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Cytometry

QC Selection

Overview of Sample Quality Controls (QC) Selection

  • Definition: Sample quality controls are substances or procedures used to monitor and validate the integrity of samples throughout the testing process
  • Purpose:
    • Verify Sample Handling: To ensure that samples are collected, transported, stored, and processed correctly
    • Assess Assay Performance: To validate the performance of flow cytometry assays using known positive and negative controls
    • Detect Contamination: To identify contamination issues that may compromise the results
  • Types of Quality Controls:
    • Internal Quality Controls (IQCs)
    • External Quality Controls (EQCs)

Internal Quality Controls (IQCs)

  • Definition: Substances or procedures that are incorporated directly into the sample or assay workflow to monitor performance
  • Purpose:
    • Monitor Sample Integrity: To assess the quality of the sample throughout the testing process
    • Detect Matrix Effects: To identify any interference from the sample matrix that may affect the assay results
    • Validate Assay Performance: To confirm that the assay is working as expected
  • Types of IQCs:
    • Cell Markers
    • Spiked Controls
    • Reagent Blanks
  • Considerations:
    • Matrix Effects: Be aware of the potential for matrix effects from the sample
    • Appropriate Range: Choose IQCs with values that are within the clinically relevant range

Cell Markers as IQCs

  • Principle:
    • Some cell markers are known to be relatively stable and consistently expressed, making them useful as internal controls for immunophenotyping assays
  • Examples:
    • CD45: A marker expressed on all leukocytes that can be used to verify proper leukocyte gating
    • CD3: A marker expressed on all T cells that can be used to verify T cell gating
  • Implementation:
    • Monitor the expression of these markers in each sample
    • Compare the expression levels to historical data or to expected values
  • What to look for:
    • Significant deviations from the expected expression levels may indicate problems with sample preparation, staining, or instrument performance

Spiked Controls

  • Principle:
    • Adding a known quantity of a reference material (e.g., cells, beads, proteins) to the sample to assess recovery and matrix effects
  • Implementation:
    • Use the recovery of the spiked control to correct for losses during sample preparation
    • Compare the results to the expected values to identify matrix effects
  • Use Cases:
    • Monitor the integrity of surface staining on leukocytes from whole blood
    • Verify cell count using enumeration beads

Reagent Blanks

  • Principle:
    • A sample that contains all of the reagents used in the assay, but no sample
    • Used to measure background signal and detect contamination
  • Implementation:
    • Run the reagent blank alongside the experimental samples
    • Subtract the signal from the reagent blank from the experimental samples
  • Use Cases:
    • Checking for any background fluorescence

External Quality Controls (EQCs)

  • Definition: Standardized samples or materials that are run periodically to assess the overall performance and accuracy of the entire flow cytometry process
  • Purpose:
    • Verify Accuracy: Assess the accuracy and reproducibility of the flow cytometer
    • Detect Systematic Errors: Identify systematic errors that may be affecting the results
    • Comply with Regulations: Meet regulatory requirements and accreditation standards
  • Types of EQCs:
    • Proficiency Testing (PT)
    • Commercially Available Control Materials
    • In-House Controls
  • Use Cases:
    • EQCs are typically used to assess the performance of the entire flow cytometry process, from sample preparation to data analysis
    • EQCs can also be used to compare the performance of different laboratories or different instruments

Proficiency Testing (PT)

  • Principle:
    • Participating in a PT program involves analyzing blinded samples provided by an external organization
    • Comparing the results to those obtained by other laboratories
  • Implementation:
    • Analyze the PT samples according to the PT provider’s instructions
    • Submit the results to the PT provider
    • Review the PT report to identify any problems and implement corrective actions
  • Helpful Tip:
    • PT programs provide a valuable way to assess the overall performance of the flow cytometry laboratory and to identify areas for improvement

Commercially Available Control Materials

  • Principle:
    • Using standardized control materials to monitor the performance of flow cytometry assays
    • These materials are typically well-characterized and have known values for the parameters being measured
  • Implementation:
    • Analyze the control materials according to the manufacturer’s instructions
    • Compare the results to the manufacturer’s specifications
    • Troubleshoot to identify if the control material is not within proper range
  • Use Cases:
    • The control products may have been previously stained for use
    • Control products may come as a mixture to simplify the QC process
    • Beads or cell preps that target instrument performance checks

In-House Controls

  • Principle:
    • Preparing and characterizing control materials within the flow cytometry laboratory
  • Implementation:
    • Collect and process samples from healthy donors or patients with known disease states
    • Characterize the samples using validated flow cytometry assays
    • Use the characterized samples as controls to monitor the performance of the assays
  • Use Cases:
    • Can be tailored to the specific needs of the flow cytometry laboratory
    • Less expensive than commercially available control materials

Analyzing Control Data and Corrective Action

  • Trend Analysis:
    • Track control data over time to identify trends or shifts in performance
    • Use control charts or other statistical methods to monitor the data
  • Corrective Action:
    • Establish corrective action procedures to address any problems identified by the control data
    • Document all corrective actions taken

Troubleshooting Sample Quality Control Issues

  • Controls Out of Range:
    • Possible Causes:
      • Reagent degradation
      • Instrument malfunction
      • Technician error
    • Troubleshooting Steps:
      • Replace suspect reagents, calibrate instruments, and review techniques
  • Inconsistent Results:
    • Possible Causes:
      • Unstable Instruments
      • Varying protocol techniques
    • Troubleshooting Steps:
      • Examine maintenance records and review operating procedures
  • Unexpected Results:
    • Possible Causes:
      • Incorrect antibody selection
      • Improper gating
      • Instrument malfunction
    • Troubleshooting Steps:
      • Check and verify markers that are used
      • Confirm correct techniques

Key Terms

  • Sample Quality Controls: Procedures to monitor the integrity of samples
  • Internal Quality Controls (IQCs): Controls incorporated into the sample
  • External Quality Controls (EQCs): Controls run periodically to assess overall performance
  • Proficiency Testing (PT): Participating in external QC programs
  • Trend Analysis: To check control charts and data