Precision Medicine
Flow Cytometric Assays

Flow Cytometry


Absolute Cell Counts and Quantitative Flow Cytometric Assays

In layman’s terms, FCs use a combination of fluidics, optics, and detectors to measure cell properties such as size, shape, granularity, and fluorescence. The fluidics system allows for the cells within the sample to be focused and delivered in a single file. The optics consist of excitation lasers and photomultiplier tubes (PMTs) and photodiodes that generate data on individual cells as they flow through the sheath fluid. Dichroic filters direct the scattered light signals to designated detectors. The filtered light signals are then transformed into digital data and processed by a computer [1]. Most FC data is analysed using traditional dot plots and/or histograms, however modern cluster data analysis is used for complex data sets [1].

Flow cytometry (FC) is a powerful tool capable of delivering both qualitative and quantitative data with high-throughput capabilities. FC techniques offer the precision to perform multi-parametric analysis of a single cell within a heterogenous pool, while simultaneously having the capacity to assess entire cell populations contained within that sample.

Quantitative flow cytometric (QFCM) assays are precise, rapid, and generates large amount of clinical and diagnostic data [2]. FCM assays provide detailed quantifiable information about cell populations and cell characteristics, such as the expression levels of cell surface markers, intracellular proteins, and other biological molecules. In a clinical QFCM, international and technical harmonisation in protocol is desirable. Each QFCM assay must take into consideration accuracy, precision, sensitivity, specificity, stability, and controls [2]. Absolute cell counts fall under the umbrella of QFCM. Unlike relative counts which expresses data as percentages or proportions of different cell populations within the sample, absolute counts refer to the determination of the actual number of cells or particles of interest per unit volume in a sample [3]. This assay can be particularly useful for clinical diagnosis and monitoring disease progression e.g., in HIV/AIDs and leukaemia. Absolute cell counts for specific immune cell subsets can be calculated by combining cell surface marker staining with volumetric measurements which require specialized FC assays [3]. Examples of conditions that benefit from absolute cell counts and QFCM for diagnosis and monitoring disease progression are HIV/AIDS, leukaemia, immunodeficiencies, transplantation, haematological disorders and cancer [4-8].

At PeploBio, we use beads to generate precise and accurate absolute counts of cells subsets in blood and tissue. Our state-of-the-art FC instrumentation comes with automated calibration processes to prevent deviations and ensures accurate measurements between samples tested across multiple days. Furthermore, our analytical team ensures optimal sample compatibility with the beads prior to batch sample processing.  

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[1] McKinnon KM. Flow cytometry: an overview. Current protocols in immunology. 2018 Jan;120(1):5-1.
[2] Mizrahi O, Ish Shalom E, Baniyash M, Klieger Y. Quantitative flow cytometry: concerns and recommendations in clinic and research. Cytometry Part B: Clinical Cytometry. 2018 Mar;94(2):211-8.
[3] Mandy F, Brando B. Enumeration of absolute cell counts using immunophenotypic techniques. Current protocols in cytometry. 2000 Jul;13(1):6-8.
[4] Maher KJ, Fletcher MA. Quantitative flow cytometry in the clinical laboratory. Clinical and Applied Immunology Reviews. 2005 Nov 1;5(6):353-72.
[5] Abraham RS, Aubert G. Flow cytometry, a versatile tool for diagnosis and monitoring of primary immunodeficiencies. Clinical and Vaccine Immunology. 2016 Apr;23(4):254-71.
[6] D'Arena G, Musto P, Cascavilla N, Dell'Olio M, Di Renzo N, Carotenuto M. Quantitative flow cytometry for the differential diagnosis of leukemic B‐cell chronic lymphoproliferative disorders. American journal of hematology. 2000 Aug;64(4):275-81.
[7] Janossy G, Jani IV, Brando B. New trends in affordable CD4+ T-cell enumeration by flow cytometry in HIV/AIDS. Clinical and Applied Immunology Reviews. 2003 Sep 1;4(2):91-107.
[8] Ward MS. The use of flow cytometry in the diagnosis and monitoring of malignant hematological disorders. Pathology. 1999 Jan 1;31(4):382-92.

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