CYTO 2026 Scientific Tutorial: Fundamental Considerations for Quantitative Fluorescence Standardization

Fundamental Considerations for Quantitative Fluorescence Standardization

Presenters:
Vera Tang, PhD, Facility Manager & Adjunct Professor, University of Ottawa
Joshua Welsh, PhD, Staff Scientist, BD Biosciences

Problem Focus/Summary:
Standardization of flow cytometry data reporting is necessary for reproducibility irrespective of the sample type across flow cytometry platforms. Cross-platform standardization as a topic is becoming increasingly important in both clinical and academic research settings. Fluorescence quantification is a method used to standardize reporting of flow cytometry data, making intra- and inter-platform comparisons possible. These are published methods, but there are currently a multitude of tools available for fluorescence standardization. In this tutorial we will identify the different tiers of standardization and then provide practical guidance on the tools necessary to achieve these different levels of standardization.

Goals: The purpose of this tutorial is to provide insight and guidance towards selecting methods and materials for fluorescence quantification, aiming to address specific standardization goals, and provide options to achieve concordance in standardized data reporting.

What this Tutorial will not do: Cover pre-acquisition variables – sample isolation/processing, staining optimization, panel design.

Learning Objectives:
To understand the different tiers of flow cytometer assay standardization from longitudinal single instrument studies to cross-platform comparison studies.
To understand the nuance in definitions for fluorescence quantification, calibration, normalization, and standardization.
To understand the practical differences between each of the fluorescence calibration units available, including cost, consistency, and ergonomics.
To demonstrate the impact of different fluorescence units on concordance in comparing cross-platform data, i.e. ERF, MESF, and ABC.

Keywords: Calibration, Quantitative, Fundamental Concepts, Hardware, Standardization, Experimental Design

CMLE Credit: 1.5