Flow cytometry is a technique that uses fluorescent-labeled antibodies to characterize cell populations by measuring cellular characteristics like surface marker expression, size, and granularity. Cells are passed individually through a flow cytometer, which uses laser beams and detectors to quantify the cells' optical properties. Immunophenotyping with flow cytometry identifies cell types by their protein marker profiles, allowing diagnosis and classification of hematological cancers.

1. FLOW CYTOMETRY
IMMUNOPHENOTYPIN
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2. OBJECTIVE
■ Appreciate the mechanism and role of flow cytometry in
diagnosis and classification of various haematological
neoplasm

3. INTRODUCTION
■ Flow cytometry (FC) measures single cells “flowing” through a detector
system
■ Flow cytometer
– Flow means – flow tract
– Cytometer – device for cell analysis
■ Applications of FC range from
– common clinical laboratory tests, such as complete blood count with
differential and monitoring of CD4 cell count in HIV patients
– to advanced, multicolor flow cytometry used to identify subtypes

4. INTRODUCTION
■ Flow cytometry is a sensitive, powerful method for simultaneously obtaining
information on various cellular processes, including expression of surface
markers, intracellular cytokine and signaling proteins
■ Measures characteristics on each cell and hence play role in characterizing
heterogeneous cell populations.

5. Flow cytometry (FC)
■ FC process begins with the selection of fluorescent-labeled antibodies
specific to cell-surface markers used to characterize the cell population of
interest.
■ These cell surface markers are usually glycoproteins called cluster of
differentiation (CD) markers, and they help differentiate cell subpopulations
■ Flow cytometry can be performed on a variety of tissues, including peripheral
blood, bone marrow aspirates, skin biopsies, and tissue culture cell lines.

6. FLOW CYTOMETRY
■ (1) Forward-
scatter detector
■ (2) side-scatter
detector
■ (3) fluorescence
detector
■ (4) filters and
mirrors
■ (5) charged
deflection plates.

7. FLOW CYTOMETRY
■ Each cell passes through several laser beams so that different optical
properties can be measured.
■ Optical information known as forward scatter (FSC) and side scatter (SSC) is
obtained based on the angle of light emitted from the analyzed cell.
■ FSC – provides information correlating with cell size.
■ SSC – information on the granularity of the cell.
– This could be useful, for example, when trying to distinguish between
lymphocytes and granulocytes

8. Immunophenotyping
■ Methods
– Flow cytometry
– Immunohistochemistry

9. Immunophenotyping
■ Immunophenotyping is a technique that uses monoclonal antibodies to
identify protein markers present in the cytoplasm, nucleus or surface
membrane of cells, thereby identifying the cell type and degree of
differentiation.
■ Immunophenotyping establishes clonality and determine pattern of
expression of cell markers and hence the determination of malignant
subtypes.
■ Examples;
– Chronic lymphocytic leukaemia exhibits CD5, CD23, CD19, CD20 markers
– Multiple myeloma, plasma cells exhibit – CD38 and CD138

10. 10
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