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Adrian Gray
Adrian Gray

A Comprehensive Review of Flow Cytometry in Neoplastic Hematology: Morphologic-Immunophenotypic Correlation, Third Edition Dow


Flow Cytometry in Neoplastic Hematology: Morphologic-Immunophenotypic Correlation, Third Edition Dow




Introduction




Flow cytometry is a powerful technique that allows the analysis of multiple parameters of individual cells in a fluid suspension. It is widely used in clinical and research settings for the diagnosis, classification, monitoring, and prognostication of hematopoietic neoplasms, such as leukemias, lymphomas, and plasma cell disorders.




Flow Cytometry In Neoplastic Hematology: Morphologic-Immunophenotypic Correlation, Third Edition Dow


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Flow cytometry in neoplastic hematology requires a multidisciplinary approach that integrates the immunophenotypic data with the morphological features and the molecular/cytogenetic abnormalities of the malignant cells. This can provide a comprehensive characterization of the disease and guide the optimal management of the patients.


One of the most comprehensive and authoritative books on this topic is Flow Cytometry in Neoplastic Hematology: Morphologic-Immunophenotypic Correlation, written by Wojciech Gorczyca, a renowned expert in the field. The third edition of this book was published in 2017 by CRC Press, Boca Raton. It covers the wide spectrum of hematopoietic tumors, with a focus on the most important clinical diagnoses, such as acute promyelocytic leukemia, identification of blasts, identification of clonal B-cell populations, distinction between chronic lymphocytic leukemia and mantle cell lymphoma, and detection of minimal residual disease. It also incorporates the latest advances and updates in the field, including the 2016 revision of the WHO classification of tumors of hematopoietic and lymphoid tissues.


In this article, we will review the main features and contents of this book, as well as its strengths and limitations. We will also discuss some of the challenges and future directions of flow cytometry in neoplastic hematology.


Principles of flow cytometry




Basic components and functions of a flow cytometer




A flow cytometer is a complex instrument that consists of four main components: a fluidics system, an optics system, an electronics system, and a computer system. The fluidics system transports the cell suspension through a narrow tube to a point where it intersects with one or more laser beams. The optics system collects the light signals emitted by the cells or by fluorescent molecules attached to them. The electronics system converts the light signals into electrical pulses that are proportional to the intensity and duration of the signals. The computer system analyzes the electrical pulses and displays them as histograms or scatter plots.


By using different combinations of lasers, filters, and detectors, a flow cytometer can measure various parameters of the cells, such as size, granularity, viability, DNA content, cell cycle status, and expression of surface or intracellular antigens. These parameters can be used to identify and quantify different cell populations, as well as to assess their functional and biological characteristics.


Data acquisition and analysis




Data acquisition is the process of collecting and storing the information generated by the flow cytometer. It involves setting the appropriate instrument settings, such as voltage, gain, compensation, and threshold, as well as selecting the parameters and regions of interest for analysis. Data acquisition can be performed in real-time or offline, depending on the availability and capacity of the instrument and the computer system.


Data analysis is the process of interpreting and presenting the information obtained by the flow cytometer. It involves applying various statistical and graphical methods to summarize and compare the data, such as histograms, scatter plots, dot plots, contour plots, density plots, quadrant analysis, cluster analysis, principal component analysis, and multidimensional scaling. Data analysis can be performed manually or automatically, depending on the complexity and variability of the data and the availability and quality of the software tools.


Quality control and standardization




Quality control is the process of ensuring that the flow cytometer and its components are functioning properly and consistently. It involves performing regular maintenance, calibration, verification, and troubleshooting of the instrument and its accessories. Quality control also involves validating and documenting the performance of the instrument and its reagents, such as antibodies, fluorochromes, beads, and buffers. Quality control can be performed internally or externally, depending on the availability and reliability of the reference materials and methods.


Standardization is the process of ensuring that the flow cytometry results are comparable and reproducible across different instruments, laboratories, operators, and protocols. It involves following standardized guidelines and recommendations for sample preparation, staining, acquisition, analysis, reporting, and interpretation of flow cytometry data. Standardization also involves participating in proficiency testing and accreditation programs to evaluate and improve the quality of flow cytometry services. Standardization can be achieved by using common terminology, nomenclature, criteria, algorithms, software tools, databases, and reference values.


Immunophenotyping of hematopoietic neoplasms




Normal hematopoiesis and immunophenotypic markers




Hematopoiesis is the process of formation and development of blood cells from hematopoietic stem cells (HSCs). HSCs are multipotent cells that can differentiate into various lineages of blood cells: erythroid (red blood cells), myeloid (granulocytes, monocytes/macrophages), lymphoid (B cells, T cells), megakaryocytic (platelets), or dendritic cells. Hematopoiesis occurs in a hierarchical manner in specific microenvironments within the bone marrow or other organs (such as thymus or spleen) under the influence of various growth factors and cytokines.


Immunophenotypic markers are molecules that are expressed on the surface or inside the cells that can be detected by flow cytometry using specific antibodies conjugated with fluorescent dyes. Immunophenotypic markers can be used to identify different cell types or stages of differentiation based on their expression pattern or intensity. Immunophenotypic markers can be classified into several categories: lineage-specific markers (such as CD19 for B cells or CD3 for T cells), differentiation/maturation markers (such as CD34 for immature cells or CD38 for plasma cells), activation markers (such as CD69 or HLA-DR), functional markers (such as CD16 for phagocytosis or CD107a for degranulation), adhesion molecules (such as CD11a or CD62L), chemokine receptors (such as CXCR4 or CCR7), cytokine receptors (such as IL-2R or IL-7R), transcription factors (such as TdT or PAX5), oncogenes (such as BCL-2 or c-MYC), tumor suppressor genes (such as p53 or Rb), apoptosis-related molecules (such as Annexin V or Caspase 3), cell cycle-related molecules (such as Ki-67 or Cyclin D1), DNA content/damage molecules (such as PI or TUNEL), or intracellular signaling molecules (such as ZAP-70 or Syk).


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