Keywords

Key Facts About FC Standardization

Aspects to be considered during the FC standardization process include the following:

  • The choice of suitable reagents is critical in assay development: antibodies, labels, and dyes. Fixative, permeabilizing, and lysing solutions

  • Antibody titration:

    • It is recommended that an antibody titration be carried out to ensure that the antibodies are used efficiently yet still remain in excess.

  • Selectivity/specificity:

    • The assay method should identify the target analyte in the presence of other components.

  • Sample collection conditions: many matrices are suitable for FC analysis. It is essential that strict criteria for sample collection, shipment, and storage be defined.

    • Factors to be considered are the choice of anticoagulant and sample handling.

  • Whenever possible, comparison with a gold standard is recommended prior to validation.

Key Facts About FC Validation

The main parameters to be included in a validation process are the following:

  • Accuracy:

    • The accuracy of an analytical procedure expresses the nearness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. In many FC assays, this value has not been established.

  • Precision:

    • Precision is defined as the nearness of individual measures of an analyte when the procedure is applied repeatedly. Intra- and inter-assay precision should be tested.

  • Stability:

    • The stability of an analyte in a given matrix under specific conditions of temperature, time, freeze-thaw cycles, etc., should be determined.

  • Detection limit:

    • The detection limit is the lowest concentration at which the analyte can be measured.

Key Facts About FC Instruments

An important part of FC techniques is control of the cytometer. Instrument factors to be considered during the validation process include the following:

  • Calibration:

    • For proper discrimination between positive and negative populations, fluorescent microbeads of a predefined fluorescent intensity can be used.

  • Quality controls:

    • Internal and external quality controls should be used, especially when multiple instruments are employed to generate data, for example, in a multicenter study.

  • Compensation controls:

    • Compensation is an electronic calculation that removes signal overlap that the optical system cannot remove. Proper compensation is vital in multicolor assays that measure the level of expression or frequency of biomarkers, especially when the protein expression level is low. Recently, manufacturers have helped to simplify the compensation process. Compensation samples and automatic compensation should be performed once a month.

  • Gating strategies to identify cell populations correctly. Automated population identification using computational methods should be developed to minimize subjectivity.

Definitions of Terms

Biomarker

A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.

Beads

Particles (usually made of polystyrene) that can be used as stable and inert standards for flow cytometric analysis. Beads can be obtained conjugated to various fluorochromes in order to standardize fluorescence detection settings and optical alignment or to calibrate fluorescence scales. They can also be conjugated to antibodies to calibrate the scale in terms of the number of binding sites.

Compensation

Compensation involves specific software or hardware manipulations that mathematically remove fluorescence overlap to simplify multicolor data interpretation and distinguish populations on a dual-parameter histogram. Since fluorochromes have a wide emission spectrum, if these fluorochromes are excited by the same laser, there will inevitably be some overlap in the emission spectra for each fluorochrome.

Fluorescence

A form of light emitted by atoms or molecules when electrons fall from excited electronic energy levels to their lower, less-energetic ground state.

Gating

A gate is a numerical or graphical boundary that can be used to define the characteristics of particles to be included for further analysis. Gating is used to identify subsets of data or populations.

Process Validation

Establishing by objective evidence that a process consistently produces a result or product meeting its predetermined specifications.

Validation

Confirmation by examination and the provision of objective evidence that the particular requirement for a specific intended use can be consistently fulfilled.

Introduction

Flow cytometry (FC) is a powerful, rapid, and cost-effective technique for monitoring multiple parameters. In this technique, cells are suspended in a fluid flow one by one through a focus of excitation light, which is scattered in patterns characteristic of the cells and their components; cells are frequently labeled with fluorescent markers so that light is first absorbed and is then emitted at altered frequencies. The size and molecular characteristics of individual cells are then measured by a sensor detecting the scattered or emitted light; this technique allows tens of thousands of cells to be examined per minute, and the data collected is processed by a computer. Some flow cytometers are also “cell sorters,” instruments that are able to selectively deposit cells from particular populations into tubes or other collection vessels. These selected cells can then be used for further experiments, cultured, or stained with another dye/antibody and reanalyzed (Givan 1992).

Flow cytometers have become inestimable instruments for cell phenotype and activity analysis both in research and in clinical laboratories. Since the 1980s, the use of FC has progressively spread from basic research to clinical diagnostic laboratories. The modern flow cytometer can offer automated high-throughput multiparameter analysis of cells (Shapiro 2003). Specific cell populations can be characterized and identified based on cell-surface antigens and intracellular and nuclear antigens. The list of parameters that can be measured by FC technology is constantly expanding. The number of useful antibodies has also progressively increased. In parallel, the number of antigens that can be assessed in a single measurement has risen dramatically owing to the availability of new multicolor digital instruments and a greater number of compatible fluorochromes providing more precise characterization of the different cellular subsets.

However, the high complexity of the panels of reagents involved has meant that greater expertise, as well as standardization of the methodologies used, is needed to correctly interpret the data obtained and minimize variability and subjectivity in the analysis. Recent progress in automated population identification using computational methods has provided an alternative to traditional gating strategies. Automated identification systems could potentially help to identify rare and hidden populations. Aspects such as reagent clones, fluorochrome conjugates, and optimally designed antibody combinations are critical issues in this methodology. Furthermore, robust protocols are needed to aid the selection of the most appropriate combinations of fluorochromes and fluorochrome-conjugated reagents in a panel, sample preparation techniques, and standard operating procedures (SOPs) to establish instrument settings, as well as the choice of the most adequate strategies for data analysis. There is also a need to introduce internal and – especially – external quality controls to control and minimize intra- and interlaboratory variability.

Because of the current requirements of the European Medicines Agency and the US Food and Drug Administration for biomarker (or a biomarker panel) validation, the methodologies involved must also be standardized and validated. As the demand for flow cytometry-based biomarkers increases in drug development and clinical monitoring, it is crucial to standardize guidance for the development and validation of FC biomarker assays.

FC offers several advantages over other similar technologies involved in biomarker analysis, such as ELISPOT or ELISA. FC provides simultaneous information about phenotypic and functional characteristics of the cells. For example, to measure the cellular expression of cytokines, ELISA measures the total amount of the secreted cytokine (without identifying which cells are synthesizing it) and ELISPOT allows the detection of a single cell that secretes a specific cytokine. FC allows individual characterization of large number of cells and can characterize the cells on the basis of the cytokine they express, rather than on the basis of their surface markers, due to the possibility of multicolor staining that can demonstrate exclusive or mutual co-expression of different cytokines in individual cells. There is increasing interest in determining which types of cell subpopulations synthesize specific cytokines, because the cell subset can determine the type of immune response (e.g., effector vs. regulatory T-cell response). Therefore, FC enables the quantification of large numbers of cells and assessment of their subset distribution, activation status, cytokine production profile, and other cellular functions. Furthermore, FC allows functional testing to help elucidate the mechanisms of action by which diseases develop as well as the mechanisms of action of drugs to be used as therapy (immunosuppressants, antitumor agents, etc.).

FC could be applied in many different fields, including oncology, hematology, transplantation, autoimmunity, tumor immunology, chemotherapy, etc. Therefore, this technique can be widely applied in the evaluation of new biomarkers. By using this technology, the effects of drugs on cell phenotypes and intracellular pathways can be monitored in multiple cell types within the same sample.

The discovery of a biomarker can be summarized as a two-stage process: in the first stage, molecules or genes are identified as candidate biomarkers; in the second stage, the candidate biomarkers undergo clinical validation. Between these two stages there may be a number of intermediate steps aimed at increasing the maximum assay utility. The main stages in the field of biomarker research are discovery, prioritization, verification, and clinical validation for implementation in routine clinical practice (Fig. 1). In the first stages of the discovery process, “omics” sciences are mainly involved through the genomic, proteomic, transcriptomic, and metabonomic analyses carried out by mass spectrometry (Maldi-TOF, LC/MS/MS, etc.), array genes, and mRNA/miRNA analysis, while in the prioritization phase, verification and clinical validation processes using FC, ELISA, and ELISPOT methods play an important role. The omics revolution has enabled the development of new experimental and analytical tools with which to study biological processes by measuring large numbers of molecular components; however, these new techniques will need further development and are not ready to be introduced into clinical practice. In the clinical validation of biomarkers, there is a need for standardized, precise, and robust methodologies that must at the same time be easy to use before they can be introduced into routine clinical practice. FC is a useful tool in this process.

Fig. 1
figure 1

Role of FC in the different stages of the process of new biomarker discovery. The main stages in biomarker research are discovery, prioritization, verification, and clinical validation for implementation in routine clinical practice. In the first stages of the discovery process, “omics” sciences are mainly involved, while in the prioritization, verification, and clinical validation process, methods such as FC, ELISA, and ELISPOT, which are easier to implement in routine practice, become more important

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If a biomarker is defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention” (National Institutes of Health [NIH] biomarker), ideally a biomarker should have the following characteristics:

  1. 1.

    Safe: the biomarker is noninvasive or has only minimal adverse effects.

  2. 2.

    Efficient: the biomarker is present and measurable early in the course of the treatment.

  3. 3.

    Measurable: the biomarker is reproducible and, if possible, quantitative.

  4. 4.

    Predictive: the biomarker shows a good correlation with changes in a defined endpoint.

In addition, biomarkers reflect (adverse) events and may thus address the safety of some treatments. In clinical development, the synergism and additive effects of combination therapy could be measured, and the systemic effect of locally administered compounds could be evaluated.

Potential predictive biomarkers clearly need to be validated using standardized operating procedures in distinct cohorts of patients before being integrated into routine clinical practice. The methodology must be robust and sufficiently reproducible to guarantee that the observed tendency in a biomarker can be related to a clinical outcome and not to imprecise methodology.

A single biomarker will probably not be sufficient to reflect all the complexities associated with biologic or pathogenic processes, or pharmacologic responses to a therapeutic intervention and a panel of distinct biomarkers will probably be needed. FC is a useful tool for this propose, because it allows multiple biologic parameters to be evaluated simultaneously and in the same sample.

Flow Cytometry in Biomarker Research

FC is recognized as an important tool in the field of the biomarker research. Because most tissues can be analyzed by FC, this technique could be applied in many different fields of research. FC allows analysis of cell subsets and their complex interplay in immunological and biological processes. However, there is a need for standardization, regulation, and validation of multiparametric FC assays. The impact of the biological matrix and the type and timing of stimulation, instrument setup, and data analysis are important considerations in the implementation of these techniques in biomarker research, in multicenter clinical trials, and subsequently in routine clinical practice (Maecker et al. 2010).

Some factors impede the widespread use of FC in clinical trials. For this methodology to be effectively standardized, a series of variables have to be taken into account, including sample handling, instrument setup, and data analysis (Table 1). Importantly, FC requires investment in training and qualified personnel, as well as in the use of the appropriate hardware and software tools to ensure the production of accurate data, which are not always available in all laboratories. Other requirements are the existence of good internal and external quality controls to evaluate sample processing and assay performance to minimize intra- and interlaboratory variability. Preserved whole blood samples with a limited phenotype that are stable for several weeks are commercially available and can be used in ambient sample analysis. Harmonization guidelines enable objective interpretation and comparison of results across clinical trials, which are necessary steps in biomarker identification and validation .

Table 1 Aspects to consider in the development and implementation of FC assays
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  • Sample handling: Many different biological matrices are subject to FC analysis: whole blood, fresh or frozen peripheral blood mononuclear cells (PBMC), cell lines, tissues, etc. It is essential that strict criteria for sample collection be defined. Treatment of the sample during and after collection is crucial. All samples must be collected under the same conditions of temperature and anticoagulant (e.g., EDTA is not suitable for functional assays requiring free calcium), if required (Kumar and Satchidanandam 2000). Whole blood is usually considered the first choice of sample for FC assays. Methods for isolating cells (e.g., lymphocytes) could cause selective loss of different cell populations and can disturb normal cell-cell interactions. In addition, whole blood assays are usually faster and require smaller samples than methods that rely on purified cells. However, because whole blood samples have limited stability and must be processed fresh, selecting an appropriate sample collection tube and shipping and storage conditions is of the utmost importance. Indeed, many assays are still performed with PBMC or cell lines because these samples can be frozen and then processed.

    Some functional assays require cell stimulation before FC analysis (e.g., some immunophenotyping, phosphoflow, intracellular cytokine staining, proliferation, etc.). The stimulus media, the source and lot of stimulation reagent, and the stimulation time and type can all influence the degree of activation (Maecker et al. 2005). This is an important point to consider. In theory, there are two ways to stimulate cells: by a nonspecific stimulus or by a specific stimulus. For example, in transplantation, a specific stimulus (with donor cells) is obviously preferable to determine the specific response to the donor. However, obtaining donor cells can sometimes be difficult; additionally, when this type of activation is used, only some T-cell clones will expand, and, in the case of intra-lymphocytary cytokine expression assays, some technical difficulties hamper their detection. Moreover, anergic patients show a very mild response to alloantigens. In contrast, a nonspecific stimulus estimates the inherent responsiveness of the recipient T cells to polyclonal activation. Therefore, this is an important aspect to take into account in FC evaluation of possible biomarkers.

  • Instrument setup: Instrument monitoring is an important part of the method. The data produced must be accurate, reproducible, and comparable between instruments and laboratories. Instrument calibration based on fluorescent microbeads of a predefined fluorescent intensity can be used to measure the capacity of the instrument to resolve negative and positive populations. For optimal instrument setup and data analysis, photomultiplier tube voltages must be established that maximize resolution sensitivity. To do this, a convenient method is Cytometric Setup & Tracking (CS&T) beads from BD Biosciences (San Jose, CA). CS&T directs the initiation of a baseline optimization procedure; for subsequent uses, the software adjusts photomultiplier tube voltages to reproduce this baseline setting. To run daily measurements, a performance check should be carried out daily; a proper compensation is vital in multicolor assays that measure the expression or frequency of biomarkers, especially when protein expression is low; manufactures have recently helped simplify the compensation process, and compensation samples and automatic compensation should be performed once a month; fluorescence minus one controls must be included in all experiments. If these controls do not take into account background staining, estimated by using isotype controls, the total number of cells (events) collected for each sample must be the same. Finally, to control antibody staining, red blood cell lysis, instrument setup and performance, and data analysis all together, BD Multi-Check Control (Becton Dickinson San Jose, CA), a stable whole blood control with assigned values that can be used to monitor the immunophenotyping process, could be useful as an intra-laboratory quality control. Assay controls are essential in the development of FC assays (not to be confused with quality controls), to guide the analyst to implement proper gating strategies to correctly identify cell populations.

FC is a highly versatile technique that allows the performance of different types of assay (Table 2):

Table 2 Types of flow cytometry assays and field application
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  • Immunophenotyping: analysis of particular cell subsets using antibodies against specific cell-surface molecules.

  • Intracellular cytokine staining: evaluation of de novo intracellular cytokine synthesis using a protein transport inhibitor and permeabilization methods from specific cell types.

  • Phosphoflow assays: measurement of phosphorylated epitopes of intracellular proteins using permeabilization methods and measurement of target drugs to allow evaluation of individual treatment response.

  • Cell proliferation: in vitro cell proliferation using fluorescent dye that is passed onto daughter cells at cell division to following rounds of cell proliferation.

  • Apoptosis: detection of porous cell membranes by uptake of fluorescent dyes that are normally impermeable.

  • Cell cycle assays: analysis of the frequency of cells in each cell cycle phase through the use of fluorescent intercalating DNA dyes.

  • Flow cytometric cell sorting: rapid isolation of pure populations of cells or particles with a desired set of biological characteristics. These populations are then available for morphological or genetic examination, as well as functional assays and therapeutics.

Immunophenotyping

Immunophenotyping is a useful tool in oncology and is mainly carried out in clinical diagnosis laboratories. It is especially relevant to the diagnosis of various hematologic neoplasms and leukemia. Indeed, this technique is currently one of the main pillars for the diagnosis and classification of leukemia and lymphoma. The list of clinically useful antibodies has progressively increased, leading to definition of complex immunophenotypic profiles (Maecker et al. 2012). In parallel, the number of antigens that can be assessed in a single measurement has increased dramatically owing to the availability of new multicolor digital instruments and a greater number of compatible fluorochromes. This has facilitated more precise identification and phenotypic characterization of specific populations in different research fields.

Immune monitoring analysis of T-cell markers in patients infected with HIV helps in the evaluation of the disease progression. Tuaillon et al. (2009) demonstrated that CD38 is of particular interest as it is a feature of activated T-memory cells, a central factor in HIV pathogenesis, and high expression of this marker correlates with a faster decrease in CD4+ T-cell counts and increase in viral load.

In renal transplantation, Xu et al. (2012) showed that immunophenotyping T-lymphocyte infiltrations in surveillance renal biopsies during the early post-transplantation period could predict acute rejection (AR) and survival. FOXP3 and Granzyme B seem to be sensitive and specific markers of regulatory and cytotoxic phenotypes, respectively. Patients with a cytotoxic phenotype had higher risk of AR, poorer renal function, and worse graft survival.

B cells are also immunophenotyped by FC as potential biomarkers. For example, B cells are being assigned an increasingly important role in the pathogenesis of chronic graft-versus-host disease (cGVHD). Kuzmina et al. (2013) recently reported that immunophenotyping monitoring of CD19 + CD21low B cells may serve as a potential diagnostic biomarker for the early onset of bronchiolitis obliterans syndrome, a progressive and often fatal complication after allogeneic hematopoietic cell transplantation, thus enabling this syndrome to be distinguished from respiratory tract infection and other organ manifestations of cGVHD of the lung, which could allow improved patient outcome. In transplantation, many studies are focused on the immune characterization of tolerant patients, defined as graft acceptance without functional impairment and sustained for years in the absence of chronic immunosuppression. Studies conducted in tolerant liver and kidney recipients showed a significant increase in both the absolute cell number and the frequency of total B cells, particularly activated, memory, and early memory B cells characterized by FC as CD19+IgD−CD38+/−CD27+. These increased B-cell numbers were associated with a significantly enriched transcriptional B-cell profile (Pallier et al. 2010). Martínez-Llordella et al. also described significantly greater numbers of circulating potentially regulatory T-cell (Treg) subsets (CD4+ CD25+ T cells and Vdelta1+ T cells) in tolerant liver recipients than in either non-tolerant patients or healthy individuals (Martínez-Llordella et al. 2007).

Immunophenotyping analysis in systemic lupus erythematosus (SLE) patients is gaining importance. Subasic et al. (2012) showed that the number of TCR molecules on the T-cell surface of SLE patients is lower than normal condition, and otherwise for these receptors CD molecules make specific connection. SLE phenotypes are characterized by double CD negativity (CD3+/−, CD4−) caused by an abnormal level of IL-2 and IL-17. T lymphocytes usually have alpha-beta and gamma-delta T-cell receptors (TCR), but, in SLE patients, a lower number of gamma-delta TCR molecules are characteristically detected in peripheral blood specimens.

Intracellular Cytokine Staining (ICS)

The pleiotropic activity of cytokines is well known. Many cytokines also seem to have paradoxical functions and to respond differently, depending on their concentrations and the microenvironments in which they are released, the cell types present in the vicinity, the timing of production, and the stage at which they are produced during an immune response.

ICS combined with immunophenotyping can be used to determine whether a specific cell population is functionally responsive to a particular agent through measurement of de novo cytokine synthesis within the Golgi apparatus (Fig. 2).

Fig. 2
figure 2

IFN-γ, IL-2, and IL-17 expression in stimulated T cells from whole blood. Heparinized blood (2 ml) was incubated for 4 h at 37 °C, 5 % CO2 with 25 ng/ml PMA and 1 μg/ml ionomycin in the presence of 10 μg/ml BFA or with BFA alone in the case of unstimulated samples. Activation protocol efficiency was evaluated in terms of CD69 expression (>95 %). A total of 20,000 CD3+ cells were acquired in each sample. Because activation with PMA + Io has been reported to downregulate CD4 expression, CD4+T cells were approximate to CD8-negative-CD3-positive T cells. Histograms were shown to correspond in this case to CD4+ and CD8+ activated cell expression of IFN-γ, IL-2, and IL-17

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This methodology is widely used in different fields. In transplantation, it has been reported that cytokine production and secretion could be modified by immunosuppressive drugs, as well as during the rejection process. Some cytokines, such as interferon (IFN)-γ, interleukin (IL)-2, and IL-10, have been identified as candidate biomarkers that correlate with graft outcome and personal response to immunosuppressive agents. Both CD4+ and CD8+ T cells participate in AR, although the rejection response is mediated mainly by activated CD8+ T cells (cytotoxic T cells), which infiltrate the graft at the time of rejection. Interest in determining which types of cell subpopulations synthesize specific cytokines is growing because the cell subset can determine the type of immune response (effector vs. regulatory T-cell response) (Benítez and Najafian 2008). FC monitoring of the frequencies of CD4+ and CD8+ effector T-cell populations and their functional role in producing inflammatory cytokines such as IFN-γ and IL-2 could be highly useful to determine the immune response to the graft in transplant recipients.

Boleslawski et al. (2004) proposed evaluation of %CD3+-CD8+-IL-2+ expression as a surrogate marker to identify patients at high risk of AR. In that study, 21 de novo liver transplant recipients treated with calcineurin inhibitors were involved and intracellular IL-2 quantification in CD8+ T cells were followed up for 6 months after transplantation. The authors found that intracellular IL-2 expression in CD8+ T cells before transplantation was closely related to the onset of AR. These results strongly correlate with those observed by Akoglu et al. (2009), who found that IL-2 expression in CD8+ T cells correlated with Banff score during organ rejection in adult liver transplant recipients. In a study of immunosuppression drug weaning in stable long-term liver transplant recipients, Millán et al. showed that soluble IFN-γ and intracellular IFN-γ and IL-2 were significantly increased in patients who rejected, suggesting that these biomarkers may prove useful to identify patients at high risk of AR (Millán et al. 2010). In addition, this group have also observed that pre- and post-transplantation evaluation of intracellular expression of IFN-γ on CD4+ and IFN-γ and IL-2 on CD8+ T cells could identify de novo liver transplant patients with a high risk of AR and high susceptibility to immunosuppressive treatment (Millán et al. 2013). Furthermore, several studies in kidney transplant recipients have shown that high frequencies of donor reactive memory T cells are associated with increased IFN-γ production, a high risk of AR in the early post-transplantation period, and poorer first-year graft function (Nickel et al. 2004; Kim et al. 2007; Bestard et al. 2008).

ICS is frequently compared to competing platforms such as ELISPOT. Both assays measure cytokine production, but ICS has clear advantages over ELISPOT: ICS allows distinct cytokines and cell-surface markers to be analyzed simultaneously, demonstrating not only functional but also phenotypic characteristics of the cells. This technique can acquire information on thousands of cells in a very short period. In addition, the introduction of techniques based on FC allows different biomarkers to be evaluated simultaneously in the same assay (e.g., IL-2 and IFN-γ). Furthermore, the ELISPOT technique requires PBMC to be isolated, causing selective loss of different cell populations and disturbing normal cell-cell interactions; measurement of intracellular cytokines using whole blood as a biological matrix is faster and requires smaller samples.

In rheumatoid arthritis, Walter et al. (2013) showed that CD4+CD45RO+CD25+CD127low Treg cells could be induced to express proinflammatory (IL-17, IFN-γ, TNF-α), as well as anti-inflammatory (IL-10), cytokines upon interaction with activated monocytes, suggesting that cytokines expressing Treg cells at sites of inflammation may still exert potent immune suppression. A study by van Roon et al. (2007), which evaluated IL7Rα expression (CD127) on CD4 T cells and CD14 monocytes/macrophages and intracellular TNF-α in patients with rheumatoid arthritis, suggested that IL-7 is an important inducer of T-cell-dependent TNF-α production in rheumatoid arthritic joints. In these diseases, anti-TNF-α treatment is used as an anti-inflammatory drug and IL-7 may be an important proinflammatory mediator; therefore, detailed analysis of the role of IL-7 in the immunopathogenesis of rheumatoid arthritis and other rheumatic diseases may lead to novel treatment strategies.

ICS is also employed in research into biomarkers of HIV. FC has been used to identify and characterize TH17 CD4+T cells in the cervical mucosa, which may play a pivotal role in transmission as this cell population appears to be particularly susceptible to HIV infection (McKinnon et al. 2011). In addition, antigen-specific production of IL-2 by CD4+T cells, either alone or in conjunction with IFN-γ, correlates with viremia control and with reduced disease progression (Nomura et al. 2006; Younes et al. 2003).

In pulmonary disease, ICS could be helpful as a diagnostic tool. DeLuca et al. (2012) showed that T cells producing IL-17 can be found in the lungs of respiratory patients in the absence of ex vivo stimulation, making IL-17 a good candidate marker of specific diseases of the lung.

Phosphoflow

Phosphoflow, a new approach, combines immunophenotyping with a functional assay by examining intracellular changes in cell-signaling pathways, such as phosphorylation of transcription factors or kinases (Schlessinger 2000). It is based on the premise that the phosphorylation state of a particular protein correlates with its biological status.

This technique is relatively easy to implement in clinical trials, because whole blood samples can be fixed in a commercial phosphoflow buffer and frozen before being shipped to the laboratory. Recent advances in the production of new phospho-specific antibodies against particular phospho-epitopes on proteins have allowed this methodology to be used to study signaling in cells that are not accessible with other biochemical techniques. Antibodies and reagents for tracking both tyrosine-phosphorylated and serine/threonine-phosphorylated signaling intermediaries in key immune signaling pathways have been developed and are now starting to be applied in a wide variety of both preclinical and clinical studies on lymphocyte responses, as well as on the functioning of cancer cells and virally infected cells.

In the FC platform, these phospho-specific antibodies are coupled to fluorophores to allow fluorescent detection and the simultaneous performance of multiple analyses. It is clear that measuring intracellular antigens is a not simple process; the approach requires extensive optimization of both the protocol and reagents used. First, the phospho-epitopes will be accessed for antibody binding; the location of the epitope within the cell may limit its accessibility. Nuclear antigens may require different fixation and permeabilization techniques from antigens in the cytosol or at the plasma membrane. It is also crucial to address the stability of phospho-epitopes in staining buffers to avoid degradation during the protocol. The selection of specific antibodies is a critical point. It is important to find those that stain the antigen of interest most efficiently and specifically. Perhaps one of the most difficult technical aspects is to maintain surface staining and scatter properties. The balance between surface and intracellular epitopes must be kept in mind.

In organ transplantation, phosphoflow has been used to generate a potential clinical assay for measuring the pharmacodynamic effects of mTOR inhibitors in heart transplant patients. S6 ribosomal protein (S6RP) is a downstream molecule of the mTOR-signaling pathway and is activated through phosphorylation by p70 ribosomal protein S6 kinase 1. The effect of mTOR inhibitors such as sirolimus or everolimus suppresses the phosphorylation of S6RP, and therefore S6RP should be a candidate target to evaluate the pharmacodynamic effects of these drugs on T-cell activation. Dieterlen et al. (2012) demonstrated that phosphoflow analysis revealed that sirolimus suppressed p-S6RP in human T cells in a dose-dependent manner with a half-maximal inhibitory concentration (IC(50)) at 19.8 nM and a maximal inhibitory effect (I(max) %) at 91.9 %. Other immunosuppressive agents, such as cyclosporin A, mycophenolic acid, and dexamethasone, are not able to inhibit mTOR-related S6RP phosphorylation. Therefore, these authors propose that personal treatment response could be identified through detection of p-S6RP by phosphoflow cytometry assay, as a method to specifically measure sirolimus- and everolimus-induced inhibition of T-cell function.

Expression of phosphorylated S6RP in cardiac biopsies has been demonstrated to correlate with antibody-mediated rejection in transplant recipients (Lepin et al. 2006).

This technique has also been used in clinical trials of metastatic melanoma. The phosphorylation state of various signaling proteins associated with the T-cell receptor was measured in CD4+ T cells and CD14+ monocytes (Comin-Anduix et al. 2010). A number of STAT family members were consistently modified in the two cell types.

These types of studies could elucidate new patterns of activated signaling pathways providing new targets for novel drugs, as well as new predictive biomarkers for treatment response.

The phosphoflow platform could be a useful tool in research into new biomarkers, such as cytokines or growth factors. Because nearly all cytokines signal through JAK-Stat pathways, analysis of Stat phosphorylation is critical to understanding how cytokines exert their effects and how they modulate gene transcription.

Another application of phospho-specific flow cytometry is in profiling disease states via their signaling status and response to particular compounds. Correlation of phospho-epitope signatures to the progression of a disease may help in the development of therapies tailored to patients in the early or late stages of a disease. For example, several tyrosine kinase receptors, including Flt-3, PDGF-R, EGF-R, and HER2, have been correlated with disease severity and prognosis in leukemias and breast cancer and are targets of drug therapy (Drevs et al. 2003).

Cell Cycle, Cell Proliferation, and Apoptosis Assays

FC using fluorescent DNA intercalating dyes, such as propidium iodide, annexin V 5-bromodeoxyuridine (BrdU), Draq5, etc., is able to evaluate the cell DNA content and the cell cycle phase in which it is found. In addition to determining the relative cellular DNA content, FC also enables the identification of cell distribution during the various phases of the cell cycle. Many drugs are developed with the aim of acting on the different phases of the cell cycle, particularly in oncology. FC has become established as a useful method to determine the relative nuclear DNA content and percentage of cycling cells of biological specimens. However, in cell cycle analysis, it is important to collect cells at the proper rate (e.g., to detect a good signal in G2/M, the rate will be below 1,000 cells per second) (Nunez 2001).

In cancer cells, apoptosis is deregulated and resistance to apoptosis has been correlated with the metastatic process. Relja B et al. reported that simvastatin is effective in inhibiting cell growth and also induces apoptosis in hepatocellular carcinoma (Relja et al. 2010).

Flow Cytometric Cell Sorting

The classification and separation of one cell type or particle from others is a fundamental task in many areas of science, as well as in biomarker research. Several techniques are available for this task; FC cell sorting provides flexible separations based on multiple parameters. It permits selections based on various levels of fluorescent, rather the complete presence or absence of the fluorescent.

The identification and isolation of specific cell populations by FC cell sorting is a useful tool in research, because it allows functional assays to be performed with these subsets, enhancing understanding of the real role of these populations in vivo, as well as their mechanism of action. San Segundo et al. used this technique to isolate T-regulatory cells in order to demonstrate their real regulatory function in mixed lymphocyte reactions and suppression assays (Fig. 3) (San Segundo et al. 2010). In cancer research, a special type of cancer cell – the cancer stem cell (CSC) – has been identified and characterized for different tumors. CSCs may be responsible for tumoral recurrence (Burkhard et al. 2012). Analysis of CSCs by multiparametric FC allows the simultaneous analysis of different cellular features with high reliability. Moreover, it enables the separation of living cells on the basis of marker expression or functional properties by fluorescence-activated cell sorting. A major advantage of this technique is its ability to isolate rare cells, which is a prerequisite for identifying small cell populations within the tumor bulk.

Fig. 3
figure 3

Representative flow cytometry profile of CD4+CD25high T cells (red dots in first dot plot). The cells show almost 100 % staining for FOXP3 (red dots in left dot plot), CD27+/CD127low (in the middle dot plot), and CD45RO+/CD62L+ (in the right dot plot)

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Flow Cytometry Limitations

Despite the advances in FC made in the last few years, this technique continues to have several limitations: one of the most important is a lack of standardization in assay and instrument setup, although numerous efforts have been made to fill this gap (Kalina et al. 2012; Wu et al. 2010; Maecker et al. 2010); standards and quality controls are also lacking for the analysis and reporting of flow data, and efforts should be made to develop automated population identification using computational methods in order to minimize subjectivity. Effective cross-training among analysts is important to generate similar assay data. The number of parameters per cell that can be measured simultaneously is limited by the number of detectors and only 6–12 color experiments are routinely performed in most laboratories. The implementation of FC in routine practice in comparison with other techniques such as ELISA (which is easier to implement and there is a greater number of validated kits for this technique) could be more difficult because analysis times may be longer and therefore the clinical response time may also be longer, especially in patients with diseases and treatments that can reduce the number of cells, thus increasing the time of acquisition and final analysis. If this methodology is used in functional assays, when the biological matrix of choice is usually whole blood because this is the matrix that best preserves physiological conditions, the samples will be processed fresh, which involves the laboratory in considerable logistic organization. Furthermore, in many cases, the rapid preparation and shipping of the samples required is not translated into rapid results for the clinician, as some of the techniques based on FC require several days for analysis. Table 3 summarizes the pros and cons of FC.

Table 3 Flow cytometry assay: pros and cons
Full size table

Advances and Future Directions

FC is under constant development and innovation. The most recent innovations combine mass spectrometry and cytometry, giving rise to machines able to analyze 45 different parameters compared with the 10 that can be analyzed by current flow cytometers (Ornatsky et al. 2010; Bandura et al. 2009). This is what is known as CyTOF® (DVS Sciences) and permits real-time quantitative analysis of individual biological cells or other microparticles. Briefly, this instrument uses isotopes of elements to label antibodies instead of the traditional fluorophore approach. After incubating the cells with antibodies, the sample is nebulized and the ions that were associated with the cell can be analyzed by mass spectrometry. This new methodology could be useful for biomarker discovery and clinical validation.

The use of FC in the field of biomarker and drug discovery and evaluation is a challenge. It is a prevalent technique in many clinical trials. There is a need to develop mathematical algorithms able to predict clinical evolution/disease progression based on the FC measurement of biomarkers. The development of new computational models for rational criteria in the interpretation of biomarker values from different centers will reduce variability in interlaboratory analysis and interpretation of the results. Correct validation of biomarkers is essential. Publication of practice guidelines for the development and validation of FC assays is a requirement (Herzenberg et al. 2006). Changes that are observed in a specific biomarker must be associated with the patient's clinical course and not with the inherent variability of the methodology.

In addition, combinations of this kind of biomarker with imaging biomarkers (PET, transient elastography, ultrasound with echo enhancer, etc.) could be useful to determine disease prognosis or treatment response. Because these biomarkers are noninvasive, they offer some advantages such as a reduced risk of sampling and severe adverse events (e.g., biopsy in transplantation), increased patient comfort, and lower cost.

Currently, there is no intelligent analysis of pharmacokinetic and pharmacodynamic data (drug concentration vs. drug effect or biological activity) and pharmacodynamic and clinical evolution . New biostatistics models should be developed to establish the most appropriate correlation between biomarkers, drug effect, and clinical outcome that would allow personalization of patients’ treatment.

There is a clear need for the implementation of new therapeutic approaches based on combined biomarker panel measurements to optimize patient care.

Potential Applications to Prognosis, Other Diseases, or Conditions

Panels of biomarkers are increasingly being used for diagnosis and determination of prognosis. The use of multiple biomarkers increases the certainty of diagnosis and predictive power for prognosis. The clinical application of FC is very extensive. FC is recognized as a key technique in the diagnosis of hematological malignancies (Brown and Wittwer 2000). Immunophenotypic analysis is critical to the initial diagnosis and classification of acute leukemia, chronic lymphoproliferative diseases, and malignant lymphomas since most of the current therapies often depend on antigenic parameters. Furthermore, immunophenotypic assays provide prognostic information not available with other techniques and allow monitoring of the clinical course of patients after chemotherapy. Another area where FC plays an important role in diagnosis and prognosis is in immunodeficiency diseases such as human immunodeficiency virus (HIV) infection. The enumeration of the absolute number of CD4+ T cells by FC together with HIV RNA levels by molecular techniques is critical for the diagnosis and prognostication of HIV infection, as well as for the management of patients receiving antiviral treatment (Hengel and Nicholson 2001).

In oncology, a variety of FC techniques have been explored to measure cell viability and apoptosis with a view to design drug treatment protocols to improve the accuracy of these drugs. New drugs such as monoclonal antibodies directed against CD25, CD20, CD52, CD45, etc., are being used (White et al. 2001). In this context, pretreatment analysis by FC is critical to confirm that the antigen is expressed by the aberrant cells, and during and after the treatment, FC is used to verify binding of the antibody and to monitor the efficacy of tumor cell eradication.

In solid organ transplantation, clinical applications of FC include pre-transplant cross-matching, HLA antibody screening, and post-transplantation antibody monitoring (Horsburgh et al. 2000; Kirmizis et al. 2012). FC has also become a useful tool to evaluate the pharmacodynamic effect of immunosuppressive therapy in order to determine the real biologic effect of specific drugs or drug combinations in the recipient. The upregulation of cytokine production and activation of surface receptors of T cells lead to T-cell proliferation, a key step during AR (Millán et al. 2013; Carey et al. 2007).

FC has been shown to have applications in microbiology (Álvarez-Barrientos et al. 2000). This technique allows single- or multiple-microbe detection (bacteria, viruses, etc.) in an easy and rapid manner. In addition, FC enables the development of quantitative procedures to assess antimicrobial susceptibility and drug cytotoxicity, as well as to evaluate different responses to antimicrobial agents.

The new generation of flow cytometers and the incorporation of innovative bioinformatics software ensure the use of FC as an indispensable tool in research into new biomarkers and their validation.

Summary Points

  • This chapter focuses on the use of flow cytometry as a platform for biomarker discovery and clinical validation.

  • Flow cytometry is used to identify and separate different types of cells based on detecting and measuring the fluorescence emitted with a laser light beam.

  • Flow cytometry has wide applications in the analysis of proteins, cytokines, and surface antigens synthesized and expressed in specific subsets of cells involved in effector and regulatory activity of immune response.

  • Multiparametric flow cytometry assays need to be standardized, regulated, and validated before they are implemented in biomarker research, in multicenter clinical trials, and subsequently in routine clinical practice.

  • Some panels of biomarkers monitored by flow cytometry may predict clinical course and individual therapeutic drug response .