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Cytometry

Hydrodaynamic Focusing

Core Concept: Hydrodynamic Focusing

  • Definition: Hydrodynamic focusing is a process that uses a sheath fluid to narrow the stream of sample fluid, forcing cells or particles to pass through an interrogation point in a single file
  • Purpose:
    • Ensures that only one cell/particle at a time passes through the laser beam, preventing coincidence (multiple events being detected as one).
    • Optimizes light scatter and fluorescence signal detection by controlling cell position
    • Increases sample throughput
  • Mechanism:
    • Sheath Fluid: A clean, particle-free fluid that surrounds the sample core
    • Pressure Differential: The sheath fluid is introduced at a higher pressure than the sample fluid
    • Nozzle/Flow Cell Design: Specific geometry of the flow cell directs and constricts the sample stream
    • Laminar Flow: The fluid dynamics are designed to create laminar flow (smooth, layered flow), preventing turbulence and mixing
    • Focused Core Stream: The higher pressure of the sheath fluid compresses the sample stream into a narrow core, typically a few micrometers in diameter

Key Factors Affecting Hydrodynamic Focusing

  • Sheath Fluid Pressure:
    • Increased pressure: Narrower core stream, higher cell velocity, increased sample throughput, lower resolution
    • Decreased pressure: Wider core stream, lower cell velocity, decreased sample throughput, better resolution
  • Sample Fluid Pressure:
    • Increased pressure: Wider core stream
    • Decreased pressure: Narrower core stream
  • Flow Cell Geometry:
    • The design of the nozzle or flow cell (e.g., size, shape, angle) significantly impacts the focusing efficiency and core stream dimensions
  • Fluid Viscosity:
    • Higher viscosity: affects the flow rate
  • Fluid Temperature:
    • Temperature changes: can affect the fluidic properties
  • Coaxial Alignment:
    • Misalignment: affects the flow profile

Sheath Fluids: Properties and Considerations

  • Composition: Typically a balanced salt solution (e.g., PBS) or deionized water with added components
  • Key Properties:
    • Purity: Must be free of particles (bacteria, debris) that could interfere with cell detection or clog the system
    • Sterility: Prevents microbial growth within the instrument’s fluidic system
    • Isotonicity: Should be isotonic to cells to prevent osmotic stress (swelling or shrinking)
    • pH: Maintained at a physiological pH (usually around 7.4) to preserve cell viability and antibody binding
    • Viscosity: Affects the flow rate and focusing efficiency; usually optimized for the specific instrument
    • Refractive Index: Can affect light scatter measurements
    • Electrical Conductivity: Important for instruments that use impedance-based cell counting
    • Compatibility: Must be compatible with the dyes and reagents used in the assay
  • Common Additives:
    • Antibiotics: To inhibit bacterial growth
    • EDTA: As a metal chelator, to prevent aggregation of cells
    • Protein (e.g., BSA): To block non-specific binding of antibodies to the flow cell
    • Surfactants: To reduce surface tension and prevent bubble formation
    • Stabilizers: To prevent the degradation of components in the sheath fluid
  • Preparation and Storage:
    • Prepared using high-quality reagents and sterile techniques
    • Filtered through a 0.2 μm filter to remove particles
    • Stored properly to prevent contamination and degradation
  • Maintenance:
    • Regularly replaced to ensure purity and prevent clogging
    • Fluid filters are replaced
    • The fluidic system is cleaned with detergent solution

Troubleshooting Fluidic Issues

  • Clogging:
    • Symptoms: Erratic flow rates, increased pressure, poor resolution, or complete blockage
    • Causes: Particulate matter in the sheath fluid or sample, cell aggregates, precipitation of reagents
    • Solutions: Filter sheath fluid and samples, use cell preparation techniques to minimize aggregates, flush the system with cleaning solutions, and replace clogged filters
  • Bubble Formation:
    • Symptoms: Erratic flow rates, unstable readings, or signal fluctuations
    • Causes: Air leaks in the fluidic system, improper degassing of sheath fluid, or surfactants in the sample
    • Solutions: Check for leaks, degas sheath fluid, adjust surfactant concentrations, and ensure proper fluid levels in reservoirs
  • Contamination:
    • Symptoms: High background noise, unexpected cell populations, or microbial growth.
    • Causes: Non-sterile sheath fluid, improper handling of samples, or contamination of the flow cell
    • Solutions: Use sterile techniques, replace contaminated sheath fluid, decontaminate the flow cell appropriate controls.
  • Pressure Issues:
    • Symptoms: Inconsistent flow rates, unstable focusing, or failure to aspirate samples
    • Causes: Blocked lines, pump malfunction, or improper pressure settings
    • Solutions: Check for blockages, inspect pump function, verify pressure settings, and calibrate the instrument
  • Carryover:
    • Symptoms: False positive results
    • Causes: Insufficient washing between samples
    • Solutions: Increase the wash volume, reduce the sample concentration, or use a carryover reduction solution

Key Terms

  • Laminar Flow: Fluid movement in smooth, parallel layers, with minimal mixing. This is essential for hydrodynamic focusing. Imagine a calm river flowing in distinct layers
  • Turbulent Flow: Fluid movement characterized by chaotic, irregular motion and mixing. We want to avoid this in hydrodynamic focusing
  • Flow Rate: The volume of fluid passing a point per unit of time (e.g., µL/min). Critical for controlling sample and sheath fluid dynamics
  • Hydrodynamic Focusing: The process of using a sheath fluid to constrain a sample stream into a narrow core, forcing cells to pass single-file through the interrogation point
  • Sheath Fluid: The fluid that surrounds and focuses the core stream
  • Flow Cell: The chamber within the flow cytometer where hydrodynamic focusing occurs and cells are interrogated
  • Isotonic: Having the same osmotic pressure as the sample. Prevents cells from swelling or shrinking
  • Refractive Index: A measure of how much light is bent when passing from one medium to another. Important for optimal light scattering
  • Electrical Conductivity: The ability of a solution to conduct electricity. Important for some cell counting methods (e.g., Coulter principle)