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Flow Cytometry – GCLP Regulations

 
 

      Flow Cytometry Information:

How to Perform Flow Cytometry Studies under GLP or GCLP

To date there has been no formal regulatory guidance on how to conduct flow cytometric assays under GLP (good lab practices) or GCLP (good clinical lab practices). However, several private groups and organizations have over the years compiled best practices and recommendations. The most often cited of these references is:

The above recommendations encompass:

  • Pre-analytical: Validation Plans, Instrument Validation, Facility, Personnel, Sample Considerations, etc.
  • Analytical: Development and Validation
  • Post-Analytical: Management Systems, Documentation

The rest of this page will only focus on the analytical aspects and sample considerations. The following is our summary interpretation of the CLSI recommendations. These recommendations should be strongly considered when developing or validating a flow method. It is important to note that, because there can be so many variations in how a flow assay is designed or conducted, each method must be assessed for the parameters that are most important for the intended application (e.g. fit-for-purpose). Depending on the parameter to be optimized, there may be no set quantitative acceptance criteria; in such a case, each parameter should be qualitatively assessed for performance based on the judgement and expertise of the principle investigator and study director.

Assay Development

Objective

  • Initial considerations for fit-for-purpose assay:
    • Type of data needed (qualitative vs semiquantitative).
    • Data source (counts, fluorescent intensity/antigen expression, percentages, etc).
    • Sample considerations (type, how to measure, sample matrix).

Panel Design

  • Select the appropriate marker(s) with well-defined properties based on scientific literature. Markers or antigens with high expression are desirable; markers selected can also be negative (dump) antigens if appropriate.
  • Select the appropriate antibodies that have been characterized for binding the target antigens with minimal cross-reactivity. Different clones may have different characteristics: ability to bind fixed targets, affinity, specificity (bind target on multiple species). The host may also be a factor if secondary antibodies are used.
  • For fluorophore selection, minimize spillover, utilize small molecule dyes if fixing, and consider brightness and fluorescence lifetime (e.g. pair brightest fluorophore with least expressed target). Minimize spread (width) of MFI peak when analyzing the target antigens (e.g. narrow peaks are better).

Matrix

  • Ensure matrix (blood, PBMC, tissue) is appropriate for endpoint.
  • Ensure method/panel works for matrix.

Antibody Titration

  • Staining Index – appropriate concentration of antibody to achieve the highest S/N ratio (specific/nonspecific).
  • Select for adequate separation between negative and positive populations.

Blocking

  • Utilize an FcR blocking reagent when necessary.
  • If secondary antibodies are used (e.g. anti-IgG), ensure that block is not targeted.

RBC Lysis

  • If needed, investigate how RBC lysis affects assay. Optimize RBC lysis as needed.

Fixatives

  • If cells are fixed before staining, ensure that the stain/antibody selection is optimized for intended use.
  • Assess sample stability and use of fixative if longer wait times between stain and analysis.
  • Optimize fixative for intracellular targets (e.g. PFA or MeOH; mild vs harsh) harsh arrest function better, but also masks target binding.

Gating

  • Determine rules for how gating is to be conducted – decide what graphs (density, histogram), what axis, in what order, and how positive/negative thresholds are set.
  • Typically start with FSC x SSC gating, then gate for single cells, then continue for the population/target of interest.

Evaluation, Optimization, and Characterization

  • Assess performance and consider different antibody clones (epitopes) if steric effects occur.
  • Optimize staining incubation time and temperature.
  • Determine minimum number of events allowable (e.g. sensitivity)
    • Number of cells needed per sample/well.
  • Determine minimal viability of starting cellular material.
  • Establish appropriate instrument conditions (thresholds, stop conditions, flow rate).
  • Determine stability and storage conditions of sample specimen (e.g. effects of storage method, temperature, and shipping), stability of processed samples (e.g. time between staining and acquisition) and stability and robustness of staining cocktail (e.g. if prepared hours ahead of time, or if small pipetting errors significantly change staining profile).
  • Determine appropriate quality controls.
Validation Parameters

Accuracy / Trueness

  • Exceedingly difficult to establish, as without a calibrated reference standard, flow assays are inherently quasi-qualitative/semiquantitative.
  • When possible, use reference standards or reference material and compare to performance by other labs, and/or compare against other technological method.

Linearity

  • Assay dependent as there may be no standard curve (reference material) for which to compare to; but a pseudo-standard curve could be prepared depending on assay needs (e.g. titer the number of cells, titer article, titer antibody stain, etc.).

Selectivity

  • Partly accomplished through panel design (e.g. testing and avoiding antibodies with cross-reactivity).
  • Test for interfering substances (as needed):
    • Other cells
    • Therapeutic
    • Soluble substances that may occur in matrix (e.g. lipids if assay is whole blood).
  • If appropriate, verify performance with healthy vs disease state specimens.
  • Include documentation from assay development that illustrates antigen/antibody choice, staining index, and gating strategy.

Precision

  • Includes: day, run, reagent lot, operator, instrument, and laboratory.
  • Assay dependent as it may be difficult to test targets at low, medium, high range as cellular material is often not predictable. Instead test multiple donors. Can also use LLOQ preparation for low sample testing.
  • Ideally a minimum of 3 reps x 3 specimens x 3 runs (plates) x 3 days x 2 operators x 2 instruments. However, specifics may vary depending on assay requirements, intended use, and available equipment/facilities.

Stability

  • Specimen Stability
    • Sample storage, collection/anticoagulant, thawing/resting effects
    • Ideally test minimum 3 samples with baseline testing at < 2hrs (if possible).
  • Process Stability
    • Time between staining and acquisition (e.g. < 1 hr, 6 hrs, overnight).
    • Storage temperature conditions (e.g. RT vs 4° C), including rapid repeated change in temperature.
    • Ideally test minimum 3 samples.

Carryover

  • Instrument Performance Qualification (PQ). Check if sample material is carried over between sample runs.
  • Run high samples (n=3), followed by low or blanks (n=3).

Reference Intervals

  • Can be determined for a normal population. Most useful for clinical (CLIA) testing; but may not be appropriate for drug development where samples are likely to be outside of normal range.
Additional Flow Cytometry Information

We have designed several informational pages with custom graphics to help explain the complex concepts of flow cytometry.

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