What investigations are needed to diagnose and classify acute leukemia?

Investigations for Acute Leukaemia

All patients suspected of acute leukaemia require bone marrow aspirate and core biopsy with comprehensive testing including morphology, flow cytometry immunophenotyping, conventional cytogenetics (karyotype), and molecular genetic/FISH studies to distinguish AML, ALL, and mixed phenotype acute leukaemia. 1

Essential Clinical Information Required

Before any testing, provide the pathologist with:

• Patient age, sex, ethnicity
• History of hematologic disorders, predisposing syndromes, prior malignancies
• Exposure to cytotoxic therapy, immunotherapy, radiotherapy, or toxic substances
• Recent growth factor therapy, transfusions, or medications that may obscure features
• Family history of hematologic disorders or malignancies
• Physical examination findings: neurologic status, mediastinal masses, cutaneous lesions, organomegaly 1

Core Diagnostic Testing Algorithm

1. Peripheral Blood Studies (Mandatory)

• Complete blood count with differential
• Peripheral blood smear review by pathologist
• If sufficient blasts present (≥20%), peripheral blood can substitute for bone marrow when bone marrow aspiration is contraindicated or yields inadequate material 1

2. Bone Marrow Examination (Mandatory)

Obtain fresh bone marrow aspirate for:

• Aspirate smears for morphologic evaluation
• Core biopsy with touch preparations
• Marrow clots if available

Critical caveat: If aspirate is unobtainable ("dry tap"), prepare touch imprint preparations from core biopsy and submit additional core unfixed in tissue culture medium for disaggregation for flow cytometry and genetic studies 1

3. Immunophenotyping (Mandatory)

• Flow cytometry immunophenotyping on bone marrow aspirate or peripheral blood
• Panel must distinguish: AML (including APL), early T-ALL, B-ALL, and acute leukaemia of ambiguous lineage
• If insufficient material for flow cytometry, use immunohistochemistry on core biopsy as alternative for limited immunophenotyping 1

4. Cytogenetic Analysis (Mandatory)

• Conventional karyotyping (cannot be replaced by molecular/FISH testing)
• Appropriate molecular genetic and/or FISH testing based on suspected subtype
• Molecular genetic/FISH does NOT replace conventional cytogenetics 1

For specific subtypes:

• Pediatric B-ALL: Test for t(12;21) ETV6-RUNX1, t(9;22) BCR-ABL1, KMT2A(MLL) translocation, iAMP21, trisomy 4 and 10 1
• Adult ALL: BCR-ABL1, KMT2A(MLL) translocation, molecular studies (PAX-5, JAK1, JAK2, IKZF1 for B-ALL; NOTCH1, FBXW7 for T-ALL) 1
• AML: Rapid FISH for PML-RARA if APL suspected, FLT3-ITD, IDH1, IDH2, NPM1, CEBPA, RUNX1, KIT (when core-binding factor AML diagnosed) 1, 2

5. Cytochemical Studies (Optional for AML)

• Myeloperoxidase (MPO) and nonspecific esterase (NSE) may assist in AML diagnosis and classification 1

6. Additional Laboratory Tests

For all patients at presentation:

• Comprehensive metabolic panel (monitor tumor lysis syndrome, particularly B-lymphoblastic lymphoma)
• Lactate dehydrogenase, phosphate, uric acid
• Coagulation panel (PT, PTT, fibrinogen) to detect disseminated intravascular coagulation in acute promyelocytic leukaemia 1

7. Cerebrospinal Fluid Evaluation

For ALL patients receiving intrathecal therapy (Mandatory):

• CSF cell count
• Cytocentrifuge preparation with blast enumeration reviewed by pathologist
• Flow cytometry may be used 1

For other acute leukaemia patients (Conditional):

• Obtain CSF when no clinical contraindication exists
• Same processing as ALL patients 1

8. Extramedullary Disease Without Marrow Involvement

If presenting with extramedullary disease alone:

• Tissue biopsy processed for morphology, immunophenotyping, cytogenetics, and molecular genetics
• Additional biopsies may be needed for fresh material for ancillary testing 1

Specimen Preservation

Preserve specimens for future studies:

• Cryopreserved cells or nucleic acid
• Formalin-fixed, nondecalcified paraffin-embedded tissue
• Unstained marrow aspirate or peripheral blood smears
• Store properly in compliant laboratory for molecular/genetic studies 1

Measurable Residual Disease Preparation

Ensure initial flow cytometry or molecular characterization is comprehensive enough to allow subsequent MRD detection 1. This is critical for monitoring treatment response and guiding therapeutic decisions.

Common Pitfalls to Avoid

1. Never rely solely on molecular/FISH testing without conventional karyotyping - you will miss important cytogenetic abnormalities 1
2. Do not delay bone marrow biopsy - peripheral blood alone is insufficient unless bone marrow is truly contraindicated or unobtainable 1
3. Ensure same physician interprets aspirate and core biopsy, or correlate interpretations if performed by different physicians 1
4. Do not forget to test for APL - rapid FISH for PML-RARA and coagulation studies are essential when suspected, as this requires immediate specific therapy 1
5. Preserve adequate material - submit additional core biopsy unfixed in tissue culture medium if initial aspirate inadequate 1