Flow cytometry

DEFINITION: Cytometry refers to the measurement of the physical or chemical characteristics of cells, or, by extension, of other biological particles. Flow cytometry is a process in which such measurements are made while the cells or particles pass, preferably in single file, through the measuring apparatus in a fluid stream.

Cancers diagnosed:Hematological (blood) cancers, including chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), acute myeloid leukemia, non-Hodgkin lymphomas, multiple myeloma

Why performed: Flow cytometry is widely used in the clinical setting for a number of cytometry-based procedures, such as immunophenotyping, cell sorting, enumeration of CD34-positive stem cell precursors, enumeration of lymphocyte subsets (B cells, T cells, and natural killer or NK cells), and fetal bleed tests in fetomaternal hemorrhage. The technique is performed on a sample of cells obtained from blood, bone marrow, or other tissue, such as a lymph node. Flow sorting extends flow cytometry by using electrical or mechanical means to divert and collect cells with one or more measured characteristics falling within a range or ranges of values set by the user.

A rapid cell sample analysis is possible with an instrument called a cytometer that can count fifty thousand cells per second. Before cytometry, cells had to be counted manually under a microscope—a labor-intensive task subject to operator error. Components of a cytometer include a fluidics system, an optical system composed of one or more monochromatic lasers or other light sources with filters to serve as an excitation beam, electronics to detect emissions from the cells, and a computer to analyze the data collected.

The flow cytometry is widely used for diagnostics and disease monitoring of hematopoietic malignancies. Immunophenotyping of hematological cancers has developed as a clinically valuable but technically complicated diagnostic procedure. It involves a variety of methodological features, in-process strategic judgments, and an extensive knowledge of clinical, morphological, and other laboratory features of the disease processes. Several internal quality-control steps are necessary to guarantee reliable results regarding instrument setup, calibration, selection, and validation of monoclonal antibody panels and process control. The data provide a wide range of hematological information essential for diagnosing blood cancers. In leukemias, for example, knowing cell lineage and maturation helps distinguish specific forms of leukemia.

Flow cytometry tests to distinguish an abnormal population of hematopoietic cells and to determine the cellular lineage of malignant cells, clonality, cellular maturation, and heterogeneity within the cancerous cell populations. Flow cytometric analysis, called immunophenotyping, allows cells to be characterized by their size, complexity, and expression patterns of surface and cellular markers. Multiparameter flow cytometry is the simultaneous use of multiple fluorochromes, which allows the completion of the full diagnostic test with fewer cells, thus reducing the specimen size needed.

Immunophenotyping by flow cytometry is used to initially diagnose hematopoietic malignancies, monitor the response to treatment administered for the malignancy, and determine the presence of minimal residual disease that may indicate recurrence of the cancer.

Patient preparation: The specimen on which the cytometry is performed is generally bone marrow aspirate, although a peripheral blood sample can also be used. Healthcare providers give the patient instructions based on the need for a bone marrow procedure rather than blood collection.

Steps of the procedure: The procedure of immunophenotyping is based on the ability of hematopoietic cells of different lineages and different levels of differentiation to present specific surface, cytoplasmic, or nuclear markers. These markers, called clusters of differentiation (CD), are molecules on the cell surface, as recognized by specific sets of antibodies, that are used to identify the cell type, stage of differentiation, and activity of a cell.

The procedure of immunophenotyping consists of three main steps: sample preparation, flow cytometric data acquisition, and analysis and interpretation of results. To prepare a sample, cells are labeled with the antibodies tagged by specific fluorochromes, which includes staining with fluorochrome-bound monoclonal antibodies, lysis of red blood cells, and fixation in a formalin-containing reagent. This staining protocol is used for all directly conjugated reagents. A whole blood lysing system prepares immunologically stained leukocytes for flow cytometry analysis. In the flow cytometric data acquisition process, cells are run through a fluidic stream so that single cells can be analyzed one at a time. Then, multiple parameters are collected on each cell and analyzed for forward-angle light scatter, ninety-degree light scatter, and fluorescent signal information from up to six different fluorochromes simultaneously. All data can be stored as list-mode files for later reanalysis.

After the procedure: Patients receive aftercare instructions according to how the sample is obtained. Healthcare practitioners communicate limitations and restrictions to the patient if sedation is used.

Risks: Potential risks of a bone marrow and aspirate or blood collection are incorporated into a consent form explained at the facility where the procedure is accomplished and signed by the patient prior to obtaining the sample for analysis.

Results: Data can be analyzed and displayed in various formats, such as single-parameter histograms, dot-plots (display of distribution of two antigens on an x-y plot), or three-dimensional plots of three antigens. Data are analyzed using a gating strategy, in which specific gates are set around the subpopulations of cells based on common parameters measured on all cells. The quantification of each cell type, its light-scatter properties, its intensity of fluorescence, and certain patterns of surface or cytoplasmic marker expression indicate hematopoietic abnormalities. The antigen expression pattern on all the cells is analyzed and compared with the distribution of normal cells. Data interpretation is based on the knowledge of specific cell phenotypes affiliated with certain types of leukemia and lymphomas. Abnormal phenotypes, however, are not always associated with disease progression. Therefore, flow cytometric analysis results must be used in conjunction with other diagnostic procedures and clinical evaluations.

Bibliography

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Movia, Dania, and Adriele Prina-Mello. Cancer Cell Culture: Methods and Protocols. Humana Press, 2023.

Porwit, Anja, and Marie-Christine Béné. Multiparameter Flow Cytometry in the Diagnosis of Hematologic Malignancies. Cambridge University Press, 2018. 

Pandey, Prerna, and Shiv Sanjeevi. Flow Cytometry and Cell Sorting. Springer, 2019.

Rees, P., et al. "Imaging Flow Cytometry." Nature Reviews Methods Primers, vol. 2, no. 86, 2022. doi.org/10.1038/s43586-022-00167-x.

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Werner, Jakub. Introduction to Flow Cytometry. Nova Science Publishers, 2019.