What is detected during immunophenotyping by flow cytometry (Fluorescence-Activated Cell Sorting)?

Flow Cytometry Detects Antigens Using Detection Antibodies

In immunophenotyping by flow cytometry, antigens are detected using detection antibodies (option B). 1

Basic Principles of Flow Cytometry

Flow cytometry is a powerful technique that allows for the identification and characterization of cells based on their physical properties and expression of specific antigens. The process works through the following mechanism:

• Cells in suspension flow through a laser beam one at a time
• Light scatter properties (forward and side scatter) provide information about cell size and granularity 2
• Fluorochrome-labeled antibodies bind to specific antigens on or within cells 1
• The fluorescence emitted is detected and quantified, allowing identification of cell populations 1

Detailed Mechanism of Antigen Detection

The detection process in flow cytometry involves:

1. Sample preparation: Cells are labeled with fluorochrome-conjugated antibodies that specifically bind to antigens of interest 1

2. Antibody binding: The antibodies used are specifically designed to recognize and bind to particular antigens on or within cells 1, 3

3. Detection: When the labeled cells pass through the laser beam:

   • The laser excites the fluorochromes attached to the antibodies
   • The fluorochromes emit light at specific wavelengths
   • Photodetectors capture this emitted light 4
   • The intensity of fluorescence correlates with the amount of antigen present 1

4. Analysis: The data is processed to identify and quantify different cell populations based on their antigen expression patterns 1

Why Other Options Are Incorrect

• Option A (fluorochromes using detection antibodies): This is incorrect because fluorochromes are the detection molecules attached to antibodies, not what is being detected 1

• Option C (antibodies using detection fluorochromes): This reverses the relationship; antibodies are conjugated to fluorochromes for detection, not the other way around 1, 3

• Option D (antibodies using detection antigens): This completely inverts the relationship; antigens are what antibodies bind to, not detection tools 1

Clinical Applications

Flow cytometry immunophenotyping is widely used in:

• Diagnosis and classification of hematologic malignancies 5
• Monitoring CD4+ T-cell counts in HIV-infected patients 1
• Detection of minimal residual disease in multiple myeloma 1
• Identification of rare antigen-specific B cells 6
• Characterization of immune cell subsets in various diseases 3

Technical Considerations

• Multiple antibodies with different fluorochromes can be used simultaneously to detect several antigens on the same cell 1
• Proper controls are essential for accurate interpretation of results 1
• The European Myeloma Network recommends using at least four detectors for optimal analysis 1
• Gating strategies using combinations of markers (e.g., CD38, CD138, CD45) are critical for accurate identification of cell populations 1

Flow cytometry has revolutionized cellular analysis by enabling the rapid, simultaneous detection of multiple antigens on individual cells, making it an indispensable tool in both clinical diagnostics and research settings.