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.