Methods for determining absolute count of particles in a sample in a flow cytometer and systems for same
Aspects of the present disclosure include methods for determining absolute count of particles in a sample in a flow cytometer. Methods according to certain embodiments include introducing a bubble into a flow stream propagating a sample having particles, irradiating the flow stream with a light source, detecting light from the irradiated particles with a photodetector, detecting the presence of the bubble in the flow stream with the photodetector and determining the absolute count of the particles in the sample based on data signals generated in response to light detected from the irradiated particles, a data signal generated when the bubble is introduced into the flow stream and data signals generated in response to the detected bubble. Systems (e.g., flow cytometers) having a light source, a light detection system and a sample line that is configured to introduce a bubble into a flow stream for practicing the subject methods are also described. Non-transitory computer readable storage medium is also provided.
1 . A method for determining absolute count of particles in a sample in a flow cytometer, the method comprising:
introducing a bubble into a flow stream propagating a sample comprising particles;
irradiating the flow stream with a light source;
detecting light from the irradiated particles with a photodetector;
detecting the presence of the bubble in the flow stream with the photodetector; and
determining the absolute count of the particles in the sample based on:
first data signals generated in response to light detected from the irradiated particles before the bubble is introduced into the flow stream and upstream from the bubble in the flow stream after the bubble has been introduced;
a second data signal generated when the bubble is introduced into the flow stream; and
third data signals generated in response to the detected bubble, wherein the third data signals are distinct from the second data signal.
2 . The method according to claim 1 , wherein introducing the bubble into the flow stream comprises retracting a sample line from a source of the sample to create an air gap in the flow stream.
3 . The method according to claim 2 , wherein the method comprises generating the second data signal when the sample line is retracted from the sample source.
4 . The method according to claim 2 , wherein the method comprises generating the second data signal at a predetermined time after the sample line is retracted from the sample source.
5 . The method according to claim 4 , wherein the second data signal is generated from 0.001 μs to 10 μs after the sample line is retracted from the sample source.
6 . The method according to claim 1 , wherein the second data signal comprises an electronic data signal.
7 . The method according to claim 1 , wherein the second data signal comprises an optical data signal.
8 . The method according to claim 1 , wherein the flow stream is continuously irradiated by the light source.
9 . The method according to claim 8 , wherein the light source comprises a laser.
10 . The method according to claim 8 , wherein the flow stream is continuously irradiated with the light source at a wavelength from 200 nm to 800 nm.
11 . The method according to claim 1 , wherein the method further comprises generating the first data signals in response to light detected from irradiated particles that are downstream from the bubble in the flow stream.
12 . The method according to claim 1 , wherein the method comprises:
continuously detecting light from particles irradiated before introducing the bubble into the flow stream;
continuously detecting light from irradiated particles upstream from the bubble in the flow stream; and
detecting light from the flow stream as the bubble passes through the interrogation field.
13 . The method according to claim 1 , wherein the detected light comprises fluorescent light.
14 . The method according to claim 1 , wherein the detected light comprises scattered light.
15 . The method according to claim 1 , wherein the presence of the bubble in the flow stream is determined based on detecting scattered light from the irradiated bubble.
16 . The method according to claim 1 , wherein the method comprises calculating the absolute count of particles in the sample using:
the second and third data signals generated in response to light detected from irradiated particles between when the bubble is introduced into the flow stream and when the bubble is detected; and
a predetermined volume.
17 . The method according to claim 1 , wherein the method further comprises generating a data signal intensity plot of the first data signals as a function of time.
18 . The method according to claim 17 , wherein the method further comprises plotting the time when the bubble is introduced into the flow stream onto the data signal intensity plot.
19 . A system comprising:
a flow cell configured to propagate a sample comprising particles in a flow stream;
a sample line in fluid communication with a source of the sample and the flow cell, wherein the sample line is configured for introducing a bubble into the flow stream;
a light source configured to irradiate the sample in the flow stream;
a light detection system comprising a photodetector for detecting light from the irradiated flow stream; and
a processor comprising memory operably coupled to the processor wherein the memory comprises instructions stored thereon, which when executed by the processor, cause the processor to calculate the absolute count of the particles in the sample based on:
first data signals generated in response to light detected from the irradiated particles before the bubble is introduced into the flow stream and upstream from the bubble in the flow stream after the bubble has been introduced;
a second data signal generated when the bubble is introduced into the flow stream; and
third data signals generated in response to the detected bubble, wherein the third data signals are distinct from the second data signal.