Flow cytometry:MPAL

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Important Note

MPAL is uncommon (2-3% of acute leukaemia). Flow cytometry provides initial evidence of MPAL. However conclusions may subsequently be modified (e.g. by results of immunohistochemistry, cytogenetics, or genetics). It is important that the provisional nature of flow cytometry assignment of MPAL is acknowledged.

Types and frequency MPAL mixed-phenotype forms
MPAL with features of Myeloid and B-lineage (MPAL M/B)	This is the most common MPAL (more than 50% of MPAL cases).
MPAL with features of Myeloid and T-lineage (MPAL M/T)	The second most frequent form (over one third of MPAL cases).
MPAL with combinations: MPAL B/T (rare) or B/T/M (very rare)	These forms are infrequent (around 10% of MPAL cases)
MPAL with defining molecular features	Following further analysis cases may be assigned additionally as: MPAL with t(9;22) (q34.1;q11.2); BCR-ABL1; MPAL with t(v;11q23.3); with KMT2A re-arrangement

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Requirements to assign lineage in mixed-phenotype forms of acute leukaemia

1. Does the case fukfil the criteria to assign myeloid lineage?
Click here to review criteria for myeloid lineage assignment

2. Does the case fulfil the criteria to assign B-lymphoid lineage
Click here to review criteria for B-lymphoid lineage assignment

3. Criteria to assign T-lymphoid lineage
Click here to review criteria for T-lymphoid lineage assignment

Notes on interpretation of B-lineage in MPAL
CD19 is considered by WHO to have high but not complete specificity for B-lineage. Its’ use requires the intensity of expression to be considered: “strong” CD19 expression should exceed 50% of the level seen on normal B progenitors in at least some of the leukaemic cells, while weaker CD19 expression has lower specificity and requires stronger additional evidence of B-lineage commitment (see Table above). Note also: (1) CD79a has frequent expression in T-LL/LBL, so should not be used to suggest B-lineage if that disorder is considered. (2) PAX5 identified by immunohistochemistry has a low evidence base in MPAL diagnosis so should be interpreted with caustion. (3) In the absence of CD19 expression, B-lineage may still be assigned if three other B antigens are expressed.

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Table 3

T-LYMPHOID LINEAGE ASSIGNMENT

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) Or CD3 by immunocytochemistry (strong)	To meet the definition of "strong" the expression intensity must exceed that of 50% of normal T lymphocytes

Notes on interpretation of T-lineage in MPAL
Expression of CD3 is required to assign T lineage in MPAL. Generally, other T-associated antigens will also be expressed:.CD7, and in the majority of cases. CD2, CD4, CD5, and CD8. Consider also that a proportion of early T precursor lymphoblastic leukaemia/lymphoma (ETP-ALL) may be difficult to distinguish from MPAL-T/M. Using an appropriate threshold for MPO positivity may be helpful and a threshold of >10% for myeloperoxidase positivity is broadly advised by WHO in this context.

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Requirements to assign acute undifferentiated leukaemia This is a difficult area where the diagnosis of acute leukaemia is suspected but no criteria for lineage assignment can be made i.e. the cases do not meet any of the criteria for myeloid, B-lymphoid, or T-lymphoid lineage given above. An important aspect of this group is to exclude the possibility of rare entities that can mimic AML: these include non-haemaotopoietic neoplasm and blastic plasma cell dendritic neoplasm.
Requirement 1
No lineage can be assigned by lineage-defining markers of myeloid, B-lymphoid or T-lymphoid lineage
Requirement 2
No more than one lineage-associated marker for any single lineage is expressed
Requirement 3
Ensure that other conditions that may resemble undifferentiated acute leukaemia are excluded: particularly consider: acute megakaryocytic leukaemia, non haematopoietic tumours, blastic plasma dendritic cell neoplasm, and rarely plasma cell leukaemias.