Quantification of Cells with Specific Phenotypic Characteristics

 Accurate enumeration of cells with specific phenotypic characteristics if of critical importance in patient care.  There are pertinent needs for cell reference materials for external measurement quality assessments for in-clinic applications such as HIV/AIDS monitoring (CD4+ cell count) and blood transfusion (CD45+ CD34+stem cell count).  NIST has contributed to the development and evaluation of the first international reference standard for CD4+ cell counting for HIV Aids monitoring, 1st WHO International Reference Reagent CD4 T-cells (human).

Due to the enormous potential and the recent success of immunotherapy (e.g., chimeric antigen receptor T-cell or CAR-T therapies) in improving patient survival rates, there are urgent needs for cell reference materials and standardized protocols to evaluate T cell functionality.  Intracellular cytokines  are crucial indicators of immune function and competence.  NIST has contributed to the characterization of a cellular reference material, NIBSC SS570, using a freeze-dried preparation of unstimulated peripheral blood mononuclear cells (PBMCs) and phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulated PBMCs, ICS Positive Control Lymphocytes, produced from healthy donors by the UK's National Institute of Biological Standards and Control (NIBSC).  A flow cytometric, rapid, single-step method has been developed and validated across different instrument platforms in three different laboratories for enumerating cytokine positive T lymphocytes.

In January 2024, the US Food and Drug Administration (FDA) released the guidance document Considerations for the Development of Chimeric Antigen Receptor (CAR) T Cell Products.  This guidance recommends including robust and validated flow cytometric assay(s) for supporting the initial investigational new drug submission for CAR-T cell Products.  Flow cytometry has been critical for establishing identity, purity, and potency for cell therapy product manufacturing with measurements supporting the approval of Biological License Applications (BLAs) by the US FDA and therapy approvals by the European Medicines Agency (EMA). B-cell control/reference materials for comparable and quantitative cytometric expression analysis are essential to support cell therapy manufacturing and immunotherapy monitoring.  NIST quantified the expression levels, and associated uncertainties, of CD19 on B cells in the instrument-independent unit of antibodies bound per cell (ABC) for three commercial lyophilized or dried PBMC preparations, shown below (left).

This work was inspired by a consensus outcome from flow cytometry workshops that called for cell reference standards with well-characterized antigen expression and immunophenotyping profiles for advanced cell manufacturing and cell therapies.  Stable and reproducible B-cell reference materials, like those evaluated by these studies, may 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, such as switching therapies or applying additional doses of the same therapy, and hence improving patient's quality of life.

To better manage patients who are receiving antigen-based immunotherapy, a quality control method was also developed using a lyophilized cell control across laboratories.  Currently, we are working on comparable and standardized antigen expression analysis across different flow cytometer platforms.  This study is designed to evaluate the performance of the following three quantification methods for quantifying CD19 expression on a lyophilized PBMC preparation across four different cytometer platforms each from a different manufacturer:  a commercial PE quantitation bead method, the approach based on CD4 as reference maker, and equivalent number of reference fluorophore (ERF)-based methodology (see above, right).  The results from this study are used to guide assay standardization studies and data analysis undertaken by the NIST Flow Cytometry Standards Consortium.

 Clinically-approved immunohistochemistry (IHC) kits for many important solid tumor biomarkers, such as HER2, PD-L1, ER, PR, Ki67, and ROS1, lack result traceability and comparability.  NIST is working closely with the Consortium for Analytical Standardization in Immunohistochemistry and the IHC community to develop a traceable and comparable measurement system.  Microbead calibration standards, prepared by covalent conjugation of fluorescein-labeled recombinant marker proteins, are critical components of the measurement system.  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 ration of fluorescein-labeled recombinant marker protein and unlabeled protein.  Fluorescence intensities of each microbead population are assigned in the ERF unit, traceable to the NIST Fluorescein Reference Solution, through a measurement service available via the NIST Flow Cytometry Standards Consortium.  These ERF-assigned microbead calibration standards, when immobilized on IHC slides, enable quantitation of instrument analytical sensitivity yielding result comparability and diagnostic accuracy. 

Disclaimer:  Certain commercial materials are identified to specify experimental results as completely as possible.  In no case does the identification of specific manufacturers, brands, or materials imply a recommendation or endorsement by the National Institute of Standards and Technology, nor does it imply that the materials are necessarily the best available for the purpose.

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