What are the diagnostic steps for suspected leukemia?

Diagnostic Workup for Suspected Leukemia

For any patient with suspected acute leukemia, obtain a fresh bone marrow aspirate with smears for morphologic evaluation, along with bone marrow trephine core biopsy, and immediately send samples for flow cytometry, conventional karyotyping, and molecular genetic testing. 1

Initial Clinical Assessment

• Document complete demographics, medical history including prior hematologic disorders, previous malignancies, cytotoxic therapy exposure, radiation exposure, family history, and ethnicity (prognostically significant in acute lymphoblastic leukemia) 2
• Perform physical examination documenting organomegaly, lymphadenopathy, cutaneous lesions, neurologic abnormalities, and tumor masses 2

Essential Laboratory Studies

Complete Blood Count and Peripheral Blood

• Obtain complete blood count with differential and review peripheral blood smear in all patients 2
• Order comprehensive metabolic panel, lactate dehydrogenase, uric acid, phosphate levels, and coagulation panel 2
• If peripheral blood contains sufficient blasts and bone marrow aspirate is inadequate or unobtainable, peripheral blood may be used for diagnosis and ancillary studies 1

Bone Marrow Examination (Core Requirement)

• Obtain fresh bone marrow aspirate for all patients suspected of acute leukemia 1
• Prepare bone marrow aspirate smears for morphologic evaluation 1
• Obtain adequate bone marrow trephine core biopsy and bone marrow trephine touch preparations 1
• If bone marrow aspirate is unobtainable ("dry tap"), prepare touch imprint preparations of core biopsy and submit an additional core biopsy unfixed in tissue culture medium for disaggregation for flow cytometry and genetic studies 1

Required Ancillary Testing (Must Be Performed Simultaneously)

Flow Cytometry Immunophenotyping

• Perform multicolor flow cytometry on bone marrow aspirate or peripheral blood sufficient to distinguish acute myeloid leukemia (including acute promyelocytic leukemia), B-lymphoblastic leukemia, T-lymphoblastic leukemia, and acute leukemia of ambiguous lineage 1
• Ensure flow cytometry analysis is comprehensive enough to allow subsequent detection of minimal residual disease 1
• If insufficient bone marrow aspirate or peripheral blood is available, use immunohistochemical studies as alternative for limited immunophenotyping 1

Cytogenetic Analysis

• Perform conventional cytogenetic analysis (karyotype) on all patients—molecular genetic and/or FISH testing does not replace conventional karyotyping 1
• Obtain appropriate molecular genetic and/or FISH testing based on suspected subtype 1

Molecular Genetic Testing

For Acute Myeloid Leukemia (all ages):

• Mandatory: FLT3-ITD testing 1
• Recommended: IDH1, IDH2, TET2, WT1, DNMT3A, TP53 for prognostic and therapeutic purposes 1
• For non-core binding factor, non-acute promyelocytic leukemia, non-myelodysplasia-related cytogenetic abnormalities: NPM1, CEBPA, and RUNX1 testing is mandatory 1

For Suspected Acute Promyelocytic Leukemia:

• Ensure rapid detection of PML-RARA is performed immediately 1
• Order coagulation studies to evaluate for disseminated intravascular coagulation 1

For Adult B-Lymphoblastic Leukemia:

• Mandatory: t(9;22)(q34.1;q11.2); BCR-ABL1 testing 1
• Recommended: KMT2A (MLL) translocations 1
• May order: PAX5, JAK1, JAK2, IKZF1, and CRLF2 overexpression 1

For Pediatric B-Lymphoblastic Leukemia:

• Mandatory: t(12;21)(p13.2;q22.1); ETV6-RUNX1, t(9;22)(q34.1;q11.2); BCR-ABL1, KMT2A (MLL) translocation, iAMP21, trisomy 4 and 10 1

For T-Lymphoblastic Leukemia:

• May order: NOTCH1, FBXW7 1

For Core Binding Factor Acute Myeloid Leukemia:

• Adults: Mandatory KIT mutation testing 1
• Pediatrics: May perform KIT mutation testing 1

Cytochemical Studies

• May request myeloperoxidase and nonspecific esterase for diagnosis and subclassification of acute myeloid leukemia 1

Cerebrospinal Fluid Evaluation

• For all acute lymphoblastic leukemia patients receiving intrathecal therapy: obtain cerebrospinal fluid sample with cell count, cytocentrifuge preparation, blast enumeration by pathologist 1
• For acute leukemia patients other than acute lymphoblastic leukemia receiving intrathecal therapy: may obtain cerebrospinal fluid when no clinical contraindication exists 1
• May use flow cytometry for cerebrospinal fluid evaluation 1

Extramedullary Disease

• For patients presenting with extramedullary disease without bone marrow or blood involvement: evaluate tissue biopsy specimen and process for morphologic, immunophenotypic, cytogenetic, and molecular genetic studies as recommended for bone marrow 1
• Perform CT scan of chest, abdomen, pelvis to determine if disease is localized or disseminated 2
• Perform PET/CT and CNS imaging if extramedullary disease suspected or significant neurologic signs/symptoms present 2

Specimen Handling

• Store cryopreserved cells or nucleic acid, formalin-fixed nondecalcified paraffin-embedded tissue, or unstained marrow aspirate/peripheral blood smears for validated molecular or genetic studies 1
• Perform molecular testing at cancer center or university-based hospital when possible 2
• Ensure specimens are properly identified and stored under appropriate conditions in laboratory compliant with regulatory/accreditation requirements 1

Reporting Timeline

• Preliminary report with basic diagnostics should be available within 48-72 hours 2
• Complete final comprehensive report should be issued within 1-2 weeks when all ancillary test results are available 2

Critical Pitfalls to Avoid

• Never rely solely on peripheral blood if bone marrow can be obtained—bone marrow aspirate is the gold standard 1
• Never substitute FISH or molecular testing for conventional karyotyping—all three are required 1
• Never delay acute promyelocytic leukemia testing—this is a medical emergency requiring immediate PML-RARA detection and coagulation studies 1
• Never perform inadequate flow cytometry panels—must be sufficient to distinguish all acute leukemia subtypes and enable minimal residual disease monitoring 1