When should flow cytometry be performed in patients with suspected or known leukemia?

Flow Cytometry Indications in Leukemia

Flow cytometry immunophenotyping must be performed on all patients with suspected acute leukemia when blasts are present on peripheral blood smear or bone marrow examination, as it is essential for distinguishing AML, B-ALL, T-ALL, and mixed phenotype acute leukemia (MPAL), and this testing directly impacts mortality through accurate diagnosis and subsequent minimal residual disease monitoring. 1, 2

Mandatory Indications for Flow Cytometry

Flow cytometry is required in the following clinical scenarios:

• Any patient with suspected acute leukemia based on peripheral blood or bone marrow morphology showing blast cells, regardless of blast percentage 1, 2
• Leukocytosis with circulating blasts detected on peripheral blood smear, where the panel must distinguish between AML, B-ALL, T-ALL, and MPAL 2
• Unexplained leukocytosis accompanied by constitutional symptoms, elevated disease markers, or cytopenias in other cell lines 2
• Bone marrow evaluation showing ≥20% blasts (or any blast percentage with specific cytogenetic abnormalities like t(8;21), inv(16), or t(15;17)) 1

Specimen Selection Algorithm

The preferred specimen is bone marrow aspirate, which should be submitted for multicolor comprehensive flow cytometry alongside morphologic examination 1, 2. The hierarchy for specimen selection follows this order:

1. Bone marrow aspirate (first choice) - provides optimal cellular material for comprehensive immunophenotyping 1, 2
2. Peripheral blood - acceptable if sufficient blasts are present (typically >5% blasts) or if bone marrow examination is contraindicated or not feasible 1, 2
3. Core biopsy tissue (unfixed, in culture media) - only if bone marrow aspirate yields a "dry tap" or is unobtainable, requiring tissue disaggregation 1, 2

Critical Diagnostic Requirements

The flow cytometry panel must be comprehensive enough to accomplish multiple diagnostic objectives simultaneously:

• Lineage determination: Must cover B-lymphoblastic markers, T-lymphoblastic markers, and myeloid markers to distinguish AML from ALL 1, 2, 3
• Subtype classification: Must identify specific immunophenotypic patterns that predict cytogenetic abnormalities and prognosis 1, 4
• Baseline establishment: The immunophenotypic profile serves as a "fingerprint" for subsequent minimal residual disease (MRD) monitoring, which is a strong recommendation that directly impacts mortality 1, 2, 4

Multicolor flow cytometry (minimum 6 colors) is highly recommended as the preferred technique compared with immunohistologic methods, though immunohistochemistry can substitute when flow cytometry has clearly established the diagnosis by routine morphologic and cytochemical criteria 1, 3, 4

Integration with Other Diagnostic Modalities

Flow cytometry does not replace other essential diagnostic tests but must be performed alongside them:

• Routine cytochemistry (peroxidase and esterase) should be carried out in conjunction with immunophenotyping 1
• Conventional cytogenetics must be performed on all patients, as karyotype provides the most important prognostic information in AML 1
• FISH and molecular testing (FLT3, NPM1, CEBPA, BCR-ABL1, KMT2A rearrangements) are required based on the acute leukemia subtype 1, 2
• Morphologic examination of at least 500 nucleated cells remains the gold standard and cannot be substituted by flow cytometry alone 1, 5

Common Pitfalls to Avoid

Flow cytometry should never replace standard differential counting and must not substitute for an inadequate bone marrow aspirate 1, 5. Several critical caveats warrant attention:

• Detection threshold limitations: Flow cytometry typically requires ≥20% positivity for most markers, and populations below this threshold may not be identified as abnormal 5
• Aleukemic presentations: Normal peripheral blood counts do not exclude acute leukemia; bone marrow assessment remains essential even when peripheral counts appear normal 5
• Ambiguous lineage cases: Some AML cases show minimal differentiation or lack typical myeloid markers, requiring comprehensive multiparameter panels on bone marrow for accurate diagnosis 5, 6
• CD34/HLA-DR negative patterns: While 80% of acute promyelocytic leukemia (APL) cases are negative for both CD34 and HLA-DR, 10% of non-APL AML also show this pattern, requiring additional testing for definitive diagnosis 6

Special Clinical Scenarios

Additional flow cytometry applications include:

• CSF evaluation: Flow cytometry has superior sensitivity over conventional cytology for detecting CNS infiltration in patients with confirmed acute leukemia 2, 7
• Minimal residual disease monitoring: The established immunophenotypic profile from diagnostic flow cytometry enables subsequent MRD detection, which is critical for treatment monitoring and predicting relapse 1, 2, 7
• Biphenotypic and undifferentiated leukemias: Flow cytometry can identify mixed phenotype acute leukemia (MPAL) and acute undifferentiated leukemia, which are not diagnosable by morphology alone 1, 6