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Atlas With Flow Cytometry Utilized In AML Today

Flow Cytometry - The Technique

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M5b M6 M7 Acute Basophhilic Leukemia Multidrug Resistance Bibliography Review Chart


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Flow cytometric techniques are now routinely utilized in clinical hematology. The diagnostic evaluation of a patient with acute leukemia, either at first diagnosis or at relapse, should include immunophenotyping in conjunction with morphology and histochemistry, cytogenetic analysis and selected molecular studies. Multiparameter flow cytometry measurements allow for the differential analysis of normal and abnormal cells within a peripheral blood or bone-marrow specimen. The characterization of leukemias by immunophenotyping is particularly helpful when the morphology is difficult to interpret. Furthermore, immune markers have led to the identification of particular leukemia subtypes, not recognized by morphologic criteria, which may have prognostic significance. The following atlas illustrates correlations between morphologic and flow cytometric evaluations in AML.

Flow Cytochemistry

Many of the commercial hematology analyzers employ flow cytochemistry in generating a five part differential. Cells are characterized by their cell size (Y axis) as well as the intensity of a cytochemical reaction for peroxidase (X axis). A second dual parameter plot defines cells on the basis of size and lobularity of the nucleus. The component subpopulations occupy different regions and form a well-recognized pattern in the normal peripheral blood subtypes of AML producing distinct changes in the "cytograms".

Templates for Cytograms
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Normal Peripheral Blood
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In normal peripheral blood, few cells fall into the large unstained cells (LUC) region. The lobularity channel has a characteristic "catepillar" appearance with the mononuclear cells (lymphocytes, monocytes) forming the head, while the tail is composed of the neutrophils and eosinophils. As expected, few (if any) basophils are detected.

Immunophenotyping

The major advantage offered by flow cytometry is that subsets of cells within a normal peripheral blood or bone marrow hematopoietic cell population can be identified by staining with antibodies that are conjugated with fluorescent dyes (fluorochromes) and recognize membrane and intracellular proteins (antigens).

If a fluorochrome (antibody) is bound to a cell, a fluorescence signal is generated when this cell passes through the laser light source of the flow cytometer. Each cell is recorded as an event and placed in a channel that is proportional to the intensity of the cell's fluorescence (fluorescence intensity is a measure of antigen density). Therefore, in a histogram (evaluation of single antibodies) or a contour plot (simultaneous evaluation of two or three antibodies), channel numbers marking the X and Y axes reflect fluorescence intensity. Below is an example of how subpopulations of normal peripheral blood lymphocytes ("gated" in an RI in a scattergram based on physical properties) can be identified by antibody staining. Lymphocytes were stained simultaneously with antibodies CD3 (T lymphocytes) and CD19 (B lymphocytes) using two fluorochromes, fluorescein isothiocyanate (FITC) for CD3 and phycoerythrin (PE) for CD19. Based on color combination (only FITC, only PE, or both FITC and PE are bound to a cell), each cell appears in a different part or quadrant of the contour plot. Cells in the left lower corner do not stain with either antibody, cells in the right lower quadrant stain only with CD3, those in the left upper quadrant stain only with CD19, and any cells in the right upper quadrant would be double stained for both CD3 and CD19. In normal peripheral blood, none of the lymphoid cells should demonstrate such double CD3/CD19 expression.

Immunophenotyping from Normal Peripheral Blood
Illustrating T and B Cells

Contour Plot
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