What does flow cytometry measure?

What Flow Cytometry Measures

Flow cytometry measures the physical and chemical characteristics of individual cells or particles suspended in a fluid stream as they pass through a laser beam, detecting properties such as size, granularity, and the presence of specific cellular components through fluorescent labeling. 1

Core Principles and Measurements

Flow cytometry operates through three main systems that work together to analyze cells:

1. Fluidics System

   • Transports the sample in a single-cell stream through the instrument
   • Allows for analysis of individual cells at rates approaching 100,000 cells per second 2
   • Enables precise positioning of cells as they pass through the sensing region

2. Optical System

   • Size and granularity detection: Measures forward scatter (FSC) and side scatter (SSC) of light
     • Forward scatter correlates with cell size
     • Side scatter reflects internal complexity/granularity
   • Fluorescence detection: Measures emission from fluorochrome-labeled antibodies bound to cellular components

3. Electronics System

   • Converts optical signals to electronic pulses
   • Processes and digitizes data for computer analysis

Specific Parameters Measured

Cell Surface Markers

• Immunophenotyping: Detection of specific antigens on cell surfaces using fluorochrome-labeled monoclonal antibodies 1
• Examples include:
  • CD markers (e.g., CD4, CD8, CD19, CD56) for identifying immune cell populations
  • Lineage-specific markers for identifying cell types

Intracellular Components

• DNA content and cell cycle analysis
• RNA content
• Cytokines and other intracellular proteins
• Signaling molecules and phosphorylation states

Functional Parameters

• Cell viability
• Apoptosis markers
• Enzyme activity
• Membrane potential
• Calcium flux

Clinical Applications

Hematology and Immunology

• HIV monitoring: CD4+ T-cell enumeration for disease progression and treatment decisions 1
• Leukemia/lymphoma diagnosis: Identification of abnormal cell populations and their phenotypes
• Multiple myeloma: Detection of abnormal plasma cells with specific phenotypic markers (CD19, CD56, CD20, CD117, CD28, CD27) 1

Minimal Residual Disease Detection

• Quantitative evaluation of residual disease after treatment
• Can detect one abnormal cell among 10,000-100,000 normal cells 1
• Critical for monitoring treatment efficacy and predicting outcomes

Other Applications

• Solid tumor analysis
• Transplantation monitoring
• Platelet function studies
• Microbiology applications

Technical Considerations

Sample Requirements

• Typically requires single-cell suspensions
• Can analyze various specimen types:
  • Whole blood
  • Bone marrow
  • Bronchoalveolar lavage fluid
  • Cerebrospinal fluid
  • Other body fluids 3

Quality Control

• Proper controls are essential for accurate analysis:
  • Instrument calibration with standardized beads
  • Fluorescence compensation to correct spectral overlap
  • Appropriate gating strategies 1

Sensitivity Limitations

• Detection typically requires at least 20-100 events (cells) for reliable identification of a cell population 1
• For minimal residual disease detection, acquiring at least 1,000 total events is recommended to achieve a sensitivity of 0.01% 1

Common Pitfalls and Caveats

• Sample quality issues: Delayed processing can affect cell viability and marker expression
• Inadequate controls: Failure to use proper controls leads to inaccurate results
• Improper gating: Incorrect gating strategies can miss rare populations or lead to false positives
• Spectral overlap: Fluorochrome emissions can overlap, requiring proper compensation
• Instrument variability: Different instruments may produce slightly different results, requiring standardization

Flow cytometry has evolved from a research tool to an essential clinical diagnostic technique, offering rapid, sensitive, and multiparametric analysis of individual cells that cannot be achieved by other methods 4, 5.