Lymphoma

Overview of Flow Cytometry Immunophenotyping in Lymphoma

• Definition: The use of flow cytometry to identify and characterize lymphoma cells based on their expression of cell surface and intracellular proteins (antigens). It’s a powerful tool, but needs to be used in the context of morphology from cytology and histology samples.
• Purpose:
  • Diagnosis of Lymphoma: Helps distinguish lymphoma from reactive lymphoid proliferations and other types of cancer
  • Classification of Lymphoma: Identifies specific subtypes of lymphoma based on the immunophenotype of the malignant cells
  • Prognosis: Can provide information about the prognosis (likely outcome) of the disease
  • Monitoring Treatment Response: Used to assess the effectiveness of treatment and detect minimal residual disease (MRD)
• Sample Types:
  • Lymph Node Biopsy: The most common sample type; a single cell suspension is prepared from the lymph node tissue
  • Bone Marrow Aspirate: Used to assess bone marrow involvement in lymphoma
  • Peripheral Blood: Can be used to evaluate for circulating lymphoma cells (e.g., in Sézary syndrome)
  • Other Tissue Biopsies: Can be used to evaluate for lymphoma in extranodal sites (e.g., spleen, liver, skin)

Common Markers Used in Lymphoma Immunophenotyping

Marker	Cell Lineage/Function	Expected Expression	Potential Significance in Lymphoma
CD45	All leukocytes	Present on most leukocytes, but with varying intensity	Used to identify and gate on leukocyte populations
CD19	B cells	Expressed on most B cells	Used to identify B-cell lymphomas
CD20	B cells	Expressed on most B cells, but expression can be lost after rituximab therapy	Used to identify B-cell lymphomas; target for rituximab therapy
CD5	T cells, subset of B cells	Expressed on most T cells; also expressed on CLL cells	Helps differentiate CLL from other B-cell lymphomas
CD10	Germinal center B cells, some T cells	Expressed on germinal center B cells; also expressed in some T-cell lymphomas	Helps identify follicular lymphoma and Burkitt lymphoma
CD3	T cells	Expressed on all T cells	Used to identify T-cell lymphomas
CD4	T Helper Cells	Expressed on T Helper Cells	Helps identify subtypes of T-cell Lymphomas such as ATLL
Kappa/Lambda	Immunoglobulin light chains (B cells)	Restricted expression (only kappa or lambda) in clonal B-cell populations	Used to assess B-cell clonality (kappa or lambda light chain restriction)
CD30	Activated lymphocytes	Expressed on activated lymphocytes; strongly expressed in Hodgkin lymphoma	Helps identify Hodgkin lymphoma and ALCL
CD15	Granulocytes, Reed-Sternberg cells (HL)	Expressed on granulocytes; also expressed on Reed-Sternberg cells in HL	Helps identify Hodgkin lymphoma
PAX5	B-cells (transcription factor)	Expressed on B-cells; but usually lost in classical Hodgkin lymphoma	Used to help identify B-cell origin
Ki-67	Proliferating cells	Measures the proliferation rate of cells	Helps differentiate aggressive from indolent lymphomas
TdT	Lymphoblasts	Terminal deoxynucleotidyl transferase; expressed in immature lymphocytes	Helps differentiate AML from ALL

• MHC (Major Histocompatibility Complex) Class II markers: *HLA-DR, DP, DQ - helps to identify antigen presenting cells such as B-cells

Key Lymphoma Subtypes and Immunophenotypic Features

• B-Cell Lymphomas:
  • Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma (CLL/SLL):
    • CD5+, CD19+, CD20dim, CD23+, FMC7-, Cyclin D1-, kappa or lambda restricted
  • Follicular Lymphoma (FL):
    • CD19+, CD20+, CD10+, BCL2+, CD5-, kappa or lambda restricted
  • Mantle Cell Lymphoma (MCL):
    • CD19+, CD20+, CD5+, Cyclin D1+, CD10-, kappa or lambda restricted
  • Marginal Zone Lymphoma (MZL):
    • CD19+, CD20+, CD5-, CD10-, BCL6-, kappa or lambda restricted
  • Diffuse Large B-Cell Lymphoma (DLBCL):
    • CD19+, CD20+, and variable expression of other markers (CD10, BCL6, MUM1)
    • Requires correlation with morphology and genetic studies
  • Burkitt Lymphoma:
    • CD19+, CD20+, CD10+, BCL6+, Ki-67 > 95%, kappa or lambda restricted
• T-Cell Lymphomas:
  • Peripheral T-Cell Lymphoma, Not Otherwise Specified (PTCL, NOS):
    • A diagnosis of exclusion - must rule out other T-cell lymphomas
    • Most are CD4+, with loss of some normal T-cell antigens
    • Clonality can be confirmed by T-cell receptor (TCR) gene rearrangement studies
  • Anaplastic Large Cell Lymphoma (ALCL):
    • CD30+, ALK-1+ (in most cases), CD4+, CD8-
  • Adult T-Cell Leukemia/Lymphoma (ATLL):
    • CD3+, CD4+, CD25+, FoxP3+

Interpreting Flow Cytometry Results

• Establish Gating Strategy:
  • Use forward scatter (FSC) and side scatter (SSC) to gate on the lymphocyte population
  • Exclude debris, doublets, and non-lymphoid cells
• Identify Cell Populations:
  • Use lineage-specific markers (e.g., CD19 for B cells, CD3 for T cells) to identify different lymphocyte populations
• Assess Antigen Expression Patterns:
  • Determine whether the cells express the expected antigens
  • Identify any aberrant antigen expression patterns (e.g., co-expression of myeloid markers on lymphoid cells)
• Evaluate Clonality:
  • Assess for light chain restriction in B-cell populations (kappa or lambda)
  • Perform T-cell receptor (TCR) gene rearrangement studies to assess for T-cell clonality

Reporting Results

• Report the percentage of cells expressing each marker
• Provide an interpretation of the results, noting any abnormal findings
• Correlate the flow cytometry results with the patient’s clinical information, morphology, and other laboratory data

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 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
• Lymphoma: A malignant disorder of lymphocytes that typically arises in lymph nodes
• Leukemia: A malignant disorder of blood-forming cells in the bone marrow
• Cell surface marker: Proteins on the surface of a cell that assist with identification.
• TdT: Terminal deoxynucleotidyl transferase; expressed in immature lymphocytes
• Clonality: The presence of a single, genetically identical population of cells