Methods for determining absolute count of particles in a sample in a flow cytometer and systems for same

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.