Flow Cytometry

Flow cytometry assays can also be used as a valuable exploratory endpoints in clinical trials to help assess a therapy’s effect on its intended target. The data generated can make all the difference in whether your therapy can advance forward. We can work with you to customize and validate PBMC flow cytometry panels to fit the context of use according to your specific program.

Flow cytometry assays help determine the functional and immunophenotypic characteristics of cell populations. Multi-color flow cytometry assays allow for the identification and quantification of different cell types in a single heterogeneous sample and provides information about the frequency and phenotypes of specific cell populations in a short time frame. These assays commonly use whole blood; however, cryopreserved PBMC samples may also be used for immunophenotyping analysis.

Due to our global footprint, we can transfer clinical flow cytometry assays to sites around the globe. Additional infrastructure allows us to accommodate the receipt of samples 24/7/365 to support the timely analysis of samples with limited stability.

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Examples of Study Endpoints Using Flow Cytometry Assays

  • Characterization of the activation, expansion, persistence, and exhaustion status of cell-based therapeutics
  • Characterization of CD8+ T cell subsets, CD4+ T cell subsets, analysis of the frequency of T cells specific for a peptide using tetramer staining, and characterization of myeloid cell subsets by the assessment of immune cell frequency, as well as phenotypic and functional markers directly ex vivo
  • Lineage markers, activation markers, and the expression of effector molecules, such as cytokines by ex vivo activation using a polyclonal- or antigen-specific stimulus (T cells) or an innate cell stimulus (e.g., LPS)
  • Receptor occupancy assessments

During method development, specific attention is paid to sample stability, which includes considerations such as sample collection (anticoagulant or fixative), sample processing (whole blood or PBMCs), shipping conditions, and the stability of the desired cell-surface markers under the optimized sample handling conditions. Validation steps for flow cytometry assays confirm the performance and reproducibility of an assay. This typically includes establishment of controls, sample collection protocols, and characterization of sample stability and critical reagents, as well as establishing a gating strategy for acquisition and data analysis, inter-/intra-assay precision, and inter-operator reproducibility. Although it is not a requirement for all clinical flow cytometry assessments, we do have capabilities to perform work in compliance with Good Clinical Laboratory Practice (GCLP). Additionally, individual sites may configure clinical assays for CLIA compliance where appropriate.

Validation of Marker Stability after Fixation by Flow Cytometry Assay

Seven charts displaying the analysis of human whole blood by flow cytometry assay to assess whether fixation affected the frequency of CD8 T cells detected in a sample or activation markers CD25 and CD69. Such information is critical in determining the logistics of handling and transporting samples from clinical sites to the flow cytometry staining and testing facility.

Having established that a flow cytometry panel is fit-for-purpose and that the markers are informative under the logistical conditions of the clinical trial, the clinical sample analysis phase can proceed. Flow cytometry analysis of cytokine expression by intracellular cytokine staining provides information on which cell types a therapeutic is modulating and can complement non-cell type specific multiplex analysis of cytokines directly in the blood by Luminex® technology.

 

Flow Cytometry Analysis

Five charts displaying PMBC flow cytometry analysis of blood samples obtained from clinical trial subjects with an autoimmune disease pre- and post- treatment to identify CD4 T cell subset activation, polarization, and proliferative responses.

Flow cytometry assays for early-phase clinical trials are powerful tools for assessing the impact of a therapy on immune cell populations and provide data to help you make informed decisions about your program. The simultaneous analysis of multiple parameters at high throughput allows for the detection of rare cell populations and changes in expression of markers of interest at a rapid rate.

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Frequently Asked Questions (FAQs) about Flow Cytometry Assays

  • How many fluorochromes can be detected in a clinical flow cytometry assay?

    Many of our sites supporting clinical flow cytometry studies can develop antibody panels for clinical use that include up to 14 fluorochromes.

  • Can you help me design a panel?

    Yes, we can provide full service technical and scientific support. We can develop panels from scratch as well as guide you and recommend relevant readouts according to your compound and its related context of use.

  • What is the regulatory level requested for a clinical flow cytometry panel?

    Flow cytometry assays are validated and used under a fit-for-purpose approach. Consequently, the degree of validation is dependent of the context of use. An exploratory end point on a clinical study will have to be evaluated in terms of sample stability, precision, and the characterization of key markers and critical reagents. While this analysis does not generally need to claim GCP or CLIA compliance, sponsors may request a flow cytometry assay validated and executed to this level of compliance at selected sites around the globe.

  • How long does it take to develop and validate a panel?

    Method development or method transfer can take 6-10 weeks. Method validation can then take 3-4 weeks depending on the complexity of the panel and cell populations.