For most applications flow cytometry is used to identify cell populations and define bivariant terms of positive and negative sub-populations according to specific biomarkers, through the binding of fluorescently tagged monoclonal antibodies (mAbs). Typically, the cutoff between these populations is set relative to a control unstained population. Since the fluorescent intensity of a signal is proportional to the amount of monoclonal antibody bound to that cell target, this signal is directly related to the expression level of that target. However, for flow cytometry endpoints to be considered truly quantitative and fulfill the rigor of clinical utility, several obstacles needed to be overcome. In this blog, we explore the rationale behind quantitative flow cytometry, and the tools that are now being implemented to help achieve standardization.
Requirements of Quantitative Flow Cytometry
Quantitative flow cytometry can be defined as the measurement of the intensity of staining of a specific biomarker on or within cells. It is typically achieved using known fluorescent standards (usually beads) that enable the true quantitation of this cellular fluorescence to the derived within the designated detection channel. There are two common units used to express this fluorescence quantification- MESF (Molecules of Equivalent Soluble Fluorochrome) and ABC (Antigen Binding Capacity), and several commercially available quantification bead kits are available to establish a calibration curve for the calculation of these. These kits are composed of a mixture of beads that are either stained with specific fluorophore intensities, or that are designed to bind defined levels of conjugates. They always include a blank to set an appropriate background/negative population. There are two common forms of bead-labeling methods- direct immunofluorescence which involves incubating the beads with the relevant mAb- conjugate; and indirect immunofluorescence where the beads are stained at the secondary antibody labeling step. The selection of bead type depends largely on the sample preparation procedure. There are several key rules to follow for quantification using beads that require mAb-labeling:
- The mAbs should always be used at saturating concentrations for both the beads and the cells. Note, these may be different for the beads and the cells- so titrate your mAbs on both of these!
- The same reagents, from the same vendor, and wherever possible the same lot, should be used at the same concentration across the experiment.
- The instrument fluorescent application settings should be maintained across the acquisition of the beads and all experimental samples.
Most quantification kits come with software to generate standard curves and support the calculation of ABC or MESF. The standard curve is generated by plotting the values of the peak channels of the blank bead and labeled beads from the cytometer against the known number of fluorochrome molecules per bead type, supplied by the manufacturer. The peak value of the test sample is obtained by acquiring the sample at the same fluorescence setting as the beads, minus the peak value of the control tube.
| Bead Kit | Type of Immunofluorescence | Features |
|---|---|---|
| Quantibrite™ from Beckton & Dickenson | Direct | Used with BD’s Cell Quanti Quest software. Beads conjugated with 4 levels of phycoerythrin (PE) – designed for use with PE-labelled antibodies for estimating ABC. |
| Quantum Simply Cellular™ (QSC) from Bangs Lab | Direct | 5 bead populations, 1 blank and four with increasing levels of Fc-specific capture antibody. Label with the same detection conjugate used to stain cells. QuickCal software determines the threshold and linearity of signal to calculate ABC for any monoclonal conjugate. |
| QIFKIT™ from Agilent | Indirect | Six-bead populations were coated with different, well-defined quantities of mouse monoclonal antibodies. Cells are labeled with primary mouse antibodies directed against the target of interest. In a separate tube, cells are labeled with an irrelevant mouse antibody (negative control). Cells and QIF beads are labeled in parallel with fluorescein-conjugated anti-mouse secondary antibodies. Saturation conditions are used for all staining procedures. |
| Quantum™ MESF Beads from Bangs Labs. | Direct and Indirect | The MESF beads are surface labeled with the same fluorophores used to stain cells for flow cytometry and are used to generate a standard curve of MESF. They are also used to assess the detection threshold, resolution, and linearity of detection. When coupled with a Simply Cellular bead, these can be used to determine F:P (Fluorescence: Protein) ratio and support the conversion of MESF to ABC. Available fluorophore: Alexa fluor 488, FITC, PE, PE-Cy5, Cy5, Alexa Fluor 647, APC. |
Table 1. Features of several common quantification bead kits used in Quantitative Flow Cytometry.
Applications of Quantitative Flow Cytometry
There are several widely used quantitative flow assay kits that are approved by the Center for Devices and Radiological Health (CDRH) for clinical use (summarized in Table 2). In addition, there are many quantitative flow cytometry Laboratory Derived Tests (LDTs) with applications in the evaluation of therapeutics, along with diagnostic use and disease monitoring- several of these are described below. Most of these tests provide an MESF readout since this unit has been formally adopted by the National Institute of Standards and Technology (NIST) and National Committee for Clinical Laboratory Standards (NCCLS) as the standard measurement of fluorescence intensity.
CD34+ Hematopoietic Stem Cell Enumeration
Flow cytometric enumeration of CD34+ cells is widely employed to determine the hematopoietic stem cell (HSC) levels in cord blood-, peripheral blood- and apheresis products, and is critically used for dosing determination for transplantation. The CD34+ antigen is stage-specific and is used to identify the early stages of HSC differentiation and can be applied for the hematopoietic reconstitutive capacity of transplant products.
Figure 1. CD34+ flow cytometry gating strategy was established using International Society of Hemototherapy and Graft Engineering (ISHAGE) guidelines. This applies a sequential Boolean gating strategy to identify the population of interest, dim CD45 expression by the CD34+, SSlow HPC. Internal reference counting beads provide the standard for enumeration.
Quantifying B-cell Antigens
Quantitative flow cytometry has been applied for the characterization of B-cell chronic lymphoproliferative disorders (CLDs), through a comparison of surface markers on B-cells from healthy versus disease state samples. Quantitative flow cytometry of B-cell CLDs profiles for CD19, CD20, CD22, CD23, CD79b, and CD5, was shown to improve the immunological criteria for the differential diagnosis of CLDs. Furthermore, this approach was shown to be valuable for the differentiation of atypical chronic lymphocytic leukemia (CLL) from mantel cell lymphoma (MCL), and hairy cell leukemia (HCL) from splenic lymphomas with villous lymphocytes (SLVL) (D’Arena et. al. 2000). A similar comparison of quantitative CD35 levels on cell populations demonstrated the clinical value of decreased CD35 expression levels in CLL versus other CLDs- supporting a testing sensitivity of 81.8% and specificity of 88.4% for the diagnosis of CLL (Shi et. al. 2021).
Minimal Residual Disease (MRD) for Acute Lymphocytic Leukemia.
For patients with leukemias, the determination of minimal residual disease is important for assessment and long-term prognosis. For ALL, the immunological detection of MRD is hampered by the fact that leukemic cells represent the malignant equivalents of normal haemopoietic precursors expressing CD10, CD19, and terminal deoxynucleotidyl transferase (TdT). Quantitative flow cytometry has been demonstrated to be a useful tool in discriminating healthy versus malignant B-cell precursors with higher TdT (>100×103), and lower CD10 (<50×103) and CD19 (<10×103) molecules per cell compared with ALL blasts. These are critical quantitative differentiators since regenerating bone marrows display a significantly higher percentage of B-cell precursors than healthy donors, at the expense of the TdT-/CD10+/CD19+ population which has the potential to be incorrectly interpreted as evidence of disease relapse if TdT is not included in the analysis (Farahat et. al. 1995).
Quantifying T-cell Antigens
Normal lymphocyte subsets naturally express differing levels of some surface antigens that can assess aberrant lymphocytes a challenge for diagnostic and prognostic evaluation. Applying a quantitative approach to multiparameter flow cytometry has been important in identifying differences in antigen expression for CD3 and CD7 within healthy and disease state leukemic T-cells, for increased accuracy in diagnosis and prognostic evaluation (Ginaldi et. al. 1995).
Exosome Profiling
Quantitative flow cytometry has applications beyond immuno-profiling that extends to the interrogation of subcellular particles such as exosomes (Nolte-t Hoen et. al. 2012) and virus particles (Vazquez, D. et. al. 2018). Nanoscale cell-derived membrane vesicles are now recognized as biomarkers for several acute and chronic diseases, as well as potential therapeutic vesicles. Flow cytometry represents one of the few analytical platforms that can explore the phenotypic heterogeneity and quantification of these unique subcellular particles, and the coupling of this characterization with accurate enumeration increases the clinical relevance of exosomes for diagnostic and prognostic applications.
Cytokine Profiling for Clinical Applications
A quantitative flow cytometry approach has been described for the interrogation of the cytokine pathways involved in the immunological dysfunction in patients undergoing hemodialysis. Many of these patients show high susceptibility to infections and malignancies and display poor T-cell responses to challenges such as vaccination. Through quantitative flow cytometry, it was determined that TNF-α was significantly elevated through the upregulation of the TNF-R2 receptor (CD120b) on monocytes and T-lymphocytes in patients on renal replacement therapy (RRT). This contrasted with the IL-2R expression between RRT and healthy control cohorts which displayed no significant differences. This level of granularity in cytokine responses in patients with RRT has resulted in more targeted therapeutic approaches to managing immunodeficiency in patients receiving intermittent hemodialysis (van Riemsdijk-van Overbeeke, I. C. et. al. 2001).
| Application | Measured Parameters | Quantitative/Qualitative | Qualification Standard Used |
|---|---|---|---|
| HIV Positive Patient Monitoring | CD4/Hgb | Quantitative | CD4 count/volume |
| Immunological assessment of patients with immune deficiency | CD3+, CD3+CD4, CD3+CD8+, CD3- CD19+, CD3- CD56+ and/or CD16+, CD45+ Low SS and CD45+ |
Qualitative and Quantitative | Beads |
| Assessment of CMV-specific immune status and risk of CMV-reactivation in immunosuppressed stem cell transplant recipients. | CMV-specific CD8 MHC tetramer or dextramers | Quantitative | Beads |
Table 2. Quantitative Flow Cytometry Testing Kits that are cleared by CDRH for clinical applications.
Final Thoughts
Flow cytometry is now widely used in clinical settings, and cellular enumeration is a significant aspect of that clinical workflow. However, there are still limitations and without standardized measures of fluorescent intensity, readouts can only be expressed in arbitrary units or as dim/intermediate/bright or negative/positive. Even when implementing tools such as quantification beads, measurements made on different instrumentation formats, at different times, and in different labs, typically cannot be directly compared. FlowMetric is a member of the NIST consortium working to introduce standards into flow cytometry applications that can help address these discrepancies with the use of reference controls, standardized reagents, and measurement procedures (Wang et. al. 2008). Nevertheless, flow cytometry standardization goes beyond these factors and includes many other aspects. In response instrument manufacturers are developing simpler and more automated approaches to flow cytometry; an example of this is the Beckman Coulters Aquios CL flow cytometer that combines sample preparation with analysis on a single platform. This system also couples premixed kits for fast and accurate cellular enumeration for clinical application including T-, B-, NK-cells, and CD4+ T-cells for HIV monitoring.
Technical advances and approaches such as these are helping to expand the adoption of quantitative flow cytometry across many areas of research and discovery (Maher, K. J., and Fletcher, M. A. 2005); from the study of signaling cascades through protein phosphorylation, to cellular aging through telomere length assessment, the translation of fluorescence signals into real mass units of intensity is expanding the application of cytometry into many new and exciting areas.
References
- D’Arena et. al. Quantitative Flow Cytometry for the Differential Diagnosis of Leukemic B-Cell Chronic Lymphoproliferative Disorders. Am. J. Hematol. 64:275–281,2000.
- Shi, Y. et. al. The potential differential diagnosis value and clinical significance of CD35 expression in B-chronic lymphoproliferative disorders. Annal of Translational Medicine Vol. 9. No. 14. 2021. doi: 10.21037/atm-21-3199
- Farahat, N., et. al. Quantitative flow cytometry can distinguish between normal and leukaemic B-cell precursors. BJ Haem vol. 91, Issue 3 pp. 640-6. 1995.
- Ginaldi, L., et. al. Differential expression of CD3 and CD7 in T-cell malignancies: a quantitative study by flow cytometry. British Journal of Hematology 93, 921-927 (1995)
- Nolte-t’ Hoen, E. N. M. et. al. Quantitative and qualitative flow cytometric analysis of nanosized cell-derived membrane vesicles. Nanomedicine: Nanotechnology, Biology and Medicine vol. 8. Issue 5. Pp.712-720. 2012. https://doi.org/10.1016/j.nano.2011.09.006
- Van Riemsdijk-van Overbeeke, I. C. et. al. Quantitative flow cytometry shows activation of the TNF‐α system but not of the IL‐2 system at the single cell level in renal replacement therapy. Nephrology Dialysis Transplantation Vol. 16. No. 7. Pp.1430-5. https://doi.org/10.1093/ndt/16.7.1430
- Maher, K. J. and Fletcher, M. A. Quantitative flow cytometry in the clinical laboratory. Clinical and Applied Immunology Reviews. Vol. 5 No. 6 pp. 353-72. 2005.
- Wang L, et. al. Toward quantitative fluorescence measurements with multicolor flow cytometry. Cytometry A 2008;73:279–288