Software Background

The FCM_PASS software has been developed to aid the interpretation and reproducibility single extracellular vesicle flow cytometry. The version 3 release of FCM_PASS allows researchers to create databases for each of their cytometers aiding their ability to track cytometer performance and catalogue experiment calibration. FCM_PASS is designed to calibrate both fluorescence and light scatter parameters and write these to .fcs files to allow sharing of raw data in standard units. The version 3 release of FCM_PASS allows integrates the MIFlowCyt-EV reporting framework as an output and auto-completes the relevant calibration fields to save researchers time.

Please contact fcmpass@nih.gov if you encounter any bugs/issues using the software or if there are any additions that would be useful to include in future updates.

Software Features

Reference Material Cataloguing - a range of reference materials are used between experiments and over time. To save time whilst maintaining complete records in the outputted calibration reports, all light scatter and fluorescence reference materials are catalogued in the software. This allows allows users of the software to not have to repeatedly input reference values such as diameters, CVs, or MESF values or useful metadata such as manufacturer, catalogue numbers or lot numbers.

Light Scatter Calibration Flow cytometer collection angle approximation – This method enables researchers to derive the collection angle of their conventional flow cytometers, simply by acquiring beads of known diameter and refractive index. Scatter-diameter plotting – Using a known collection angle, or utilising the above feature, researchers can approximate the scatter parameter sensitivity of their flow cytometer irrespective of refractive index. This method also enables approximation of particle refractive index and/or particle diameter with the correct controls. Particle diameter approximation – Using the plotted ‘scatter-diameter’ curve it is possible to interpolate raw SSC data with the curves to produce particle diameter distributions. Particle refractive index approximation – Using the plotted ‘scatter-refractive index’ curve it is possible to interpolate diameter data with the curves to produce particle refractive index distributions. Using this feature would require calibrating a fluorescence parameter to approximate diameter.

Refractive index auto-adjustment - When cataloguing the reference materials for light scatter calibration in the software users are able to pick a composition of polystyrene, silica, or other. Polystyrene and silica refractive indicies are automatically updated based on the light scatter wavelength the user chooses to model using published dispersion data for polystrene and silica, respectively. All other refractive indices are also updated with respect to the dispersion properties of water to maintain their scattering ratios. These can however been updated manually if more accurate data exists.

Fluorescence calibration – Converting arbitrary flow cytometer units to known reference values, such as molecules of equivalent soluble fluorophore (MESF), is a method of fluorescence standardization. The FCM_PASS software includes a tool to perform fluorescence regression and write the data to flow cytometry .fcs files for downstream analysis and reporting.

Write calibrated data to .fcs - along with outputting calibration plots the FCMPASS software makes it possible to write all of the calibrated data directly to the .fcs file for downstream analysis and sharing with the aim to increase standardization and transparency in reporting of data.

MIFlowCyt-EV output - upon calibration of the files a spreadsheet is outputted that contains a MIFlowCyt-EV framework with the fields related to calibration auto-completed. To further aid the transparency and reproducibility of the calibrations all parameters and metadata used for fluorescence and light scattering calibration are also included in separate sheets.