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Material Measurement Laboratory

Biosystems and Biomaterials Division

FCSC Working Group 2

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Flow Cytometry Assay Standardization

Working Group 2 (WG2) designs and conducts interlaboratory studies for the development of standards, assay control materials, and best practices protocols that achieve flow cytometry assay standardization and that generate high-quality reference datasets.  Activities in progress include:

  1. An interlaboratory study (WG2-ILS1) on standardization of a T-/B-/monocyte/NK-cell (TBMNK) assay for cell count and cell health  Data submission is complete with contributions from 17 Consortium member organizations across 21 sites and 42 different instrument platforms that include imaging, spectral, and conventional flow cytometers. The goals of WG2-ILS1 are:

    • Determine variability across a complex study design including healthy-donor cryopreserved peripheral blood mononuclear cells (PBMCs), synthetic cell mimics, cytometer calibration and reference counting beads, and fluorescently-labeled antibodies.
    • Collect "real-world" flow cytometric assay results in SI-traceable units including cells per microliter (cell count) and ERF units (CD marker expression).
    • Populate a repository of assay results for centralized consensus analysis and generation of high-quality reference flow cytometry data.

    WG2-ILS1 has already produced deliverables and outcomes that include:

    • Four standard operating procedures (SOPs) that address
      • SOP 1 - instrument setup, characterization and standardization
      • SOP 2 - preparation of three compensation matrices
      • SOP 3 - preparation and measurement of study samples and controls
      • SOP 4 - preliminary data analysis and reporting including gating strategies for population reporting

    • Curated collection of thousands of .FCS files recorded using common materials and standardized methods

       

      Fishbone-like diagram showing variability contributions, from left to right, first for cell and control samples and instrument platforms, second for each antibody clone and panel compensation samples, third for cell counting approach and dead cell and fluorescence minus one controls, and, finally, fourth data acquisition and analysis and standardized file naming and storage. These contributions point to the end result of a standardized assay and data file collection.
      Fishbone-like diagram for FCSC WG2-ILS1 sources of variability and applied controls, through SOPs, leading to an overall standardized assay and data file collection

       

  2. An interlaboratory study (WG2-ILS2) study on phenotyping of engineered T-cell subsets and lineages as well as chimeric antigen receptor (CAR) expression analysis  This is study is in an active planning stage and is expected to commence in the first half of 2025.

 

Bioscience, Biomanufacturing, Cell biology, Gene delivery systems, Health, Cell and gene therapy, Precision medicine, Regenerative medicine and advanced therapy, Metrology, Amount of substance, Standards and Reference materials

Contacts

Consortium PI and WG2 Technical Lead

Created November 29, 2024