Due to its ability to analyze multiple parameters across different cell types within a sample, flow cytometry can provide very rich and clinically valuable data sets from even small volumes of blood. However, flow cytometry is a challenging platform to master, and requires significant investment into equipment and technical training. So, for many researchers, outsourcing flow cytometry to a Contract Research Organization (CROs) is both cost-effective and the best way to ensure the highest quality of data from their samples.

So, what types of flow cytometry applications are the most outsourced to CROs?

Immunophenotyping

One of the most routine and yet powerful applications of flow cytometry is immunophenotyping. This involves the analysis of white blood cells by measuring the presence or absence of key biomarkers/antigens, coupled with the characteristic differential light scatter properties of cells. Combined with cell counting beads, immunophenotyping by flow cytometry can be used to enumerate specific cell subsets and has many applications across clinical studies including the diagnosis of leukemias and lymphomas and the stratification of patient responses within clinical trials. The reliability of immunophenotyping analysis though is dependent on several factors including the quality of the sample and the staining and processing reagents, instrument performance, and the quality of the analyses. It is therefore always recommended to work with a CRO that has an established Quality Management System underpinned by Standard Operating Procedures across all workflow applications to ensure the quality and reproducibility of the analysis.

Immunophenotyping for clinical diagnostic applications may require CLIA or CAP accreditation, whereas patient monitoring during clinical trials, may be accomplished under the GLP and GcLP guidelines. FlowMetric Life Sciences provides immunophenotyping services under all of these compliance requirements.

Cell Type Common Identifying Biomarker
B-lymphocytes CD19, CD20, CD22, CD79a, immunoglobulin light chains (κ or λ)
T-lymphocytes CD2, CD3, CD5, CD7, CD4 or 8
Myeloid MPO, CD13, CD33
Natural Killer CD16, CD56
Immature Precursors CD34, CD117, TdT
Platelets CD41, CD42b, CD61
Red Blood Cells CD235a
Monocytic differentiation markers C11b, CD14, CD33, HLA-DR

Table 1. Basic markers used for Immunophenotyping applications. Flow cytometry is now the preferred method for lineage assessment, maturational characterization of malignancies, heterogeneity, and quantification of subpopulations (Rothe and Schmitz).

Figure 1. Example of a TBNK report for an immunophenotyping panel performed under CLIA certification, CAP accreditation. The report summarizes both the absolute numbers of T-, B- and NK cells as well as their % within the population.

Cell Sorting

One specialized flow cytometry application is fluorescence-activated cell sorting or FACS that enables specific target cell populations to be collected based on their surface antigens and light scatter properties, into enriched populations. Cell sorting has many applications ranging from genomic analysis of specific cell types to the functional analysis of CAR-T cells. The sorting of some rarer cell types, such as Tregs or TDC can take considerable expertise to optimize, and as with all cell sorting applications, is a balance between purity and yield of the sorted population. CROs such as FlowMetric who offer cell sorting, have optimized methods and staining panels to ensure successful cell sorts for many different target populations.

Receptor Occupancy

Receptor Occupancy assays are used to quantify the binding of therapeutic molecules to their target receptors on the surface of cells. These assays are used to complement pharmacodynamic and pharmacokinetic studies to establish the PKPD relationship and are used in dosing studies to evaluate the quantity of drug needed to attain therapeutically effective drug-receptor occupancy and establish Minimal Biological Effect Level (MABEL). Long-term maximal RO may indicate overdosing and potential toxicity and is, therefore, a frequently reported parameter in phase I and II clinical trials to support efficacy dosing selections and informed Go/No-Go decisions. During phase III clinical trials, RO is often used to help define population PK characteristics (Stewart et. al. 2016).

Figure 2. Summary of the principles of RO assays by flow cytometry. The three most common RO assay measures i. Free Receptor; ii. Total Receptor and iii. Bound Receptor.

There is no one formula for RO assays and each has its own idiosyncrasies and range from measuring the number of cell surface receptors bound with the anti-receptor drug, to more complex scenarios such as receptor internalization and shedding events induced by drug binding. Careful reagent selection is critical to assay performance and the optimal assay endpoint.

  1. The determination of free receptors requires a competitive antibody or fluorescently labeled drug molecule that is used to label receptors that do not have the drug bound. There are considerations when selecting the detection antibody- if the affinity is much lower than that of the drug, then it may measure free RO, whereas a much higher affinity to the receptor compared with the drug may push this towards the total receptor. So, it is important to understand the kinetics of the binding of both the drug and detection reagents with the target receptor.
  2. Total receptor measurements require the use of non-competitive antibodies that bind to the receptor regardless of the presence or absence of the drug. Another approach that is now widely used to determine the total available target receptors is the use of supersaturating drug levels in vitro, and then the monitoring over time of drug bound with an anti-drug antibody.
  3. For so-called True RO measurements, the direct detection of the bound drug to the receptor is often the method of choice. The detection of bound receptors can be determined by a number of different ways- either with labelled- drug, -anti-drug antibodies or -antibody targeting epitopes only presented when the drug is bound to the receptor. As with free receptor detection, it is important to consider the binding kinetics, and ensure that the detection agent does not impact the affinity of the drug binding.

The determination of which of these assay formats to use is largely driven by the mechanism of action of the drug, coupled with the availability of reagents. Antagonistic drugs typically rely on free-receptor RO assays that measure the receptors available to the drug. Drugs that work through up or downregulation of receptors are analyzed using total RO. This enables researchers to consider several factors such as the ablation of receptor-expressing cells, the up or down-regulation of receptor expression, and mobilization of receptor-expressing cells.

Typically, a directly labelled drug is the best reagent for RO assays but when this is not available, then a fluorescently labeled anti-drug antibody (ADA) may be adopted. However, it is important to consider the potential for ADA to biased results and use non-neutralizing anti-idiotypic antibodies or anti-Fc of the drug. ADA interference may be avoided by using detection reagents that compete with the drug for the target receptor but that have no other similarity with the drug and so are unaffected by ADA effects.

Receptor Modulation is also a feature of receptor function that can be measured using RO flow cytometry methods. It is also an important consideration for RO assays on clinical trial samples; these frequently require shipping to central labs, during which time internalization, shedding, capping, or proteolytic degradation of the receptors may occur, and biased RO endpoints.

When developing an RO experimental plan, the FlowMetric team strives to develop a robust RO assay approach that utilizes a multiplexed strategy to detect both free and bound receptor forms and takes into consideration the MOA of the drug and the binding kinetics of all components. All of these factors should be captured within the validation plan outlined between the sponsor and the CRO.

Functional Cellular Assays

Another area where flow cytometry support services can provide great value is with functional cellular assays. These are specialized assays that couple cellular subset labeling with functional profiling (Rabinovitch and Robinson, 2002; O’Gorman 2001). Below are some examples of functional assays that are provided by the FlowMetric team, and their applications in different areas of research.

Intracellular Cytokine staining (ICS)

ICS is an extremely versatile, high-resolution method that allows researchers to examine the intracellular cytokine production profiles in different cell types, in response to various stimuli. Cytokines are key cellular communicators and play central roles in immune responses- from the anti-viral production of IFN-γ and TNF-α to the induction of T-cell proliferation by IL-2. ICS involves several steps that must be optimized for different matrices whether cultured cells, fresh blood, frozen PBMCs to isolated TILs. ICS is an important tool in vaccine and immunotherapy development for the evaluation of epitopes to induce the desired immune responses.

Figure 3. Step by step summary of the process for ICS by flow cytometry. Since cytokine signatures are temporal in nature, it is recommended that experimental parameters are optimized for each cell type and stimulation condition to ensure the optimum signal-to-noise ratio associated with specific cytokine responses.

ICS can provide much more detailed cytokine profiles than ELISPOT, however, it should be considered that cellular cytokine responses are invoked by a range of stimuli, and determining antigen-specific responses requires careful experimental design and execution. FlowMetric has the instrumentation in place to develop and execute ICS assays that utilize up to 20 parameters (18 colors), providing plenty of flexibility to researchers for intracellular cytokine staining to be added with cell surface markers in one streamlined panel.

Phosphoflow Assays

Phosphorylation assays using flow cytometry, also known as phosphoflow represent the highly sensitive analysis of intracellular signaling events through the detection of phosphorylated proteins within cells. Phosphosflow assays are used to examine cellular responses to cytokines, transcription factors, and other cell signaling and metabolic processes. Unlike lysate-based approaches, phosphoflow assays facilitate the detection and analysis of heterogeneous signaling responses in complex biological matrices, with single-cell resolution. Rich data measuring multiple phosphorylation targets may be obtained from limited sample sizes, enabling high-resolution comparative analysis of the phenotypic and functional differences with multiple cell types, including the rare cell populations within samples. However, since these phosphorylation events can be transitory in nature, careful experimental design and specialized reagent and buffer selection are required.

Figure 4. P-STAT-5 Activation Profiles. Which Lymphocyte Respond to IL-2?

Activation Profiles for a. CD4+, b. CD8+ T Cells, and c. CD19+ B Cells. The data is displayed as histogram overlays of the MFI signaling profiles of the phosphorylation marker STAT-5: unstimulated cells (solid grey), and after stimulation with 100ng/ml IL-2 (black outlined white peaks). The data was collected from human whole blood samples. This analysis demonstrates the T-cell responses to IL-2 includes phosphorylation of STAT-5. This response it not observed in the B-cells.

T Cell Activation/Exhaustion Assays

T cell activation and exhaustion are pivotal biological events of the immune system, and flow cytometry is one of the tools most frequently used to identify and monitor both Activated and Exhausted T-cells. Commonly used T cell activation markers include: CD69, CD71, CD25, coupled with increased cytokine production measured with ICS. T Cell Exhaustion occurs after chronic antigen exposure to virus or cancer and is identifiable through decreased cytokine production, and the display of specific immunoregulatory markers such as TIM-4, LAG-3, PD-1, CTLA-4- also called immune checkpoints, that bind to tumor cells or antigen-presenting cells but are inactive or dysfunctional.

A growing number of investigators are now testing drugs that serve as immune checkpoint inhibitors, that reverse the exhaustion of T cells, making these T cells active and functional, and able to attack tumor cells. T cell activation and cell exhaustion assays by flow cytometry help us to monitor drugs that serve as checkpoint inhibitors or blockade, which are proven to be clinically beneficial to patients with advanced cancer. FlowMetric has developed and validated several high dimensional flow cytometry panels to interrogate T cell activation and cell exhaustion for research and clinical applications.

Figure 5A.

Figure 5B.

Figure 5C.

Figure 5. Immune Checkpoint Monitoring for Identification of Immune Cell Subsets.

PMBCs from normal healthy donors were stained with a 16-color T Cell Exhaustion Panel and analyzed by flow cytometry. (A) and (B) Assessment of expression levels of an important checkpoint and activation molecules on CD4+ and CD8+ T cells. (C) T Reg cells were identified as CD25high CD127low and FoxP3+. Expression was induced by stimulating cells with anti-CD3, anti-CD28 coated beads.

Cell Degranulation Assays

NK Cells are cytotoxic lymphocytes that play a key role in the defense against viral infections and cancer. Cell Degranulation Assays evaluate the cytotoxic function of NK cells, which are important when testing therapeutic candidates for the treatment of viral infections and intervention for cancer remission. CD8 T cells with CD107 (LAMP-1) molecules and other cells like eosinophils have degranulation activities that can be assessed with Cell Degranulation Assays. FlowMetric’s Cell Degranulation Assays can be used to monitor the effectiveness of therapeutic candidates and for assessing clinical conditions such as the diagnosis of immunodeficiency syndromes.

Cell Proliferation Assays

Cell proliferation assays are useful to determine cell division and measure the expanding population. While for decades the methodology for cell proliferation was extremely cumbersome, using radioactive molecules 3H-thymidine to be integrated in the genetic material of new or “daughter” cells, the use of flow cytometry allows us to implement cell proliferation assays without radioactivity. With flow cytometry data analysis, we can generate accurate information to quantify the proliferation rate, the metabolic activity of the cells and even assess different antigens expressed when cell proliferation occurs.

Figure 6. The principal of dye dilution assays for Cell Proliferation. Parental cells are labeled with tracking dye on day 0. Flow Cytometry analysis can reveal successively dimmer peaks representing each generation of cells from that parental generation.

Examples of applications of cell proliferation assays:

  1. testing safety- if your pharmaceutical drug is safe, it should not induce cell proliferation,
  2. testing efficacy of a pharmaceutical drug that aims to inhibit tumor cell growth,
  3. during CAR-T cells development, CAR-T cells are transferred into patients with cancer, when developing these T cells, we need to make sure they get activated upon recognition of antigen in tumor and start proliferating to kill tumor cells,
  4. testing clonal expansion in T and B cells, upon re-call with specific antigen,
  5. developing diabetes drugs with islet cells (cells that produce insulin and stop proliferating with high blood glucose levels.

Bromodeoxyuridine (BrdU) incorporation has long been used to assess DNA synthesis in vivo and in vitro. The foundation of this method is the incorporation of BrdU as a thymidine analog into the nuclear DNA during the S-phase of the cell cycle, which can be tracked using antibody probes. It is much safer and easier to use than older systems such as 3H-thymidine incorporation, and although considered toxic, the concentration that BrdU is used for labeling is typically not an issue.

Figure 7. Jurkat cells were pulsed with 10µM BrdU for 1 hour, using the acid denaturation method, and stained with anti-BrdU-APC. Cells were stained with 7-AAD for DNA analysis.

Figure 8. PBMCs were stimulated with 1:2 ratio of CD3/CD28 Dynabeads (Beads:Cells) for 3 days in 10% FBS RPMI 1640 complete media +P/S with 0.1µg/ml CD3 and 30 IU IL-2.

Figure 8A. PBMCs unstimulated (Left) and Stimulated (Right), gated on CD3+ T Cells. With stimulation cells undergo multiple generations of proliferation as shown by the Cell Trace Violet Staining which is also Ki67+ compared to unstimulated cells.

Figure 8B. PBMCs gated on CD3+ T cells, overlay of unstimulated (shaded peak) and stimulated (blue line) cells.

Final Thoughts

There is a growing need for flow cytometry assays to support in drug development initiatives, largely due to their multiplex nature and subsequent rich data sets. However, the barriers to entry and time needed to develop flow cytometry assays means that outsourcing to specialized CRO such as FlowMetric, will ultimately save both time and money, all while ensuring the robustness of your data.

The development of novel high-performance fluorophores and cellular probes, coupled with the commercial availability of monoclonal antibodies against an increasing number of antigens means that flow cytometry can provide analytical solutions across a wide range of cellular functions. Working with a CRO such as FlowMetric who provides the highest quality of flow cytometry services, ensures that your research team is tapped into state-of-the-art flow cytometry instrumentation, methodology, and analytics to help accelerate your project goals.

References

  • Rothe, G., Schmitz, G. European Working Group on Clinical Cell Analysis (EWGCCA). Consensus Document on Leukemia Immunophenotyping (1996).
  • J.J. Stewart et.al. Cytometry Part B (Clinical Cytometry) 90B: 110-116 (2016) Role of Receptor Occupancy Assays by Flow Cytometry in Drug Development. https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.21355
  • Rabinovitch, P. S and Robinson, J. P. Current Protocols in Cytometry (May 2002). Overview of Functional Cell Assays. https://doi.org/10.1002/0471142956.cy0901s21
  • O’Gorman, M. R. Clin. Lab. Med. 21 (4) 779-94. (2001) Clinically Relevant Functional Flow Cytometry Assays. PMID: 11770287