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Updated: Aug 8, 2026
Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up
Published on: March 26, 2018
Detection of intracellular lymphoid differentiation antigens by flow cytometry in acute lymphoblastic leukemia
1Department of Haematology, Westmead Hospital, New South Wales, Australia.
Insights
Flow cytometry reliably detects intracellular CD3 and CD22, crucial markers for distinguishing early T and B cell lineages in acute lymphoblastic leukemia (ALL). This method aids in the differential diagnosis of acute leukemia.
Area of Science:
- Immunology
- Hematology
- Cell Biology
Background:
- Accurate classification of acute leukemia subtypes is essential for effective treatment.
- Distinguishing between B-cell and T-cell acute lymphoblastic leukemia (ALL) is critical for therapeutic strategies.
- Immunophenotyping plays a key role in the diagnosis and subclassification of leukemias.
Purpose of the Study:
- To evaluate the utility of intracellular CD3 and CD22 detection by flow cytometry in the differential diagnosis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL).
- To assess the reliability of cytoplasmic CD3 and CD22 as markers for early T and B cell lineage identification in leukemia.
Main Methods:
- Flow cytometry was used to analyze fixed and permeabilized cells from AML, ALL patient samples, and leukemia cell lines.
- Cells were stained with monoclonal antibodies against CD3 and CD22 using indirect immunofluorescence.
- Detection of intracellular antigens was performed on leukemia blasts.
Main Results:
- Intracellular CD22 was detected in >20% of blasts in all 20 precursor-B ALL cases analyzed (mean 55%).
- Intracellular CD3 was detected in all 8 precursor-T ALL cases but not in precursor-B ALL or AML.
- Intracellular CD22 was absent in precursor-T ALL and AML, while intracellular CD3 was absent in precursor-B ALL and AML.
Conclusions:
- Intracellular CD3 and CD22 are reliable and specific markers for identifying early T and B cell lineages, respectively, in acute lymphoblastic leukemia.
- Flow cytometric detection of these intracellular antigens is a valuable tool for the differential diagnosis and classification of acute leukemia.
- This technique can be readily incorporated into routine immunophenotyping workflows for improved leukemia diagnosis.
Abstract:
The value of flow cytometric detection of the intracellular lymphoid differentiation antigens CD3 and CD22 in the differential diagnosis of acute leukemia was assessed in cases of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and leukemic cell lines. Cells were fixed in 0.25% paraformaldehyde at 4 degrees C for 60 min, permeabilized with 0.2% Tween 20 at 37 degrees C for 15 min, then stained with CD3 or CD22 monoclonal antibodies by indirect immunofluorescence. Cytoplasmic CD22 was detected on greater than 20% (mean 55%; range 20-87%) of blasts from all 20 cases of precursor-B ALL analyzed. The percentage of cells with cytoplasmic CD22 was greater than that with membrane CD22 in all except 2 cases of precursor-B ALL. Cytoplasmic CD22 was not detected in 8 cases of precursor-T ALL, 4 T-leukemia cell lines, or in 7 cases of AML. In contrast, cytoplasmic CD3 was detectable by flow cytometry in all 8 cases of precursor-T ALL, but not in precursor-B ALL, pre-B leukemia cell lines, or in AML. These results confirm that cytoplasmic CD3 and CD22 are excellent markers of the early T and B lineages in ALL and can be reliably detected by flow cytometry. This technique should be a valuable addition to routine immunophenotyping for classification of acute leukemia.
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