Method and apparatus for orienting sperm in a fluid stream

• US 8,206,988 B2
• Filed: 05/12/2011
• Issued: 06/26/2012
• Est. Priority Date: 03/28/2003
• Status: Active Grant

• Current Assignee: Inguran LLC
• Original Assignee: Inguran LLC
• Inventors: Durack, Gary, Wallace, Jeffrey D., Vandre, Gary P., Westfall, Lon A., Hatcher, Jeremy T., Nayak, Niraj V.
• Primary Examiner(s): Wallenhorst, Maureen
• Application Number:
  US13/106,727
• Publication Number:
  US 20110215111A1
• Time in Patent Office: 411 Days
• Field of Search: 436/63, 436/164, 436/165, 436/43, 436/50, 436/55, 436/180, 422/67, 422/68.1, 422/73, 422/82.05, 422/501, 422/509, 422/515, 435/2, 435/287.1, 435/288.7, 435/286.4, 435/366
• US Class Current: 436/63
• CPC Class Codes: A01N 1/0284 Temperature processes, i.e. using a designated change in temperature over time
• CPC Class Codes: C12N 5/06 Animal cells or tissues; Human cells or tissues preservation of living cells or tissues A01N1/02
• CPC Class Codes: C12N 5/061 Sperm cells, spermatogonia
• CPC Class Codes: C12N 5/0612 sorting of gametes, e.g. according to sex or motility
• CPC Class Codes: C12Q 1/02 involving viable microorganisms
• CPC Class Codes: C12Q 3/00 Condition responsive control processes apparatus therefor C12M1/36; controlling or regulating in general G05
• 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 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 15/147 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 2015/1406 Control of droplet point
• CPC Class Codes: G01N 2015/1415 Control of particle position
• CPC Class Codes: G01N 2021/6439 with indicators, stains, dyes, tags, labels, marks
• CPC Class Codes: G01N 21/63 optically excited
• 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 33/48 Biological material, e.g. blood, urine G01N33/02, G01N33/26, G01N33/44, G01N33/46 take precedence; Haemocytometers counting blood corpuscules distributed over a surface by scanning the surface G06M11/02
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: Y10T 436/10 : Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive
• CPC Class Codes: Y10T 436/12 : Condition responsive control
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2525 : Stabilizing or preserving
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/255 : including use of a solid sorbent, semipermeable membrane, or liquid extraction
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A flow cytometry nozzle and method for orienting sperm cells in a fluid flow path including an interior surface of a nozzle. The interior surface of the nozzle having a first, a second and a third axially tapered region for progressively accelerating the speed of a fluid stream in a downstream direction toward a nozzle exit. At least two of the regions having generally elliptical cross sectional shapes oriented in different directions for applying torsional forces to a fluid stream passing there through, tending to bring sperm cells in the stream into a desired orientation.

Claims

1. A flow cytometry apparatus nozzle to sort sperm cells in a fluid stream by chromosome content comprising:

a. a nozzle body having a fluid stream entry portion and a fluid stream exit portion and an interior surface separating both portions along which a fluid stream flows when operating to sort sperm cells;

b. the interior surface of the nozzle comprising;

a first axially tapered region having a first angle of taper;

a second axially tapered region having a second angle of taper, the second axially tapered region adjacent to and downstream of the first axially tapered region; and

a third axially tapered region having a third angle of taper, the third axially tapered region adjacent to and downstream of the second axially tapered region, wherein the angle of taper of the second axially tapered region is different than the angle of taper of the first and third axially tapered regions, and wherein at least two of the axially tapered regions have generally elliptical cross sectional shapes oriented in different directions for applying torsional forces to the fluid stream passing there through, the torsional forces tending to bring sperm cells in the stream into a desired orientation.

2. The nozzle as claimed in claim 1, wherein the generally elliptical cross sectional shape of one of the said at least two regions is oriented at about 90 degrees relative to the generally elliptical cross sectional shape of the other of the said at least two regions.

3. The nozzle as claimed in claim 1, wherein the generally elliptical cross sectional shapes having major axises of the first and second regions are oriented in substantially the same direction to define a first torsional zone, and the generally elliptical cross sectional shape of the third region, constitutes a second torsional zone, which is oriented at about 90 degrees relative to the generally elliptical cross sectional shapes of the first and second regions.

4. The nozzle as claimed in claim 3, wherein the first torsional zone has an axial length of 3.0-4.5 mm and the second torsional zone has an axial length of 3.5-5.0 mm.

5. The nozzle as claimed in claim 1, wherein the third axially tapered region tapers at an angle of 42-48 degrees.

6. The nozzle as claimed in claim 1, wherein all of the regions have generally elliptical cross sectional shapes.

7. The nozzle as claimed in any claim 1, wherein the nozzle body has an exterior surface with a non-reflective coating thereon.

8. The nozzle as claimed in claim 1, further comprising a mount for mounting the nozzle in a position pointing upward in its normal attitude of operation to direct the fluid stream along an upward trajectory.

9. The nozzle as claimed in claim 8, wherein the nozzle body is rotatable on the mount on a longitudinal axis of the body.

10. The nozzle as claimed in claim 8 wherein the angle of the taper in the second region is greater than that of the first region but less than that of the third region.

11. The nozzle as claimed claim 1, wherein interior surface of the nozzle includes a fourth non tapered region coaxial with and downstream from the first, second and third axially non-tapered regions, the fourth tapered region being adjacent to the third axially tapered region.

12. A method of orienting animal sperm cells in a flow cytometry apparatus, the method comprising the steps of:

introducing a fluid containing sperm cells into a fluid stream;

directing the fluid stream containing the sperm cells under a pressure in the range of 20 to 40 psi through a nozzle body having three or more adjacent axially tapered regions and an exit orifice with a diameter in the range of 50-70 microns;

accelerating the speed of the fluid stream in a downstream direction toward the nozzle orifice through a first axially tapered region having a first angle of taper, a second axially tapered region having a second angle of taper and a third axially tapered region having a third angle of taper, wherein the second angle of taper is different than the first angle of taper and the third angle of taper; and

applying a torsional force to the fluid stream tending to bring the cells into a desired orientation as they pass through the orifice at a rate in the range of 30,000 to 50,000 sperm cells per second with a generally elliptical cross section shape in at least one of the axially tapered regions.

13. The method as claimed in claim 12, wherein the nozzle body has two torsional zones defined by interior surfaces of the nozzle body in the three or more axially tapered regions.

14. The method as claimed in claim 12, wherein the nozzle body has a flow axis through the nozzle orifice, and wherein at least some of the interior nozzle surfaces have generally elliptical shapes which are rotated relative to one another about the axis.

15. The method as claimed in claim 12, wherein the method further includes directing the stream along an upward non-vertical trajectory.

16. The method as claimed in claim 12, wherein the method further includes rotating the nozzle body on a central longitudinal axis of the nozzle body while directing the stream.

17. An apparatus for classifying and sorting sperm cells according to chromosomal DNA characteristics of the cells, comprising:

a nozzle system for delivering a fluid stream containing the cells to a first location and for causing the stream to break into droplets at a second location, the nozzle system including a nozzle having an interior surface configured to exert a force on the cells tending to bring them into a desired orientation before reaching the first location, wherein the interior surface of the nozzle comprises;

a first axially tapered region having a first angle of taper;

a second axially tapered region having a second angle of taper, the second axially tapered region adjacent to and downstream of the first axially tapered region; and

a third axially tapered region having a third angle of taper, the third axially tapered region adjacent to and downstream of the second axially tapered region, wherein the taper of the second axially tapered region is different than the taper of the first and third axially tapered regions, and wherein at least two of the regions having generally elliptical cross sectional shapes oriented in different directions for applying torsional forces to the fluid stream passing there through, tending to bring sperm cells in the stream into a desired orientation;

an optics system for directing a beam of electromagnetic radiation to intersect the fluid stream at the first location so that cells in the stream pass through the beam resulting in emissions of electromagnetic radiation from the cells; and

a photodetector for detecting and converting at least some of the emissions into electrical signals; and

a system for classifying the cells according to the DNA characteristics and for sorting the droplets according to the classification of cells contained in the droplets.