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Cytometry

Immunophenotyping

Overview of Immunophenotyping

  • Definition: Immunophenotyping is the process of identifying and quantifying different cell populations based on the expression of specific cell surface or intracellular markers using antibodies and flow cytometry
  • Purpose:
    • Cell Identification: To identify and enumerate different cell types in a sample
    • Disease Diagnosis: To diagnose and classify diseases based on the abnormal expression of cell markers
    • Therapeutic Monitoring: To monitor the response to treatment and detect minimal residual disease
    • Research: To study the immune system and investigate the mechanisms of disease
  • Key Applications:
    • Immunologic Evaluations
    • Hematologic Disorders

Immunologic Evaluations

  • Definition: Assessment of the immune system’s components, function, and overall health.
  • Purpose:
    • Identify Immune Deficiencies: To diagnose primary and secondary immunodeficiencies
    • Monitor Immune Status: To monitor the immune status of patients undergoing immunosuppressive therapy or transplantation
    • Assess Immune Function: To assess the function of immune cells in response to stimuli
  • Commonly Assessed Cell Populations:
    • T Cells:
      • CD4+ T Helper Cells: Coordinate immune responses and activate other immune cells
      • CD8+ Cytotoxic T Cells: Kill infected or cancerous cells
      • Regulatory T Cells (Tregs): Suppress immune responses and maintain tolerance
    • B Cells:
      • Naive B Cells: Have not yet encountered an antigen
      • Memory B Cells: Have encountered an antigen and are ready to respond quickly upon re-exposure
      • Plasma Cells: Secrete antibodies
    • Natural Killer (NK) Cells:
      • Innate immune cells that kill infected or cancerous cells
      • CD56 dim cells: are cytotoxic
      • CD56 bright cells: secrete cytokines
    • Monocytes and Macrophages:
      • Phagocytic cells that engulf and digest pathogens and debris
    • Dendritic Cells:
      • Antigen-presenting cells that activate T cells
  • Immunophenotyping Panels:
    • Basic T Cell Panel: CD3, CD4, CD8
    • B Cell Panel: CD19, CD20, IgD, IgM
    • NK Cell Panel: CD3, CD16, CD56
    • Treg Cell Panel: CD3, CD4, CD25, CD127
  • Example Applications:
    • HIV/AIDS: Monitoring CD4+ T cell counts to assess immune status
    • Autoimmune Diseases: Identifying and quantifying autoreactive T cells or B cells
    • Transplant Monitoring: Monitoring immune cell populations to detect rejection
    • Allergy Testing: Identifying and quantifying allergic immune responses

Hematologic Disorders

  • Definition: Diseases that affect the blood, bone marrow, and lymphatic system
  • Purpose:
    • Diagnosis: To diagnose and classify hematologic malignancies, such as leukemia, lymphoma, and myeloma
    • Prognosis: To predict the course of the disease and the response to treatment
    • Minimal Residual Disease (MRD) Monitoring: To detect residual cancer cells after treatment and predict relapse
  • Commonly Assessed Cell Populations:
    • Myeloid Cells:
      • Granulocytes (Neutrophils, Eosinophils, Basophils): Involved in innate immune responses
      • Monocytes: Precursors to macrophages
      • Dendritic Cells: Antigen-presenting cells
      • Erythroid Cells: Red blood cell precursors
    • Lymphoid Cells:
      • B Cells: Produce antibodies
      • T Cells: Coordinate immune responses and kill infected cells
      • NK Cells: Kill infected or cancerous cells
    • Stem Cells:
      • Hematopoietic Stem Cells (HSCs): Give rise to all blood cell types
      • Progenitor Cells: More differentiated cells that are committed to a specific lineage
  • Immunophenotyping Panels:
    • Acute Leukemia Panel: CD34, CD117, CD13, CD33, CD19, CD10, CD7, TdT
    • Chronic Lymphoproliferative Disorder Panel: CD5, CD19, CD20, CD23, CD103, FMC7
    • Multiple Myeloma Panel: CD38, CD138, CD45, CD56, CD19
  • Example Applications:
    • Acute Myeloid Leukemia (AML): Identifying and classifying AML based on the expression of myeloid markers and genetic mutations
    • Chronic Lymphocytic Leukemia (CLL): Diagnosing and staging CLL based on the expression of lymphoid markers and disease-specific markers
    • Multiple Myeloma: Monitoring MRD after treatment to predict relapse

Sample Preparation for Immunophenotyping

  • Whole Blood:
    • Lysis of Red Blood Cells: Use of lysing buffer to remove red blood cells
    • Surface Staining: Incubation with fluorochrome-conjugated antibodies to label cell surface markers
  • Bone Marrow Aspirate:
    • Density Gradient Separation: Use of density gradient media to separate cells based on density
    • Surface Staining: Incubation with antibodies to label cell surface markers
  • Lymph Node Biopsy:
    • Mechanical Disaggregation: Use of mechanical methods to disrupt tissue and create single-cell suspensions
    • Enzymatic Digestion: Use of enzymes to further digest the tissue and release cells
  • Sample Handling:
    • Storage: Should be stored at an optimal temperature and tested as soon as possible to ensure best results
    • Cell concentration: Should be at an optimal concentration to prevent any instrument malfunction
    • Quality: Should be of proper quality to produce the most accurate results.

Gating Strategies for Immunophenotyping

  • Forward Scatter (FSC) vs. Side Scatter (SSC):
    • Used to distinguish between different cell populations based on size and granularity
  • CD45 vs. Side Scatter (SSC):
    • Used to distinguish between different leukocyte populations
  • Hierarchical Gating:
    • Used to identify specific cell subsets based on multiple markers
  • Boolean Gating:
    • Used to combine multiple markers to define complex cell populations

Controls for Immunophenotyping

  • Isotype Controls:
    • Used to assess non-specific antibody binding
  • Fluorescence Minus One (FMO) Controls:
    • Used to identify gating boundaries and account for spread
  • Compensation Controls:
    • Used to correct for spectral overlap
  • Viability Dyes:
    • Used to exclude dead cells from analysis

Troubleshooting Immunophenotyping Assays

  • Low Event Count:
    • Possible Causes:
      • Sample loss
      • Instrument malfunction
      • Incorrect gating
    • Troubleshooting Steps:
      • Optimize sample collection and preparation
      • Inspect instrument for malfunctions
      • Verify gating strategy
  • High Background Noise:
    • Possible Causes:
      • Non-specific antibody binding
      • Autofluorescence
      • Contamination
    • Troubleshooting Steps:
      • Use blocking reagents
      • Reduce autofluorescence
      • Clean samples
  • Unexpected Results:
    • Possible Causes:
      • Incorrect antibody selection
      • Improper sample preparation
      • Instrument malfunction
    • Troubleshooting Steps:
      • Verify antibody specificity
      • Review sample preparation protocols
      • Inspect instrument for malfunctions