Flow cytometry for high throughput screening

• US 6,890,487 B1
• Filed: 02/10/2000
• Issued: 05/10/2005
• Est. Priority Date: 09/30/1999
• Status: Expired due to Term

• Current Assignee: Scientific Technologies Incorporated
• Original Assignee: Agency For Science Technology and Research
• Inventors: Kuckuck, Frederick W., Sklar, Larry A., Edwards, Bruce S.
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): GABEL, GAILENE
• Application Number:
  US09/501,643
• Time in Patent Office: 1,916 Days
• Field of Search: 422/50, 422/55, 422/63, 422/65, 422/68.1, 422/81, 422/82, 422/82.08, 422/93, 422/100, 436/286.5, 436/286.6, 436/287.1, 436/287.3, 436/287.6, 436/288.4, 436/288.5, 436/52, 436/53, 436/54, 436/165, 436/172, 436/180, 435/286.5, 435/286.6, 435/287.1, 435/287.3, 435/287.6, 435/288.4
• US Class Current: 422/93
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: G01N 15/01 specially adapted for biological cells, e.g. blood cells investigating sedimentation of particle suspensions in blood G01N15/05
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1436 the optical arrangement forming an integrated apparatus with the sample container, e.g. a flow cell
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 2015/1006 for cytology
• CPC Class Codes: G01N 2015/1402 Data analysis by thresholding or gating operations performed on the acquired signals or stored data
• CPC Class Codes: G01N 2015/1413 Hydrodynamic focussing
• CPC Class Codes: G01N 2015/1477 Multiparameters
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 2201/06113 Coherent sources; lasers
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/085 Flow Injection Analysis
• CPC Class Codes: G01N 35/1095 for supplying the samples to flow-through analysers for a specific analyser see relevant groups, e.g. under G01N15/00, G01N21/00, G01N27/00, G01N30/00, H01J49/00
• CPC Class Codes: Y10T 436/117497 with a continuously flowing sample or carrier stream
• CPC Class Codes: Y10T 436/118339 with formation of a segmented stream
• CPC Class Codes: Y10T 436/119163 with aspirator of claimed structure
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention, provides a flow cytometry apparatus for the detection of particles from a plurality of samples comprising: means for moving a plurality of samples comprising particles from a plurality of respective source wells into a fluid flow stream; means for introducing a separation gas between each of the plurality of samples in the fluid flow stream; and means for selectively analyzing each of the plurality of samples for the particles. The present invention also provides a flow cytometry method employing such an apparatus.

Claims

1. A flow cytometry apparatus for the detection of particles from a plurality of samples comprising:

means for moving the plurality of samples comprising particles from a plurality of respective source wells into a fluid flow stream, said means for moving the plurality of samples comprising a pump;

means for introducing a separation gas between each of said plurality of samples in said fluid flow stream; and

a flow cytometer for hydrodynamically focusing said fluid flow stream and selectively analyzing said particles in each of said plurality of samples as said fluid flow stream passes through said flow cytometer.

2. The flow cytometry apparatus of claim 1, wherein said means for moving said plurality of samples further comprises an autosampler.

3. The flow cytometry apparatus of claim 2, wherein said autosampler includes a probe and said flow cytometry apparatus includes a means for exposing a probe tip of said probe to a jet of gas to remove liquid from said probe tip.

4. The flow cytometry apparatus of claim 2, wherein said autosampler includes a probe having a conical tip.

5. The flow cytometry apparatus of claim 2, wherein said autosampler includes a hydrophobic probe.

6. The flow cytometry apparatus of claim 5, wherein said probe comprises a hydrophobic material.

7. The flow cytometry apparatus of claim 5, wherein said probe is coated with a hydrophobic material.

8. The flow cytometry apparatus of claim 1, wherein said pump comprises a peristaltic pump.

9. The flow cytometry apparatus of claim 8, wherein a portion of said fluid flow stream passing through said peristaltic pump is contained within a high speed multi-sample tube.

10. The flow cytometry apparatus of claim 8, further comprising a single length of tubing extending from said autosampler to said flow cytometer.

11. The flow cytometry apparatus of claim 10, wherein said single length of tubing comprises a high speed multi-sample tube.

12. The flow cytometry apparatus of claim 11, wherein said high speed multi-sample tube comprises a poly vinyl chloride tube.

13. The flow cytometry apparatus of claim 11, wherein said high speed multi-sample tube comprises a poly vinyl chloride tube having an inner diameter of about 0.02 inches and a wall thickness of about 0.02 inches.

14. The flow cytometry apparatus of claim 8, wherein a portion of said fluid flow stream passing through said peristaltic pump is contained within a tube having an internal diameter of 0.02 inches.

15. The flow cytometry apparatus of claim 1, wherein said separation gas comprises air.

16. The flow cytometry apparatus of claim 1, wherein said plurality of samples are homogenous.

17. The flow cytometry apparatus of claim 1, wherein said plurality of samples are heterogeneous.

18. The flow cytometry apparatus of claim 1, wherein said particles comprise biomaterials.

19. The flow cytometry apparatus of claim 18, wherein said biomaterials are fluorescently tagged.

20. The flow cytometry apparatus of claim 1, further comprising a well plate including said plurality of respective source wells.

21. The flow cytometry apparatus of claim 20, wherein said well plate includes at least 96 source wells.

22. The flow cytometry apparatus of claim 20, wherein said well plate includes at least 384 source wells.

23. The flow cytometry apparatus of claim 20, wherein said well plate includes at least 1536 source wells.

24. The flow cytometry apparatus of claim 20, wherein said well plate includes wells having a conical shape.

25. The flow cytometry apparatus of claim 20, wherein said well plate is mounted in an inverted position.

26. The flow cytometry apparatus of claim 1, further comprising a means for injecting a buffer fluid between adjacent samples in said fluid flow stream so that said adjacent samples are separated by two bubbles of separation gas and said buffer fluid located between said two bubbles of separation gas.

27. The flow cytometry apparatus of claim 1, wherein at least one said plurality of samples includes a drug present therein.

28. The flow cytometry apparatus of claim 1, wherein a portion of said fluid flow stream passing through said pump is contained within a tube having an internal diameter of 0.02 inches.

29. A flow cytometry apparatus for the detection of particles from a plurality of samples, comprising:

a pump for moving the plurality of samples comprising particles from a plurality of respective source wells into a fluid flow stream;

separation means for introducing a parameter-controlled separation gas between each of said plurality of samples in said fluid flow stream; and

a flow cytometer for hydrodynamically focusing said fluid flow stream and selectively analyzing said particles in each of said plurality of samples as said fluid flow stream passes through said flow cytometer, said separation gas having parameters of predetermined values to permit the hydrodynamic focusing of said fluid flow stream in said flow cytometer,

said separation means including tubing of a given size for introducing separation gas aliquots of a controlled volume into said fluid flow stream with said samples flowing at a specific rate, said given size and said controlled volume and said specific rate all being predetermined to permit the hydrodynamic focusing of said fluid flow stream in said flow cytometer.

30. The flow cytometry apparatus of claim 29, wherein said separation means includes tubing extending from said pump and having compression characteristics allowing said pump to move samples separated by gas through said tubing at a speed of at least 6 samples per minute without causing adjacent samples to mix with one another.

31. The flow cytometry apparatus of claim 30, wherein said tubing has an inner diameter of less than about 0.03 inch.

32. The flow cytometry apparatus of claim 31, wherein said inner diameter is greater than about 0.01 inch.

33. The flow cytometry apparatus of claim 29, wherein said given size is less than about 0.03 inch.

34. The flow cytometry apparatus of claim 29, wherein said specific rate is at least six samples per minute.