HIGH THROUGHPUT FLOW CYTOMETRY SYSTEM AND METHOD

• US 20100105074A1
• Filed: 10/27/2009
• Published: 04/29/2010
• Est. Priority Date: 10/27/2008
• Status: Active Grant

• Current Assignee: Nodality Inc.
• Original Assignee: Nodality Inc.
• Inventors: Purvis, Norman, Cordiero, James, Rosen, David, Palazzo, Adam, Covey, Todd
• Granted Patent: US 9,034,257 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 2035/00435 Refrigerated reagent storage
• CPC Class Codes: G01N 2035/00752 bar codes
• CPC Class Codes: G01N 2035/0091 GUI [graphical user interfaces]
• CPC Class Codes: G01N 33/5008 for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor
• CPC Class Codes: G01N 35/00722 Communications; Identification
• CPC Class Codes: G01N 35/00732 Identification of carriers, materials or components in automatic analysers
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• 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

Abstract

The invention provides systems, compositions, kits and methods for automated processing of biological samples and analysis using a flow cytometer.

Claims

1. An automated system for processing and analysis of samples using a flow cytometer, comprising:

a. one or more microplate holder;

b. alternate positions where a microplate can be processed;

c. one or more reservoirs and/or one or more wash stations

d. a microplate handling apparatus for moving said microplate between said microplate holder and said alternate positions;

e. a dispensing head for withdrawing and/or dispensing liquids from said microplate, said one or more reservoirs, and/or said one or more wash stations;

f. a flow cytometer; and

g. a computer system including a processor, a display unit, and a memory unit that controls the microplate holder, the microplate handling apparatus, the dispensing head, and the flow cytometer, wherein;

i. said microplate handling apparatus selects said microplate from said microplate holder and relocate it to a first position, wherein said microplate;

ii. said dispensing head dispenses reagents into said microplate and/or withdraws liquid from said microplate; and

iii. said microplate handling apparatus selects said microplate from a first position and relocate it to a flow cytometer for analysis.

2. The system of claim 1, whereby the memory unit contains executable computer program instructions and a graphical user interface for controlling operation of the microplate holder, the microplate handling apparatus, the dispensing head, and the flow cytometer.

3. The system of claim 2, whereby the graphical user interface provides display at least a first window, a second window, and a third window;

a. wherein the first window provides a set of open fields for entering source microplate data;

b. wherein the second window provides at least one list of processing variables associated with a sample; and

c. wherein the third window provides a set of open fields for entering destination microplate data.

4. The system of claim 3, whereby at least one list of processing variables associated with the sample comprise:

a. an operator;

b. a modulator;

c. a fixative;

d. a binding element;

e. a buffer; and

f. an instrument such as a dispensing head, incubator, or flow cytometer.

5. The system of claim 2, further comprising a barcode scanner that:

(i) scans a barcode on the microplate and (ii) automatically associates this information with information entered in the windows of the graphical user interface.

6. The system of claim 1, wherein said system is used for the analysis of a plurality of cell signaling states, phosphoflow cytometry, analysis of a cell receptor function and/or a combination thereof.

7. The system of claim 6, wherein said system is used for the analysis of a drug transporter function.

8. The system of claim 7, wherein said system is used for the analysis of the function of a drug transported selected from the group consisting of—

glycoprotein (MDR1), MDR—

associated protein and breast cancer resistance protein.

9. The system of claim 1, whereby a second microplate is processed.

10. The system of claim 9, whereby a plurality of additional microplates is processed.

11. The system of claim 1, further comprising a microplate stage, a centrifuge, an incubator, a freezer, a refrigerator or a combination thereof.

12. The system of claim 1, wherein said dispensing head is a micropippettor.

32. A method for automated processing samples and analysis using a flow cytometer, comprising:

a. providing a system according to any of the preceding claims;

b. loading one or more biological samples into a microplate;

c. withdrawing and/or dispensing liquids from one or more reservoirs, one or more wash stations and/or one or more reagent containers to perform a protocol, wherein said protocol comprises;

(i) contacting said one or more biological samples with one or more modulators;

(ii) determining an activation level of at least one activatable element in said one or more biological samples, wherein said activation level is determined by a process comprising the binding of a binding element which is specific to a particular activation state of said activatable element; and

d. analyzing said biological sample using a flow cytometer.

33. The method of claim 32 wherein said one or more reagent containers comprises a reagent selected from the group consisting of modulators, inhibitors, binding elements, buffers, fixatives, and permebializers.

34. The method of claim 32, wherein said protocol further comprises counting cells, fixing the biological sample and/or permeabilizing the biological sample.

35. The method of claim 32, wherein said microplate further comprises a barcode.

36. The method of claim 34, further comprising scanning the barcodes and associating said barcode with a microplate processing information.

37. The method of claim 32, wherein said binding element comprises an antibody, recombinant protein, or fluorescent dye.

38. The method of claim 37 wherein said antibody is specific to a particular phosphorylation state of said activatable element.

39. The method of claim 32, wherein said protocol is performed in a volume of about 50 uL to about 100 uL.

40. The method of claim 32, wherein said microplate is a 96 well plate.

41. The method of claim 40, wherein the analysis of said 96 well plate is performed in about 1.5 hours.

42. A method for automated processing samples and analysis using a flow cytometer, comprising:

a. providing a system according to any of the preceding claims;

b. loading a biological sample a microplate;

c. withdrawing and/or dispensing liquids from one or more reservoirs, one or more wash stations and/or one or more reagent containers to perform a protocol, wherein said protocol comprising;

(i) contacting said biological sample with one or more modulators;

(ii) determining a function of a cell receptor; and

d. analyzing said biological sample using a flow cytometer.

43. The method of claim 42 wherein said one or more reagent containers comprises a reagent selected from the group consisting of modulators, binding elements, inhibitors, fluorescent dyes, buffers, fixatives, and permebializers.

44. The method of claim 42, wherein said protocol further comprises counting cells, fixing the biological sample and/or permeabilizing the biological sample.

45. The method of claim 42, wherein said microplate further comprises a barcode.

46. The method of claim 45, further comprising scanning the barcodes and associating said barcode with a microplate processing information.

47. The method of claim 42, wherein said protocol further comprises (iii) determining an activation level of at least one activatable element, wherein said activation level is determined by a process comprising the binding of a binding element which is specific to a particular activation state of the particular activatable element.

48. The method of claim 47, wherein said binding element comprises an antibody, recombinant protein, or fluorescent dye.

49. The method of claim 48, wherein said antibody is specific to a particular phosphorylation state of said activatable element.

50. The method of claim 42, wherein said cell receptor is a drug transporter.

51. The method of claim 50, wherein said drug transporter is selected from the group consisting of—

glycoprotein (MDR1), MDR—

associated protein and breast cancer resistance protein.

52. A kit comprising:

a. one or more microplates comprising;

(i) one or more modulators;

(ii) one or more binding elements; and

b. a software, wherein said software is capable of uploading an operating a protocol onto any of the system of claim 12.

53. The kit of claim 52, wherein said one or more microplates further comprise:

(iii) one or more fixatives, wherein said fixatives can be used to fixing a biological sample;

(iv) one or more buffers; and

/or

(v) one or more permeabilizers, wherein said permeabilizers can be used to permeabilize a biological sample.

54. The kit of claim 52, wherein said binding element comprises an antibody, recombinant protein, or fluorescent dye.

55. The kit of claim 54, wherein said antibody is specific to a particular phosphorylation state of said activatable element.

56. A kit comprising:

a. one or more microplates comprising;

(i) one or more modulators;

(ii) one or more inhibitors, wherein said one or more inhibitors inhibit a cell receptor; and

(iii) one or more fluorescent dyes, wherein said one or more fluorescent dyes are substrates of said cell receptor; and

b. a software, wherein said software is capable of uploading an operating a protocol onto any of the system of claim 12.2 and/or 12.3.

57. The kit of claim 56, wherein said cell receptor is a drug transporter.

58. The kit of claim 57, wherein said drug transporter is selected from the group consisting of—

glycoprotein (MDR1), MDR—

associated protein and breast cancer resistance protein.

13. A system, comprising:

a computer system including a processor, a display unit, and a memory unit containing executable computer program instructions causing;

i. a microplate handling apparatus to select a first microplate from a microplate holder and relocate it to a first position, wherein said first microplate comprising one or more biological samples;

ii. a dispensing head to dispense reagents into said first microplate and/or withdraw liquid from said microplate; and

iii. the microplate handling apparatus to select the first microplate from a first position and relocate it to a flow cytometer for analysis.

14. The system of claim 13, further comprising a microplate stage, a centrifuge, an incubator, a freezer, a refrigerator or a combination thereof.

15. The system of claim 13, wherein said dispensing head is a micropippettor.

16. The system of claim 13, whereby the computer system causes a second microplate to be processed.

17. The system of claim 16, whereby the computer system causes a plurality of additional microplates to be processed.

18. An automated system for processing samples and analysis using a flow cytometer, comprising:

a. a microplate containing biological samples and a microplate barcode;

b. a microplate holder to store said microplate;

c. alternate positions where said microplate can be processed;

d. one or more reservoirs and/or wash stations

e. a microplate handling apparatus for moving microplates between the microplate holder and said alternate positions;

f. a dispensing head for withdrawing and/or dispensing liquids from microplates, reservoirs, or wash stations, the dispensing head being in operable connection to the microplate holder;

g. one or more reagent containers, wherein said one or more reagent containers comprises one or more reagents selected from the group consisting of modulators, binding elements, buffers, permebealizers and fixatives, wherein said one or more reagents are added to the microplates containing biological samples prior to analysis with a flow cytometer;

h. a flow cytometer operably connected to an autosampler, so that said one or more biological samples may be automatically moved from said microplate to the flow cytometer;

i. a barcode reader for scanning the microplate barcodes so that the microplate barcode can be associated with microplate processing information; and

j. a computer system including a processor, a display unit, and a memory unit, wherein

i. the memory unit comprises an executable computer program instructions and a graphical user interface for controlling an operation of the microplate holder, the microplate handling apparatus, the dispensing head, the flow cytometer, and associating the microplate barcodes with said microplate processing information; and

ii. the graphical user interface provides display at least a first window, a second window, and a third window for entering said microplate processing information; and

19. The system of claim 18, wherein the computer system causes a second microplate to be processed.

20. The system of claim 18, wherein the computer system causes a plurality of additional microplates to be processed.

21. The system of claim 18 further comprising:

k. a centrifuge to spin said microplate during sample preparation, wherein the centrifuge is operable connected to the microplate handling apparatus; and

l. an incubator to hold said microplate at a specified temperature, wherein the incubator is operable connected to the microplate handling apparatus, the dispensing head, and the microplate holder;

22. A system comprising:

a. a housing;

b. a dispensing head comprising a plurality of independently controlled micropipettors;

c. a multi-position rotating stage comprising a plurality of positions wherein at least one position has vortexing capabilities and another position has heating and/or cooling capabilities, and wherein all positions are individually presented for interacting with the independently controlled micropipettors;

d. a user interface;

e. means for uploading an operating protocol onto the system.

23. The system of claim 22, whereby said means for uploading the operating protocol is a computer readable tangible medium.

24. The system of claim 22, wherein the operating protocol performs the following method:

a. contacting said biological sample with one or more modulators;

b. determining an activation level of at least one activatable element, wherein said activation level is determined by a process comprising the binding of a binding element which is specific to a particular activation state of the particular activatable element.

25. The system of claim 24, wherein said binding element comprises an antibody, recombinant protein, or fluorescent dye.

26. The system of claim 25 wherein said antibody is specific to a particular phosphorylation state of said activatable element.

27. The system of claim 22, wherein said protocol further comprises counting cells, fixing the biological sample and/or permeabilizing the biological sample.

28. The system of claim 22, wherein the operating protocol performs the following method:

a. contacting said biological sample with one or more modulators;

b. determining the function of a cell receptor, wherein said function is determined by a process comprising contacting said biological sample with one or more fluorescent dyes, wherein said one or more fluorescent dyes are substrates of said cell receptor.

29. The system of claim 28, wherein said determining the function of a cell receptor further comprises contacting said biological sample with one or more inhibitors, wherein said one or more inhibitors are inhibitors of said cell receptor.

30. The system of claim 28, wherein said cell receptor is a drug transporter.

31. The system of claim 30, wherein said drug transporter is selected from the group consisting of—

glycoprotein (MDR1), MDR—

associated protein and breast cancer resistance protein.