11/12/2012

Diagnostic Hematology (LM357)

Dr. Mohammad A. Audeh

First semester
Nov 12th 2012

Leukemia

Is a group of malignant (neoplastic) disorders that are

characterized by the clonal expansion and accumulation of one or

more blood cell line(s) with eventual involvement of all

hematopoietic organs and other organs.

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Leukemia: Origin
• Leukemia is a result of two or more transforming somatic mutations
within the genome of HSC or multipotential progenitors/precursors

Activation of specific proto-oncogene (BCL-2)

De-activation of tumor suppressor genes (eg. p53)

A clone of cells with characteristics of a malignant cell.

􀁻 Prolonged life (immortal)/resistance to apoptosis
􀁻 Growth factor independent growth
􀁻 Insensitivity to growth-inhibitory signals (increased proliferation rate)
􀁻 Ability to invade and metastasize
􀁻 Ability to stimulate angiogenesis
􀁻 Blockage of intracellular differentiation

Leukemia: Etiology
• Etiology: causes of genomic mutations include (not limited to):
A. Environmental B. Genetic

Strong Correlation – Inherited genetic predisposition

– Viral agents (family history of malignancy)
(e.g. HTLV and HIV)
– Ionizing radiation – Chromosomal abnormalities such
– Chemicals/drugs as chromosomal breaks, duplication
(e.g. Benzene) and translocations
(e.g. Down syndrome and Fanconi’s anemia)
Moderate Correlation
– Smoking – Immunologic abnormalities
– Alcohol consumption
– Patient age

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Leukemia: Classification
 Based on the clinical and path-physiologic characteristics:
• Acute:
- Characterized by the clonal expansion and accumulation of immature
leukocytes (blood cells) in hematopoietic organs

• Chronic:
- Clonal expansion and accumulation of mature, but abnormal leukocytes in
hematopoietic organs

 Based on the affected blood cell lines:

• Myelogenous: Myeloid cell line
• Lymphocytic: Lymphoid cell line

Leukemia: Classification

Acute Myeloid Leukemia (AML)

Acute Lymphoid Leukemia (ALL)

Chronic Myeloid Leukemia (CML)

Chronic Lymphoid Leukemia (CLL)

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Leukemia: Diagnosis

• Preliminary hematological analysis: CBC and blood film examination

- Normocytic normochromic anemia (mild to severe)
- Platelets count: reduced to normal
- WBC’s count: decreased to markedly decreased mature leukocytes
- The appearance of abnormally circulating blood cells (malignant cells)

• BM aspirate and/or biopsy

- B.M. hyperactivation and hyperplasia
- Blasts: represents greater than 30%

Leukemia: Diagnosis
• Cytochemistery:
- Myeloperoxidase and SBB are positive in myeloblast while negative in lymphoblasts)
- PAS is positive for lymphoblast (Coarse) while negative in myeloblast except in AML-M6
that is characterized by fine positive staining)

•Immunological markers (Surface, Cytoplasmic & Nuclear ~)

- TdT: differentiate Myeloblasts from lymphoblasts (B & T precursors)
- CD markers :e.g. AML: CD13/CD33/CD117
ALL (Precursor B): CD19/CD22/CD79a
ALL (T): CD7/CD3/CD2

• Cytogenetics: specific Chromosomal abnormalities

- Philadelphia chromosome (t(9;22)): common in CML
- t(8;21): AML-M2 - t(1;19), t(8;22), t(11,14): ALL

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Leukemia: Clinical features

• Symptoms reflects B.M. failure to produce normally functioning
blood cells
- Features of anemia: Weakness, shortness of breath, malaise and pallor
- Features of leukopenia: infections (septicemia, pneumonitis, skin and mucosa)
- Features of thrombocytopenia: Bleeding and increased hemorrhagic tendency

• Symptoms reflects B.M. hyperplasia and excessive accumulation,

infiltration and proliferation of malignant cells
- Bone and Joint pain - Hepatosplenomegaly
- Lymphadenopathy - Neurological (nausea, vomiting and headach)
- Gingival hypertrophy (hyperplasia) and oral lesions

Leukemia: Classification

 According to cell counts and number of abnormal cells in the

peripheral blood.
• Leukemic – High white count, many circulating abnormal cells
(redundant)
• Subleukemic- normal to slightly increased white count, few
circulating abnormal cells.
• Aleukemic – Decreased white count, very rare if any circulating
abnormal cells. Abnormal cells found in bone marrow.

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Acute Leukemia: Clinical features

Skin purpura Ocular hemorrhages manifestations

(petechiae, ecchymoses, or hematomas)

Acute Leukemia: Clinical features

Gingival hypertrophy (hyperplasia) Leukemia cutis (nodules or ulcers) that

represents skin infiltration by blast
cells.

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Acute Leukemia: Clinical features

Conjunctiva and skin palor (a result of eryhtrocytopenia)

Acute Leukemia: Clinical features

multiple abscesses
of liver

Extensive fungal
infection of tongue

extensive lobar
pneumonia

Perirectal abscess

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Acute Leukemia: Clinical features

Lymphadenopathy

Pyoderma gangrenosum: painful skin lesion that may evolve in

large ulcers

Clubbing of fingers can herald the onset of various malignancies

including AML.

Acute Myeloid Leukemia (AML)

• Diagnosed when approximately 20% of BM nucleated cells are
myeloblasts (based on WHO-modified FAB classification)

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AML-M0
• AML with no evident morphological differentiation
>>> i.e. no characteristic myeloid features
• Bone marrow contains 30 to 90% blast forms that are granule free
(clear cytoplasm).
• The blasts seen in AML and ALL are very similar; with a high nuclear
cytoplasmic ratio, immature chromatin, nucleoli, and a variable
amount of cytoplasm
• <3% of blasts are MPO and SBB positive
• Blasts are positive for CD13 and CD33 markers

AML-M0

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AML-M1
• Poorly differentiated myeloblasts (more than 90% of NEB)
• MPO & SBB are positive (>3 but < 50%)
• NSP is with at least 20% positivity
• Auer rods (may present but rarely seen)

AML-M1

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AML-M2
• AML with maturation (AML-M2)
• Similar to those of M1 except evidence of maturation up to the
promyelocyte stage (represents more than 10% of blasts)
• > 50% of leukemic cells are MPO and SBB positive.
• 10-20% positivity for NSE
• Auer rods are occasionally seen
• M1-M2 combined type of AML: commonest (50% of cases)

AML-M2

- MPO stains reveal strong granular

cytoplasmic staining in many leukemic blasts
-Auer rods presence (Arrow)

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AML-M2

AML-M3
• Acute promyelocytic leukemia (AML-M3)
• Promyelocytes with heavy granulation
• Auer rods frequently seen (Faggot cells: cells with bundles of AR)
• Cells are strongly positive with MPO and SBB
• Negative with NSE
• t(15;17) is a unique and common feature of AML-M3.
• AML-M3 variant: showing the characteristic bilobed hypogranular
promyelocytes
• AML-M3 and DIC

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AML-M3

AML-M3 variant: showing the AML-M3: hypergranular

characteristic bilobed hypogranular promyelocytes, one of which contains a
promyelocytes. giant granule.

AML-M4
• Acute myelomonocytic leukemia (AML-M4)
• Cells are with granulocytic and monocytic differentiation
• 20-80% of cells are positive for MPO, SBB and NSE.
• Differential diagnosis: serum lysozyme level > 3 times
• AML-M4 with Eosinophilia

- Eosinophils represents > 5% of nonerythroid cells

- immature eosinophils positive for SE and PAS

- 16q deletion or inversion are common.

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AML-M4
MPO

NSE

AML-M4 Eos variant

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AML-M5
• Acute monoblastic leukemia (AML-M5).
• > 80% of NEC are monocytic lineage
• Two subclasses: Poorly (M5a) & well (M5b) differentiated

- M5a: Predominance of monoblasts

- M5b: Predominance of promonocytes and monocytes

• > 80% of cells are positive for NSE

• Cells are negative for MPO and SBB (weakly positive in monoblast).

AML-M5

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AML-M5

AML-M5b

PB smear with large mature monocytes

that have low N:C ratios, lobulated nuclei BM aspirate smear shows clusters of
with moderately closed chromatin pattern monocytic blasts that have a more immature
and neutral staining, and finely granular, appearance than do the circulating blasts.
vacuolated cytoplasm.

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AML-M6
• Acute eythroblastic leukemia (erythroleukemia).
• Hypercellularity of B.M. with marked erythroid hyperplasia
• > 50% of ANC are of erythroid lineage
• Blasts commonly show megaloblastoid characterestic features
including multi-nucleation, cytoplasm vaculation
• Most erythroid precursors are positive for PAS (Fine staining)
• Negative to weakly positive for MPO, SBB and NSE

AML-M6

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AML-M6
Differential diagnosis of AML (M6) against MDS (RAEB)

B.M. aspirate

Erythroblast % of ANC

< 50% > 50%

Blasts % of ANC

≥ 30% < 30% ≥ 30%

AML MDS AML

AML-M7
• Acute megakaryoblastic leukemia
• Uncommon form
• Extensive proliferation of megakaryoblasts and ~cyte.
• >50% of blasts are megakaryoblasts
• Blasts identified by platelets peroxidase
• Negative for MPO and SBB, while positive for PAS.

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AML-M7

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Acute Lymphocytic Leukemia (ALL)

• predominantly a disease of Children

• Based on FAB classification, ALL is categorized into three catergories
ALL-L1, ALL-L2 and ALL-L3
• ALL blasts, in particular the L2 morphologic variants, can look very
much like typical AML blasts Therefore, immunohistochemistry
and immunophenotyping are almost always necessary to distinguish
ALL from AML.

ALL-L1

Blast in ALL-L1 are with high N/C ratio, a delicate diffuse

chromatin pattern and small prominent nucleoli.

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ALL-L2

The blasts are larger than those of L1, have more plentiful cytoplasm
and are more pleomorphic.

ALL-L3

L3 blasts cells are fairly regular in shape with strong basophilic

cytoplasm and prominent cytoplasmic vaculation

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