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Quantitative Flow Cytometry Measurements

Summary

Flow cytometry is a widely used technique for single-cell and particle analysis. For flow cytometry to be used in a clinical, industrial, or research setting, measurements must be made precisely and with sufficient measurement assurance. Our objective is to develop reference materials, methodology and procedures to enable quantitative measurements of biological substances such as cells, extracellular vesicles, proteins, and nucleic acids.  By providing quantitative flow cytometry measurement solutions, we ensure that researchers and clinicians can produce more reliable data, develop better drugs, and provide better treatment to patients in the clinical setting.

Description

Flow cytometry is an essential tool for basic immunological research, the clinical discovery of potential therapeutics, development, and approval of drugs and devices, disease diagnosis, and therapeutic treatment and monitoring. For example, flow cytometry is commonly used in pre-clinical and clinical trials for evaluating the safety/efficacy of drugs including engineered T-cells. In HIV/AIDS monitoring, accurate measurement of CD4+ cell counts using flow cytometry is the key to ensuring that patients receive the appropriate antiretroviral treatment (ART). However, the measurements made on different instrument platforms at different times and places often cannot be compared. Discrepancies between and among measurements introduce uncertainty in diagnostic and therapeutic decisions and impede advances in basic science. We collaborate with other government agencies, industry, academia, professional societies, standards organizations, and field experts to accelerate the standardization of flow cytometry measurements with the use of reference controls and standards and measurement procedures. 

Current Projects

1. Flow Cytometry Standards Consortium – The objective of the consortium is to collaboratively develop reference standards including biological reference materials, reference data, reference methods, and service for assigning the equivalent number of reference fluorophores (ERF) to calibration microspheres and assessing the associated uncertainties and utilities. This is the first step towards reliable quantitative measurements in flow cytometry. The ERF assignment service under the consortium has recently been expanded to extracellular vesicle (EV) and virus sized particles.

NISTSRM1934
Schematic for the ERF value assignment of a commercial calibration microsphere based on the NIST published standard operating procedure using NIST SRM 1934 and NIST Research Reference Materials, and a calibrated laser-based fluorescence spectrometer. Microspheres with assigned ERF values enable the quantitative standardization of the fluorescence intensity scale and performance characterization of flow cytometers. ERF values for a particular fluorescence channel under a specified set of instrument conditions are comparable between instruments and over time, even when different manufacturers’ calibration beads are used.

Two interlaboratory studies have been planned under the consortium [Working Group (WG) 1 and 2]. The WG1 study is aimed to measure several different sets of ERF calibration beads and an unknown biological sample to compare calibration results across different ERF beads sets, instruments and laboratories. The results of this study will establish how effective ERF calibration beads are for obtaining instrument-independent results using quantitative flow cytometry. The objective of the WG2 study is to standardize an assay on cell count and health that is important to gene and cell therapy manufacturing. The study is aimed to evaluate various assay and instrument control materials as well as assay reagents and establish an assay standard operating procedure enabling result comparability across different instruments.   

To learn more about the Flow Cytometry Standards Consortium, click here.

2. Quantification of Cells with Specific Phenotypic Characteristics – A Broad International Collaborative Effort for the Development of Human Blood Cell-based Reference Materials and Controls

(I) Accurate enumeration of cells with specific phenotypic characteristics is of critical importance in inpatient care. There are pertinent needs for cell reference materials for external measurement quality assessments in areas, such as HIV/AIDS monitoring (CD4+ cell count) and blood transfusion (CD45+CD34+ stem cell count) in clinics.  Our scientists have produced and evaluated the first international reference standard for CD4+ cell counting for HIV/AIDS monitoring (WHO BS/10.2153). Accurate measurement of CD4+ cells is the key to ensuring that patients receive the appropriate anti-retroviral treatment (ART) once their CD4+ cell count falls below 350 cells per microliter.

(II) Due to the enormous potential and the recent success of immunotherapy in clinics, there are urgent needs for cell reference materials and standardized protocols to evaluate T cell functionalities. Intracellular cytokines are crucial indicators of immune function and competence. Our scientists have generated and evaluated a cellular reference material using a freeze-dried preparation of unstimulated (NIBSC code: SS570) peripheral blood mononuclear cells (PBMCs) and phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulated PBMC (NIBSC code:15/272), obtained from healthy blood donors. A flow cytometric-based, the rapid single-step method has been developed and validated across different instrument platforms in three different laboratories for enumerating cytokine positive T lymphocytes.

Fig1p2

 

3. Quantitative Measurements of Immuno-Oncology Markers and Disease Biomarkers – (I) PD-L1 (programmed cell death 1 ligand 1) expression has been identified as a predictive diagnostic marker to select patients that may benefit from anti-PD-1 (programmed cell death 1) therapies such as nivolumab, pembrolizumab, atezolizumab, and durvalumab. Currently, there are multiple qualitative PD-L1 assays on the market, either FDA approved, or laboratory-developed tests, involving various antibodies, to assess the expression of PD-L1 by immunohistochemistry (IHC) using chromogenic methods. Due to the lack of result traceability and comparability using these clinical PD-L1 IHCs, we are working with the IHC community to use the quantification scheme established in flow cytometry and mass spectrometry to undertake the traceability and comparability issue in IHC. A calibrator system consists of a set of cell-sized glass microbeads covalently attached with fluorescein-conjugated recombinant PD-L1 protein and unlabeled peptide. Each glass microbead population is defined by a specific ratio of fluorescein-labeled recombinant PD-L1 protein and unlabeled peptide. The fluorescence intensity in the units of ERF of each microbead population is measured based on the NIST SRM 1934 under the Flow Cytometry Quantitation Consortium. Cultured cells of the human epidermal receptor protein-2 (HER2) breast cancer cell lines, e.g. MDA-MB-361, MDA-MB-453, and SK-BR-3 with different expression levels of PD-L1 that are quantified using quantitative mass spectrometry would enable the transformation of PD-L1 copy number to the calibration established by the microbead calibrators using flow cytometry. These bead calibrators serve as the calibration system for both flow cytometry and IHC. The translation of PD-L1 copy number per cell onto the glass bead calibration enables a quantitative and comparable measure of analyte PD-L1 in IHC.

PDL1
Schematic for PD-L1 quantification in IHC (right image) that is ultimately traceable to HER2+PD-L1+ breast cancer cell references with known PD-L1 copy number per cell (left picture). Glass bead calibrators coated with fluorescein-labeled recombinant PD-L1 to which anti-PD-L1 antibodies are bound serve as an essential link for the measurement traceability established using flow cytometry (left picture) to IHC (middle and right pictures).

(II)Flow cytometry has been critical for establishing identity, purity, and potency for cell therapy product manufacturing and associated data to support the approval of Biological License Applications by the U.S. FDA and the approval by the EMA. It is essential to establish B-cell reference control materials for comparable and quantitative cytometric expression analysis to assist cell therapy manufacturing and immunotherapy monitoring. Our scientists are quantifying the expression levels of CD19 on B cells in the instrument independent unit of antibodies bound per cell (ABC) as well as their respective associated uncertainties for three commercial lyophilized or dried-down PBMC preparations. The work is inspired by a consensus outcome from flow cytometry workshops that call for cell reference standards with well-characterized antigen expression and immunophenotyping profiles for advanced cell manufacturing and cell therapies. We envision that the PBMC-based materials in this study will be useful as expression analysis reference markers for quantifying disease and immunotherapy relevant B cell markers, e.g. CD19, CD20, and CD22. Quantitative measurement of these biomarkers of B-cell malignancies with high confidence is critically important for the determination of proper treatment options and regimens, e.g. switching drugs and applying a second dose of the same drug, and hence improving patient’s quality of life.

4. Development of Process Control Materials and Protocols for Reliable Measurements of Extracellular Vesicle and Lentivirus Using Flow Cytometry – Extracellular vesicles (EVs), which are biologically active lipid bilayer membranes, have been at the forefront of life science research due to their significant role in both physiological and pathological processes. Research on EV’s role in these processes has been primarily focused on exosomes (30–150 nm) or microvesicles (200–1,000 nm).  In order to rapidly analyze these materials, flow cytometry has become one of the critical characterization tools due to its high-throughput and multi-parameter analysis capabilities. However, the analysis of EVs by flow cytometry has been limited, not only due to their small sizes and weak signals from fluorescent labels but also by the limited experience level of many instrument users.  Therefore, detailed procedures to fine-tune the flow cytometer through careful calibration and APD (or PMT) setting adjustment using known control materials have become key in obtaining reliable and meaningful data. We present an example of a flow cytometry method using a CytoFlex flow cytometer. EV phenotype was characterized by comparing a general lipid membrane staining (PC5.5) with an antibody staining (Y585-PE) of multiple EV-specific membrane proteins called tetraspanins (CD9, CD63 and CD81). As expected, the A549-derived EVs are positive for both the tetraspanin stain and the lipid membrane stain. Furthermore, data-independent acquisition mass spectrometry (DIA-MS) confirmed these A549-derived EVs contained 132 of the known 138 EV-specific proteins including tetraspanins.

PC5.5 vs. Y585-PE
Left figure: Dot plot of PC5.5 vs. Y585-PE displaying background control of saline buffer with minimal signal in the right upper quadrant; Middle figure: Dot plot of PC5.5 vs. Y585-PE displaying A549-derived EVs stained with both lipid membrane (PC5.5) and tetraspanin (Y585-PE) in the gated region; Right figure: The analysis of DIA-MS data found 2392 total proteins/EV sample for A549-derived EVs of which 132 proteins match the EV-specific proteins.

5. Proteomic and Genomic Analysis of CRISPR/Cas9 Engineered Cells and Cell Stability – A commonly used genome editing system, being used for improving protein, cell, and gene therapies is CRISPR/Cas9. While this tool has great potential, long-term data about its genomic and phenotypic stability and off-target effects that may arise during the editing process are sparse. Since the entire cell can be used in a patient with cell and gene therapies, cell characterization is essential for safety. In support of the production of safe and effective CRISPR/Cas9 engineered protein, cell and gene therapies, the objective of this work is to study both on-target and off-target effects of CRISPR/Cas9 using flow cytometry on a B-lymphoblast cell line, GM24385, whose genome sequence has been well characterized. Importantly, this project enables us to extend our flow cytometry capability to measure transcriptomes and proteomes of interest simultaneously at a single-cell level.

Pic5
Left two plots: Live lymphocytes of cultured GM24385 cells are gated in a dot plot of FSC vs. SSC; CD19+ cells under live lymphocyte gate are shown exclusively in the histogram of CD19 PE. Right two plots: After CRISPR/Cas9 editing of CD19 genome to lockout CD19 expression, CD19- cells become dominant in the histogram under the live lymphocyte gate.

6. Rare Events Quantitation Using Quantum Cytometer  Cancer cell and engineered therapeutic cell heterogeneity, long recognized as an important clinical determinant of patient outcomes, is poorly understood at a molecular level, mostly due to the current limitation of rare event quantitation at a single-cell level. PCR-based approaches require extracted DNA from patient samples, resulting in average values and losing information on the cellular and population heterogeneity. Other methods such as IHC and fluorescence in situ hybridization (FISH) produce qualitative or sub-quantitative results at best. The quantum cytometer has the potential to detect rare events in a single cell in a high-throughput and quantitative manner. We collaborate with Dr. Sergey Polyakov at the Physical Measurement Laboratory of NIST to count gene copy numbers in single cells. NIST possesses 5 different viral copy numbers (VCN) Jurkat cell lines with 0 – 4 copies of Green Fluorescent Protein (GFP) reporter integrated at various genomic loci. For instance, VCN4 means 4 copies of unique sequence integrated at 4 known ectopic sites on the genome with GFP reporter of Jurkat cell, VCN0 means 0 copy (background cells), and so on. Using the unique sequence information of VCN, we are designing FISH probes to distinguish the signal intensities from various VCN cell lines with varying copy numbers. The traditional FISH analysis utilizes microscopy which severely limits the number of samples that can be analyzed and is time-consuming. We intend to utilize Flow-FISH to analyze samples in high throughput fashion by combining the power of flow cytometer with FISH analysis.  Orthogonal studies to count the number of copies are also being devised by combining CRISPR-based targeting of Quantum dots (QD) labeled guide RNAs (gRNA) to assess the integrated sequence in collaboration with Sergey’s group. The output in these experiments is expected to be through the use of QD-based flow cytometry. This method will be providing an absolute quantification through direct counting of the number of quantum dots per cell. This flow-FISH cytometric assay can be expanded in the future for simultaneous detection of rare event gene mutation and protein biomarkers at a single cell level for molecular and conventional pathology. Moreover, it will be applicable to other significant NIST programmatic areas in regenerative medicine, engineering biology, and precision medicine, such as genomic editing on and off-target counts at a single-cell level.

7. Quantitative and Traceable Serology and Neutralization Assays for COVID-19 Detailed project information is provided in the Serology and Neutralization Assays for COVID-19.

Major Accomplishments

  • Produced and evaluated jointly the first international reference standard for CD4+ cell counting for HIV/AIDS monitoring (WHO BS/10.2153). Accurate measurement of CD4+ cells is the key to ensuring that patients receive the appropriate anti-retroviral treatment (ART) once their CD4+ cell count falls below 350 cells per microliter.
  • Developed the unit of an equivalent number of reference fluorophores (ERF) for fluorescence value assignments of microsphere calibration materials.
  • Formed the Flow Cytometry Quantitation Consortium with the capability to provide an ERF value assignment service to consortium members that produce microspheres for flow cytometer calibration. The measurement services have been provided to BD Biosciences, Spherotech, a joint working group of three international professional societies, Medical Discovery Partners LLC, Beckman Coulter, and Thermo Fisher Scientific.
  • SRM 1934, Fluorescent dyes for quantitative flow cytometry (visible spectral range).
  • Research Reference Materials, Preparation and Concentration Determination of Pacific Orange, Alexa Fluor 700, and Alexa Fluor 750 Solutions for Quantitative Flow Cytometry.
  • USP Chapter <127> Flow Cytometric Enumeration of CD34+ Cells.
  • Supported the development of the WHO international standard and reference panel for anti-SARS-CoV-2 antibody and the human SARS-CoV-2 serology standard produced by NIH/NCI Serology Science Network and Frederick National Laboratory for Cancer Research.
  • Established the Flow Cytometry Standards Consortium working towards instrument standardization, cell and gene therapy assay standardization, and flow cytometric data analysis and data standards.
  • CLSI H62 Validation of Assays Performed by Flow Cytometry
  • Development and Validation of Measurement Traceability for In Situ Immunoassays
Created April 9, 2016, Updated June 1, 2022