Acute leukemia can be classified based on aggressiveness, lineage, and site of involvement. Diagnosis requires morphology identification of blasts, cytochemistry including MPO and esterase staining, immunophenotyping by flow cytometry, and genetic studies like cytogenetics and molecular genetics. Cytogenetics identifies prognostic groups in AML and distinguishes secondary from de novo disease. Molecular genetics detects cryptic abnormalities and monitors minimal residual disease. Together, these analyses classify acute leukemias and guide treatment decisions.

1. Overview to diagnosis of Acute
Leukemia
By:
Ahmed Makboul Ahmed
M.B.B.Ch, M.Sc
Assistant Lecturer, Clinical Pathology Department, South Egypt Cancer Institute

2. INTRODUCTION:
All hematopoietic and lymphoid neoplasms can be described according to
three major characteristics:
• Aggressiveness: Acute versus Chronic.
• Lineage: Lymphoid versus Myeloid.
• Predominant Site of Involvement: Blood and Bone Marrow versus Tissue.

3. 1. Aggressiveness: ACUTE vs. CHRONIC
• Hematologic neoplasms can be divided into acute and chronic types based on two
characteristics: survival and maturation:
SURVIVAL:
• Acute: Survival measured in weeks or a few months (without effective therapy).
• Chronic: Survival measured in years.
With effective therapy for many of the acute neoplasms, the survival difference between
acute and chronic neoplasms has been greatly diminished or eliminated.
MATURATION:
• Acute: Predominance of immature cells (blasts)
• Chronic: Predominance of mature cells

4. 2. Lineage: Lymphoid vs. Myeloid
• This division relates to the first step in differentiation of the hematopoietic stem
cell into the CFU-L (colony-forming unit-lymphoid) and the CFU-GEMM (colony-
forming unit-granulocyte-erythrocyte-megakaryocyte-macrophage).
• Neoplasms derived from the CFU-L are designated lymphoid; those derived from
the CFU-GEMM are myeloid.

5. 3. Predominant Site of Involvement: Blood and Bone
Marrow vs. Tissue
• Neoplasms with predominant blood and bone marrow involvement are
called leukemia.
• Neoplasms with predominant tissue involvement are called lymphomas if
composed of lymphocytes and granulocytic sarcomas (sometimes called
chloromas or extramedullary myeloid tumors) if composed predominantly of
myeloid cells (now called myeloid sarcoma).

6. Definition:
• Acute Leukemia is a malignant disease characterized by clonal expansion of hematopoietic precursors
(Blasts) in the bone marrow BM, peripheral blood PB or other tissues. The most important factor is the
presence of ≥ 20% blasts in the peripheral blood or bone marrow.
• According to age, acute leukemia can be divided into childhood acute leukemia in patients < 15 years,
adult acute leukemia in patients > 15 years and acute leukemia at elderly age in patients > 60 years.
In the latter group the response to therapy is inferior.
• According to cell type, acute leukemia is divided into 2 main groups, acute myeloid leukemia AML
forming 80% of adult cases and acute lymphoblastic leukemia ALL forming 80% of childhood cases.

7. Prerequisite laboratory techniques for diagnosis of
Acute Leukemia:
They include:
1. Morphology.
2. Cytochemistry.
3. Immunophenotyping.
4. Genetic studies: Cytogenetic analysis & Molecular genetic analysis.

8. 1. Role of Morphology:
I. Differential count:
• Manual differential counts of blood films and bone marrow films should be performed in all
patients.
• The WHO expert group advises a 200 – cell differential count on a blood film and a 500 – cell
differential count on the bone marrow film in an area as close to a particle and as undiluted with
blood as possible. Performing a 500 – cell differential count on the bone marrow is particularly
important if the percentage of blast cells is at the level of 20% which is the critical level for the
diagnosis of acute leukemia.
• The blast % derived from the bone marrow aspirate correlates with an estimate of the blast % in
the trephine biopsy, although focal clusters or sheets of blasts are regarded as disease
progression.

9. II. Identification of blasts/blast equivalents:
1. Myeloblasts:
Size: They are large cells.
Nucleus:
- Blasts commonly have more than 2 nucleoli.
- Nuclear folding with lipping is more in
myeloblasts while nuclear clefting is more in
lymphoblasts.
- Prominent nucleoli.
Cytoplasm:
- The cytoplasm is smooth, abundant,
homogeneous and usually pale at the periphery. It
may contain Azurophilic granules or Auer rods. The
presence of Auer rods should be mentioned.

10. 2. Monoblasts vs Promonocytes:
Criteria Monoblast Promonocyte
Size: Large. Intermediate to large.
Nucleus: o Round nuclear contours.
o Fine chromatin.
o Prominent nucleolus.
o Smaller, variably prominent nucleoli.
o Gently lobulated nuclei with delicate
nuclear folding.
o More even/dispersed chromatin
pattern than typical monocyte.
Cytoplasm: o Abundant variably basophilic
cytoplasm.
o Fine cytoplasmic azurophilic
granules.
o No Auer rods.
o May see cytoplasmic vacuoles.
o Abundant, lightly basophilic
cytoplasm.

11. Monoblast Promonocyte

12. 3. Abnormal promyelocytes:
• Blast equivalent in acute promyelocytic leukemia.
• Has 2 variants:
a). Typical (Hypergranular) variant:
oAbnormal promyelocytes with irregular and often
bilobed nuclei.
oNumerous large intracytoplasmic granules and
granules covering nuclei.
oAbnormal cells with numerous Auer rods (faggot
cells) can be identified in majority of cases.
Hypergranular APL

13. b). Microgranular (Hypogranular) variant:
oAbsent or scant cytoplasmic granules by light
microscopy.
oPresence of abundant submicroscopic
granules highlighted by strong
myeloperoxidase reactivity.
oFrequent bilobed nuclei (sliding plates).
oRare faggot cells present in most cases.
Microgranular APL

14. 4. Erythroblasts:
• Blast equivalent in pure erythroid leukemia.
• Size: Variably sized, small to large.
• Nucleus:
oRound nucleus.
oFine/immature chromatin.
oProminent nucleolus.
• Cytoplasm:
oDeeply basophilic cytoplasm.
oCytoplasmic vacuoles.

15. 5. Megakaryoblasts:
• Size: They are of medium to large size
• Nucleus: Nucleus is round, slightly irregular or
indented with fine reticular chromatin and one
to three nucleoli.
• Cytoplasm: The cytoplasm is basophilic,
agranular and may show distinct blebs or
pseudopod formation.
• In some cases blasts are predominantly small
with high nuclear cytoplasmic ratio resembling
L1 lymphoblasts. Large & small blasts may be
present in the same patient.

16. Lymphoblasts:
Criteria L1 blasts L2 blasts
Size: Small blasts. Large blasts.
Cytoplasm: Scant cytoplasm. Moderate cytoplasm and may have
vacuoles.
Nucleus: o Condensed chromatin.
o Indistinctive nucleoli.
o Dispersed chromatin.
o Variable nucleoli.

17. L1 Blasts L2 Blasts

18. L3 Blasts

19. 2. Cytochemistry:
i. Myeloperoxidase (MPO):
• Myeloperoxidase is an enzyme located in the
azurophil (primary) granules of myeloid cells.
• MPO positivity appears as coloured granules in the
cytoplasm of cells mainly at the site of enzyme activity
(Golgi zone).
• All the stages of neutrophil series show MPO
positivity.
• In monocyte series azurophil granules are smaller and
MPO activity stains less strongly and appears late
during maturation.
• MPO is never seen in lymphoblasts.
• The main use of MPO is to distinguish AML from ALL.
• MPO is positive in AML subtypes M1, M2, M3 (strong
positivity), and M4

20. ii. Sudan black B (SBB):
• Phospholipids in the membrane of neutrophil granules
are stained by SBB. SBB positivity parallels that of MPO
in neutrophil series.
iii. Estrases:
1. Chloroacetate esterase (CAE) (a.k.a. specific
esterase):
• The reaction is present in all cells of neutrophil series
(though less sensitive than MPO and SBB) and is
negative in monocyte series.
• It is commonly used in combination with non-specific
esterase (NSE) for diagnosis of leukemia with both
myeloid and monocyte components (AML M4). Both
esterases (CAE for myeloid and NSE for monocytic
components) can be demonstrated in the same blood
film; this is called as combined or double esterase
reaction.

21. 2. Non-specific esterase (NSE) reaction
(usually demonstrated by ANAE or ANBE):
• α-naphthyl acetate esterase is an enzyme that is
present in large quantities in monocytic cells.
• It is present in small amounts in myeloid and lymphoid
cells.
• The non-specific esterase reaction is intensely and
diffusely positive in monocyte series and is sensitive to
sodium fluoride.
• ANAE/NASDA gives a characteristically strong diffuse
reaction in monocytic leukemias (M4 & M5) and gives a
localized pattern in M6 & M7.
• Esterase reaction is sensitive to inhibition by sodium
fluoride in monocytes. It is partially inhibited in M4 and
totally inhibited in M5.

22. iv. Periodic acid schiff’s reaction (PAS):
• Periodic acid is an oxidising agent that transforms
glycols and related compounds to aldehydes.
• The aldehyde groups then along with Schiff’s reagent
form an insoluble red-or magenta-coloured
compound.
• In hematopoietic cells, positive reaction is due to the
abundance of glycogen in cytoplasm.
• In ALL-B lineage, PAS stain shows characteristic
pattern of block positivity.
• In T cell-ALL and in L3 subtype of ALL, PAS reaction
is negative.
• PAS positivity is also seen in monoblasts (in AML M5)
and in erythroblasts (in AML-M6); however, in these
cells small blocks of positive material are present
against a diffusely positive cytoplasmic background.

23. v. Acid phosphatase stain:
• It is used for recognition of Golgi zone in
T- lineage ALL.
• Both PAS & acid phosphatase stains are
no longer indicated in suspected ALL
unless there is no access to
immunophenotyping.

24. 3. Flow cytometric Immunophenotyping:
Role of immunophenotyping in acute leukemia:
1. IPT is necessary for lineage assignment and to detect mixed phenotype acute
leukemia.
2. IPT is also important in distinguishing between:
• Minimally differentiated AML and ALL.
• Myeloid blast phase and lymphoid blast phase in CML.
3. A secondary role for IPT is in monitoring minimal residual disease. This is achieved by
defining a leukemia related phenotype in an individual patient. For this purpose, an
extended antibody panel is needed.
4. If there is a possibility of a monoclonal antibody being used in therapy e.g. Myelotarg;
anti CD33 in AML, then it should be included in the diagnostic panel.

25. 4. Genetic studies:
I. Cytogenetic analysis:
-When resources permit, it should be carried out in all patients for the following reasons:
1. AML can be assigned to certain prognostic groups so treatment can be modified accordingly. e.g.
good risk cytogenetic groups which include:
◦ t(8;21)(q22;q22): it occurs predominantly in AML M2 and some cases of AML M4.
◦ Inv16 (p13; q22) or t (16; 16) (p13; q22): it occurs in AML M4 with notable association with abnormal
eosinophilia.
◦ t (15;17)(q24;q21): it is specific for AML M3 & M3 variant. The response to all retenoic acid ATRA is
remarkable.
2. It facilitates the recognition of secondary leukemia e.g. therapy related AL and the distinction between
the alkylating agent related leukemia involving chromosomes 5 & 7 and the topoisomerase II interactive
drug related leukemia involving chromosome 11 q23 e.g. t(4;11)(q21;q23).
3. Cytogenetic analysis is essential if the WHO classification of AML is to be used.

26. A case of AML with normal karyotype (46,XY)

27. - Cytogenetic analysis in ALL is difficult due to the low mitotic index of the
leukemic blasts and the poor chromosome morphology. This can be
overcome by using molecular genetic analysis.

28. II. Molecular genetic analysis:
• Molecular genetic analysis only permits the detection of abnormalities that are specifically
sought whereas conventional cytogenetic analysis can assess all chromosomes. So molecular
genetic analysis supplements cytogenetic analysis and does not replace it.
• Techniques used in molecular genetic analysis include Fluorescence in situ hybridization
(FISH) & RT-PCR.
• A potential role for molecular genetics is monitoring of MRD e.g. monitoring the transcription
of fusion gene e.g PML-RARA fusion gene.
• For prognosis, identification of abnormal gene expression e.g. FLT3 expression, FLT3-ITD
mutations are associated with an adverse outcome. Some cases of AML with an apparently
normal karyotype carry mutations in nucleophosmin (NPM) gene, have a favorable prognosis.

29. Left panel: 2 red and 2 green (2R 2G) signal pattern correspond to normal PML (red) and RARA
(green).
Right panel: FISH analysis using probes directed to PML (green) and RARA (red), demonstrate
intact PML and RARA signals (red and green) and 2 fusion signals (yellow).

30. Example of quantitative RT-PCR using TaqMan probes and primers designed to amplify 3
PML-RARA fusion transcripts demonstrate high copy numbers of PML-RARA short form
transcript (Blue). The amplification plot in black represents ABL1 internal control gene.

31. Acute Myeloid Leukemia Genetic Risk Classification (European LeukemiaNET 2017 risk
stratification):
Risk Status Cytogenetic Molecular Abnormalities
Favorable: o Core binding factor AML: AML with
t(8;21), AML with inv(16)/t(16;16).
o Acute promyelocytic leukemia with PML-
RARA.
Normal karyotype with:
o NPM1 mutation and absence of FLT3-ITD
mutation.
o Biallelic CEBPA mutation.
Intermediate: o AML with t(9;11).
o Normal karyotype.
o Other non-defined.
o Core binding factor AML with KIT mutation.
o Mutated NPM1 with FLT3-ITD.
Unfavorable: o Complex (> 3 clonal abnormalities).
o AML with inv(3)/t(3;3) (GATA2-MECOM).
o t(11q23-KMT2A gene) other than t(9;11).
o AML with t(6;9) (DEK-NUP214).
o AML with t(9;22).
o Monosomal karyotype.
o Monosomy 5, del(5q).
o Monosomy 7, del(7q).
Normal karyotype with:
o FLT3-ITD mutation.
o TP53 mutation.
o Mutated RUNX1.
o Mutated ASXL1.
o Wild type NPM1 with FLT3-ITD.

32. Favorable Intermediate Unfavorable
Hyperdiploidy (> 50 chromosomes,
especially with trisomy 4, 10, 17).
t(5;14) (IL3-IGH). Hypodiploidy (< 45 chromosomes).
t(12;21) (ETV6-RUN1). t(1;19) (TCF3-PBX1) t(9;22) (BCR-ABL-1).
Normal karyotype. KMT2A rearrangement; t(4;11)
Any other abnormalities not in
favorable or unfavorable
categories.
B-ALL with iAMP21
BCR-ABL1-like B-ALL
Complex abnormalities

33. Molecular genetic analysis has the following advantages over conventional
cytogenetic analysis:
1. It can confirm the presence of specific fusion gene in patients with variant
rather than classical translocation.
2. It can yield results when cytogenetic analysis fails or gives normal
metaphases.
3. It can detect relevant abnormalities that are not detected by cytogenetic
analysis (i.e. cryptic) e.g. ETV6-RUNX1 (TEL-AML1) fusion gene in B-lineage ALL
associated with cryptic t(12;21).
4. It can detect submicroscopic abnormalities e.g. GATA1 mutations in AML-M7 in
Down syndrome or internal tandem duplication of FLT3 in multiple morphological
subtypes of AML.
5. It gives rapid results so clinical decisions can be made.