Cell analysis methods

• US 6,251,615 B1
• Filed: 02/18/1999
• Issued: 06/26/2001
• Est. Priority Date: 02/20/1998
• Status: Expired due to Fees

• Current Assignee: Allvivo, Inc.
• Original Assignee: Cell Analytics Inc.
• Inventors: Oberhardt, Bruce J.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/252,206
• Time in Patent Office: 859 Days
• Field of Search: 422/55, 422/58, 422/61, 422/73, 422/82.05, 435/7.2, 435/7.1, 435/7-1-, 435/287.1, 435/287.3, 435/7.92, 435/173.4, 435/173.5, 435/173.7, 435/287.6, 435/288.7, 435/326, 435/971, 435/973, 436/164, 436/524, 436/800, 436/807, 436/517, 436/518, 436/34, 436/52, 436/56, 436/63, 436/64, 436/147, 436/172, 436/537, 436/546, 356/39, 356/73, 356/315
• US Class Current: 435/7.21
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1475 using image analysis for extracting features of the particle
• CPC Class Codes: G01N 15/1484 microstructural devices
• CPC Class Codes: G01N 2015/0073 Red blood cells
• CPC Class Codes: G01N 2015/008 White cells
• CPC Class Codes: G01N 2015/012 Red blood cells
• CPC Class Codes: G01N 2015/016 White blood cells
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1486 Counting the particles
• CPC Class Codes: G01N 33/5094 for blood cell populations red blood cells G01N33/80
• CPC Class Codes: Y10S 435/971 Capture of complex after antigen-antibody reaction
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream

Abstract

A method of analyzing cells in a carrier solution comprises the following steps: (a) Introducing the carrier solution into a conduit having a surface portion (preferably a substantially flat surface portion). The carrier solution has the cells suspended therein. (b) Allowing the cells to settle on the surface portion, the surface portion including at least one imaging field. In an alternate embodiment, one or more discreet capture zones (e.g., formed from an affinity species immobilized on the substrate or a textured region on the substrate) are formed on the surface portion, and this step (b) comprises capturing the cells in the capture zone. (c) Sequentially interrogating a plurality of the cells in the imaging field with emitted light. (d) Processing resultant light from the imaging field. (e) Generating digital information for each of the plurality of cells from the resultant light. (f) Generating a response file for each of the plurality of cells from the digital information. The response file generated in the method can be used for the sizing, enumeration, characterization, and/or classification of cells in the sample. Apparatus for carrying out the foregoing method is also disclosed, along with methods and apparatus employing reciprocal flow techniques.

Claims

1. A method of analyzing cells in a carrier solution, comprising:

(a) introducing said carrier solution into a conduit having a surface portion wherein the surface portion has a discreet capture zone formed therein, said carrier solution having a plurality of cells suspended therein;

(b) contacting said plurality of said cells to said surface portion, said surface portion containing at least one imaging field;

(c) sequentially interrogating said plurality of cells in said imaging field with at least two different types of emitted light;

(d) processing resultant light from said imaging field for each of said at least two different types of emitted light;

(e) generating digital information for each of said plurality of cells from said resultant light for each of said at least two different types of emitted light; and

then

(f) generating a response file for each of said plurality of cells from said digital information for each of said at least two different types of emitted light;

and wherein said sequentially interrogating step is followed by the steps of;

altering the temperature of said cells in said capture zone; and

then repeating said sequentially interrogating step.

2. The method according to claim 1, wherein said introducing step is carried out by flowing said solution through said conduit.

3. The method according to claim 1, wherein said contacting step is carried out by allowing said cells to settle on said surface portion.

4. The method according to claim 1, wherein said surface portion has a first discrete capture zone formed thereon, with said capture zone positioned in said imaging field, and wherein said contacting step is carried out by binding said cells to said first capture zone.

5. The method according to claim 1, wherein said at least two different types of emitted light differ in a property selected from the group consisting of frequency, intensity, direction of travel with respect to said cells, and combinations thereof.

6. The method according to claim 1, wherein said resultant light is selected from the group consisting of light reflected by, absorbed by, scattered by, transmitted through, generated by molecules associated with said cells, and generated by molecules displaced by said cells.

7. The method according to claim 1, wherein said carrier solution comprises a biological fluid or buffered sample medium.

8. The method according to claim 1, wherein said surface portion is a flat surface portion.

9. The method according to claim 1, wherein said processing step comprises an optical detection step followed by an electronic processing step.

10. The method according to claim 1, wherein said response file includes the location and boundaries of each of said plurality of cells.

11. The method according to claim 1, further comprising staining said cells following said contacting step.

12. The method according to claim 1, further comprising generating a histogram plot of parameters for said plurality of cells from said response files.

13. The method according to claim 1, further comprising generating a cell scatter or cell distribution diagram from said response files.

14. The method according to claim 1, further comprising determining viability for each of said plurality of cells from said response files.

15. The method according to claim 1, further comprising determining the proliferation index of said plurality of cells from said response files.

16. The method according to claim 1, further comprising determining the incidence of apoptosis of said cells from said response files.

17. The method according to claim 1, further comprising counting said cells from said response files.

18. The method according to claim 1, further comprising determining the DNA content of said cells from said response files.

19. The method according to claim 1, further comprising detecting specific cytoplasmic or cell surface markers from said response files.

20. The method according to claim 1, further comprising determining the activation state of said cells from said response files.

21. The method according to claim 1, further comprising classifying said plurality of cells according to type from said response files.

22. The method according to claim 1, wherein said cells are live cells.

23. The method according to claim 4, said surface portion having at least a second discrete capture zone formed thereon to provide a plurality of different discrete capture zones, each positioned in an imaging field;

and wherein said sequentially interrogating step is repeated for each of said discrete capture zones.

24. The method according to claim 4, wherein said cells are blood cells, and wherein said blood cells bind to said capture zone.

25. The method according to claim 1, wherein the flow of said cells in said solution during said contacting step is modified by feedback from said resultant light or said response files.

26. The method according to claim 1, wherein said sequentially interrogating step is followed by altering the rate of flow of said solution through said conduit;

and then repeating said sequentially interrogating step.

27. The method according to claim 1, further comprising lysing cells bound to said capture zone, and then analyzing nucleic acid released from said lysed cells;

and wherein said lysing follows said contacting step.

28. The method according to claim 1, further comprising transiently permeabilizing said cells to release a portion of the contents thereof, while retaining nucleic acid for subsequent analysis therein;

and wherein said cells are transiently permeabilized after said contacting step.

29. The method according to claim 4, wherein said capture zone comprises an affinity species immobilized on said surface portion.

30. The method according to claim 4, wherein said capture zone comprises a textured segment of said surface portion.

31. The method according to claim 1, further comprising permeabilizing cells bound to said first capture zone;

and wherein said permeabilizing step follows said contacting step.