional image of the sample corresponding to a second one of the wavelengths;   analyzing the images to identify a set of representative red blood cells;   determining an area of each of the red blood cells in the set;   determining a color value of each of the red blood cells in the set; and   for each one of the red blood cells in the set, identifying the red blood cell as a reticulocyte if the area of the red blood cell exceeds an area cutoff value and the color value of the red blood cell is less than a color cutoff value,   wherein the color value of each one of the red blood cells comprises a difference between optical density values of the red blood cell at the two illumination wavelengths.   
     
     
         2 . The method of  claim 1 , wherein one of the wavelengths is between 400 nm and 475 nm, and the other wavelength is between 575 nm and 600 nm. 
     
     
         3 . The method of  claim 1 , wherein determining the color value of each of the identified red blood cells comprises, for each one of the red blood cells:
 determining a set of pixels associated with the cell;   determining optical densities corresponding to the first wavelength for each one of the set of pixels;   determining optical densities corresponding to the second wavelength for each one of the set of pixels;   determining a mean optical density corresponding to the first wavelength for the set of pixels;   determining a mean optical density corresponding to the second wavelength for the set of pixels; and   calculating a difference between the mean optical densities to determine the color value of the cell.   
     
     
         4 . The method of  claim 1 , wherein determining the area of each of the red blood cells in the set comprises, for each one of the red blood cells:
 determining a set of pixels associated with the cell; and   determining the area of the cell by counting the number of pixels in the set.   
     
     
         5 . The method of  claim 1 , wherein determining the area of each of the red blood cells in the set comprises, for each one of the red blood cells:
 determining a set of pixels associated with the cell;   determining a polygon that circumscribes the set of pixels; and   determining the area of the cell by calculating an area of the polygon.   
     
     
         6 . The method of  claim 1 , wherein the color cutoff value is based on a percentile of a distribution of the color values of the red blood cells. 
     
     
         7 . The method of  claim 6 , wherein the percentile corresponds to a 70th percentile within the distribution of color values of the red blood cells. 
     
     
         8 . The method of  claim 6 , wherein the color cutoff value corresponds to a sum of the percentile and a color offset value. 
     
     
         9 . The method of  claim 8 , further comprising determining the percentile and the color offset value based on a set of training data in which the numbers of reticulocytes are known. 
     
     
         10 . The method of  claim 1 , wherein the area cutoff value is based on a percentile of a distribution of the areas of the red blood cells. 
     
     
         11 . The method of  claim 10 , wherein the percentile corresponds to a 20th percentile of the distribution of areas of the red blood cells. 
     
     
         12 . The method of  claim 10 , wherein the area cutoff value corresponds to a sum of the percentile and an area offset value. 
     
     
         13 . The method of  claim 12 , further comprising determining the percentile and the area offset value based on a set of training data in which the numbers of reticulocytes are known. 
     
     
         14 . The method of  claim 1 , further comprising excluding the red blood cell from the representative set if a standard deviation of optical densities of pixels associated with the red blood cell at one of the two wavelengths is larger than a cutoff value. 
     
     
         15 . The method of  claim 1 , further comprising excluding the red blood cell from the representative set if a standard deviation of optical densities of pixels associated with the red blood cell at a wavelength different from the two wavelengths is larger than a cutoff value. 
     
     
         16 . The method of  claim 1 , further comprising, for each of the red blood cells identified as reticulocytes, determining a volume of the reticulocyte. 
     
     
         17 . The method of  claim 16 , further comprising determining the volume of the reticulocyte based on integrated optical densities of the reticulocyte corresponding to multiple illumination wavelengths. 
     
     
         18 . The method of  claim 17 , further comprising determining a mean reticulocyte volume parameter for the sample. 
     
     
         19 . The method of  claim 1 , further comprising, for each of the red blood cells identified as reticulocytes, determining a hemoglobin content of the reticulocyte. 
     
     
         20 . The method of  claim 19 , further comprising determining the hemoglobin content of the reticulocyte based on a weighted combination of an area of the reticulocyte, volumes of the reticulocyte corresponding to multiple illumination wavelengths, and integrated optical densities of the reticulocyte corresponding to the multiple illumination wavelengths. 
     
     
         21 .- 42 . (canceled)