Acoustic Cytometry Methods and Protocols

• US 20140147860A1
• Filed: 06/27/2012
• Published: 05/29/2014
• Est. Priority Date: 06/27/2011
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Kaduchak, Gregory, Ward, Michael, Bradford, Jolene, Dubbels, Bradley, Seredick, Barbara, Kerndt, Rickie, Haataja, Kristi, Anderson, April, Melquist, Penny, Langsdorf, Christopher, Hicks, Justin, Kihn, Kathleen
• Application Number:
  US14/129,777
• Publication Number:
  US 20140147860A1
• Time in Patent Office: Days
• US Class Current: 435/7.21
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 2015/142 Acoustic or ultrasonic focussing
• CPC Class Codes: G01N 21/6408 with measurement of decay time, time resolved fluorescence
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/56966 Animal cells

Abstract

Various embodiments disclosed herein comprise acoustic cytometry based methods, kits, computer software methods and systems to analyze a variety of bioparticles. In one embodiment, a method for analyzing bioparticles comprises: acoustically focusing one or more bioparticles through an interrogation zone; optically exciting the one or more bioparticles in the interrogation zone with an excitation source; detecting an optical signal from the bioparticles; and analyzing the optical signal to characterize at least one quality or quantity parameter of the bioparticles. Properties of biomolecules that may be analyzed include but are not limited to cell proliferation analysis, live/dead cell discrimination, cell cycle analysis, basic phenotyping, immunophenotyping, rare-event detection, apoptosis, phagocytosis, pinocytosis, detection of phosphoproteins, detection of one or more cellular markers, detection of one or more intracellular marker, detection of cancer cells, detection of pathological markers on a cell, microbial cell analysis and/or picophytoplankton analysis.

Claims

1. A method for analyzing bioparticles comprising:

acoustically focusing one or more bioparticles through an interrogation zone;

optically exciting the one or more bioparticles in the interrogation zone with an excitation source;

detecting an optical signal from the bioparticles; and

analyzing the optical signal to characterize at least one quality or quantity parameter of the bioparticles.

2. The method of claim 1, wherein the bioparticle is a cell, an organelle, a protein, a peptide, or a nucleic acid.

3. The method of claim 1 wherein the bioparticle is labeled.

4. The method of claim 1 wherein the bioparticle is intrinsically fluorescent.

5. The method of claim 1, wherein the type of analysis performed on the bioparticles is one of cell proliferation analysis, live/dead cell discrimination, cell cycle analysis, basic phenotyping, immunophenotyping, rare-event detection, apoptosis, phagocytosis, pinocytosis, detection of phosphoproteins, detection of one or more cellular markers, detection of one or more intracellular marker, detection of cancer cells, detection of pathological markers on a cell, microbial cell analysis or picophytoplankton analysis.

6. The method of claim 5, wherein cell proliferation analysis further includes subjecting the bioparticles to a cell proliferation stimulus prior to the acoustic focusing step.

7. The method of claim 5, wherein immunophenotyping analysis further includes labeling the bioparticles with one or more conjugated antibodies prior to the acoustic focusing step.

8. The method of claim 7, wherein certain optical signals are indicative of a particular immunophenotype.

9. The method of claim 7, wherein the labeled bioparticles are cells which are labeled with multiple conjugated antibodies.

10. The method of claim 7, wherein the cells are blood cells.

11. The method of claim 7, wherein the cells are human blood cells.

12. The method of claim 11, wherein the human blood cells may be immunophenotyped based on the expression of a CD45 marker, a CD3 marker, a CD4 marker, a CD8 marker, a CD19 marker or a CD56 marker.

13. The method of claim 11, wherein the human blood cells may be immunophenotyped as T-cells, B-cells, NK-cells, CD3 T-cells, CD19B-Cells, CD56-NK cells, CD4 T-helper cells, CD8 T-suppressor cells lymphocytes and combinations thereof.

14. The method of claim 9 further comprising performing a multi-color immunophenotyping.

28. The method of claim 5, wherein different optical signals correspond to different cell cycle phases.

29. The method of claim 28, further comprising quantifying the percentage of cells in one or more cell cycle phases.

30. The method of claim 5, wherein different optical signals correspond to different types of microbial events.

31. The method of claim 30, wherein the microbes are intrinsically fluorescent.

32. The method of claim 30, wherein detection of one or more optical signals are indicative of microbial cell events selected from the group consisting of microbial viability, number of microbial cells, detection of gram positive status of a microbe, detection of gram negative status of a microbe, microbial membrane potential, microbial metabolism and combinations thereof.

33. The method of claim 32, wherein microbial viability comprises detecting live microbial cells separately from dead microbial cells.

15. A method for detecting phosphoproteins on a cell disposed within a fluid medium, comprising:

stimulating or inhibiting the cell with a kinase or a kinase inhibitor respectively to phorsporylate or de-phosphorylate one or more proteins on the cell;

contacting the cell with one or more antibody specific to detect the one or more phosphorylated protein;

acoustically focusing the cell in the fluid medium;

optically exciting the cell with an excitation source;

detecting an optical signal from the cell; and

analyzing the optical signal, wherein the optical signal is indicative of the presence or absence of the one or more phosphorylated protein.

16. A method for detecting fluorescent protein expression on a cell disposed within a fluid medium, comprising:

transfecting the cell with one or more fluorescent proteins;

acoustically focusing the cell in the fluid medium;

optically exciting the cell with an excitation source;

detecting one or more optical signals from the cell; and

analyzing the optical signal,

wherein the detection of an optical signal corresponding to one or more fluorescent protein is indicative of the presence of expression of the one or more fluorescent proteins and the absence of an optical signal corresponding to one or more fluorescent protein is indicative of the absence of expression of the fluorescent protein.

17. The method of claim 16, wherein the detection of an optical signal corresponding to one or more fluorescent protein is indicative of successful transfection.

18. The method of claim 16, wherein the detection of a first optical signal corresponding to a first fluorescent protein and the detection of a second optical signal corresponding to a second fluorescent protein is indicative of transfection of the cell by the first and the second fluorescent proteins.

19. The method of claim 16, wherein analyzing the optical signal further comprises analyzing the percentage of cells transfected with the one or more fluorescent proteins.

20. The method of claim 16, wherein the fluorescent protein is a red fluorescent protein, a green fluorescent protein, a blue fluorescent protein, a yellow fluorescent protein.

21. A method for detection a rare event within a population of cells, the method comprising:

acoustically focusing the population of cells;

optically exciting the population of cells with an excitation source;

detecting one or more optical signals from the population of cells; and

analyzing the optical signal,

wherein the detection of an optical signal corresponding to a rare event is indicative of the presence of the rare event and the absence of an optical signal corresponding to a rare event is indicative of the absence of the rare event.

22. The method of claim 21, wherein the rare event is the detection of a rare subset of cells within the population of cells.

23. The method of claim 22, wherein the rare subset of cells comprises less than 5% the population of cells.

24. The method of claim 22, further comprising identification of the rare subset of cells.

25. The method of claim 22, comprising the detection of plasmocytoid dendritic cells.

26. The method of claim 22, comprising the detection of CD34+ cells from a population of peripheral blood cells.

27. The method of claim 22, comprising detecting human mesenchymal cells, angiogenic cells, circulating endothelial cells or circulating hematopoietic progenitor cells in human blood.

34. A method for detecting cell apoptosis, the method comprising:

acoustically focusing one or more cells disposed within a fluid;

optically exciting the one or more cells with an excitation source;

detecting one or more optical signals from the cells; and

analyzing the detected optical signals to identify morphological or biochemical changes that are indicative of cell apoptosis.

35. The method of claim 34, wherein an optical signal corresponding to detecting an apoptotic event in the cell is indicative of an apoptotic cell and the absence of an optical signal corresponding to detecting an apoptotic event in the cell is indicative of the absence of apoptosis.

36. The method of claim 34, wherein the optical signal corresponding to detecting an apoptotic event comprises detecting a change in the cells mitochondrial membrane potential, a change in the cells mitochondrial redox potential, a change in the protein composition in the cells plasma membrane and combinations thereof.

37. The method of claim 34, wherein an optical signal corresponding to detecting translocation of phosphatidylserine (PS) from the inner leaflet of the plasma membrane of the cell to the outermembrane of the plasma membrane of the cell is indicative of an apoptotic cell.