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US 10,705,007 B2Granted
Flow measurement and control for improved quantification of particles in flow cytometry
AssigneeEssen Instruments, Inc.
InventorsKathy L. Rowlen, Matthew Ferris
Life of this patent
Estimating the term…
Abstract
A flow cytometry system and method for quantification of fluorescently labeled virus particles smaller than 1 micron in size measures and controls sample fluid flow rate to an investigatory flow cell at a rate below 5000 nanoliters per minute and calculates in real time particle concentration in the sample fluid using measured flow rated data flow rate data and with correction for sample fluid flow rate fluctuations.
Claims19
A flow cytometry 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 sample fluid 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;
- 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 optically 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;
- and
- 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;
- 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.
10 more elements
A flow cytometry system to determine a number of virus particles in a fluid volume, the system comprising:
- a sample reservoir to contain a sample fluid having a plurality of particles to be counted, wherein the particles are of a size less than 1 micron and are individually fluorescently labeled virus particles;
- at least one compressed fluid supply in fluid communication with the sample reservoir;
- a sample pressure regulator in fluid communication with the at least one compressed fluid supply, and the sample reservoir to regulate a pressure of the sample reservoir;
- a sheath reservoir to contain a sheath fluid, the at least one compressed fluid supply being in fluid communication with the sheath reservoir;
- a sheath pressure regulator in fluid communication with the at least one compressed fluid, supply and the sheath reservoir to regulate a pressure of the sheath reservoir;
- a flow cell, the flow cell being in fluid communication with both the sample reservoir and the sheath reservoir;
- a sample flow sensor configured to determine sample fluid flow rate to the flow cell, the sample flow sensor being coupled to the sample pressure regulator through a feedback control loop configured to alter the pressure of the sample reservoir to maintain a substantially constant sample fluid flow rate to the flow cell of less than 5000 nanoliters, per minute;
- and
- an excitation and detection system optically coupled to the flow cell to interrogate sample fluid flow through the flow cell, the excitation and detection system comprising a light source configured to impinge cm the sample fluid flow in the flow cell and a detection and analysis system to receive and process an optical signal to determine whether a said individually fluorescently labeled virus particles is present in the sample fluid, the detection and analysis system configured to:
- detect only fluorescent response from the sample fluid flow through the flow cell;
- count a number of fluorescent response detection events that are indicative of the presence of a said individual fluorescently-labeled virus particle in the sample fluid flow through the flow cell;
- and
- calculate 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 determined by the flow sensor that correspond with the volume of the sample fluid;
- and
- wherein the calculation of the concentration of virus particles comprises correcting by the excitation and detection system for sample fluid flow rate fluctuations using the sample fluid flow rate data.
12 more elements
Dependencies are inferred from the claim text.