Immunophenotyping Of Leukemias

Overview Of Diagnostic Utility

• Flow cytometry provides definitive method for distinguishing leukemic cell lineages.
• Analyzes surface and cytoplasmic antigens to classify blasts.
• Essential for monitoring minimal residual disease throughout therapy.

Acute Lymphoblastic Leukemia

Accounts for majority of pediatric acute leukemias. Differentiated into B-lineage and T-lineage.

B-Cell Acute Lymphoblastic Leukemia

Comprises approximately 85% of cases.

Marker Category	Specific Antigens	Diagnostic And Prognostic Implications
Primary Lineage Markers	CD19, CD10, CD22, cCD79a	Define B-cell lineage.
Secondary Markers	CD20, CD45, kappa, lambda	CD20 expression less common in children.
Favorable Prognostic Markers	Presence of CD10, CD19	Indicates favorable outcome.
Adverse Prognostic Markers	Absence of CD10	Associated with mixed lineage leukemia (MLL) translocations; poor outcome.
Mature B-Cell (Burkitt)	CD19, CD20, CD21, surface Ig+	L3 morphology; necessitates intensified chemotherapy.

T-Cell Acute Lymphoblastic Leukemia

Comprises approximately 15% of cases.

Subtype	Specific Antigens	Clinical Features
Standard T-ALL	cCD3, CD2, CD4, CD5, CD7, CD8, CD1a, CD45	Older age, high leukocyte count, mediastinal mass, CNS involvement risk.
Early T-Cell Precursor (ETP)	CD1a-, CD8-, CD5- (or dim)	Originates from early thymocytes retaining stem cell features.
ETP Myeloid/Stem Markers	CD34, CD117, HLA-DR, CD13, CD33, CD11b, CD65	Requires one or more myeloid/stem markers for diagnosis.

Acute Myeloid Leukemia

Diagnosis requires comprehensive panels evaluating myeloperoxidase and monocytic differentiation.

General Myeloid Profile

• Primary diagnostic markers: cMPO, CD13, CD33, CD117, CD15.
• Additional markers: CD11b, CD14, CD16, CD34, CD45, HLA-DR.

Correlation With French-American-British Subtypes

Surface marker expression dictates morphological subtype.

Subtype	HLA-DR	CD11b	CD13	CD14	CD15	CD33	CD34	Specific Defining Markers
M1 / M2	+		+		+	+	+
M3 / M3V			+		+	+
M4 / M5	+	+	+	+	+	+	+
M6	+		+			+	+	Glycophorin (+)
M7	+		+			+	+	CD41 (+), CD42 (+), CD61 (+).
M0	+		+			+	+

Acute Leukemias Of Ambiguous Lineage

Mixed phenotype acute leukemias (MPAL) display multiple lineages on single blast population.

• Defined by strict immunophenotypic criteria.

Target Lineage	Required Flow Cytometry Diagnostic Criteria
Myeloid	Myeloperoxidase (MPO) positivity.OR Monocytic differentiation (requires $\ge$ 2): NSE, CD11c, CD14, CD64, Lysozyme.
T-Lineage	Strong cytoplasmic CD3 OR Surface CD3.
B-Lineage	Strong CD19 AND $\ge$ 1 strong expression of: CD79a, cytoplasmic CD22, CD10.OR Weak CD19 AND $\ge$ 2 strong expression of: CD79a, cytoplasmic CD22, CD10.

Chronic Myeloid Leukemia

Clonal disorder originating in pluripotent hematopoietic stem cells.

• Diagnostic Method: Flow cytometry plays secondary role to cytogenetics and molecular testing.
• Key Molecular Marker: Diagnosis relies on detecting Philadelphia chromosome t(9;22)(q34;q11) yielding BCR-ABL1 fusion gene.
• Peripheral Blood/Marrow Profile: Hypercellularity displaying complete spectrum of granulocyte precursors.
• Blast Percentage: Blasts typically constitute <2-5% in chronic phase; $\ge$ 10-19% indicates accelerated phase; $\ge$ 20% indicates blast crisis.

Juvenile Myelomonocytic Leukemia

Rare aggressive overlap myelodysplastic/myeloproliferative neoplasm.

• Cellular Proliferation: Hyperproliferation of monocytic and granulocytic cells.
• Immunophenotypic/Morphologic Profile: Blood smear reveals striking monocytosis with immature myeloid forms and dysplasia.
• Diagnostic Criteria: Requires absolute monocytosis >1000/mm3.
• Blast Percentage: Blasts constitute <20% in blood and bone marrow.
• Molecular Markers: Diagnosis relies on somatic or germline mutations driving hyperactive RAS signaling (PTPN11, KRAS, NRAS, NF1, CBL) rather than specific flow cytometry markers.