Flow cytometry in clinical pathology

Abstract

Flow cytometry has had an impact upon all areas of clinical pathology and now, in the 21st century, it is truly coming of age. This study reviews the application of flow cytometry within clinical pathology with an emphasis upon haematology and immunology. The basic principles of flow cytometry are discussed, including the principles and considerations of the flow-cell and hydrodynamic focusing, detector layout and function, use of fluorochromes and multicolour flow cytometry (spectral overlap and colour compensation), alongside the strategies available for sample preparation, data acquisition and analysis, reporting of results, internal quality control, external quality assessment and flow sorting. The practice of flow cytometry is discussed, including the principles and pitfalls associated with leukocyte immunophenotyping for leukaemia and lymphoma diagnosis, immune deficiency, predicting and monitoring response to monoclonal antibody therapy, rare event detection and screening for genetic disease. Each section is illustrated with a case study. Future directions are also discussed.

Cell Surface Profiling Using High-Throughput Flow Cytometry: A Platform for Biomarker Discovery and Analysis of Cellular Heterogeneity

“…These include ease of use, the ability to rapidly analyze very large cell numbers, analysis of rare populations of cells, and the ability to obtain multi-parameter information on individual cells, which is particularly important for heterogeneous cell samples. The robustness and utility of flow cytometry is illustrated by the large number of clinical applications for which it is now being used around the world [9]. Traditionally, flow cytometry assays are performed on individual samples with a panel composed of up to 11 antibodies at a time that are known to be useful for a particular diagnosis or identification of specific cell types.…”

“…FC is typically used to analyze up to 11 markers at a time, with complex analysis being possible through the use of overlapping panels [7] allowing identification and analysis of subpopulations of cells within complex mixtures. Indeed, such flow cytometry assays are now used clinically in several areas such as diagnosis and monitoring of hematological malignancies [8], [9], illustrating the power of this approach.…”

Promise, Progress, and Pitfalls in the Search for Central Nervous System Biomarkers in Neuroimmunological Diseases: A Role for Cerebrospinal Fluid Immunophenotyping

“…Due to methodological advances, 53 multi-color (polychromatic) flow cytometry (11 – 12 colors) allows a broader range of immune cells to be phenotyped in a single but sufficient CSF sample. Not without shortcomings, 54 multiparameter flow cytometry is “truly coming of age” in the 21 st century, 55 providing a treasure-trove of other potential biomarkers beyond the scope of this review. 9 The 50-fold concentration of the CSF cell pellet obviates the need for higher volume requirements in controls, whose CSF leukocyte counts are only 1–3/cu mm.…”

Flow Cytometry Bioinformatics

“…As the number of markers measured by flow cytometry increases, the number of scatter plots that need to be investigated increases exponentially (some markers need to be investigated several times for each group of cells to resolve high-dimensional differences between cell types that appear to be similar in most markers) [36]. To address this issue, principal component analysis has been used to summarize the high-dimensional datasets using a combination of markers that maximizes the variance of all data points [37].…”