Uncertainty Quantification of Fluorescence Signals in Flow Cytometry Part I: An Analytical Perspective Beyond Q and B

Area of Science:

• Metrology
• Uncertainty Quantification (UQ)
• Flow Cytometry

Background:

• Traditional flow cytometry analysis struggles with accurate estimation of detection efficiency (Q) and background (B).
• Existing methods often yield unstable estimators and negative background values due to approximations and noise amplification.
• Comparing instruments based on current performance metrics remains challenging.

Purpose of the Study:

• To develop a robust data analysis strategy for flow cytometry measurements.
• To accurately estimate detection efficiency (Q) and background (B) while accounting for gain-dependent and independent effects.
• To establish a rigorous framework for comparing cytometer performance and defining limits of detection/quantification.

Main Methods:

• A global data analysis strategy combining measurements from different instrument gains.
• Simultaneous accounting for gain-independent background effects.
• Quantification of various noise sources and their impacts.

Main Results:

• Stable and reliable estimates of detection efficiency (Q) and background (B).
• Quantification of the relative impact of different noise sources.
• A unified explanation for the shortcomings of existing analysis methods.
• Rigorous definition of instrument-specific limits of detection and quantification.

Conclusions:

• The proposed method enables direct, sample-independent comparison of flow cytometers.
• It provides crucial information for optimizing cytometer performance by minimizing instrument-induced uncertainties.
• Experimental validation was performed on commercial and NIST-developed instruments.