Fluidics

Fluidics: The Foundation of Flow Cytometry

The fluidics system is the heart of any flow cytometer. It’s responsible for:

• Transporting cells or particles from the sample tube to the interrogation point
• Focusing those cells into a single-file stream for accurate measurement
• Maintaining a stable and controlled flow environment

Without a well-designed and properly functioning fluidics system, the optical and electronic components wouldn’t have anything to analyze effectively!

The Three Pillars of Fluidics

1. Hydrodynamic Focusing:
   • What it is: A technique that uses a sheath fluid to constrict the sample stream into a narrow core, forcing cells to pass through the laser beam one at a time
   • Why it’s important: Prevents coincidence (multiple cells being detected as one), optimizes signal detection, and improves resolution
   • Key factors: Sheath fluid pressure, sample fluid pressure, flow cell geometry, and laminar flow
2. Sheath Fluids:
   • What they are: Clean, particle-free fluids that surround the sample core in hydrodynamic focusing
   • Why they’re important: Enable hydrodynamic focusing, maintain cell viability, and prevent contamination
   • Key properties: Purity, sterility, isotonicity, pH, viscosity, refractive index, electrical conductivity, and compatibility with reagents
3. Sample Delivery Systems:
   • What they are: The mechanisms used to transport the sample from its container to the flow cell
   • Why they’re important: Ensure precise flow control, minimize sample loss, enable automation, and reduce carryover
   • Types:
     • Syringe Pump Systems: Precise flow control, limited sample volume
     • Pressure-Based Systems: Larger sample volumes, automated sampling
     • Vacuum-Based Systems: Simple design, automated sampling (but less common now)
     • Acoustic Focusing Systems: Label-free focusing, gentle cell handling

How They Work Together

Imagine a well-orchestrated dance:

1. Sample Delivery introduces the sample (containing cells) into the fluidics system using a syringe pump, pressure, vacuum, or acoustic method
2. The Sample meets the Sheath Fluid
3. Hydrodynamic Focusing uses the sheath fluid to squeeze the sample stream into a narrow core
4. This focused stream of cells passes single-file through the Interrogation Point, where lasers and detectors do their work

Importance of Fluidics in Flow Cytometry

• Accuracy: Ensures that each cell is analyzed individually, improving the accuracy of cell counts and signal measurements
• Resolution: Improves the ability to distinguish between different cell populations by minimizing coincidence and optimizing signal detection
• Reproducibility: Provides a stable and controlled flow environment, enhancing the reproducibility of results
• Throughput: Enables high-throughput analysis by rapidly processing samples and acquiring data
• Sensitivity: Optimizes signal detection, allowing for the detection of rare cell populations or weak signals

Troubleshooting Fluidic Issues

Common problems and solutions:

• Clogging: Filter samples and sheath fluid, use cleaning solutions
• Bubble Formation: Check for leaks, degas sheath fluid
• Contamination: Use sterile techniques, replace contaminated fluids
• Pressure/Vacuum Issues: Check for blockages, inspect pump/vacuum function
• Carryover: Increase wash volume, use appropriate cleaning solutions

Key Takeaways

• Fluidics is a critical component of flow cytometry, responsible for transporting, focusing, and maintaining a stable flow environment for cells
• Hydrodynamic focusing uses a sheath fluid to constrict the sample stream, ensuring single-file cell analysis
• Sheath fluids must be pure, sterile, isotonic, and compatible with cells and reagents
• Sample delivery systems transport the sample to the flow cell, with different systems offering varying degrees of precision, automation, and throughput
• Proper maintenance and troubleshooting of the fluidics system are essential for reliable flow cytometry results.