Measurement of Microsphere Concentration Using a Flow Cytometer with Volumetric Sample Delivery

Area of Science:

• Quantitative Flow Cytometry
• Microsphere Characterization
• Biophysical Measurement Techniques

Background:

• Accurate microsphere concentration determination is essential for assigning Equivalent Reference Fluorophores (ERF) units in quantitative flow cytometry.
• Existing methods may lack precision due to variations in sample delivery and buffer composition.

Purpose of the Study:

• To evaluate a syringe-based sample delivery system for precise microsphere concentration measurement.
• To establish an optimized protocol for accurate microsphere quantification using flow cytometry.

Main Methods:

• Calibrated syringe volume delivery and draw modes to identify systematic volume-dependent bias.
• Measured microsphere concentrations using lyophilized microspheres and fluorescein microspheres in various buffer solutions (PBS, PBS+BSA, PBS+TWEEN 20).
• Developed a corrected volume calculation by subtracting a 13 µL offset derived from calibration.

Main Results:

• Syringe volume calibration revealed a -13 µL offset and a systematic volume-dependent bias (slope 0.93).
• Phosphate-buffered saline with 0.05% bovine serum albumin (PBS+BSA) buffer yielded the most consistent count standard concentration.
• The optimized procedure, using PBS+BSA and a corrected delivered volume, yielded results consistent with certified values and relative measurements, with ~4% uncertainty.

Conclusions:

• A syringe-based flow cytometer system can accurately measure microsphere concentrations when accounting for volume bias.
• Using PBS+BSA buffer and a corrected delivered volume (indicated volume - 13 µL) is recommended for optimal microsphere quantification.
• The developed method provides a reliable and accurate approach for microsphere concentration determination in flow cytometry applications.