Acute Leukemia of Ambiguous Lineage:

1. Entities excluded from this diagnosis:

  • AML with t(8;21), inv(16) and PML-RARA fusion
  • AML with myelodysplasia-related changes
  • Therapy related AML
  • Leukemia with FGFR1
  • CML in blast crisis

2. Acute undifferentiated leukemia:

  • Express no more than one membrane marker for any given lineage
  • Lack expression of CyCD3, MPO, CyCD22, CyCD79 or CD19
  • Lack features of cells of other lineages such as megakaryocytes and plasmacytoid dendritic cells

3. Mixed-phenotype acute leukemia (acute bilineage or bilineal leukemia):

  • Separate populations of blasts of more than one lineage
  • Requirement for lineage assignment is not as stringent as cases with single blast population

4. Mixed-phenotype acute leukemia (acute biphenotypic leukemia):

  • Single population of blasts coexpressing antigens of more than one lineage (see Fig 1).

Requirement for assigning more than one lineage to a single blast population:

Myeloid lineage:

  • MPO (by flow, IHC or cytochemistry); or
  • ≥ 2 monocytic markers: non-specific esterase, CD11c, CD14, CD64 and lysozyme

T-cell lineage:

  • Strong cytoplasmic CD3 (by flow); or surface CD3
  • CD3 expression need to be strong: brightest cCD3+ blasts should reach the intensity of the normal T-cells (strong expression is not required for T-ALL diagnosis)

B-cell lineage:

  • Strong CD19 with ≥1 of strongly expressed CD79a, CyCD22, CD10; or
  • Weak CD19 with ≥2 of strongly expressed CD79a, CyCD22, CD10
AL_ambiguous_1 AL_ambiguous_2 AL_ambiguous_3 AL_ambiguous_4

Fig 1.Mixed-phenotype acute leukemia, B/myeloid. Blasts (red) are variably positive for B-cell markers (CD19, CyCD79a and CD22), positive for CD13 and partially positive for CD117 and MPO. T-cells (dark blue) can be used as negative control. Normal B-cells (green) can be used as positive control.

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