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

Key Terms

Immunophenotyping: Identifying and quantifying cell populations based on cell surface or intracellular markers
Immunologic Evaluations: Assessing the immune system’s components and function
Hematologic Disorders: Diseases that affect the blood, bone marrow, and lymphatic system
Cell Surface Marker: A protein or other molecule expressed on the surface of a cell that can be used to identify it
Antibody: A protein that binds specifically to a target molecule
Fluorochrome: A fluorescent dye that emits light when excited by a specific wavelength of light
Gating Strategy: A set of criteria used to identify specific cell populations
Control: A sample that is used to validate the staining protocol or to correct for background noise

Optimization

Functional Assays