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Classifying acute leukemia

15 min

Skills you'll build3 skills
  • Name the cytogenetic and molecular tests needed to classify a suspected AML
  • Name the immunophenotype and genetic tests needed to classify a suspected ALL
  • Assign a cell population's lineage from its full flow cytometry marker pattern

At the bench: Recognize abnormal cell findings that need further review

Try first

Try first

A marrow shows 30% myeloblasts and marked multilineage dysplasia. The karyotype is normal, and sequencing has not been run. Can you assign an AML type from this?

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Genetic classification of AML

WHO5 classifies AML first by defining genetic abnormalities. Most genetically defined AML types have no fixed minimum blast percentage when the lesion is present and the clinicopathologic findings support AML.1 AML with BCR::ABL1 and AML with a qualifying CEBPA mutation still require at least 20% blasts, and so does AML, myelodysplasia-related (AML-MR), which needs that blast percentage plus a specified myelodysplasia-related cytogenetic or molecular abnormality or a qualifying history of a myelodysplastic neoplasm.1 Morphologic dysplasia alone does not establish AML-MR, however many lineages it involves. ICC generally requires at least 10% blasts for genetically defined AML, so state the framework applied when the two differ.2

Genetic classification of B-ALL

WHO5 classifies B-lymphoblastic leukemia/lymphoma (B-ALL) by its defining genetic abnormality: fusions such as BCR::ABL1 or ETV6::RUNX1, a KMT2A rearrangement, and chromosome-number categories such as high hyperdiploidy.3 A full genetic workup that finds no recurrent abnormality yields the category B-ALL not otherwise specified (B-ALL, NOS). When testing is limited, the laboratory can apply the family-level term B-ALL not further classified (B-ALL, NFC).3,4 A film shows lymphoblasts but no genotype. Assigning a genetic category from morphology reports an abnormality that has not been tested.

Flow cytometry marker patterns

Lineage assignment depends on the whole marker pattern and its intensity on a gated population. The gate is set on a logarithmic CD45-versus-side-scatter plot, which orients the analysis.5 CD13, CD33, and CD117 are myeloid-associated markers that can also appear on lymphoblasts, and TdT and CD34 mark immaturity in more than one lineage.5 A single positive marker can misassign a blast population. Flow cytometry supports lineage and clonality, and genetic testing classifies the leukemia.5

Follow the bench demonstration

Sources for this step
References
  1. Khoury JD, Solary E, Abla O, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: myeloid and histiocytic/dendritic neoplasms. Leukemia. 2022;36(7):1703-1719. doi:10.1038/s41375-022-01613-1
  2. Arber DA, Orazi A, Hasserjian RP, et al. International Consensus Classification of myeloid neoplasms and acute leukemias: integrating morphologic, clinical, and genomic data. Blood. 2022;140(11):1200-1228. doi:10.1182/blood.2022015850
  3. Alaggio R, Amador C, Agnarsson I, et al. The 5th edition of the World Health Organization Classification of Haematolymphoid Tumours: lymphoid neoplasms. Leukemia. 2022;36(7):1720-1748. doi:10.1038/s41375-022-01620-2
  4. Steinemann D, Dawidowska M, Russell LJ, Harrison CJ, Göhring G. Genetic alterations in lymphoblastic leukaemia/lymphoma: a practical guide to WHO HAEM5. Med Genet. 2024;36(1):39-45. doi:10.1515/medgen-2024-2007
  5. Keohane EM, Preston MM, Mirza KM, Walenga JM, eds. Rodak's Hematology: Clinical Principles and Applications. 7th ed. Elsevier; 2025. Accessed September 26, 2026. https://www.us.elsevierhealth.com/rodaks-hematology-9780323936507.html

Watch one

Read each decision and its explanation.

A marrow aspirate shows 24% myeloblasts. The karyotype shows an inv(16) rearrangement, and flow cytometry shows CD13, CD33, and CD117 positivity with monocytic and granulocytic maturation markers in subpopulations. What tests confirm the AML category, and can it be classified now?

  1. Check the blast percentage. At 24% it is above the 20% threshold that several AML categories require.

    The blast percentage is checked first because several AML categories have their own fixed minimum.

  2. Review the flow pattern. Myeloid markers with monocytic and granulocytic subpopulations are consistent with a core-binding-factor category, and the genetic result establishes it.

    Flow cytometry supports lineage. The defining lesion comes from genetic testing.

  3. Confirm the rearrangement. Request FISH or RT-PCR for CBFB::MYH11, the fusion associated with inv(16).

    inv(16) can be subtle on a karyotype, and FISH or RT-PCR confirms the fusion that defines the category.

  4. Assign the final category after the confirmatory result returns. The karyotype suggests the category but does not establish it.

    The category is assigned only when the defining evidence is available.

Request FISH or RT-PCR to confirm CBFB::MYH11. The 24% blast count meets the requirement, and the category is assigned once the fusion is confirmed.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A marrow shows 25% myeloblasts with multilineage dysplasia and a normal karyotype. Sequencing is pending. What does WHO5 require before AML, myelodysplasia-related (AML-MR) can be assigned?

Correct. WHO5 requires at least 20% blasts plus a specified myelodysplasia-related cytogenetic or molecular abnormality, or a qualifying history of MDS or MDS/MPN. The eight MDS-related genes are SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2, so the pending sequencing result can meet the genetic criterion.

Incorrect. Under WHO5, morphologic dysplasia alone does not establish AML-MR, however many lineages it involves.

Incorrect. The 10% rule applies when dysplasia is used to diagnose a myelodysplastic neoplasm (MDS). For AML-MR, WHO5 needs the genetic or history criterion regardless of the extent of dysplasia.

Hint
  1. Check what WHO5 requires beyond the blast percentage and the dysplasia already seen.
  2. Sequencing of the eight MDS-related genes can supply the missing evidence.
  3. A documented history can substitute for the genetic finding.

Review WHO5 genetic categories

Problem 2 of 3 · Hints available

Large blasts with prominent nucleoli and irregular nuclear membranes are seen on a film. How is lineage assigned?

Incorrect. The larger lymphoblast form can be confused with AML blasts. Morphology establishes that a blast process is present, and lineage comes from immunophenotyping and genetics.

Correct. Lymphoblasts and myeloid blasts can overlap in appearance. Immunophenotyping establishes lineage, and genetic findings complete the classification.

Incorrect. TdT marks immaturity in both B- and T-lineage lymphoblasts, so it shows a lymphoblast process without assigning its lineage.

Hint
  1. Morphology cannot distinguish this population's lineage from a myeloblast population.
  2. One marker is common to both B- and T-lineage lymphoblasts and cannot assign lineage.
  3. Cytoplasmic or surface CD3 is lineage-defining for one of the two lineages.

Review Morphology and immunophenotype

Problem 3 of 3 · On your own

A blast population is CD117 positive and CD34 positive, with no other markers yet reported. Can lineage be assigned from this?

CD117 is myeloid-associated and can also appear on lymphoblasts. A single positive marker can misassign a blast population.

Assigned lineage from a single marker

Markers overlap between lineages: CD13, CD33, and CD117 are myeloid-associated and can also appear on lymphoblasts, and TdT and CD34 mark immaturity in more than one lineage. A lineage call rests on the whole marker pattern and its intensity on a gated population, so a single positive marker can misassign a blast population.

CD117 and CD34 both mark immaturity in more than one lineage. Lineage depends on the whole gated marker pattern, and one or two markers cannot establish it.

CD34 shows that the population is immature. It does not assign a lineage.

Review Flow cytometry immunophenotyping

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • A marrow aspirate from a man with a 3-week history of fatigue and easy bruising shows 35% blasts.
  • The blasts have fine chromatin and scant cytoplasm, with no Auer rods seen.
  • Flow cytometry shows CD19, cytoplasmic CD79a, TdT, and HLA-DR positivity. Surface immunoglobulin is absent. CD13 and CD33 are negative.
  • The karyotype is 46,XY, with no rearrangement detected.
  • RT-PCR for ETV6::RUNX1 and BCR::ABL1 is pending.
Decision 1 of 3

What does the flow pattern support?

CD13 and CD33 are negative, and the positive markers, CD19, cytoplasmic CD79a, and HLA-DR, are B-lineage markers.

CD19, cytoplasmic CD79a, and TdT with negative CD13 and CD33 support B-lineage lymphoblasts. Absent surface immunoglobulin fits this immature stage. A mature B-cell process would express it.

The karyotype adds genetic classification. The marker pattern already supports the lineage.

Review Flow cytometry immunophenotyping

Decision 2 of 3

The normal karyotype is back. Can the WHO5 genetic category be assigned now?

A normal karyotype does not rule out a cryptic fusion. RT-PCR for ETV6::RUNX1 and BCR::ABL1 is still pending and can detect rearrangements a karyotype can miss.

Named a genetic ALL category from the film

WHO5 B-ALL categories rest on defining genetic abnormalities such as BCR::ABL1, ETV6::RUNX1, or KMT2A rearrangement, found by cytogenetic or molecular testing. The film shows lymphoblasts and no genotype, so naming a category from it reports an abnormality nobody measured.

ETV6::RUNX1 and BCR::ABL1 can each occur with a normal karyotype. The category is assigned only after the pending results, or, if both are negative, after the full genetic workup is complete.

Morphology does not establish a genetic category. ETV6::RUNX1 requires a positive molecular or cytogenetic result.

Review WHO5 classification

Decision 3 of 3

A colleague suggests reporting this as AML, myelodysplasia-related, because myeloblast processes are more familiar to the referring service. What do you do?

The flow pattern supports a B-lineage process, and AML-MR is a myeloid category. Reporting it as AML-MR would report a lineage the evidence does not support.

CD13 and CD33 are negative, and the B-lineage markers are positive. The report states the lineage the evidence supports, and the genetic category is assigned after the pending RT-PCR results.

The panel already includes CD19, CD79a, TdT, HLA-DR, CD13, and CD33, enough to support B-lineage. A repeat panel does not change what these markers already show.

Review Flow cytometry immunophenotyping

The marker pattern of CD19, cytoplasmic CD79a, and TdT positive with CD13 and CD33 negative supports a B-lineage lymphoblastic process. The normal karyotype does not exclude a cryptic fusion, so the WHO5 genetic category is assigned after the pending RT-PCR results.

For reference

Sources checked