What is included in the diagnostic workup for leukemia or lymphoma using flow cytometry and immunophenotyping?

Flow Cytometry and Immunophenotyping in Leukemia/Lymphoma Diagnosis

Flow cytometry immunophenotyping is an essential component of the diagnostic workup for leukemia and lymphoma, requiring a comprehensive multicolor panel to identify cell lineage, establish clonality, detect aberrant antigen expression, and enable subsequent minimal residual disease monitoring. 1

Core Components of Flow Cytometry Immunophenotyping

Sample Requirements and Processing

• Bone marrow aspirate is the preferred specimen for flow cytometry analysis in acute leukemia, though peripheral blood can be used if sufficient blasts are present 1, 2
• Fresh, unfixed specimens must be processed promptly to preserve cell viability and antigen expression 1
• If bone marrow aspirate is inadequate, a second core biopsy can be submitted unfixed in tissue culture medium for disaggregation 1

Essential Immunophenotypic Panels

For Lymphoma (NHL/CLL/SLL)

• Pan-B and Pan-T cell antigens form the initial screening panel 1
• Specific markers for flow cytometry include: kappa/lambda light chains, CD19, CD20, CD5, CD23, and CD10 1
• Cyclin D1 assessment or FISH for t(11;14) is mandatory when using flow cytometry alone to exclude mantle cell lymphoma, as both CLL/SLL and MCL are CD5+ B-cell tumors 1
• The typical CLL/SLL immunophenotype is CD5+, CD10−, CD19+, CD20 dim, surface immunoglobulin dim, CD23+, CD43+/−, and cyclin D1− 1

For Acute Lymphoblastic Leukemia (ALL)

• Multicolor comprehensive flow cytometry panel must be performed on bone marrow aspirate or peripheral blood 1
• B-ALL requires assessment of: B-cell lineage markers, CD10, CD19, CD20, plus evaluation for aberrant myeloid antigen expression 1
• T-ALL requires: T-cell lineage markers including CD3, CD5, CD7, and assessment of maturation stage 1
• The panel must be comprehensive enough to allow subsequent MRD detection, which is critical for treatment monitoring 1

For Acute Myeloid Leukemia (AML)

• Myeloid lineage markers including CD13, CD33, CD117, HLA-DR, and myeloperoxidase (MPO) are essential 1
• Maturation markers such as CD34 and CD38 help determine blast maturity 1
• Aberrant antigen expression (such as lymphoid markers on myeloid blasts) should be documented for MRD monitoring 1

Complementary Diagnostic Studies Required

Flow cytometry does not function in isolation—it must be integrated with morphology, cytogenetics, and molecular studies for accurate diagnosis 1:

• Morphologic examination of bone marrow aspirate smears, touch preparations, and core biopsy remains foundational 1, 2
• Cytochemical stains (myeloperoxidase and nonspecific esterase) may assist in AML diagnosis and classification 1
• Conventional karyotyping must be performed on all acute leukemia cases, as cytogenetics provide the most important prognostic information 1
• FISH studies are performed based on suspected subtype—for example, BCR-ABL1 in ALL, PML-RARA in acute promyelocytic leukemia, or specific translocations in lymphoma 1
• Molecular genetic testing including next-generation sequencing for mutations (FLT3-ITD, NPM1, CEBPA, IDH1/2, TP53) is essential for risk stratification and targeted therapy selection 1

Critical Diagnostic Algorithms

For Suspected Lymphoma

Excisional or incisional lymph node biopsy is the gold standard—fine needle aspiration alone is insufficient for initial diagnosis 1:

• FNA combined with core biopsy plus flow cytometry and molecular studies may suffice only when lymph nodes are inaccessible, particularly for CLL diagnosis 1
• Flow cytometry of peripheral blood is adequate for CLL diagnosis without requiring bone marrow biopsy 1
• For SLL diagnosis, lymph node biopsy showing architectural effacement is required 1

For Suspected Acute Leukemia

The diagnostic threshold is ≥20% blasts in bone marrow or peripheral blood, with exceptions for specific cytogenetic abnormalities 1, 2:

• If peripheral blood contains sufficient blasts, flow cytometry, FISH, and molecular studies can be performed on blood alone 2
• Bone marrow examination (aspirate and biopsy) remains standard for definitive diagnosis and complete characterization 2

Special Circumstances and Monitoring

Cerebrospinal Fluid Evaluation

• CSF examination with flow cytometry is mandatory for ALL patients receiving intrathecal therapy 1
• Cell count, cytospin preparation, and blast enumeration by a pathologist are required 1
• Flow cytometry may be performed on CSF to enhance diagnostic sensitivity 1

Extramedullary Disease

• Tissue biopsy must be processed for morphology, immunophenotyping, cytogenetics, and molecular studies identical to bone marrow evaluation 1
• Fresh tissue should be submitted for flow cytometry when extramedullary involvement occurs without marrow disease 1

Minimal Residual Disease Detection

• Flow cytometry analysis must be designed at diagnosis to enable MRD detection during follow-up 1
• This requires documentation of leukemia-associated immunophenotypes (aberrant antigen expression patterns) at initial diagnosis 1

Common Pitfalls to Avoid

• Never rely on flow cytometry alone for lymphoma diagnosis—architectural assessment requires tissue biopsy 1
• Hodgkin lymphoma cannot be detected by standard flow cytometry due to the rarity of Reed-Sternberg cells and reactive background 3
• Immunohistochemistry on paraffin sections may be necessary when flow cytometry specimens are inadequate, though this provides limited immunophenotyping 1
• Always exclude mantle cell lymphoma when evaluating CD5+ B-cell populations by assessing cyclin D1 or t(11;14) 1
• Specimen quality is critical—inadequate aspirates or delayed processing compromise flow cytometry results 3, 4