Method and apparatus for cell counting and cell classification

• US 5,556,764 A
• Filed: 05/02/1994
• Issued: 09/17/1996
• Est. Priority Date: 02/17/1993
• Status: Expired due to Term

• Current Assignee: Biometric Imaging, Inc. (Becton, Dickinson & Co)
• Original Assignee: Biometric Imaging, Inc. (Becton, Dickinson & Co)
• Inventors: Sizto, Ning L., Dietz, Louis J.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Tanigawa, Gary
• Application Number:
  US08/236,645
• Time in Patent Office: 869 Days
• Field of Search: 356/244, 356/246, 356/427, 382/128, 382/133, 382/134, 250/432 R, 250/458.1, 250/459.1, 250/461.1, 250/461.2, 435/7.1, 435/7.2, 435/7.21, 435/7.22, 435/7.23, 435/7.24, 435/7.25, 435/805, 435/288.7, 435/968, 435/973, 422/50, 422/55, 422/57-59, 422/68.1, 422/73, 422/82.05, 436/172
• US Class Current: 435/7.24
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G06V 20/69 Microscopic objects, e.g. biological cells or cellular parts
• CPC Class Codes: Y10S 435/968 High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/805 Optical property

Abstract

A method and an apparatus for analyzing a material within a container, such as blood within a capillary in a volumetric cytometry system provides for detecting the edges of the container, counting the cells within the container, characterizing the cells within the container, and evaluating channels of data which contain information relevant to more than one of the detectable characteristics of the cells. A scanner scans a container of material including certain cells. Sampling circuitry is coupled to the scanner to generate scanned images of the material in the container. Two or more scanned images are generated based on fluorescence data from dyes that have overlapping spectra. The two scanned images are processed using a linear regression analysis among corresponding pixels in the scanned images near certain cells to characterize relative contents of two fluorescing dyes in a target cell. Target cells are identified from the scanned images using processing resources which identify a peak sample within a neighborhood, and compare the amplitude of the peak with the amplitude of pixels on the perimeter of the neighborhood. Upon identifying a target cell in this manner, data from the plurality of scanned images corresponding to the identified cell are saved for further analysis.

Claims

1. A method for analyzing a sample containing cells to detect and characterize target cells having first detectable characteristics and second detectable characteristics in a fixed volume capillary that contains a fluorescent background which exhibits background characteristics, the method comprising:

scanning the fixed volume capillary containing the sample to generate a first channel of data and a second channel of data, wherein the first channel of data includes the first detectable characteristics and first background characteristics and the second channel of data includes the second detectable characteristics and second background characteristics;

sampling the first and the second channels of data to produce corresponding first and second sets of pixel values;

loading the first and the second sets of pixel values into a pixel map buffer;

analyzing the first and the second sets of pixel values in the pixel map buffer to identify a peak event indicative of the target cell;

saving a first neighborhood of pixel values from the first set of pixel values and a second neighborhood of pixel values from the second set of pixel values for the peak event;

performing linear regression to correct for detection of the second detectable characteristics in the first channel and for detection of the first detectable characteristics in the second channel;

filtering the first neighborhood of pixel values and the second neighborhood of pixel values for the peak event using predetermined peak characteristic so as to adjust for the first and the second background characteristics;

generating a first intensity value from the first neighborhood and a second intensity value from the second neighborhood for the peak event; and

comparing the first intensity value and the second intensity value for the peak event to a first threshold to classify the target cell.

2. The method of claim 1, further comprising processing the first and the second sets of pixel values to calculate a background index, wherein the step of analyzing the first and the second sets of pixel values includes identifying the peak event which exceeds a second threshold and wherein the first threshold and the second threshold are determined as a function of the background index.

3. The method of claim 1, wherein the capillary has edges and at least one channel of data has information relevant to the edges of the capillary, the method further comprising processing at least one set of pixel values corresponding to at least one channel of data to detect the edges of the capillary, and wherein the step of analyzing the first and the second sets of pixel values in the pixel map buffer includes ignoring pixels outside the edges of the capillary.

4. The method of claim 1, wherein the target cells are blood cells.

5. The method of claim 1, wherein the target cells are leukocytes and the sample contains red blood cells.

6. The method of claim 1, wherein the target cells are labeled with a fluorophore excitable in a range of 600 to 1,000 nanometers.

7. A method for analyzing a sample of cells in a capillary that contains fluorescent matter which exhibits background characteristics so as to identify and classify a plurality of subsets of cells having detectable characteristics, the method comprising:

scanning the capillary containing the sample to generate two channels of data, each channel of data containing information relevant to two detectable characteristics and to the background characteristics;

sampling the two channels of data to produce two sets of pixel values representing information relevant to the two detectable characteristics and to the background characteristics;

loading the two sets of pixel values into a pixel map buffer;

analyzing the two sets of pixel values to identify a peak event;

saving two neighborhoods of pixel values corresponding to the peak event;

performing linear regression on the two neighborhoods of pixel values to discriminate the two detectable characteristics in the two channels for the peak event;

filtering the two neighborhoods of pixel values using predetermined characteristics of the peak event so as to adjust for the background characteristics;

generating two intensity values for the peak event; and

classifying the peak event as one of a plurality of subsets of cells by comparing the two intensity values to a first threshold.

8. The method of claim 7, further comprising processing the two sets of pixel values to calculate a background index, wherein the step of analyzing the two sets of pixel values includes identifying the peak event which exceeds a second threshold, and wherein the first threshold and the second threshold are determined as a function of the background index.

9. An apparatus for analysis of a sample containing cells to detect and characterize target cells having detectable characteristics in a fluorescent background that has background characteristics, the apparatus comprising:

a scanner which scans the sample to generate two channels of data, each channel containing information relevant to two detectable characteristics and the background characteristics;

means for sampling the two channels of data to generate two streams of pixel values;

a processing system having memory, said processing system being coupled to said means for sampling and having first resources which load the two streams of pixel values into a pixel map buffer, which analyze the two streams of pixel values to identify a peak event indicative of the target cell and which save two neighborhoods of pixel values for the peak event; and

second resources which classify the peak event based upon the two neighborhoods of pixel values, said second resources configured to perform linear regression between the two neighborhoods of pixel values to discriminate the two detectable characteristics in the two channels, to filter the two neighborhoods of pixel values using predetermined characteristics of peak events so as to adjust for the background characteristics, to generate two intensity values corresponding to the two neighborhoods, and to classify the target cell by applying the two intensity values to a first threshold.

10. The apparatus of claim 9, wherein said second resources are furthered configured to detect an edge of the sample and ignore pixel values outside the detected edge.

11. The apparatus of claim 9, wherein said second resources are further configured to generate two intensity values for the target cell independent of neighborhoods related to other peak events.

12. The apparatus of claim 9, wherein said processing system is further configured to process the two streams of pixel values to calculate a background index and to identify the peak event using a second threshold, wherein the first threshold and the second threshold are determined as a function of the background index.

13. The apparatus of claim 9, wherein the sample contains blood cells.

14. The apparatus of claim 9, wherein the target cells are leukocytes and the sample contains red blood cells.

15. The apparatus of claim 9, wherein the target cells are labeled with a fluorophore excitable in a range of 600 to 1,000 nanometers.