What is the diagnostic approach for acute lymphoblastic leukemia (ALL)?

Diagnostic Approach for Acute Lymphoblastic Leukemia

The diagnosis of ALL requires bone marrow examination with at least 30% blast cells, combined with comprehensive flow cytometry immunophenotyping, conventional cytogenetic analysis (karyotype), and molecular genetic testing to establish lineage and identify prognostically important genetic abnormalities. 1

Initial Clinical and Laboratory Assessment

Essential First Steps

• Obtain complete blood count (CBC) with manual differential and peripheral blood smear review to identify circulating blasts and establish baseline values 2
• Document relevant clinical history including ethnicity (Hispanics have worse outcomes and higher rates of Philadelphia-like ALL), environmental exposures, recent growth factor therapy, transfusions, or medications that might obscure features 2
• Perform physical examination focusing on neurologic findings, presence of mediastinal masses, lymphadenopathy, hepatosplenomegaly, and cutaneous lesions 2

Critical Laboratory Tests at Presentation

• Comprehensive metabolic panel, lactate dehydrogenase, phosphate, and uric acid levels to assess for tumor lysis syndrome risk, particularly in B-lymphoblastic lymphoma 2
• Coagulation panel (PT, PTT, fibrinogen) to detect early disseminated intravascular coagulation 2

Bone Marrow Examination (The Definitive Diagnostic Step)

Sample Collection and Processing

• Obtain fresh bone marrow aspirate for morphologic evaluation with aspirate smears, touch preparations, cell clots, and core biopsy 2
• If bone marrow aspirate is unobtainable (dry tap) or patient is clinically unstable, peripheral blood can be used if sufficient blasts are present (>20%), with touch imprint preparations of core biopsy evaluated 2
• Submit additional core biopsy unfixed in tissue culture medium for disaggregation for flow cytometry and genetic studies if aspirate is inadequate 2

Common Pitfall: Do not skip bone marrow examination in favor of peripheral blood alone unless medically contraindicated—bone marrow provides superior material for comprehensive ancillary testing 2, 3

Essential Ancillary Testing (Must Be Performed Simultaneously)

Flow Cytometry Immunophenotyping

• Perform multicolor comprehensive flow cytometry panel on bone marrow aspirate (or peripheral blood if bone marrow unavailable) to distinguish B-ALL, T-ALL, AML, and mixed phenotype acute leukemia 2
• Ensure panel is comprehensive enough to allow subsequent minimal residual disease (MRD) detection 2
• If insufficient material for flow cytometry, immunohistochemical studies can serve as alternative for limited immunophenotyping 2

Cytogenetic Analysis

• Conventional karyotyping must be performed on bone marrow—this is mandatory and cannot be replaced by FISH or molecular testing alone 2, 1
• FISH and molecular genetic testing augment but do not substitute for karyotype 2

Molecular and Genetic Testing by Age Group

For Adult B-ALL:

• Mandatory: t(9;22)(q34.1;q11.2)/BCR-ABL1 testing by FISH and/or RT-PCR 2, 1
• Recommended: KMT2A (MLL) translocations 2
• Consider: PAX5, JAK1, JAK2, IKZF1 mutations and CRLF2 overexpression for prognostic stratification [2, @15@]

For Pediatric B-ALL:

• Mandatory: t(12;21)(p13.2;q22.1)/ETV6-RUNX1, t(9;22)/BCR-ABL1, KMT2A (MLL) translocation, iAMP21, and trisomy 4 and 10 2, 1
• Molecular studies for PAX5, JAK1, JAK2, IKZF1 for B-ALL 2, 1

For T-ALL (All Ages):

• NOTCH1 and FBXW7 mutations 2, 1

Critical Point: Philadelphia chromosome-positive ALL (BCR-ABL1) has dramatically improved outcomes with tyrosine kinase inhibitors, making this the single most important genetic abnormality to identify for treatment planning 4, 5

Cerebrospinal Fluid Evaluation

• Obtain lumbar puncture with CSF cell count, cytology examination with cytocentrifuge preparation, and blast enumeration by pathologist for all ALL patients, as CNS involvement affects treatment 2
• Flow cytometry on CSF is recommended for enhanced sensitivity 2, 1
• Immunohistochemistry with TdT stain can be performed 2, 1

Timing Consideration: CSF should be obtained before or at initiation of intrathecal therapy 2

Extramedullary Disease Assessment

• Imaging of mediastinum, liver, spleen, lymph nodes, and other potential sites of involvement 2
• If extramedullary disease presents without bone marrow or blood involvement, tissue biopsy must be processed for morphologic, immunophenotypic, cytogenetic, and molecular genetic studies identical to bone marrow workup 2, 6

Specimen Handling and Storage

• Properly identify and store cryopreserved cells, nucleic acids, or formalin-fixed paraffin-embedded tissue under appropriate conditions for future diagnostic, prognostic, or therapeutic purposes 2, 1
• This allows for additional testing as new prognostic markers emerge 2, 1

What NOT to Do

• Do not rely on cytochemical studies (Sudan Black B, periodic acid-Schiff, acid phosphatase) as routine tests for ALL—these are not recommended and are primarily useful for AML diagnosis 1
• Do not perform additional bone marrow biopsy of extramedullary sites if complete ancillary studies have been performed on positive peripheral blood or bone marrow samples 2
• Do not use peripheral blood for conventional karyotyping as the primary specimen—bone marrow is required 2, 1