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, methodologies, and procedures to enable quantitative measurements of biological substances such as cells, extracellular vesicles, viruses, virus-like particles, proteins, and nucleic acids.  By providing quantitative flow cytometry measurement solutions, we ensure that researchers and clinicians can produce more reliable data enabling AI/ML applications, develop better engineered products and drugs, and provide better treatment to patients in the clinic.

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 and efficacy of therapeutics including engineered T-cells. In HIV/AIDS monitoring, accurate measurement of CD4+ cell counts using flow cytometry is key to ensuring that patients receive appropriate antiretroviral treatment (ART). However, measurements made on different instrument platforms, at different times, and/or in different places often cannot be compared. Measurement discrepancies 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 through the use of reference controls, standards, and measurement procedures. 

Current Projects

1. NIST Flow Cytometry Standards Consortium (FCSC)
The objectives of the Consortium are to collaboratively develop standards, which include biological reference materials, reference data, reference methods, and measurement service for assigning the equivalent number of reference fluorophores (ERF) to calibration microspheres, assessing the associated measurement uncertainties, and demonstrate the utility of these tools. 

The Consortium has recently extended its activities through August 1, 2029.  Four working groups (WGs) have been established to accomplish the Consortium's objectives.  These working groups currently include:

• Working Group 1 (WG1) - ERF-based instrument calibration and standardization
• Working Group 2 (WG2) - Flow cytometry assay standardization
• Working Group 3 (WG3) - Data repository and centralized data analysis
• Working Group 4 (WG4) - Gene delivery systems

Working Group 5 (WG5) - Artificial intelligence and machine learning applications is planned to leverage the multiple high-quality datasets resulting from Consortium interlaboratory studies. Learn more about the NIST Flow Cytometry Standards Consortium.

2. Quantification of Cells with Specific Phenotypic Characteristics and Biomarkers of Immuno-Oncology and Diseases
Quantitative flow cytometry plays a pivotal role in advancing cell and gene therapies by providing precise, reproducible and standardized data that are critical for diagnostics, therapeutic development, and regulatory compliance. Ongoing innovations and standardization continually expand this role for human health, and NIST is conducting active projects including:

• Accurate enumeration of cells with specific phenotypic characteristics
• Comparable and standardized antigen expression analysis across different cytometer platforms
• Standardization of solid tumor biomarkers.

Learn more about NIST's phenotyping and biomarkers projects.

3. Sub-Micrometer Particle Standards for Quantitative Flow Cytometry
Measuring number concentrations and size distributions of sub-micrometer bioparticles, such as extracellular vesicles (EVs), viruses and virus-like particles (VLPs) is of growing importance in clinical diagnostics, biotherapeutics and biotechnologies.  The number of commercially-available instruments, including flow cytometers, that can make these measurements is also increasing.  Sub-micrometer particle standards are required to improve the accuracy and reproducibility of these techniques.  Calibration microsphere suspensions of cell-sized (2 to 10 mm diameter) beads with known number concentrations and fluorescence intensities have been available to the quantitative flow cytometry community for years.  Now, similar sub-micrometer sized spheres are needed for instrument quality control/calibration and for biological applications.  A consensus-based protocol using high-accuracy, validated techniques has been developed at NIST to assign certified number concentration values to sub-micrometer particle suspensions.  Learn more about NIST's sub-micrometer particle standards projects.

4. Physical and Functional Characterization of Lentiviral Vectors

Lentiviral vectors (LVVs) serve as primary tools for stable gene delivery needed in producing cell and gene therapies (CGTs)—a therapeutic modality that may account for hundreds of billions of U.S. dollars in global revenue within the next decade.  LVVs are used to engineer chimeric antigen receptor T cells (CAR-T) for therapies treating many cancers and genetic disorders.  LVVs are enveloped, single-stranded RNA viruses that are capable of infecting both dividing and non-dividing cells.  Measuring LVV titers and quality attributes is important for predicting the functionality of the LVVs and and the potency of the resulting CAR-T treatment.  Currently, physical and functional characteristics of LVVs vary significantly between vendors and batches, and measurements vary by methodology, platform, and location.  NIST is conducting projects using both flow cytometry and orthogonal methods to compare precise measurements of LVVs across materials and methodologies.  Learn more about NIST's LVV characterization projects.

5.  Development of Process Control Materials and Protocols for Reliable Measurements of Extracelluar Vesicles Using Flow Cytometry

Extracellular vesicles (EVs) are lipid bilayer bioparticles involved in various physiological and pathological processes, making them potential disease biomarkers and therapeutic vehicles.  Research primarily focuses on exosomes (30 to 150 nm diameter) and micro-vesicles (200 to 1,000 nm diameter).  Flow cytometry is a critical tool for analyzing EVs.  The method's high-throughput and multi-parameter capabilities allow for quantitative measurements of EV count, size, cargo, and functionality.  However, challenges remain due to the small sizes of EVs, limitations of current fluorescent labels, limited expertise in applying flow cytometry methods to EVs.  Advances are also needed in isolation techniques that yield high-quality EV materials and in precise calibration of flow cytometers using control materials to increase measurement reliability and comparability.  NIST is conducting projects across six focus areas to enhance EV measurement reliability.  Learn more about NIST's EV measurement projects.

6. Rare Event Quantitation using Imaging Flow Cytometry

Heterogeneity, both in cancer cells and in engineered cell therapies, is long recognized as an important clinical determinant of health outcomes but remains poorly understood at a molecular level.  However, identifying the entirety of heterogeneity in a cell population is currently limited by capability to quantify rare genomic events at a single-cell level.  PCR-based methods generally require DNA extracted from many cells taken from a patient sample and, thereby, produce aggregate results that can mask cellular heterogeneity.  Immunohistochemistry and fluorescence in situ hybridization (FISH) better preserve cellular heterogeneity within samples but only produce qualitative or quasi-quantitative results at best with very low throughput.

NIST, in collaboration with Lentigen Technology, Inc., has extensively characterized a series of Jurkat cell lines each with a known, genomically-integrated, GFP-reporter vector copy number (VCN) ranging from 1 to 4 vector copies.  (For example, the GFP-VCN4 line carries four unique vector copies integrated at four distinct ectopic sites within the Jurkat cell genome.) We are using the unique sequence information of the Jurkat GFP-VCN cell line series to design FISH probes that distinguish signal intensities as a function of VCN.  In addition, unlike low-throughput, traditional FISH analysis using microscopy, we combine FISH with flow cytometry, a.k.a. Flow-FISH, to conduct high-throughput measurements.  This quantitative work contributes to future applications of Flow-FISH for simultaneous detection of rare event gene mutations and/or protein biomarkers at the single-cell level for conventional and molecular pathology.  NIST is conducting this project along with others in rare event quantification by imaging flow cytometry to enable new single-cell measurements in regenerative medicine, precision medicine, and engineering biology.

7. Quantitative and Traceable Serology and Neutralization Assays for SARS-CoV-2

The global response to the COVID-19 pandemic stimulated rapid advances in diagnostic, surveillance, vaccine, and therapy development.  Critical measurements underpinning much of these efforts are serological assays that assess the complex patient responses to SARS-CoV-2, and globally-traceable reference materials for assay control/validation and analysis methods for assay harmonization have been urgently needed.  To address these needs, NIST has developed a rapid, multiplexed, sensitive, flow-cytometry based serological assay, BSL-3 sparing neutralization assays, and a method for harmonizing results from different assay platforms.  Learn more about these NIST projects in Serology and Neutralization Assays for COVID-19.

Major Accomplishments

• Contributed to the development and evaluation of the first international reference standard for CD4+ cell counting for HIV/AIDS monitoring (WHO BS/10.2153).
• 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.  
• Developed SRM 1934, Fluorescent dyes for quantitative flow cytometry (visible spectral range).
• Developed a set of Research Reference Materials for quantitative flow cytometry.  These included, preparation and concentration determination of Pacific Orange, Alexa Fluor 700, and Alexa Fluor 750 solutions.
• Participated on expert committee for 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, gene delivery system measurement harmonization, and flow cytometric data analysis, metadata management, and data standards.
• Participated on the document development committee for CLSI H62 Validation of Assays Performed by Flow Cytometry
• Collaborated on Development and Validation of Measurement Traceability for In Situ Immunoassays