Instrument QC

Overview of Instrument Quality Control (QC)

Definition: Instrument quality control (IQC) refers to the procedures and practices used to ensure that a flow cytometer is operating according to its specifications and is producing accurate and reliable data
Importance:
- Data Accuracy: To ensure that the data accurately reflects the biological system being studied
- Reproducibility: To ensure that experiments can be reproduced over time
- Compliance: To comply with regulatory requirements and accreditation standards
Key Aspects of Instrument Quality Control:
- Optical Alignment
- Detector Calibration
- Fluidics System Checks
- Electronic Stability

Definition: The process of ensuring that the lasers, lenses, mirrors, and detectors in the flow cytometer are properly aligned
Importance:
- Optimal Signal: To maximize the amount of light that reaches the detectors
- Consistent Performance: To ensure consistent performance of the flow cytometer over time
- Accurate Measurements: To ensure that the fluorescence and scatter measurements are accurate
Methods:
- Visual Inspection:
  - Inspect the optical components for any signs of damage or misalignment
  - Look for any obstructions or debris in the light path
- Alignment Beads:
  - Use alignment beads to optimize the position of the lasers and detectors
  - Adjust the alignment until the beads are centered in the flow cell and the signal intensity is maximized
- Software Tools:
  - Use software tools to monitor the alignment of the optical system over time
  - The software may provide guidance on how to adjust the alignment
Troubleshooting Common Optical Misalignment Problems:
- Weak Signals:
  - Make sure there are no obstructions, and verify laser function.
- Variability:
  - Use the QC beads to assess instrument performance.
  - Clean, repair, or replace parts, as needed.
General notes:
- Optical alignment should occur when there are any known issues or at a fixed interval (e.g. weekly)

Definition: The process of adjusting the voltages of the detectors (PMTs or APDs) to ensure that they are responding linearly to changes in light intensity
Importance:
- Accurate Quantification: To ensure that the fluorescence values accurately reflect the expression of the target antigens
- Data Comparison: To allow for comparison of data from different experiments or different instruments
Methods:
1. Calibration Beads:
  - Use a set of calibration beads with known fluorescence intensities
  - The calibration beads are typically labeled with a range of fluorescence intensities that span the range of the flow cytometer
2. Voltage Adjustment:
  - Run the calibration beads on the flow cytometer
  - Adjust the detector voltages until the fluorescence intensity values of the beads match their known values
3. Linearity Check:
  - Verify that the detectors are responding linearly to changes in light intensity
  - This can be done by plotting the fluorescence intensity values of the calibration beads against their known MESF values
Linearity of Voltage should be carefully checked

Definition: The process of verifying that the fluidics system is functioning properly
Importance:
- Stable Flow Rate: To ensure a stable and consistent flow rate, which is essential for accurate cell counting and sizing
- Proper Focusing: To ensure that the cells are properly focused, which is essential for optimal signal intensity and resolution
- Prevention of Clogging: To prevent clogging of the flow cell or tubing, which can disrupt the flow of cells
Methods:
- Flow Rate Measurement:
  - Measure the flow rate using a graduated cylinder and a timer
  - Compare the measured flow rate to the expected flow rate
- Bubble Check:
  - Inspect the tubing and flow cell for air bubbles
  - Remove any air bubbles that are present
- Pressure Check:
  - Monitor the pressure readings in the fluidics system
  - Ensure that the pressure is within the acceptable range
Examples of tests for fluidics system checks:
- Verify that the sheath fluid is at the appropriate level
- Remove any air bubbles
- Clean the instrument with detergent, especially after running sticky samples

Definition: Assessing the stability of the flow cytometer’s electronics, which can affect the accuracy and reproducibility of the measurements
Importance:
- Ensure that the data is consistent.
- To allow results to be properly interpreted and to troubleshoot other aspects.
Electronic stability can be affected by:
- Room temperature fluctuation
- Variances from normal conditions
How to troubleshoot:
- Assess the flow rate and stability with calibration beads before and after running samples

Weak Signals:
- Possible Causes:
  - Misaligned optics
  - Low laser power
  - Incorrect detector settings
- Troubleshooting Steps:
  - Align the optical system
  - Check laser power
  - Adjust detector settings
High Background Noise:
- Possible Causes:
  - Contaminated reagents
  - Incorrect instrument settings
  - Electrical interference
- Troubleshooting Steps:
  - Use clean reagents
  - Optimize instrument settings
  - Reduce electrical interference
Variable Results:
- Possible Causes:
  - Inconsistent technique
  - Instrument instability
  - Sample heterogeneity
- Troubleshooting Steps:
  - Standardize IQC protocols
  - Calibrate instrument
  - Increase sample size

Instrument Quality Control (IQC): The process of ensuring that a flow cytometer is operating according to its specifications
Optical Alignment: Ensuring that the lasers, lenses, mirrors, and detectors are properly aligned
Detector Calibration: Adjusting the voltages of the detectors to ensure that they are responding linearly to changes in light intensity
Fluidics System Checks: Verifying that the fluidics system is functioning properly
Electronic Stability: Assessing the stability of the flow cytometer’s electronics
Calibration Beads: Beads with known fluorescence intensities that are used to calibrate the flow cytometer