Flow Cytometry:Myeloid lineage assignment

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Requirements to assign myeloid lineage 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: lysozyme or non-specific esterase in malignant cells (enzyme cytochemistry)

Notes on interpretation of MPO positivity
The WHO classification advises that MPO expression is assessed by its intensity of expression and includes some flexibility in interpretation. The is based on several considerations:
(1) The techniques used to detect MPO do not have the same sensitivity: immunohistochemistry > flow cytometry > enzyme-cytochemistry. This means diagnosis of MPAL may be method dependent – expressing MPO as intensity allows expression to be compared with that of normal cells detected using the same method. For flow cytometry an expression level that is at least half as intense as normal neutrophils provides greater diagnostic confidence.
(2) MPO is not fully specific for myeloid lineage in all cases: this is particularly the case when expression is uniform and dim, so an element of subjectivity is present when interpreting (particularly when detected by more sensitive techniques such as immunocytochemistry).
(3) Context of MPO may be important: Applying a threshold of >10% cells being positive for MPO may improve specificity. Detecting variability of expression level of MPO by blast cells may suggest partial maturation and support myeloid lineage origin (often together with variable light scatter). The presence of other myeloid markers may provide greater confidence that MPO is lineage specific. Similarly, be aware of common patterns of aberrancy that are associated with specific alternative diagnoses: Dim (weak) expression of MPO may be a feature of otherwise typical B-LL/LBL; Burkitt-like entities may have strong MPO staining however other investigations will confirm their nature and other myeloid markers will be absent.

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