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| '''Important Note'''
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| 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.
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| <div style="width: 95%"; font-size:90%>
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| {| class="wikitable"
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| !colspan="2" style = "background:lightgrey; font-size:90%; border:solid"| '''Types and frequency''' MPAL mixed-phenotype forms
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| |-
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| !colspan="1" style = "background:#FFFFF0;border:solid; font-size:90%;"| MPAL with features of Myeloid and B-lineage (MPAL M/B)
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| !colspan="1" style = "background:white;border:solid; font-size:90%;"| This is the most common MPAL (more than 50% of MPAL cases).
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| |-
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| !colspan="1" style = "background:#FFFFF0;border:solid"| MPAL with features of Myeloid and T-lineage (MPAL M/T)
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| !colspan="1" style = "background:white; border:solid; font-size:90%;"| The second most frequent form (over one third of MPAL cases).
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| !colspan="1" style = "background:#FFFFF0;border:solid"| MPAL with combinations: MPAL B/T (rare) or B/T/M (very rare)
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| !colspan="1" style = "background:white; border:solid; font-size:90%;"| These forms are infrequent (around 10% of MPAL cases)
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| !colspan="1" style = "background:#FFFFF0;border:solid"| MPAL with defining molecular features
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| !colspan="1" style = "background:white; border:solid; font-size:90%;"| Following further analysis cases may be assigned additionally as:</br>
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| <span style="font-size:90%;">MPAL with t(9;22) (q34.1;q11.2); BCR-ABL1; </span>
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| <span style="font-size:90%;">MPAL with t(v;11q23.3); with KMT2A re-arrangement</span>
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| |}
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| </div>
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| ----
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| <div style="width: 95%"; font-size:90%>
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| {| class="wikitable"
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| !colspan="2" style = "background:lightgrey; font-size:90%; border:none"| '''Requirements to assign lineage in mixed-phenotype forms of acute leukaemia
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| |}
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| </div>
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| Assignment to a lineage requires that cases meet specific criteria. These are given below (see also the associted notes).</br></br>
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| 1. '''Does the case meet the criteria necessary to assign myeloid lineage'''</br>
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| This requires definite evidence of granulocytic maturation (MPO expression) or sufficient evidence to assign monocytic lineage.</br>
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| For full details of requirements use the Table: [[#MPAL_Table1|Myeloid lineage assignment (table)]]
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| 2. '''Criteria to assign B-lymphoid lineage''' This is based on definite evidence of CD19 together with sufficient supporting evidence of B-lymphoid maturation. For full details go to Table: [[#MPAL_Table2|B-Lymphoid lineage assignment (table)]]
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| 3. '''Criteria to assign T-lymphoid lineage''' This is based on definite evidence of definite CD3 expression by the neoplastic cells. Consideration of other markers of T-lineage is not considered. For full details go to Table: [[#MPAL_Table3|T-Lymphoid lineage assignment (table)]]
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| 4. '''Criteria to assign undifferentiated acute leukaemia''' This is based on definite evidence of definite CD3 expression by the neoplastic cells. Consideration of other markers of T-lineage is not considered. For full details go to Table: [[#MPAL_Table4|Undifferentiated lineage assignment (table)]]
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| ----
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| <div id="MPAL_Table1">'''Table 1'''</div>
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| '''MYELOID LINEAGE ASSIGNMENT'''
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| <span id="anchor_0"></span>.
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| <div style="width: 100%; border: 1px solid black; font-size:100%">
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| {| class="wikitable" style="color:black; background-color:#ffffff;" cellpadding="0"
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| !colspan="2" <span style="font-size:90%; font-color:navy; style="text-align: left; border: 1px solid black; background:pale gray">|'''Requirements to assign myeloid lineage in MPAL'''</br></span>
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| <div class="mw-collapsible mw-collapsed" data-expandtext="Click for explanation" data-collapsetext="Hide explanation">
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| <div class="mw-collapsible-content">
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| <span style="font-size:80%; text-align:left;"></br>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).</br>
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| </span>
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| </div></div></div>
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker option 1'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;" |'''Demonstrate expression of Myeloperoxidase (MPO)'''
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| |colspan="1" style = "font-size:90%;"|[[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).
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker option 2'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;" |'''Demonstrate clear evidence of monocytic lineage'''
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| |colspan="1" style = "font-size:90%;|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)
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| |-
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| |}
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| </div>
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| <span style="font-size:85%;>'''Notes on interpretation of MPO positivity in MPAL'''</br>
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| 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:</br>
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| (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.</br>
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| (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).</br>
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| (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.</span>
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| ----
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| <div id="MPAL_Table2">'''Table 2'''</div>
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| '''B-LYMPHOID LINEAGE ASSIGNMENT'''
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| <span id="anchor_0"></span>.
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| <div style="width: 100%; border: 1px solid black; font-size:100%">
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| {| class="wikitable" style="color:black; background-color:#ffffff;" cellpadding="0"
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| !colspan="2" <span style="font-size:90%; font-color:navy; style="text-align: left; border: 1px solid black; background:pale gray">|'''Requirements to assign B-lymphoid lineage in MPAL'''</br></span>
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| <div class="mw-collapsible mw-collapsed" data-expandtext="Click for explanation" data-collapsetext="Hide explanation">
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| <div class="mw-collapsible-content">
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| <span style="font-size:80%; text-align:left;"></br>For B-lymphoid lineage assignment the key lineage-marker is CD19. However, this marker has recognised expression in many cases of otherwise typical AML cases so additional criteria of expression intensity it is required that other markers must be expressed in addition to allow B-lineage assignment. Rarely CD19 is absent in which case a laregr number o other B-cell markers is required.</br>
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| </span>
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| </div></div></div>
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker option 1'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;"|'''Required''' [[CD19|Strong expression of CD19]]</br>'''Required also''' ONE of either: [[CD10]], [[CD22]], or [[CD79a]] (surface or cytoplasmic)
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| |colspan="1" style = "font-size:90%;"|To meet the definition of "strong" the expression, the intensity of CD19 '''must exceed''' that of 50% of normal B lymphocytes
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker option 2'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;"|'''Required''' [[CD19|Weak expression of CD19]]</br>'''Required also''' TWO of either: [[CD10]], [[CD22]], or [[CD79a]] (surface or cytoplasmic)
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| |colspan="1" style = "font-size:90%;"|To meet the definition of "weak" the expression intensity of CD19 must be '''less than''' that of 50% of normal B lymphocytes
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker option 3'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;"|[[CD19|CD19 is not expressed]]</br>'''In this rare occurrence then requires''' THREE of either: [[CD10]], [[CD22]], or [[CD79a]] (surface or cytoplasmic)
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| |colspan="1" style = "font-size:90%;"|This is a rare occurrence and should be carefully considered
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| |-
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| |}
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| </div>
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| <span style="font-size:85%;>'''Notes on interpretation of B-lineage in MPAL'''</br>
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| 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.</br>
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| </span>
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| ----
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|
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| <span id="anchor_0"></span>.
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| <div id="MPAL_Table3">'''Table 3'''</div>
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| '''T-LYMPHOID LINEAGE ASSIGNMENT'''
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| <div style="width: 100%; border: 1px solid black; font-size:100%">
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| {| class="wikitable" style="color:black; background-color:#ffffff;" cellpadding="0"
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| !colspan="2" <span style="font-size:90%; font-color:navy; style="text-align: left; border: 1px solid black; background:pale gray">|'''Requirements to assign T-lymphoid lineage in MPAL'''</br></span>
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| <div class="mw-collapsible mw-collapsed" data-expandtext="Click for explanation" data-collapsetext="Hide explanation">
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| <div class="mw-collapsible-content">
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| <span style="font-size:80%; text-align:left;"></br>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.</br>
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| </span>
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| </div></div></div>
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Marker requirement'''
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| |-
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| |colspan="1" style = "font-size:90%; color:black;"|'''Either''' [[CD3|CD3 surface or cytoplasm (strong)]] '''Or''' [[CD3|CD3 by immunocytochemistry (strong)]]
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| |colspan="1" style = "font-size:90%;"|To meet the definition of "strong" the expression intensity '''must exceed''' that of 50% of normal T lymphocytes
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| |-
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| |}
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| </div>
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| <span style="font-size:85%;>'''Notes on interpretation of T-lineage in MPAL'''</br>
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| 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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| </span>
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| ----
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|
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| <span id="anchor_0"></span>.
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| <div style="width: 100%; border: 1px solid black; font-size:100%">
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| {| class="wikitable" style="color:black; background-color:#ffffff;" cellpadding="0"
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| !colspan="2" <span style="font-size:90%; font-color:navy; style="text-align: left; border: 1px solid black; background:pale gray">|'''Requirements to assign acute undifferentiated leukaemia'''</br></span>
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| <div class="mw-collapsible mw-collapsed" data-expandtext="Click for explanation" data-collapsetext="Hide explanation">
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| <div class="mw-collapsible-content">
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| <span style="font-size:80%; text-align:left;"></br>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.</br>
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| </span>
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| </div></div></div>
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| |-
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Requirement 1'''
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| |colspan="2" style = "font-size:90%; color:black;"|No lineage can be assigned by lineage-defining markers of myeloid, B-lymphoid or T-lymphoid lineage
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Requirement 2'''
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| |colspan="2" style = "font-size:90%; color:black;"|No more than one lineage-associated marker for any single lineage is expressed
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| |colspan="2" style = "font-size:90%; color:black; background:#ddeee1" |'''Requirement 3'''
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| |-
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| |colspan="2" style = "font-size:90%; color:black;"|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.
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| |-
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| |}
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| </div>
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