Table 1.

Staged approach to standardization of flow cytometric assays

Three stan dardization stages	Four flow cytometry standardization categories
	Equipment	Antibody panels	Antibodies/staining reagents	Analysis/reporting
Fundamental	Different instruments Performance of instrument calibration using CS&T and application settings (BD), FSP (Beckman Coulter), or SpectroFlo (Cytek Aurora) beads Challenge: requires thorough data comparisons across different types of instruments	Different antibody panel at each site, albeit same marker(s) to define cell types. Check full panel stain on samples relevant to the trial (e.g., subjects with T1Ds) Challenge: different clones can lead to differences in MFI and percentage of positive cells detected	Perform antibody titrations and optimization of other key reagents. Ensure regents are used within expiry dates Challenge: time- and resource-consuming to check titration of different antibody lots	Similar gating strategy and the use of appropriate controls. Report all deviations from protocol that may impact analysis Challenge: a rigid analysis may preclude unexpected results
Desirable	Similar instruments, despite different configurations (e.g., BD LSRFortessa 3 vs. 4 lasers) Back-up instruments at each site Challenges: requires thorough data comparisons across different instruments; some sites will limit and prioritize markers due to lack of number of parameters	Similar panels across sites that use same markers and clones albeit a slight difference in marker/fluorochrome combination Challenge: different fluorochromes usually affect marker staining sensitivity; must be taken into account	Marker evaluation across platforms to ensure that populations of interest can be detected by cytometers at each clinical trial site. Challenge: time- and resource-consuming	Predefined gating strategy and data acceptance criteria along with normalization to controls Challenge: a rigid analysis may preclude unexpected results
Ideal	Identical instruments with the same configuration Levy-Jennings plots for cross-validating QC across sites & over time Challenge: same instrument can differ in daily performance and sensitivity; some comparisons of data is desirable	Identical panels with consideration to minimize fluorochrome spillover and spread Challenge: there may be lot-to-lot variability between identical antibodies. Consider purchasing lots in bulk	Use of lyophilized antibody tubes to reduce or eliminate lot-to-lot variation and pipetting error Perform lot-to-lot crossovers when using wet antibodies Challenge: when several panels are used for a study, lot-to-lot crossover testing can become tedious, expensive, and not feasible	Use of automatic gating and independent analyses of same samples/data by two different sites. Internal controls to assess technical versus biological variation Challenge: a rigid analysis may preclude unexpected results
Case study	Desirable Site B: 5-laser BD LSRFortessa Site C: 3-laser BD LSRFortessa Ideal improvement: use of same instrument with identical configuration to allow for the use of identical flow panels	Desirable Similar five flow panels using same antibody clones Ideal improvement: identical instrument used with identical flow panels	Desirable Wet antibody titrated with documentation of reagent lot numbers Ideal improvement: validate the use of antibody master mixes and/or identify where possible the use of lyophilized antibody tubes to reduce pipetting	Ideal Identical gating strategy, data analyzed by site each and all data reviewed by coordinating site Operator and time between sample collection and processing were documented to assess technical variation

The application of three stages of standardization (fundamental, desirable, and ideal) to four categories (equipment, antibody panel design, antibodies/staining reagents, and analysis/reporting) when designing flow cytometric assays in a clinical trial and when applied to a case study with challenges noted.