Flow measurement and control for improved quantification of particles in flow cytometry
The present invention relates to methods that enable improved accuracy for quantitative particle counting in a flowing liquid stream. The methods of the present invention utilize the real-time measurement of flow rates and flow rate control through feedback mechanisms to improve quantification, and this improved quantification translates to more accurate particle counting. In certain embodiments, particles being counted are biological particles in a liquid sample, such as viruses.
1. A method for analyzing sample fluid for presence of free virus in solution, the method comprising:
flowing a sample fluid comprising liquid and a plurality of particles of a size smaller than 1 micron, wherein the particles are individual fluorescently-labeled virus particles from a sample reservoir under pressure from a compressed gas applied to the sample reservoir to and through a flow cell, comprising controlling the sample fluid flow rate to the flow cell at less than 5000 nanoliters per minute;
measuring the sample fluid flow rate to the flow cell with a sample flow sensor coupled with a sample pressure regulator through a sample feedback control loop;
the controlling the fluid sample flow rate comprising feedback control through the sample feedback control loop of the sample pressure regulator to control pressure of the compressed gas applied to the sample reservoir based on measured sample fluid flow rate from the sample flow sensor during the measuring;
merging the sample fluid flow with a sheath fluid flow prior to the flow cell;
providing the sheath fluid flow from a sheath reservoir under pressure from compressed gas applied to the sheath reservoir;
prior to the merging, measuring the sheath fluid flow rate with a sheath flow sensor coupled with a sheath pressure regulator through a sheath feedback control loop;
controlling the sheath fluid flow rate to the merging, the controlling the sheath fluid flow rate comprising feedback control through the sheath feedback control loop of the sheath pressure regulator to control the pressure of the compressed gas applied to the sheath reservoir based on measured sheath fluid flow rate from the measuring the sheath fluid flow rate;
interrogating the sample fluid flow through the flow cell, the interrogating comprising:
excitation of the sample fluid flow in the flow cell by a light source of an excitation and detection system coupled with the flow cell; and
detection by a detection and analysis system of the excitation and detection system of only fluorescent response of the sample fluid flow in the flow cell to the excitation;
counting by the detection and analysis system a number of fluorescent response detection events occurring during the detection that are indicative of the presence of a said individual fluorescently-labeled virus particle in the sample fluid flow;
calculating by the detection and analysis system a concentration for virus particles in a volume of the sample fluid interrogated in the flow cell using the counted number of detection events and measured sample fluid flow rate data from the measuring that correspond with the volume of the sample fluid, wherein the detection and analysis system performs the counting and the calculating in real time or near real time of the interrogating; and
displaying concentration results of the calculating in the real time or near real time;
wherein the individual fluorescently-labeled virus particles within the fluid sample are fluorescently labeled with different fluorescent species for virus nucleic acid and virus protein, and the detection comprises detecting on separate detection channels for fluorescent responses of the different fluorescent species; and
wherein the calculating a concentration for virus particles comprises correcting for sample fluid flow rate fluctuations using the measured sample fluid flow rate data.