System and method of flow cytometry and sample handling
DC CAFCFirst Claim
1. A method of flow cytometry sample processing, comprising the steps of:
- a. establishing a sheath fluid;
b. injecting a sample into said sheath fluid at an injection point;
c. establishing a single torsional surface in a nozzle having a central axis around which a torque is applied;
d. generating single torsional hydrodynamic forces from said single torsional surface;
e. orienting said sample with said single torsional hydrodynamic forces;
f. exiting said sample from said nozzle;
g. analyzing said sample.
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Abstract
An improved nozzle system for a flow cytometer and accompanying methods have been invented for a high efficiency orientation and sorting process of a flat sample and dedicates items such as equine or bovine sperm cells. This improved nozzle system comprises a nozzle with a novel interior surface geometry that can both gently accelerate the cells and can include an elliptical-like, single torsional interior surface element within the nozzle, i.e., a single torsional orientation nozzle. The elliptical-like, single torsional interior surface element may have a laminar flow surface and may produce the simplest flow path for applying minimal forces which act in either an accelerative nature or orienting hydrodynamic forces, namely, the single torsional orientation forces, to orient a flat sample such as animal sperm cells into a proper direction for an analyzing and efficiently sorting process in clinical use, for research and for the animal insemination industry.
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Citations
74 Claims
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1. A method of flow cytometry sample processing, comprising the steps of:
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a. establishing a sheath fluid;
b. injecting a sample into said sheath fluid at an injection point;
c. establishing a single torsional surface in a nozzle having a central axis around which a torque is applied;
d. generating single torsional hydrodynamic forces from said single torsional surface;
e. orienting said sample with said single torsional hydrodynamic forces;
f. exiting said sample from said nozzle;
g. analyzing said sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74)
a. subjecting said sample to a first axial motion surface in a nozzle;
b. transitioning to a second axial motion surface in said nozzle;
c. subjecting said sample to said second axial motion surface in said nozzle wherein said first and said second axial motion surfaces transition with a maximal acceleration differentiation;
d. coordinating said maximal acceleration differentiation to maintain functionality of said sample over its length; and
e. affirmatively limiting said maximal acceleration differentiation to maintain functionality of said sample over its length.
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6. A method of flow cytometry sample processing as described in claim 5 wherein said step of subjecting said sample to a first axial motion surface in a nozzle comprises the step of subjecting said sample to a first axial acceleration surface in said nozzle and wherein said step of subjecting said sample to said second axial motion surface in said nozzle comprises the step of subjecting said sample to a second axial acceleration surface wherein said first and said second axial motion surfaces transition with a maximal acceleration differentiation.
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7. A method of flow cytometry sample processing as described in claim 6 wherein said nozzle creates acceleration values though its internal surface and wherein said acceleration values are selected from a group comprising:
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not more than about 0.16 m/sec per micron, not more than about 0.05 m/sec per micron away from the vicinity of the exit orifice, not more than about 0.10 m/sec per micron away from the vicinity of the exit orifice, not more than about 0.13 m/sec per micron away from the vicinity of the exit orifice, not more than about 0.16 m/sec per micron in the vicinity of the exit orifice, not more than about 0.20 m/sec per micron in the vicinity of the exit orifice, not more than about 0.23 m/sec per micron in the vicinity of the exit orifice, not more than about 100×
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3 m/sec per micron at a distance of more than 300 um away from the exit orifice,not more than about 50×
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3 m/sec per micron at a distance of more than 300 um away from the exit orifice,not more than about 25×
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3 m/sec per micron at a distance of more than 300 um away from the exit orifice,such acceleration values with respect to axial location as do not discontinuously change along a central axis, not more than about 100,000×
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6 m/sec per micron2,not more than about 10,000×
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6 m/sec per micron2,not more than about 2,000×
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6 m/sec per micron2,not more than about 1,100×
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6 m/sec per micron2,not more than about 100,000×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 50,000×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 10,000×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 5,000×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 1,000×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 300×
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6 m/sec per micron2 away from the vicinity of the exit orifice,not more than about 200×
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6 m/sec per micron2 at a distance of more than 300 um away from the exit orifice,not more than about 100×
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6 m/sec per micron2 at a distance of more than 300 um away from the exit orifice,such rate of change of acceleration values with respect to axial location as do not discontinuously change along a central axis, and such rate of change of acceleration values with respect to axial location as do not change signs along a central axis away from the vicinity of the exit orifice.
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8. A method of flow cytometry sample processing as described in claim 5 wherein said single torsional hydrodynamic forces and said maximal acceleration differentiation combine and are affirmatively chosen to maintain functionality of said sample over its length.
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9. A method of flow cytometry sample processing as described in claim 8 wherein said step of transitioning to a second axial motion surface in said nozzle comprises the step of subjecting said sample to a unitary surface.
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10. A method of flow cytometry sample processing as described in claim 9 wherein said step of transitioning to a second axial motion surface in said nozzle comprises the step of subjecting said sample to a unitary exit orifice.
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11. A method of flow cytometry sample processing as described in claim 5 and further comprising the steps of:
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a. forming drops around said sample after it has exited said nozzle; and
b. sorting said drops at a rate selected from the group comprising at least 500 sorts per second, at least 1000 sorts per second, and at least 1500 sorts per second.
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12. A method of flow cytometry sample processing as described in claim 5 and further comprising the step of pressurizing said nozzle at a pressure of at least 50 psi.
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13. A method of flow cytometry sample processing as described in claim 11 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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14. A method of flow cytometry sample processing as described in claim 12 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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15. A method of flow cytometry sample processing as described in claim 5, 9, 10, 11, or 12 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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16. A method of creating a sexed sperm specimen comprising the step of producing a sexed sperm specimen as described in claim 13 or 14 and wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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17. A method of creating a sexed sperm specimen comprising the step of producing a sexed sperm specimen as described in claim 16 wherein said step of injecting sperm cells into said sheath fluid comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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18. A method of creating a mammal comprising the step of producing a sexed sperm specimen as described in claim 13 or 14 and wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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19. A method of creating a mammal comprising the step of producing a sexed sperm specimen as described in claim 18 wherein said step of injecting sperm cells into said sheath fluid comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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20. A method of flow cytometry sample processing as described in claim 4 wherein said step of smoothly varying said ellipticity of said tapered single torsional interior surface comprises the step of decreasing the ellipticity of said tapered single torsional interior surface downstream from said injection point.
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21. A method of flow cytometry sample processing as described in claim 4 wherein said step of smoothly varying said ellipticity of said tapered single torsional interior surface comprises the steps of:
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a. increasing the ellipticity of said tapered single torsional interior surface downstream in said nozzle;
b. reaching an ellipse-like demarcation location; and
c. decreasing the ellipticity of said tapered single torsional interior surface downstream from said ellipse-like demarcation location.
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22. A method of flow cytometry sample processing as described in claim 20 and further comprising the steps of:
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a. laminarly flowing said sheath fluid within said nozzle;
b. subjecting said sheath fluid to a conical zone;
c. subjecting said sheath fluid to a cylindrical zone;
d. creating an exit stream having a circular cross section;
e. forming drops from said exit stream; and
f. sorting said drops.
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23. A method of flow cytometry sample processing as described in claim 21 and further comprising the steps of:
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a. laminarly flowing said sheath fluid within said nozzle;
b. subjecting said sheath fluid to a conical zone;
c. subjecting said sheath fluid to a cylindrical zone;
d. creating an exit stream having a circular cross section;
e. forming drops from said exit stream; and
f. sorting said drops.
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24. A method of flow cytometry sample processing as described in claim 22 wherein said step of subjecting said sheath fluid to a conical zone and said step of subjecting said sheath fluid to a cylindrical zone both comprise the step of utilizing a unitary surface.
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25. A method of flow cytometry sample processing as described in claim 22 wherein said step of subjecting said sheath fluid to a conical zone, and said step of subjecting said sheath fluid to a cylindrical zone, and said step of creating an exit stream having a circular cross section all comprise the step of utilizing a unitary surface.
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26. A method offlow cytometry sample processing as described in claim 20 wherein said ellipticity has a ratio of a major axis to a minor axis at said injection point, and further comprising the step of optimizing said ratio for said sample to maintain functionality of said sample.
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27. A method of flow cytometry sample processing as described in claim 26 wherein said step of optimizing said ratio for said sample comprises the step of setting said ratio at 2.2.
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28. A method of flow cytometry sample processing as described in claim 22 wherein said ellipticity has a ratio of a major axis to a minor axis at said injection point, and further comprising the step of setting said ratio at 2.2.
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29. A method of flow cytometry sample processing as described in claim 24 wherein said tapered single torsional interior surface has cross section areas, and wherein said step of smoothly varying said ellipticity of said tapered single torsional interior surface further comprises the step of decreasing the cross section areas downstream from said injection point.
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30. A method of flow cytometry sample processing as described in claim 29 wherein said ellipticity has a major and a minor axis and wherein said step of smoothly varying said ellipticity of said tapered single torsional interior surface comprises the step of making said major and a minor axis progressively become equal downstream.
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31. A method of flow cytometry sample processing as described in claim 23 wherein said step of subjecting said sheath fluid to a conical zone comprises the step of subjecting said sheath fluid to a conical zone for a length for said sample as it travels downstream to maintain functionality of said sample.
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32. A method of flow cytometry sample processing as described in claim 31 wherein said step of subjecting said sheath fluid to a conical zone for a length for said sample as it travels downstream to maintain functionality of said sample comprises the step of subjecting said sheath fluid to a 0.3 mm long conical zone.
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33. A method of flow cytometry sample processing as described in claim 31 wherein said step of subjecting said sheath fluid to a cylindrical zone comprises the step of subjecting said sheath fluid to a cylindrical zone for a length for said sample as it travels downstream to maintain functionality of said sample.
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34. A method of flow cytometry sample processing as described in claim 32 wherein said step of subjecting said sheath fluid to a cylindrical zone for a length for said sample as it travels downstream to maintain functionality of said sample comprises the step of subjecting said sheath fluid to a 0.15 mm long cylindrical zone.
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35. A method of flow cytometry sample processing as described in claim 3 wherein said step of establishing a tapered single torsional interior surface in said nozzle comprises the step of gradually tapering said tapered single torsional interior surface.
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36. A method of flow cytometry sample processing as described in claim 35 wherein said step of gradually tapering said tapered single torsional interior surface comprises the step of setting a taper at about 23°
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37. A method of flow cytometry sample processing as described in claim 21 wherein said step of increasing the ellipticity of said tapered single torsional interior surface downstream in said nozzle and decreasing the ellipticity of said tapered single torsional interior surface each comprises the step of setting a taper at about 23°
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38. A method of flow cytometry sample processing as described in claims 24 or 25 wherein said step of utilizing a unitary surface comprises the step of utilizing a unitary ceramic surface.
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39. A method of flow cytometry sample processing as described in claim 24 wherein said step of utilizing a unitary surface comprises the step of establishing a nozzle having a height of about 13 mm and an outer diameter of about 6 mm.
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40. A method of flow cytometry sample processing as described in claim 23 wherein said step of creating an exit stream having a circular cross section comprises the step of creating an exit stream having a diameter of about 0.07 mm, and wherein said step of smoothly varying said ellipticity of said elliptical-like, single torsional interior surface comprises the step of establishing a mouth of about 5.25 mm in diameter.
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41. A method of flow cytometry sample processing as described in claim 39 wherein said step of creating an exit stream having a circular cross section comprises the step of creating an exit stream having a diameter of about 0.07 mm, and wherein said step of smoothly varying said ellipticity of said elliptical-like, single torsional interior surface comprises the step of establishing a mouth of about 5.25 mm in diameter.
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42. A method of flow cytometry sample processing as described in claim 39 wherein said step of subjecting said sheath fluid to a conical zone comprises the step of subjecting said sheath fluid to a conical zone having an inner diameter at a top of said conical zone of about 0.19 mm.
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43. A method of flow cytometry sample processing as described in claim 41 wherein said step of subjecting said sheath fluid to a conical zone comprises the step of subjecting said sheath fluid to a conical zone having an inner diameter at a top of said conical zone of about 0.19 mm.
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44. A method of flow cytometry sample processing as described in claim 20 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of assisting in orienting said sample at said injection point.
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45. A method of flow cytometry sample processing as described in claim 44 wherein said step of assisting in orienting said sample at said injection point comprises the step of creating a beveled flow near said injection point.
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46. A method of flow cytometry sample processing as described in claim 45 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of establishing a beveled tip having circular mouth with a diameter of about 0.01 mm.
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47. A method of flow cytometry sample processing as described in claim 45 and further comprising the step of aligning said beveled flow with said tapered single torsional interior surface in said nozzle.
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48. A method of flow cytometry sample processing as described in claim 1 wherein said step of orienting said sample with said single torsional hydrodynamic forces comprises the step of minimally torquing said sample.
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49. A method of flow cytometry sample processing as described in claim 48 wherein said sample travels a distance after accomplishing said step of generating single torsional hydrodynamic forces from said single torsional surface and before accomplishing said step of exiting said sample from said nozzle and further comprising the step of minimizing said distance.
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50. A method of flow cytometry sample processing as described in claim 49 wherein said step of exiting said sample from said nozzle occurs at an exit orifice and wherein said step of minimizing said distance comprises setting the distance from said injection point to said exit orifice at about 6 mm.
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51. A method of flow cytometry sample processing as described in claim 11 wherein said sample is oriented as described in any of claims 28, 32, 34, 40, 42, or 50.
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52. A method of flow cytometry sample processing as described in claim 1 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells in a sperm compatible buffer into said sheath fluid.
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53. A method of flow cytometry sample processing as described in claim 52 and further comprising the steps of:
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a. forming drops around said sperm cells after they have exited said nozzle; and
b. sorting said drops.
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54. A method of flow cytometry sample processing as described in claim 52 and further comprising the step of collecting said sperm cells after accomplishing said step of sorting said drops.
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55. A method of flow cytometry sample processing as described in claim 53 wherein said step of injecting sperm cells in a sperm compatible buffer into said sheath fluid comprises the step of injecting sperm cells in a sperm compatible buffer into said sheath fluid selected from a group consisting of equine sperm cells and bovine sperm cells.
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56. A method of flow cytometry sample processing as described in claim 55 wherein said sample is oriented as described in any of claims 28, 32, 34, 40, 42, or 50.
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57. A method of creating a sexed sperm specimen comprising the step of producing a sexed sperm specimen as described in any of claims 1, 22, 25, 26, 28, 32, 34, 36, 43, 45, 48, 49, 55, or 56.
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58. A method of creating a mammal comprising the step of producing a sexed sperm specimen as described in any of claims 1, 22, 25, 26, 28, 32, 34, 36, 43, 45, 48, 49, 55, or 56.
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59. A method of flow cytometry sample processing as described in claim 20, 25, 28, 32, 34, 36, 41, 43, 46, or 50 and further comprising the steps of:
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a. subjecting said sample to a first axial motion surface in a nozzle;
b. transitioning to a second axial motion surface in said nozzle;
c. subjecting said sample to said second axial motion surface in said nozzle wherein said first and said second axial motion surfaces transition with a maximal acceleration differentiation;
d. coordinating said maximal acceleration differentiation so as to maintain functionality of said sample over its length; and
e. affirmatively limiting said maximal acceleration differentiation so as to maintain functionality of said sample over its length.
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60. A method of flow cytometry sample processing as described in claim 59 wherein said single torsional hydrodynamic forces and said maximal acceleration differentiation combine and are affirmatively chosen so as to maintain functionality of said sample over its length.
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61. A method of flow cytometry sample processing as described in claim 60 wherein said step of transitioning to a second axial motion surface in said nozzle comprises the step of subjecting said sample to a unitary surface.
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62. A method of flow cytometry sample processing as described in claim 61 wherein said step of transitioning to a second axial motion surface in said nozzle comprises the step of subjecting said sample to a unitary exit orifice.
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63. A method of flow cytometry sample processing as described in claim 59 and further comprising the steps of:
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a. forming drops around said sample after it has exited said nozzle; and
b. sorting said drops at a rate selected from the group comprising at least 500 sorts per second, at least 1000 sorts per second, and at least 1500 sorts per second.
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64. A method of flow cytometry sample processing as described in claim 59 and further comprising the step of pressurizing said nozzle at a pressure of at least 50 psi.
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65. A method of flow cytometry sample processing as described in claim 63 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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66. A method of flow cytometry sample processing as described in claim 64 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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67. A method of flow cytometry sample processing as described in claim 59 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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68. A method of flow cytometry sample processing as described in claim 62 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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69. A method of flow cytometry sample processing as described in claim 63 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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70. A method of flow cytometry sample processing as described in claim 64 wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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71. A method of creating a sexed sperm specimen comprising the step of producing a sexed sperm specimen as described in claim 65 and wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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72. A method of creating a sexed sperm specimen comprising the step of producing a sexed sperm specimen as described in claim 69 and wherein said step of injecting sperm cells into said sheath fluid comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
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73. A method of creating a mammal comprising the step of producing a sexed sperm specimen as described in claim 65 and wherein said step of injecting a sample into said sheath fluid at an injection point comprises the step of injecting sperm cells into said sheath fluid.
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74. A method of creating a mammal comprising the step of producing a sexed sperm specimen as described in claim 69 and wherein said step of injecting sperm cells into said sheath fluid comprises the step of injecting sperm cells selected from the group comprising bovine sperm cells and equine sperm cells into said sheath fluid.
Specification