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Hematology

Lymphocyte Subsets

Overview of Lymphocyte Subset Analysis by Flow Cytometry

  • Definition: Flow cytometry immunophenotyping is used to identify, quantify, and characterize lymphocyte populations (T cells, B cells, and NK cells) based on the expression of specific cell surface markers
  • Clinical Significance:
    • Assessment of Immune Function: Provides information about the number and proportions of different lymphocyte subsets, which is essential for evaluating immune competence and detecting immune deficiencies
    • Diagnosis and Monitoring of Immunodeficiency Disorders: Used to diagnose and monitor patients with HIV/AIDS, severe combined immunodeficiency (SCID), and other immunodeficiency disorders
    • Monitoring Immunosuppressive Therapy: Used to monitor the effects of immunosuppressive drugs in transplant recipients and patients with autoimmune diseases
    • Diagnosis and Monitoring of Lymphoproliferative Disorders: Can help identify and characterize abnormal lymphocyte populations in leukemia and lymphoma
  • Key Cell Types:
    • T Lymphocytes (T cells):
      • Helper T Cells (CD4+ T Cells)
      • Cytotoxic T Cells (CD8+ T Cells)
      • Regulatory T Cells (Tregs)
    • B Lymphocytes (B Cells)
    • Natural Killer (NK) Cells

Cell Surface Markers and Their Significance

The following table outlines the key cell surface markers used to identify and characterize lymphocyte subsets:

Marker Cell Type/Lineage Function Expression Pattern
CD45 All Leukocytes Protein tyrosine phosphatase; regulates leukocyte activation Expressed on all leukocytes, but with varying intensity
CD3 T Cells Part of the T cell receptor (TCR) complex; involved in T-cell activation Expressed on all T cells
CD4 Helper T Cells Coreceptor for MHC class II; involved in T-cell activation Expressed on helper T cells; also expressed on some monocytes and dendritic cells
CD8 Cytotoxic T Cells Coreceptor for MHC class I; involved in T-cell cytotoxicity Expressed on cytotoxic T cells and some NK cells
CD19 B Cells B-cell receptor complex component; involved in B-cell activation Expressed on most B cells
CD20 B Cells Calcium channel; involved in B-cell activation Expressed on mature B cells; expression can be lost after rituximab therapy
CD56 (NCAM) NK Cells, Some T Cells Neural cell adhesion molecule; involved in cell-cell interactions Expressed on NK cells and some T cells
CD16 (FcγRIII) NK Cells, Neutrophils Fc receptor for IgG; involved in antibody-dependent cell-mediated cytotoxicity (ADCC) Expressed on NK cells and neutrophils
CD25 (IL-2Rα) Activated T and B Cells Part of the IL-2 receptor; involved in lymphocyte proliferation Expressed on activated T and B cells, regulatory T cells
CD103 Regulatory T Cells Alpha E integrin, plays a role in trafficking and homing T regulatory cells

Common Lymphocyte Subset Combinations

  • T Cell Analysis:
    • CD45, CD3: To identify all T cells
    • CD4: To identify helper T cells
    • CD8: To identify cytotoxic T cells
    • CD4:CD8 Ratio: A useful marker for classifying types of T-Cells
    • CD25 and FoxP3 can identify regulatory T cells
  • B Cell Analysis:
    • CD45, CD19: To identify all B cells
    • Kappa and Lambda Light Chains: To assess for clonality in B-cell populations
  • NK Cell Analysis:
    • CD45, CD3-: Used to exclude T cells
    • CD56 or CD16: To identify NK cells

Applications of Lymphocyte Subset Analysis

  • HIV/AIDS:
    • Monitoring CD4+ T Cell Count: The CD4+ T cell count is a key indicator of immune function in HIV-infected individuals
    • Low CD4+ T cell counts indicate immune deficiency and increased susceptibility to opportunistic infections
    • Monitoring Treatment Response: Used to assess the effectiveness of antiretroviral therapy (ART) by monitoring increases in CD4+ T cell counts
  • Severe Combined Immunodeficiency (SCID):
    • Diagnosis: Flow cytometry is used to identify the absence or severe reduction of T cells, B cells, and/or NK cells in infants with suspected SCID
    • T-B+NK+ SCID: Absence of T cells, but presence of B and NK cells
    • T-B-NK+ SCID: Absence of T cells and B cells, but presence of NK cells
    • T-B+NK- SCID: Absence of T cells and NK cells, but presence of B cells
    • T-B-NK- SCID: Absence of T cells, B cells, and NK cells
  • Autoimmune Disorders:
    • Monitoring Immunosuppressive Therapy: Flow cytometry can be used to monitor the effects of immunosuppressive drugs on lymphocyte subsets in patients with autoimmune disorders (e.g., rheumatoid arthritis, systemic lupus erythematosus)
    • Assessment of Disease Activity: Changes in lymphocyte subset numbers or activation markers may correlate with disease activity
  • Transplant Monitoring:
    • Chimerism Analysis: Used to assess the proportion of donor and recipient cells in the blood and bone marrow after hematopoietic stem cell transplantation (HSCT)
    • Detection of Graft-versus-Host Disease (GVHD): Flow cytometry can be used to identify activated T cells that are involved in GVHD

Reporting Results

  • Report the absolute counts and percentages of each lymphocyte subset (e.g., CD4+ T cells, CD8+ T cells, B cells, NK cells)
  • Include the reference range for each parameter
  • Note any abnormal results or deviations from normal lymphocyte subset distributions
  • Correlate the laboratory findings with the patient’s clinical information

Key Terms

  • Flow Cytometry: A technique for identifying and quantifying cells based on their surface markers
  • Immunophenotyping: Using flow cytometry to identify cell surface markers
  • Gating: Selecting a population of cells for analysis based on their light scatter properties and/or expression of specific markers
  • 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
  • T Lymphocyte (T Cell): A type of lymphocyte that mediates cell-mediated immunity
  • Helper T Cell (CD4+ T Cell): A type of T cell that helps activate other immune cells
  • Cytotoxic T Cell (CD8+ T Cell): A type of T cell that kills infected or cancerous cells
  • B Lymphocyte (B Cell): A type of lymphocyte that produces antibodies
  • Natural Killer (NK) Cell: A type of lymphocyte that kills infected or cancerous cells without prior sensitization