Uncertainty Quantification of Fluorescence Signals for Cytometry Part II: Comparison of Serial and Traditional Flow...

Area of Science:

• Bioanalytical instrumentation
• Quantitative measurement science

Background:

• Flow cytometers are essential for bioanalysis, with ongoing advancements driving new systems.
• NIST developed a serial microcytometer for per-object uncertainty quantification, necessitating improved benchmarking methods.

Purpose of the Study:

• To develop and apply new metrics for quantitative comparison of flow cytometers, addressing limitations of conventional methods.
• To establish robust metrics for instrument performance and uncertainty quantification (UQ) in flow cytometry.

Main Methods:

• Developed an improved model building upon conventional UQ models for enhanced comparability.
• Defined metrics including upper limit of linearity, limit of background, limit of detection, and noise-to-signal ratio.
• Utilized a serial microcytometer to separate instrument- and population-dependent contributions to UQ.

Main Results:

• The NIST serial microcytometer demonstrated performance comparable to conventional analytical flow cytometers.
• The study successfully separated component-level contributions to UQ.
• Established per-object uncertainty as a key metric for data utilization.

Conclusions:

• The developed metrics provide robust methods for evaluating flow cytometer performance.
• Per-object uncertainty quantification enhances the classification and application of cytometry data in research and clinical contexts.
• Serial microcytometry facilitates detailed UQ analysis, advancing the field of bioanalytical measurements.