Leukemia

Overview of Flow Cytometry Immunophenotyping in Leukemia

Definition: Flow cytometry immunophenotyping is a critical tool for identifying and classifying leukemias. It allows for the rapid and accurate characterization of leukemic cells based on their expression of cell surface and intracellular antigens (markers).
Importance: Distinguishing between different types of leukemia (acute vs. chronic, myeloid vs. lymphoid) and identifying specific subtypes within each category.
- Lineage Assignment (Myeloid, B-lymphoid, T-lymphoid)
- Defining the Stage of Maturation Arrest
- Identifying Aberrant Antigen Expression
- Prognostic Stratification
- Monitoring Minimal Residual Disease (MRD)

Key Markers Used in Leukemia Immunophenotyping

Here’s a breakdown of commonly used markers, with the caveat that specific panels will vary between labs:

Marker	Lineage/Cell Type	Notes
CD45	All Leukocytes	Used to identify and gate leukocyte populations; expression levels vary with cell type and maturation
Myeloid Markers
CD13	Myeloid	Expressed on most myeloid cells
CD33	Myeloid	Expressed on most myeloid cells
CD117	Myeloid, some lymphoid	Expressed on myeloid cells and some B and T cell precursors
Myeloperoxidase (MPO)	Myeloid, Granulocytic	Expressed in myeloblasts, promyelocytes, and neutrophils; not expressed in lymphoblasts
CD14	Monocytes/Macrophages	Helps identify monocytic differentiation
CD64	Monocytes/Macrophages	Helps identify monocytic differentiation
CD11b	Monocytes/Granulocytes	Helps identify monocytic and granulocytic differentiation
B-Lymphoid Markers
CD19	B cells	Expressed on most B cells
CD20	B cells	Expressed on mature B cells; expression can be lost after rituximab therapy
CD10	Germinal center B cells	Also expressed on some T cells and in certain lymphomas; used in ALL to assess maturation
CD34	B/T precursor cells
TdT	Lymphoblasts	Terminal deoxynucleotidyl transferase; expressed in immature lymphocytes
Kappa/Lambda Light Chain	Mature B cells	Used to assess B-cell clonality (kappa or lambda light chain restriction)
T-Lymphoid Markers
CD3	T cells	Expressed on all T cells
CD4	Helper T cells	Expressed on helper T cells
CD8	Cytotoxic T cells	Expressed on cytotoxic T cells
CD5	T cells, subset of B cells	Expressed on most T cells; also expressed on CLL cells
CD7	T cells
Other Markers
HLA-DR	Antigen-presenting cells	Expressed on B cells, monocytes, and dendritic cells
CD38	Activated lymphocytes	Increased expression on activated cells and in some leukemias
CD15	Reed-Sternberg cells	Also can be found in some AML.
CD30	Hodgkin and some T-cell Lymphoma cells

Key Leukemia Subtypes and Immunophenotypic Features

Acute Myeloid Leukemia (AML)
- Key Immunophenotypic Features:
  - Myeloid Blast Population: Cells are positive for one or more myeloid markers (CD13, CD33, CD117, MPO)
  - Absence of Lymphoid Markers: Blasts are typically negative for T-cell markers (CD3, CD5, CD7) and B-cell markers (CD19, CD20)
  - Variable Expression of CD34 and HLA-DR: May be positive or negative, depending on the AML subtype
- Lineage Assignment: MPO is the best and most reliable marker to separate out ALL (Acute Lymphoblastic Leukemia) from AML. CD34 can be used to measure the cell population if MPO is not present.
- Specific AML Subtypes:
  - AML with t(8;21)(q22;q22); RUNX1-RUNX1T1:
    - Often expresses CD34, CD117, CD13, and MPO
    - May also express CD19
  - AML with inv(16)(p13.1q22) or t(16;16)(p13.1;q22); CBFB-MYH11:
    - Often expresses CD117, CD13, CD33, MPO, and CD11b
    - May also express CD2
  - Acute Promyelocytic Leukemia (APL) with t(15;17)(q24.1;q21.1); PML-RARA:
    - Typically expresses CD33 and MPO
    - Lacks HLA-DR and CD34 expression
  - AML with mutated NPM1:
    - Usually expresses CD34 and HLA-DR
    - Lacks CD14 and CD11b expression
  - AML with mutated CEBPA:
    - Displays variable and often aberrant marker expression
Acute Lymphoblastic Leukemia/Lymphoma (ALL/LBL)
- General Features:
  - Lymphoblasts express markers associated with B- or T-cell lineage, but not myeloid markers
  - TdT (terminal deoxynucleotidyl transferase): a marker of immature lymphocytes, is usually positive
- B-Lymphoblastic Leukemia/Lymphoma (B-ALL/LBL): * Common Markers: CD19+, CD10+, CD34+, TdT+, HLA-DR+ * Cytoplasmic CD79a and/or CD22 * Surface Ig is usually negative (except for pre-B ALL) * WHO Classification Further Divides B-ALL based on Genetics * Common Genetic Abnormalities Include: t(9;22), t(12;21), t(4;11)
- T-Lymphoblastic Leukemia/Lymphoma (T-ALL/LBL): * Common Markers: CD3+, CD7+, TdT+, CD1a+ (often), CD34+ * May Express CD4, CD8, or both * Often involves the thymus * WHO Classification divides further based on genetics, with NOTCH1 mutations being the most common

Important Considerations

Aberrant Antigen Expression: Leukemic cells may express antigens that are not normally found on cells of that lineage. This is very helpful in diagnosis.
- Example: Myeloid markers (e.g., CD13, CD33) on lymphoblasts in B-ALL
Antigen Co-expression: Cells can express antigens from multiple lineages, particularly in acute leukemias.
Antigenic Modulation: Expression of certain antigens can be altered by treatment, so assessing pre-treatment samples is ideal
Isotype Controls: These are used to differentiate specific from non-specific antibody staining. This is especially useful for confirming the presence of low-level antigen expression
“Antigenic Fidelity”: Term used to describe the correlation of expected staining and protein production by a specific cell.

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
Leukemia: A malignant disorder of blood-forming cells in the bone marrow
Lymphoma: A malignant disorder of lymphocytes that typically arises in lymph nodes
Blast Cells: Immature, abnormal cells seen in acute leukemia
Lineage Assignment: Determining the cell type that a leukemia or lymphoma is derived from (e.g., myeloid, B-lymphoid, T-lymphoid)
Aberrant Antigen Expression: Expression of antigens that are not normally found on cells of that lineage

Flow Cytometry

Lymphoma