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Quantitative Flow Cytometry Measurements

Summary

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

Description

Flow cytometry image

Flow cytometry is an essential tool for basic immunological research, 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/effectiveness of drugs including engineered T-cell. 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 Quantitation Consortium – The objective of the consortium is to collaboratively develop reference materials 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.

ERF Values
The schematic for ERF value assignments is based on the published standard operating procedure using NIST SRM 1934 and calibrated laser-based CCD fluorimeter. The microspheres with assigned ERF values enable the standardization of the fluorescence intensity scale of flow cytometers in quantitative ERF unit and standardization of cytometer performance characterization.

 

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

2. Quantification of Cells with Specific Phenotypic Characteristics – Accurate enumeration of cells with specific phenotypic characteristics is of critical importance in patient 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.

Dot Plot of CD4 cells
A dot plot of Side Scatter Channel (SSC) vs. FITC Fluorescence Channel showing CD4+ cell population gated in red, TruCount beads, CD4 negative cell population and monocyte population.

3. Characterization of Commercially Available Human Peripheral Blood Mononuclear Cell (PBMC) Preparations for Quantitative Flow Cytometry  Multiplexed flow cytometry assays are routinely used in clinics for disease diagnosis, vaccine trials, and immunotherapies. Biological cell reference materials have been recognized as phenotypic benchmarks for quantitative and reproducible measure of patient characteristics in longitudinal studies and/or across locations. The objective of this project is to evaluate the expression levels of multiple biomarkers on different human blood cell subtypes from three commercially available candidate PBMC preparations and their utilities as cell reference controls for quantitative flow cytometry.

CD4 quantification
Gating strategies illustrated for the two CD4 quantification methods, conventional flow cytometry (top) and CyTOF (bottom).

4. Fluorescently Labeled Monoclonal Antibody Metrology for Quantitative Flow Cytometry – A significant effort is underway to express flow cytometer measurements on a cell in terms of the number of antibodies bound to the cell. A critical part of this effort is the identification of a reference cell with a known number of bound antibodies (e.g., CD4+ T lymphocytes). A properly calibrated fluorescence intensity(FI) scale allows the comparison of the FIs from the test cell and the reference cell, and the assignment of the number of antibodies bound to the test cell. This procedure requires careful characterization of antibody binding affinities as well as any changes in fluorescence upon binding.  The development of an antibody qualification matrix including binding affinity, staining index, and fluorescence yield (also called effective fluorophore(s) per antibody molecule) is hugely important for estimating the sensitivity and reproducibility of any flow cytometer assay.

5. Quantitative Measurements of Cell Functional Markers and Disease Biomarkers – For quantifying expression levels of cell functional markers, e.g. cytokines and chemokines, and disease biomarkers, e.g., CD20, ZAP-70, and CD38, multiplexed cytometric assays are being developed to take advantage of the known CD4 expression on healthy human T lymphocytes as a reference biomarker control. The CD4 expression levels on T helper cells from healthy individuals and a commercially available lyophilized PBMC product have been quantitatively measured using flow and mass cytometry, and mass spectrometry in our cell characterization project and are expressed in the unit of antibodies bound per cell (ABC). These biomarker expression results in the unit of ABC are quantitative and comparable across different instrument platforms and locations, and essentially are instrument independent.

Quantifying CD20 expression level in the unit of ABC
CD20 is a very useful biomarker for B-cell chronic lymphocytic leukemia (CLL). The quantification of CD20 is based on known CD4 expression on T helper cells from Cyto-Trol control cells, both stained in APC. The whole blood sample was stained with CD45 FITC, CD19 PE-Cy7 and CD20 APC, and Cyto-Trol was stained with CD45 FITC, CD3 V450 and CD4 APC in two separate sample tubes. After staining and washing, the two samples were combined in a single tube and run on a calibrated flow cytometer. (A) two individual lymphocyte gates (CD45+ and Low SSC) were drawn as ‘Cyt’ for Cyto-Trol cells and ‘Lymph’ for unknown whole blood sample in CD45 FITC vs. SSC-A; (B) gated on ‘Cyt’, T cells were identified in a dot plot of CD45 FITC vs. CD3 V450; (C) under T-cell gate, CD4 histogram shows the positive CD4+ gate, which was used to obtain respective MFI value of CD4; (D) gated on ‘Lymph’, B cells were identified in a dot plot of CD45 FITC vs. CD19 PE-Cy7; (E) gated on B cells, CD20 histogram shows the positive CD20+ gate that was used to obtain the MFI value of CD20. With measured MFI values of CD20 and CD4, CD20 expression in ABC can be obtained. The use of CD4 expression as the reference control drastically reduces the variability of CD20 expression measurements and enables quantitative measure of CD20 expression that is independent of cytometer platforms used.

6. Genomic Material Measurements Using Flow Cytometry – Researchers studying genetic profiles of different cell subsets currently have two different technology options available to them: affinity bead-based separation and cell sorting.  Both affinity bead-based separation and cell sorting methods suffer from major limitations that have thus far limited clinical, therapeutic, and diagnostic advancements. With the advent of more quantitative technologies to measure isolated genomic material, improved microscopy functionality, and more powerful flow cytometry instrumentation, we are just beginning to break the barriers that previously limited us in quantitative genomic measurements. While our group is currently leveraging all of these different quantitative methodologies to measure genomic material, the cornerstone of our technology lies in our flow cytometry capabilities. This technology will allow us to decisively measure genomic material within intact cells while simultaneously cross referencing these measurements to specific cellular subsets.

 

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 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.


NIST-NRC and NIH/NIST NRC Postdoctoral Fellowship Opportunities:

  1. Quantitative Flow Cytometry Measurements (RO#: 50.64.41.B6740)
  2. Multiplexed Assays for Cell-based Production of Biopharmaceuticals (RO#: 50.64.41.B8223)

WHO/BS/10.2153
SRM 1934
ERF value assignment service under the Flow Cytometry Quantitation Consortium
USP Chapter <127> Flow Cytometric Enumeration of CD34+ Cells

Created April 9, 2016, Updated July 13, 2017