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, viruses, proteins, and nucleic acids.  By providing quantitative flow cytometry measurement solutions, we ensure that researchers and clinicians can produce more reliable data, develop better engineered products and drugs, and provide better treatment to patients in the clinical setting.

Description

Flow cytometry is an essential tool for basic biotechnological and 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.

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 conducted 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.

 

3. Quantitative Measurements of Immuno-Oncology Markers and Disease Biomarkers 
(I)  Due to the lack of result traceability and comparability using clinically approved immunohistochemistry (IHC) kits for many important solid tumor biomarkers such as HER2, PD-L1, ER, PR, Ki67, and ROS1, we are working closely with the Consortium for Analytical Standardization in Immunohistochemistry and IHC community to develop a traceable and comparable measurement system. Microbead calibration standards are critical components of the measurement system and are made of fluorescein-labeled recombinant marker proteins that are covalently conjugated to microbeads. For each oncology marker, a microbead calibration standard consists of a set of cell-sized microbeads with different fluorescence intensity populations. Each microbead population is defined by a specific ratio of fluorescein-labeled recombinant marker protein and unlabeled protein. The fluorescence intensity of each microbead population in the unit of ERF is measured based on the NIST SRM 1934 through a measurement service under the Flow Cytometry Standards Consortium. These microbead calibration standards immobilized on the IHC slides enable quantification of instrument analytical sensitivity, result comparability, and diagnostic accuracy.  

(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. At present, synthetic B-cell mimics are being investigated in comparison with lyophilized PBMCs. 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 stable and reproducible B-cell reference materials evaluated by these studies will be impactful as expression analysis reference markers for quantifying disease and immunotherapy relevant B cell markers, e.g., CD19, CD20, CD22, and CD23. 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.

Left plot: CD19 expression levels in unit of antibodies bound per cell (ABC) and associated standard deviations at ~95% confidence level for the three PBMCs preparations (PBMC-A, -B, and -C) using a computational method for the two quantification approaches of flow cytometry, QuantiBrite PE calibration (light grey bar) and CD4 cell reference calibration (grey bar), and the two-flow cytometry method combined uncertainty analysis (red bar). The CyTOF quantification was conducted on a selected subset of the PBMC samples. The CD19 expression levels and associated standard deviations obtained using two CyTOF quantification methods, EQ4 calibration (blue bar) and ABC bead calibration (green bar) are also displayed within the flow cytometric results from the combined uncertainty analysis. Right plot: Relative variation (i.e., total variance divided by the mean squared) in typical CD19 measurement by PBMC manufacturer and calibration method. The relative contributions of each source of uncertainty for each PBMC preparation are indicated by the different colors.

4. Development of Process Control Materials and Protocols for Reliable Measurements of Extracellular Vesicle and Lentivirus Using Flow Cytometry
(I) Extracellular vesicles (EVs) are biologically active lipid bilayer biomolecules. EVs are involved in a variety of physiological and pathological signaling processes and have spurred tremendous interest in their broad application as potential biomarkers, regenerative medicines and therapies, and drug delivery vehicles. Research on EV’s role in such processes has been primarily focused on exosomes (30–150 nm) or microvesicles (200–1,000 nm).  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. Flow cytometry can provide quantitative and reproducible measurements of the count, size, cargo, and functionality of EVs. However, the analysis of EVs by Flow Cytometry has been limited due to their small sizes, limitations of fluorescent labels, and the experience level of instrument users.  Therefore, detailed procedures to fine-tune the Flow Cytometer through careful calibration using known control materials are key to obtaining reliable and meaningful data. To develop measurement assurances around EV technology, the Flow Cytometry research at NIST is focused on several research areas: 1) Develop standardized measurement systems for determining the molecular composition and biological activity of EVs by Flow Cytometry; 2) Develop EV reference materials to validate EV measurements and maintain measurement assurance throughout EV production and characterization; 3) Develop standardized cell-based platforms for EV-based therapeutics; 4) Develop robust EV isolation and analytical characterization measurements for the analysis of human blood cancer-derived EVs.

 

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.

(II) Lentivirus-based gene delivery system is widely used for developing effective and safe cell and gene therapies. The characterization of the critical quality attributes (CQAs) is essential for their safe and sound applications. Flow cytometry along with other orthogonal technologies is utilized to conduct cutting edge measurements of lentiviral vectors including physical titer, viral packaging information, potential contamination, and biological activity and potency.    

5. Proteomic and Genomic Analysis of CRISPR/Cas9 Engineered Cells and Cell Stability 
CRISPR/Cas9 is a commonly used genome editing system being deployed for engineering proteins, and for cell and gene therapies. While this tool has great potential, long-term data about the genomic and phenotypic stability and off-target effects are sparse. Since the entire cell can be used in a patient with cell and gene therapy products, cell characterization is essential for safety. We have been utilizing CRISPR/Cas9 to support the production and characterization of safe and effective engineered proteins, and other cell and gene therapy products to study both on- and off-target effects of CRISPR/Cas9 using Flow Cytometry on GM24385 cells, a B-lymphoblast whose genome sequence is well characterized. Importantly, this project enables us to extend our Flow Cytometry capability to measure transcriptomes and proteomes simultaneously at a single-cell level in high throughput fashion.

The Flow Cytometry group is also interested in utilizing the targeting feature of CRISPR/ Cas9 for the study of genomic copy numbers and rare events. To this end, studies for counting the number of genomic copies are also being devised by combining CRISPR-based targeting of quantum dots (QD) labeled Cas9 with guide RNAs (gRNA) to assess integrated sequences at genomic loci. By combining the power of robust QD fluorescence with the high throughput acquisition of a Flow Cytometer, direct counting of the number of QDs per cell is feasible for quantification of absolute genomic counts. These projects have tremendous significance in cell and gene therapy space and for rare event genomic measurements.

6. Rare Events Quantitation Using Quantum Cytometer
Cancer cell and engineered therapeutic cell heterogeneity are long recognized as an important clinical determinant of patient outcomes but are 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 cumulative values and losing information on cellular and population heterogeneity. Other methods like Immunohistochemistry and fluorescence in situ hybridization (FISH) produce qualitative or sub-quantitative results at best and are time-consuming. NIST possesses different viral copy numbers (VCN) Jurkat cell lines with various copies of GFP reporter integrated at genomic loci. For instance, VCN4 means 4 copies of unique sequence integrated at 4 known ectopic sites on the genome with GFP reporter in Jurkat cell, VCN0 means 0 copies (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 an Imaging Flow Cytometer with FISH analysis. 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, rare event quantification by imaging flow cytometry will be applied to other significant NIST programmatic areas in regenerative medicine, engineering biology, and precision medicine 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, the 2^nd WHO international standard for anti-SARS-CoV-2 immunoglobulin and Reference Panel for antibodies to SARS-CoV-2 variants of concern, 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, metadata management, and data standards.
• CLSI H62 Validation of Assays Performed by Flow Cytometry
• Development and Validation of Measurement Traceability for In Situ Immunoassays

RELATED PUBLICATIONS

• Standardization of flow cytometric detection of antigen expression
• Number Concentration Measurements of Polystyrene Submicrometer Particles
• Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis
• Evaluation protocol for CRISPR/Cas9-mediated CD19 knockout GM 24385 cells by flow cytometry and Sanger sequencing
• Expanding NIST's Calibration of Fluorescent Microspheres for Flow Cytometry to More Fluorescence Channels and Smaller Particles
• Methodology for evaluating and comparing fluorescence measurement capabilities: Multi-site study of 23 flow cytometers
• Comparison of volumetric and bead-based counting of CD34 cells by single-platform flow cytometry
• Quantitative Fluorescence Measurements with Multicolor Flow Cytometry
• Quantitative Flow Cytometry Measurements in Antibody Bound per Cell Based on CD4 Reference
• Assignment of the Number of Equivalent Reference Fluorophores to Dyed Microspheres
• Quantification of Cells with Specific Phenotypes I: Determination of CD4+ Cell Count per &#181;L in Reconstituted Lyophilized Human PBMC Pre-labelled with Anti-CD4 FITC Antibody
• Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry
• NIST/ISAC standardization study: variability in assignment of intensity values to fluorescence standard beads and in cross calibration of standard beads to hard dyed beads
• Size and Number Concentration Measurements of Polystyrene Nanoparticle Suspensions using Several Different Techniques