U01.01.038 Flow cytometry

Flow cytometry is a powerful laboratory technique used to assess various characteristics of individual cells within a sample. It allows for the evaluation of size, granularity, and protein expression (immunophenotype) of cells by utilizing fluorescently labeled antibodies specific to surface or intracellular proteins. This technique is commonly applied in the study of hematologic abnormalities and immunodeficiencies.

How Flow Cytometry Works

Tagging Cells with Antibodies:
- Cells are labeled with antibodies that specifically target surface or intracellular proteins.
- These antibodies are conjugated to unique fluorescent dyes.
Laser Analysis:
- The sample is passed through a laser beam, which focuses on individual cells.
- The intensity of light scatter and fluorescence emitted from the tagged antibodies is measured.
Data Representation:
- The data is plotted in either a histogram (one measure) or a scatter plot (two measures).
- The scatter plot allows visualization of cell populations based on multiple characteristics.

Flow Cytometry Data Interpretation: Quadrant Analysis

Left Lower Quadrant: Cells negative for both CD8 and CD3.
Right Lower Quadrant: Cells positive for CD8 but negative for CD3. This quadrant may be empty if all CD8+ cells also express CD3.
Left Upper Quadrant: Cells positive for CD3 but negative for CD8.
Right Upper Quadrant: Cells positive for both CD8 and CD3.

Quadrant	Characteristics	Cell Type Examples
Left Lower	CD8- / CD3-	T-cell negative for both markers
Right Lower	CD8+ / CD3-	CD8+ T-cells (no CD3 expression)
Left Upper	CD8- / CD3+	CD3+ T-cells (no CD8 expression)
Right Upper	CD8+ / CD3+	CD8+ T-cells expressing both markers

Applications of Flow Cytometry

1. Hematologic Abnormalities

Leukemia: Identifying abnormal cell populations based on immunophenotyping.
Paroxysmal Nocturnal Hemoglobinuria: Analyzing cell surface markers to assess defects in cell populations.
Fetal RBCs in Maternal Blood: Detecting fetal cells in maternal circulation.

2. Immunodeficiencies

HIV: Assessing CD4+ T-cell count, a key marker of immune function in HIV patients.

Points to Remember

Fluorescent Labels: Used to tag antibodies for precise identification of proteins on the cell surface or within the cell.
Laser: Induces fluorescence in labeled antibodies, enabling the detection of specific cellular markers.
Scatter Plots: Provide a detailed view of multiple parameters (e.g., size, granularity, protein expression) simultaneously.
Clinical Utility: Essential in diagnosing hematologic conditions and evaluating immune function.

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