Flow Cytometry in Acute Leukemia: Essential Diagnostic and Monitoring Tool
Flow cytometry immunophenotyping is mandatory for the initial diagnostic workup of all patients with suspected acute leukemia and must be performed on bone marrow aspirate (or peripheral blood if sufficient blasts are present) alongside morphologic examination, conventional cytogenetics, and molecular testing. 1, 2
Critical Role in Initial Diagnosis
Primary Diagnostic Functions
Flow cytometry serves three essential purposes at diagnosis that cannot be achieved by morphology alone:
Lineage determination: The flow cytometry panel must be comprehensive enough to distinguish AML (including APL), early T-ALL, B-ALL, and acute leukemia of ambiguous lineage in all patients. 1, 2
Subtype classification: Multiparameter flow cytometry (minimum 6 colors) identifies complex antigenic profiles associated with specific molecular defects and well-defined biology, providing more precise classification than morphology alone. 3, 4
MRD monitoring preparation: The pathologist or treating clinician must ensure that flow cytometry analysis is comprehensive enough at diagnosis to allow subsequent detection of minimal residual disease by documenting leukemia-associated immunophenotypes. 1, 2
Specimen Requirements and Processing
Preferred specimen: Bone marrow aspirate is the gold standard for flow cytometry analysis in acute leukemia, though peripheral blood can be used if sufficient blasts are present (typically >20%). 1, 2
Alternative when aspirate inadequate: If bone marrow aspirate is unobtainable or yields a dry tap, an additional core biopsy may be submitted unfixed in tissue culture medium for disaggregation for flow cytometry and genetic studies. 1
Processing requirements: Fresh, unfixed specimens must be processed promptly to preserve cell viability and antigen expression. 2
Specific Diagnostic Applications
Acute Myeloid Leukemia (AML)
Flow cytometry provides critical diagnostic information in AML:
APL differentiation: The combined absence of CD34 and HLA-DR has high predictive value for acute promyelocytic leukemia—80% of APL cases are negative for both markers, while 58% of non-APL AML cases are positive for both. 5
Minimally differentiated AML (M0): Flow cytometry is especially useful for identifying AML M0 based on lack of cytoplasmic CD3 and CD79a expression combined with expression of one or more myelomonocytic-associated antigens and/or myeloperoxidase. 5
Genetically-defined subtypes: Specific cytogenetic abnormalities like t(8;21), inv(16), and t(15;17) have distinctive immunophenotypic profiles that flow cytometry can identify, providing rapid diagnostic and prognostic insights before molecular results are available. 6, 4, 7
Acute Lymphoblastic Leukemia (ALL)
B-ALL characterization: Flow cytometry identifies CD20 expression (present in 24% of precursor B-ALL cases) and CD34 status (absent in 29% of cases), both of which have prognostic implications. 5
T-ALL identification: CD5 and CD7 are the most sensitive antigens present in all T-ALL cases aside from cytoplasmic CD3. 5
TdT-negative variants: Flow cytometry is particularly valuable for correctly identifying TdT-negative ALL and unusual variants such as transitional B-cell ALL and biphenotypic acute leukemias. 5
Role in CSF Evaluation
ALL patients: For patients with ALL receiving intrathecal therapy, flow cytometry should be used in CSF evaluation for superior sensitivity over conventional cytology, particularly for detecting low levels of CNS infiltration. 1, 8
Other acute leukemias: For patients with acute leukemia other than ALL, the pathologist may use flow cytometry in CSF evaluation when clinically indicated. 1
Technical requirements: Cell count must be performed with examination and enumeration of blasts on cytocentrifuge preparation reviewed by a pathologist. 1, 8
Extramedullary Disease Assessment
Tissue biopsy processing: For patients presenting with extramedullary disease without bone marrow or blood involvement, tissue biopsy specimens must be processed for immunophenotypic studies (along with morphologic, cytogenetic, and molecular genetic studies) identical to bone marrow evaluation. 1, 2
Fresh material requirement: Additional biopsies may be indicated to obtain fresh material for flow cytometry and other ancillary testing. 1
Minimal Residual Disease (MRD) Monitoring
The flow cytometry analysis at diagnosis must be designed to enable MRD detection during follow-up—this is a strong recommendation that directly impacts treatment decisions and outcomes. 1, 2
Documentation requirement: Leukemia-associated immunophenotypes must be documented at initial diagnosis to serve as the baseline for subsequent MRD monitoring. 2
Clinical significance: MRD status is a key predictor of relapse and outcomes, making the initial comprehensive flow cytometry analysis critical for long-term management. 7
Common Pitfalls to Avoid
Do not use flow cytometry to replace morphologic blast counting: Flow cytometry should not substitute for an inadequate bone marrow aspirate or replace standard differential counting of at least 500 nucleated cells. 3
Do not rely on peripheral blood alone: Normal peripheral blood counts do not exclude acute leukemia—bone marrow assessment with flow cytometry is essential even when peripheral counts appear normal, as many patients present with aleukemic leukemia. 3
Do not use insufficient panels: The flow cytometry panel must be multiparametric (minimum 6 colors) and comprehensive enough to distinguish all acute leukemia subtypes and enable subsequent MRD detection. 1, 2, 3
Do not delay specimen processing: Fresh specimens must be processed promptly as delayed processing compromises cell viability and antigen expression, leading to inaccurate results. 2
Immunohistochemistry limitations: If sufficient bone marrow aspirate or peripheral blood material is not available for flow cytometry, immunohistochemical studies may be used as an alternative but provide only limited immunophenotyping compared to multiparameter flow cytometry. 1