Description
This advanced lecture note on Flow Cytometry in Pathology provides a comprehensive overview of the technique for tertiary students in Biomedical Sciences, Histopathology, and Laboratory Medicine. Authored by Ephraim Imhotep Zulu, BSc BMS, MSc Path, from the Department of Biomedical Sciences at the University of Zambia's School of Health Sciences, the material explains the principle of flow cytometry as a method for rapidly analyzing the physical and chemical characteristics of individual cells or particles suspended in a fluid stream as they pass a laser beam. It details how fluorochrome-conjugated antibodies or DNA stoichiometric dyes are used to tag specific cellular components, with emitted light detected and converted into digital data displayed as dot plots or histograms. The note lists key fluorochromes like Propidium Iodide for DNA and FITC for antibodies, along with their excitation and emission wavelengths. It outlines suitable samples, including fine needle aspirates and effusion fluids, and covers major applications such as DNA ploidy analysis, cell cycle assessment, and immunophenotyping for diagnosing and classifying lymphoid neoplasms. A significant focus is on interpreting DNA histograms to identify diploid, aneuploid, and S-phase cell populations for prognostic evaluation in cancers like bladder and prostate carcinoma. The lecture compares flow cytometry with immunocytochemistry and discusses limitations, including the lack of morphological correlation, high instrument cost, and potential issues with scanty or admixed cell populations. This resource is essential for diploma and degree students to understand the clinical utility, technical execution, and analytical interpretation of flow cytometry in modern diagnostic pathology. Download this lecture to gain a thorough understanding of this powerful quantitative analytical tool.
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Ephraim Imhotep Zulu, BSc BMS, MSc Path University of Zambia School of Health Sciences Dept. of Biomedical Sciences Cellular Pathology Lecture #22 Advanced Histological Techniques: Flow Cytometry in Pathology Lecture Outline • Principle of Flow Cytometry • Fluorochrome Dyes Used used in FCM • Samples for Flow Cytometry • Targets of Applications • DNA Content & Ploidy Analysis • Clinical Application • Limitations of FCI Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Learning Objectives: At the end of this lecture, the student is expected to: • Understand the principle of flow cytometry • Know the clinical applications of flow cytometry in Pathology • Appreciate the advantages and limitations of flow cytometry in Pathology • Appreciate the Fluorochrome Dyes Used used in FCM • Understand how flow cytometry is used in DNA Content & Ploidy Analysis Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Flow Cytometry Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Synopsis • Flow Cytometry (FCM) is the method by which the various characteristics of individual particle or cells are studied. • FCM provides us very quick assessment of cell surface antigens, DNA content and intracellular proteins. • It is now a well-established technique for the diagnosis and classification of lymphoid neoplasms, identification of malignant cells in effusion cytology and other body fluids. Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Principle of Flow Cytometry • Single dissociated cells in liquid medium are essential for flow cytometry. The specific component of the cell is identified by the antibody tagged with a fluorescent dye. • Similarly DNA can also be stained by a DNA stoichiometric dye. • The single cells rapidly pass in front of a laser beam, and the laser beam of particular wave length hits the cell. The individual cells absorb the light and emit light of different wavelength. • The emitted light is detected by the photomultiplier tube and is converted into a digital pulse. The intensity of the digital signal is stored in the computer and expressed in a relative scale known as channel. The result of the events is expressed as dot plot or histogram. Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Single cell Emitted fluorescence Laser beam Photomultipler tube Computer Histogram Dot plot Fig. 17.1 Schematic diagram of basic principle of flow cytometry is highlighted in the picture. The single cells are hit by the laser beam of particular wavelength. The emitted light is detected by the photomultiplier tube and is converted into a digital pulse. The result of the events is expressed as dot plot or histogram Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Electron moves in higher orbit Exited state Ground state Flurochrome dye Excitation wave length Electron jumps back and emits light Emission wave length Hit by laser beam Fig. 17.2 The diagram shows the principle of fluorochrome dye activation. When a fluorochrome compound absorbs quantum of light, the electron moves from lower orbit to the higher orbit, and the compound remains in excited state. When the compound comes back to its ground state, the electron returns to its original orbit and the compound emits the quantum of light of lower wavelength with different colour Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Fluorochrome Dyes Used used in FCM Table 17.1 Fluorochrome dye used in DNA Flurochrome Excitation maximum (nm) Emission maximum (nm) Propidium iodide 305, 540 Ehidium bromide Hoechst 33342 Hoechst 33258 Diamidinophenylindole (DAPI) Acridine orange 530 (DNA), 640 (RNA) Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Table 17.2 Fluorochrome dye for conjugating with antibody Fluorochrome Excitation maximum (nm) Emission maximum (nm) Fluorescein isothiocyanate (FITC) 488 Phycoerythrin (PE) Allophycocyanin (APC) Rhodamine Red-X Texas Red® PE-Cy7® 488 Peridinin chlorophyll (PerCP) nm nano micrometre, 10−9 m Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY Samples for Flow Cytometry Cytology Samples • The various types of cytology specimens for FCM include: • 1. Fine needle aspiration cytology materials: Lymph node, breast, lung, prostate, etc. • 2. Exfoliative samples: Effusion fluid, CSF, bladder wash Wednesday, July 27, 2022 Ephraim Zulu - HISTOPATHOLOGY
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