Flow Cytometer Coincident Event Detection Using Trigger Time Intervals
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Solution Overview
Problem
Current flow cytometry systems fail to adequately address crosstalk between lasers and detectors, which increases as the trend towards multi-color protocols and increased speed requirements, leading to compromised data fidelity and confounding artifacts.
Innovation Solution
Methods and systems for evaluating flow cytometer data to identify coincident events by generating a time interval between particle detections and comparing it to a measurement window or standard value to determine simultaneous irradiation, allowing for the identification and modification of potentially suspect data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multi-color protocols and increased speed requirements are implemented, then productivity and versatility are improved, but crosstalk between lasers and detectors increases leading to compromised data fidelity
Solution Approach 1:
The system performs preliminary evaluation of coincident events by calculating time intervals between particle detections and comparing them to expected values before final data analysis. This preliminary action identifies potential crosstalk artifacts early, allowing for their correction or exclusion before they compromise the overall data fidelity in multi-color, high-speed flow cytometry experiments.
2Adaptability or versatility
If more colors are included in flow cytometric protocols, then adaptability and versatility are improved, but crosstalk between lasers and detectors increases creating confounding artifacts
Solution Approach 1:
The system implements feedback by continuously monitoring detection events, calculating time intervals between successive particle detections, and comparing these intervals to expected values based on flow cytometer operating parameters. When coincident events are detected through this feedback mechanism, the system can identify and correct for crosstalk artifacts, thereby maintaining data accuracy in multi-color protocols.
Solution Approach 2:
The time interval calculation serves as an intermediary mechanism that indirectly detects crosstalk events without requiring direct measurement of laser-to-detector interference. By using the timing between particle detections as a mediator, the system can identify coincident events that cause crosstalk in multi-color experiments without needing to directly measure the crosstalk itself.
3Productivity
If flow cytometers operate at higher speeds, then productivity is improved, but the occurrence of coincidence events increases
Solution Approach 1:
The system addresses the speed-accuracy trade-off by dynamically adjusting evaluation parameters based on the flow cytometer's operating speed. By changing the time interval thresholds and evaluation criteria according to the specific operating conditions, the system maintains accurate coincidence detection even at higher processing speeds where particles pass through the interrogation region more quickly.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Improves data quality by identifying and mitigating coincident events, thereby enhancing the accuracy and reliability of flow cytometric data.
Implementation Method 1
To characterize the components of the flow stream, the flow stream is irradiated with light. Variations in the materials in the flow stream, such as morphologies or the presence of fluorescent labels, may cause variations in the observed light
Implementation Method 2
the presence of fluorescent labels, may cause variations in the observed light
Data Source
AI summary
Methods for evaluating flow cytometer data for the presence of a coincident event are provided. Methods of interest include receiving flow cytometer data associated with a first particle, where the received flow cytometer data is obtained from a detector following the irradiation of the first particle and a second particle in a flow stream by first and second light sources, respectively. Methods also include receiving first and second trigger signals associated with the first and second particles, respectively. In addition, the subject methods include generating a time interval for the first and second particles based on a difference between the first and second trigger signals, and evaluating the flow cytometer data for the presence of a coincident event based on the generated time interval by determining if the first and second particles are irradiated by the first and second light sources simultaneously. Systems and computer-readable media are also provided.