Immunology chapter 11

Overview of Flow Cytometry

Flow cytometry is a sophisticated analytical technique that integrates immunological methods with hematological analysis to diagnose and analyze various hematological disorders. It operates under high complexity testing regulations and is predominantly utilized in larger hospitals, specialized clinics, and research laboratories due to its intricate procedures and requirement for advanced instrumentation.

Fundamentals of Laser Technology

Definition of LASER:

• Light Amplification by Stimulated Emission of Radiation. This technology is fundamental in flow cytometry, where precise light manipulation is essential for accurate measurements.

Characteristics of Laser Light:

• The laser light encompasses a broad spectrum, from ultraviolet to infrared, including the visible light range.

• It exhibits highly concentrated energy, allowing for the sorting of atoms and molecules, resulting in powerful energy waves essential for the process of analysis.

• Various media can be employed, including gases, liquids, or crystals; in the context of flow cytometry, the fluid typically consists of diluted liquid blood or bone marrow within a specialized sheath fluid.

Photon Interaction:

• Photons interact with atoms and stimulate their emission, creating a cascade of energy that ultimately results in a powerful laser beam. Such interactions are critical in exciting fluorescent tags in cells.

• In flow cytometers, lasers serve as the primary and most reliable light source, ensuring optimal performance during analyses.

Principles of Cell Cytometry

Basic Components:

The essential components that facilitate flow cytometry include:

• Fluid dynamics to ensure proper fluid stream manipulation.

• Optics for managing the flow of light and capturing fluorescent signals.

• Laser science that underpins the excitation methods.

• High-speed computers for data processing and analysis.

• TAG fluorochrome conjugated monoclonal antibodies, which are pivotal for marking specific cell types for identification.

Fluorochromes:

• These are fluorescent molecules that act as distinctive tags for cells. They absorb light at shorter wavelengths and re-emit it at longer wavelengths, thereby enhancing detection sensitivity.

Excitation and Emission:

• Once fluorochromes are excited by the laser light, electrons jump to a higher energy level, and as they return to their original state, they emit light at a longer wavelength.

• During flow cytometry, stained cells are injected into the laser beam one by one through a process called hydrodynamic focusing, allowing for precise measurement and analysis.

Detection of Light:

• Sensors positioned at a 90-degree angle from the laser beam capture the light that is scattered by the cells, providing critical data on cell granularity and size. This helps in accurately enumerating different cell populations.

Applications of Flow Cytometry

Cell Identification:

• Flow cytometry analyzers are capable of identifying the major categories of white blood cells, including lymphocytes, monocytes, and neutrophils.

• It can provide absolute counts of each cell type and highlight abnormalities or changes in the cell populations, which is crucial for diagnosing various diseases

Flow Cell Immunophenotyping:

• This specific application employs monoclonal antibodies that are directed against specific CD markers on cells. These CD markers are vital for a deeper understanding of cellular populations and can reveal important information about cell lineage and maturation stages.

  • Examples of CD Markers include:

    • CD4: T-helper cells, critical in immune response.

    • CD34: Stem cells, useful in identifying immature cells.

• Immunophenotyping is essential for sorting cells into subpopulations, which can be subjected to further testing for more accurate diagnostics.

The Sorting Process

• During the sorting process, cells are marked with fluorescent monoclonal antibodies and then passed through a carefully regulated stream of sheath fluid.

• When exposed to the laser, the fluorescent tags emit light of specific colors, enabling straightforward identification and categorization of cells.

• Electrostatic charges may be applied to the droplets that contain cells, guiding them into distinct collection streams based on their electric charges and further improving sorting efficiency.

CD Markers in Flow Cytometry

• Definition of CD: Cluster Designation (CD) markers are molecules found on the surface of cells that serve to identify specific cell types and their functional states.

• Common CD markers:

  • CD3: Represents T cells, crucial for immune function.

  • CD8: Corresponds to cytotoxic T cells and suppressor cells, pivotal in regulating immune responses.

  • CD19: Identifies B cells that play a significant role in antibody production.

Applications of Immunophenotyping

• Cancer Diagnosis: Approximately 80% of immunophenotyping applications are utilized in oncology, aiding in the diagnosis of malignancies, monitoring treatment efficacy, and tracking for potential recurrences.

• HIV Monitoring: This technique allows for detailed assessment of T cell subsets, which are vital for evaluating immune responses and monitoring disease progression, with particular focus on CD4 counts.

• Basic Panels: Typical CD marker panels include a repertoire of markers such as CD3, 4, 8, 19, along with additional markers like CD16, CD56, and anti-CD4/CD14 to differentiate between lymphocytes and monocytes.

Sample Preparation for Flow Cytometry

Sample Types:

• Samples used for analysis can include whole blood (ideally collected in EDTA tubes), bone marrow (preferably in heparinized tubes), and various other body fluids. It is crucial to ensure samples are free from hemolysis or clotting to guarantee accurate results.

• Tagged monoclonal antibodies are added to the samples just prior to analysis to allow effective labeling of the target cells for identification and sorting.

Summary and Figures

• Visual representations of flow cytometry methods (such as diagrams illustrating laser interactions, cell tagging, and sorting mechanisms) greatly aid in understanding complex processes involved.

• Flow cytometry plays an integral role in the detailed analysis of cellular components in research and clinical settings, reaching applications in immunology, oncology, and beyond.