Flow Cytometry

Overview of Flow Cytometry Immunophenotyping

Definition: A powerful and versatile technique that allows for the identification, characterization, and quantification of cells based on their expression of specific cell surface markers (antigens)
Principle:
- Cells are labeled with fluorescently labeled antibodies that bind to specific cell surface or intracellular antigens
- The labeled cells are passed through a laser beam in a flow cytometer
- The instrument measures the amount of light scattered and fluorescence emitted by each cell
- The data are analyzed to identify and quantify different cell populations
Clinical Significance:
- Diagnosis and Classification of Hematologic Malignancies: Essential for diagnosing and classifying leukemias and lymphomas
- Monitoring Minimal Residual Disease (MRD): Used to detect small numbers of residual cancer cells after treatment
- Assessment of Immune Function: Used to enumerate lymphocyte subsets (e.g., CD4+ T cells, CD8+ T cells) and assess immune cell activation
- Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH): Detects deficiency of GPI-linked proteins on blood cells
- Transplant Monitoring: Used to assess chimerism and detect graft-versus-host disease (GVHD)

Basic Principles of Flow Cytometry

Sample Preparation:
- Whole Blood or Bone Marrow Aspirate: Samples are collected in EDTA (purple-top) tubes or heparin tubes
- Cell Separation: RBCs are lysed to remove them, and the remaining cells are washed and resuspended in buffer
Antibody Labeling:
- Antibodies: Monoclonal antibodies specific for different cell surface or intracellular antigens are used
- Fluorochromes: Each antibody is conjugated to a fluorescent dye (fluorochrome) that emits light at a specific wavelength when excited by a laser
- Antibody Combinations: Multiple antibodies with different fluorochromes are often used simultaneously to identify multiple markers on the same cell (multicolor flow cytometry)
- Incubation: The cells are incubated with the antibody cocktail, allowing the antibodies to bind to their target antigens
Flow Cytometry Analysis:
- Flow Cell: The labeled cells are passed through a flow cell in a single stream
- Laser Excitation: A laser beam illuminates the cells as they pass through the flow cell
- Light Scatter: The instrument measures:
  - Forward Scatter (FSC): Proportional to cell size
  - Side Scatter (SSC): Proportional to cell granularity and internal complexity
- Fluorescence Detection: Detectors measure the intensity of fluorescence emitted by each fluorochrome
Data Analysis:
- Gating: Cell populations are identified and gated based on their FSC and SSC properties
  - Sequential Gating: The cells are first gated based on FSC and SSC to isolate the population of interest (e.g., lymphocytes, blasts), and then further gated based on the expression of specific markers
  - “Backgating”: Starting with the specific population and looking to the source based on side and forward scatter.
- Data Display: Data are typically displayed as dot plots or histograms, showing the expression of different markers on each cell population
- Quadrant Statistics: Quadrant markers are placed on dot plots to divide the cell population into four quadrants, and the percentage of cells in each quadrant is calculated
  - Allows quantification of cells that are positive or negative for two different markers

Applications of Flow Cytometry Immunophenotyping

Leukemia and Lymphoma Diagnosis and Classification
- Acute Leukemias:
  - Lineage Assignment
  - Subtyping of AML
- Chronic Lymphoproliferative Disorders
- Minimal Residual Disease (MRD) Monitoring
Lymphocyte Subset Analysis
- CD4+ T Cell Count
- CD8+ T Cell Count
- B Cell Quantification
- NK Cell Quantification
Paroxysmal Nocturnal Hemoglobinuria (PNH)

Quality Control

Instrument Calibration and Standardization:
- Regular calibration of the flow cytometer to ensure accurate and reproducible results
- Use of standardized reagents and protocols
Compensation:
- Correction for spectral overlap between different fluorochromes
- Using compensation controls to set appropriate compensation values
Gating Strategies:
- Use consistent and reproducible gating strategies
- Include appropriate controls to identify and exclude non-specific staining

Factors Affecting Accuracy

Sample Handling:
- Collect blood in EDTA (purple-top) tubes
- Process samples within 24 hours of collection to minimize cell degradation
Antibody Selection:
- Use high-quality, well-characterized antibodies
- Select antibodies that are known to be sensitive and specific for GPI-linked proteins
Instrument Calibration:
- Perform daily instrument calibration and quality control procedures
Compensation:
- Properly compensate for spectral overlap between different fluorochromes
Gating Strategy:
- Use a consistent and well-defined gating strategy to accurately identify cell populations
Data Analysis:
- Ensure that the data analysis is performed by trained personnel

Key Terms

Flow Cytometry: A technique for identifying and quantifying cells based on their surface markers and other characteristics
Immunophenotyping: Using flow cytometry to identify cell surface markers
Gating: Selecting a population of cells for analysis based on their forward and side scatter characteristics
Fluorochrome: A fluorescent dye used to label antibodies or other molecules
Antibody: A protein that binds to a specific antigen
Antigen: A molecule that is recognized by an antibody
Leukemia: A malignant disorder of blood-forming cells
Lymphoma: A malignant disorder of lymphocytes that typically arises in lymph nodes
Minimal Residual Disease (MRD): Small numbers of residual cancer cells that remain after treatment
Paroxysmal Nocturnal Hemoglobinuria (PNH): An acquired stem cell disorder characterized by deficiency of GPI-anchored proteins

Heinz Body

Leukemia