Automated and accurate drop delay for flow cytometry

Patent metadata

Field	Value
Publication number	US-10508990-B2
Application number	US-201715616712-A
Country	US
Kind code	B2
Filing date	Jun 7, 2017
Priority date	Jun 7, 2012
Publication date	Dec 17, 2019
Grant date	Dec 17, 2019

Abstract

Disclosed is an automated method and apparatus for automatically setting a drop delay period by detecting calibration particles in a waste stream. The drop delay is incremented over a series of drop delays and the number of calibration particles in the waste stream is detected for each drop delay. The drop delay is selected which has the least number of calibration particles in the waste stream.

First claim

What is claimed is:

1. A method of selecting a desired drop delay in a flow cytometer, wherein the desired drop delay is representative of an elapsed time between when particles pass through an interrogation point in a stream formed by the flow cytometer and when a droplet containing the particles separates from the stream at a droplet separation point, the method comprising:
operating the flow cytometer to provide a stream with calibration particles to form droplets during a plurality of drop delay periods that each have a different selected drop delay from a range of potential selected drop delays, wherein the selected drop delay governs the timing of intervals when a charge is applied to the stream during the drop delay period by the flow cytometer to create charged droplets;
deflecting the charged droplets during the plurality of drop delay periods;
collecting non-deflected droplets during each drop delay period in waste tubing to form a waste stream in the waste tubing;
illuminating the waste stream in the waste tubing to cause scattered light or fluorescent emissions of the calibration particles in the waste stream associated with each drop delay period;
sensing the scattered light or fluorescent emissions from the calibration particles with a detector in association with each drop delay period;
collecting data regarding a number of calibration particles sensed in the waste stream in association with each of the plurality of drop delay periods using the sensed scattered light or fluorescent emissions;
determining which selected drop delay from the range of potential selected drop delays results in the lowest rate of calibration particles in the waste stream based on the number of calibration particles sensed in the waste stream; and
using the determined selected drop delay from the range of potential selected drop delays as the desired drop delay.

2. The method of claim 1 , further comprising using pattern recognition techniques to compare the number of calibration particles sensed in the waste stream during each drop delay period to determine which selected drop delay of the different selected drop delays results in the lowest rate of calibration particles in the waste stream.

3. The method of claim 1 , further comprising using statistical analysis techniques to compare the number of calibration particles sensed in the waste stream during each drop delay period to determine which selected drop delay of the different selected drop delays results in the lowest rate of calibration particles in the waste stream.

4. The method of claim 1 , further comprising using the detector to also detect scattered light or fluorescent emissions from calibration particles at the interrogation point.

5. The method of claim 4 , further comprising:
collecting the scattered light or fluorescent emissions using collection optics; and
transmitting the scattered light or fluorescent emissions through a fiber optic cable to the detector.

6. The method of claim 1 , wherein the waste tubing has an internal diameter larger than an exit orifice diameter of a nozzle used to produce the stream.

7. The method of claim 1 , wherein operating the flow cytometer with calibration particles during a plurality of drop delay periods further comprises mixing the calibration particles with a sheath fluid.

8. The method of claim 7 , wherein the sheath fluid comprises electrolytes.

9. The method of claim 7 , further comprising flowing the mixed calibration particles and sheath fluid through a nozzle so that the mixed calibration particles and sheath fluid exiting the nozzle form the stream passing through the interrogation point, wherein the stream has a substantially uniform velocity and flow.

10. The method of claim 9 , wherein the charged droplets are charged by applying an electrical charge to the nozzle just before each charged droplet separates from the stream.

11. The method of claim 9 , wherein deflecting the charged droplets during the plurality of drop delay periods comprises electrostatically deflecting the charged droplets using charged plates.

12. The method of claim 9 , further comprising vibrating the stream of exiting the nozzle via a piezoelectric generator such that a droplet separation point occurs at a consistent location along the stream.

13. The method of claim 1 , further comprising pumping the waste stream through the waste tubing so that the waste stream does not back up in the waste tubing.

14. The method of claim 1 , wherein determining which selected drop delay results in the lowest rate of calibration particles in the waste stream comprises displaying an amount of calibration particles in the waste stream for each drop delay period to allow an operator of the flow cytometer to select a drop delay period having the lower rate of calibration particles in the waste stream.

15. The method of claim 1 , wherein collecting data regarding the number of calibration particles sensed in the waste stream comprises receiving electrical signals transmitted by the detector at a controller, wherein each electrical signal corresponds to the detection of a fluorescent emission.

16. The method of claim 15 , wherein collecting data regarding the number of calibration particles sensed comprises incrementing a counter when received electrical signals transmitted by the detector exceed a threshold value.

17. The method of claim 1 , wherein sensing the scattered light or fluorescent emissions comprises passing the scattered light or fluorescent emissions through a filter before the scattered light or fluorescent emissions reach the detector, wherein the filter is configured to remove light that does not correspond to the fluorescent emissions.

18. The method of claim 1 , further comprising:
operating the flow cytometer with calibration particles to provide the stream and form droplets during a plurality of second drop delay periods that each have a different second selected drop delay from a second range of potential second selected drop delays, wherein the second selected drop delay governs the timing of intervals when a charge is applied to the stream during the second drop delay period by the flow cytometer to create charged droplets and the second range of potential second selected drop delays is centered about the determined selected drop delay;
deflecting charged droplets during the plurality of second drop delay periods;
collecting non-deflected droplets during each second drop delay period in the waste tubing to form a waste stream in the waste tubing;
illuminating the waste stream in the waste tubing to cause scattered light or fluorescent emissions of the calibration particles in the waste stream associated with each second drop delay period;
sensing the scattered light or fluorescent emissions from the calibration particles with a detector in association with each second drop delay period;
collecting data regarding a number of calibration particles sensed in the waste stream in association with each of the plurality of second drop delay periods using the sensed scattered light or fluorescent emissions;
determining which second selected drop delay from the second range of potential second selected drop delays results in the lowest rate of calibration particles in the waste stream based on the number of calibration particles sensed in the waste stream; and
using the determined second selected drop delay from the second range of potential second selected drop delays as a new desired drop delay.

19. The method of claim 1 , wherein illuminating the wast

Assignees

• Bio Rad Laboratories

Inventors

• Fox Daniel Nelson
• Hunter Susan
• Gaskill-Fox Nathan Michael
• Raley Kevin P
• Miles Richard A

Classifications

• G01N21/64 Primary

  Fluorescence; Phosphorescence · CPC title

• G01N15/1459

  the analysis being performed on a sample stream · CPC title

• G01N2015/1406

  Control of droplet point · CPC title

• G01N2015/1006

  for cytology · CPC title

• G01N15/1012 Primary

  Calibrating particle analysers; References therefor · CPC title