MPAL

Assignment of category to ambiguous cases in acute leukaemia:

Final lineage requires integration of additional molecular features; assignments made by flow are therefore "provisional" pending full molecular assessment.‎ Four categories may be assigned:

• My/B
• My/T**
• T/B
• Undiff

MPAL with t(9;22) (q34.1;q11.2); BCR-ABL1) MPAL with t(v;11q23.3); with KMT2A re-arrangement

Flow cytometry is only one element in assigning a diagnosis of MPAL but conclusions may subsequently be modified by results of immunohistochemistry, cytogenetics, or genetics. It is important therefore that an element of uncertainty is acknowledged in assigning a diagnosis of MPAL by flow cytometry, acknowledging that any may be subject to modification if other disease-defining characteristics emerge.

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Requirements to assign myeloid lineage in MPAL The assignment of myeloid lineage recognises MPO expression to be a defining marker of myeloid lineage. However MPO is expressed only by around 80% of AML cases. The assignment of myeloid lineage can therefore also be made if monocytic lineage can be established, requiring at least 2 of 5 possible lineage markers to be detected (although only 3 of these can be established by flow cytometry).
Marker option 1
Demonstrate expression of Myeloperoxidase (MPO)	Myeloperoxidase expression alone may be sufficient to establish myeloid lineage, but be aware of the limitations: (1) intensity should be at least half of that of mature neutrophils in at least of proportion of cells measured by the same method; (2) there are circumstances where judgement is required (see notes).
Marker option 2
Demonstrate clear evidence of monocytic lineage	If MPO is not demonstrated then myeloid lineage may still be assigned through demonstration of monocytic features. This can be assigned by the detection of at least two of the following features: By flow cytometry: CD11c, CD14, CD64; by other approaches: raised lysozyme in serum or urine or non-specific esterase in malignant cells (enzyme cytochemistry)

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NOTE Interpretation of MPO positivity in MPAL The assignment of myeloid lineage recognises MPO expression to be a defining marker of myeloid lineage. However MPO is expressed only by around 80% of AML cases. The assignment of myeloid lineage can therefore also be made if monocytic lineage can be established, requiring at least 2 of 5 possible lineage markers to be detected (although only 3 of these can be established by flow cytometry).

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Requirements to assign B-lymphoid lineage in MPAL For B-lymphoid lineage assignment the key lineage-marker is CD19. However, this marker has recognised aberrant expression in AML cases so additional criteria of expression intensity it is required that other markers must be expressed in addition to allow B-lineage assignment.
Marker option 1
Required Strong expression of CD19	To meet the definition of "strong" the expression intensity must exceed that of 50% of normal B lymphocytes
In addition 1 of: CD10, CD22, or CD79a (surface or cytoplasmic)	If CD19 is strong then B-lineage can be assigned if there is at least ONE of these additional markers
Marker option 2
Required Weak expression of CD19	To meet the definition of "weak" the expression intensity must be less than that of 50% of normal B lymphocytes
In addition 2 of: CD10, CD22, or CD79a (surface or cytoplasmic)	If CD19 is weak then B-lineage can be assigned only if there are at least TWO of these additional markers

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Requirements to assign T-lymphoid lineage in MPAL For T-lymphoid lineage assignment the key lineage-marker is CD3. Provided that this marker meets the intensity criteria then it is T-lineage defining in MPAL. This expression can be determined either by CD3 detection by flow cytometry OR by immunocytochemistry on trephine.
Marker requirement
Either CD3 surface or cytoplasm (strong)	To meet the definition of "strong" the expression intensity must exceed that of 50% of normal T lymphocytes
Or CD3 by immunocytochemistry (strong)	For immunohistochemistry it is important a non-zeta chain reagent is used