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HAEMATOLOGY

ACUTE LEUKAEMIA

Leukemia is a malignant proliferation of cells.


The cells arise from a single haemopoeitic stem cell.
A series of events rather than a single alteration is responsible for the malignant transformation.
Causes of acute leukemia:
A number of studies have been done to establish patterns in the incidence of acute leukemia. Results
of these (as at 2002) include:
Children in England living in the vicinity of power stations have a high incidence of ALL.
African countries have a much lower incidence of ALL than Europe or U.S.A., but Africa has more
AML.
South Africa seems to have a high incidence of Acute Promyelocytic Leukemia (APL-M3).
Some causes of acute leukemia may be:
1. Radiation: After the atomic bombs in Japan there was an increase of Acute leukemia which
was often preceded by an MDS syndrome.
2. Chemicals: Chemotherapeutic drugs and toxins such as benzene have been implicated.
Patients often suffer from MDS prior to developing leukemia and have multiple cytogenic
abnormalities.
3. Viral: Certain viruses have been associated with the development of leukemia, e.g. Epstein
barr and HTLV-1.
4. Genetic: Certain genetic abnormalities have also been associated with Acute leukemia, e.g.
Fanconis anemia, Downs Syndrome and various immunodeficiency syndromes.
Clinical features:
Clinical features are usually associated with the proliferation of leukemic cells.
Symptoms include: Fatigue, weakness, bleeding and bruising, fever and infection.
Each sub-type of leukemia does however have its own particular symptoms.
Diagnosing Acute leukemia:
Acute leukemia is diagnosed when one of the following is present:
> 30 % (WHO: > 20 %) of the total nucleated cells on the Bone marrow are blasts.
In the case of Erythroleukemia: Erythroblasts comprise > 50 % of nucleated BM cells.
Myeloblasts comprise > 30 % of non-erythroid BM cells.
Acute promyelocytic leukemia: The predominating cells are promyelocytes.

Blast cells:
The FAB group have described two types:
1. Type 1 (classic blast): These blasts lack granules, have uncondensed smooth chromatin,
high nuclear-cytoplasmic ratio and prominent nucleoli.
2. Type 2: These cells are exactly the same but have a few azurophilic granules and a lower
nuclear-cytoplasmic ratio.
Promyelocytes: Eccentric nucleus, a golgi zone, denser chromatin and numerous granules.
Promyelocytes with these features apart from granules are called hypogranular or agranular.
Classifying Acute leukemia:
Acute leukemias differ in their pathogenesis, history and prognosis.
Broadly, acute leukemia can be divided into Acute myeloid leukemia (AML) and Acute lymphoid
leukemia (ALL).
Each of these two groups comprise of different subgroups.
Distinguishing between AML and ALL blasts:
MYELOID
NUCLEAR-CYTOPLASMIC
RATIO
NUCLEI
NUCLEOLI
CYTOPLASM
CYTOPLASMIC
INCLUSIONS

Not high

Fine chromatin
35
Basophilic
Auer rods,
azurophilic granules

LYMPHOID
High

Denser chromatin (can be clumped)


12
Deeply basophilic
None

Some blasts may be indistinguishable by morphology alone and need further tests such as
cytochemistry, immunophenotyping and cytogenetics.

THE FAB CLASSIFICATION


Myeloid subtypes:
1. M1: Acute Myeloid Leukaemia Without Maturation
Accounts for 15 20 % of all AML.
Clinical:
Characterised by either rapid or gradual onset.
Symptoms include: Fever, fatigue and bleeding
Bone tenderness
Hepatomegaly and splenomegaly
Localised tumor masses consisting of myeloblasts known as chloromas.
Laboratory:
FBC: Anaemia and thrombocytopaenia.
White cells are very increased.
Peripheral smear: Increase in myeloblasts. Can have auer rods.
Monocytes comprise < 1 % of cells.
Bone Marrow:
FAB CRITERIA: M1
Blasts: > 90 % of non-erythroid cells.
>/= 3 % of blasts must be positive with the myeloperoxidase or sudan black stain.
The bone marrow maturing component must be < 10 % of non-erythroid cells.
Other tests which will contribute:
Chloroacetate esterase.
Immunophenotyping: CD13 and CD33.
Electron Microscopy.
Cytogenetics.
2. M2: Acute Myeloid Leukaemia With Maturation
Accounts for +/- 30 % of all AML.
Clinical:
Haemorrhagic symptoms bruising, petechiae.
Hepatomegaly and splenomegaly.
Laboratory:
FBC: Anaemia and thrombocytopaenia.
Increased White cell count often > 100 X 109 /l.
Peripheral smear: Increase in myeloblasts.
Maturation of granulocytes is often seen with the appearance of promyelocytes.
Bone Marrow:
FAB CRITERIA: M2
Blasts: 30 89 % of non-erythroid cells.
Maturing granulocytes (promyelocytes to neutrophils) comprise > 10 % of non-erythroid cells.
Monocytic cells: < 20 % of non-erythroid cells.
Cytochemistry: Sudan black: > 3 % positive.

Myeloperoxidase: > 3 % positive.


Chloroacetate esterase: positive.
Immunophenotyping: CD13+, CD33+.
In M2 often increased CD34+.
Electron Microscopy: Demonstrates granules and auer rods.
Cytogenetics: t(8;21)
A minority of patients have a predominance of eosinophils or basophils. These cases have been
called: M2-Eosinophilia and M2-Basophilia.
Sometimes dysplasia is seen.
3. M3: Acute Promyelocytic Leukaemia
Clinical:
The main clinical symptom is bleeding which is caused by disseminated intravascular coagulation
(DIC). This needs prompt treatment. The DIC is caused by a release of tissue factor from the
granules of the promyelocyte.
Other symptoms include: fatigue, infection, hepatomegaly and splenomegaly.
Laboratory:
FBC: Anemia and thrombocytopaenia. (Often pancytopaenia.)
White cells can be variable, ranging from very low to very high.
A low count is more common.
Myeloperoxidase printout is shifted to the right.
Peripheral blood: Promyelocytes dominate.
They are heavily granulated with many auer rods, often grouped as faggots.
Nuclei are often bi-lobed or dumb-bell shaped.
Bone Marrow:
FAB CRITERIA: M3
< 30 % blast cells.
Majority of cells are promyelocytes.
Cytochemistry:
Myeloperoxidase
Sudan black
Chloroacetate esterase

Very strongly positive, with auer rods.

Cytogenetics: t(15;17)
Immunophenotyping: Cells lack immature markers CD34 and HLA-DR.
Express myeloid markers CD13 and CD33.
M3-Variant
Promyelocytes lack granules: Hypogranular or agranular.
Often called microangular APL.
White cell count is often high.
Electron microscopy reveals cells packed with granules.
M3 variant can be confused with monocytic leukaemia.
Therefore further tests need to be done:
Cytochemistry: Non-specific esterase.
Immunophenotyping: Monocytic markers.
Electron microscopy: Detection of granules.

4. M4: Acute Myelomonocytic Leukaemia


Clinical:
Fatigue, weakness, bruising and infection.
Gum hypertrophy with oral ulcers.
Skin infiltration.
Enlarged spleen.
Central nervous system (CNS) involvement.
Laboratory:
FBC: Anemia, reduced platelets.
White cells: variable, rarely exceeding 100 X 109 /l.
Peripheral blood:
Increased proliferation of granulocytes and monocytic cells.
Monocyte count can exceed 5 X 109 /l.
Early myeloid cells predominate but monocytic cells are +/_ 20 % of cells.
Auer rods may be present.
(Monocytes have lower peroxidase activity than granulocytes, so not very prominent on automated
counter.)
Bone Marrow:
FAB CRITERIA: M4
Blasts > 30 % of non-erythroid cells.
Bone marrow granulocytic component (from blast to neutrophil) must be > 20 % of non-erythroid
cells.
A significant monocytic component must be demonstrated by one of the following:
1. BM monocytic cells (monoblast to monocyte) must be > 20 % of non-erythroid cells and
peripheral blood monocytes must be > 5 X 109 /l.
OR
2. BM monocytic component must be > 20 % of non-erythroid cells and must be confirmed
with either: Cytochemistry: Non-specific esterase.
Serum or urine muraminidase.
OR
3. The bone marrow can resemble a M2 but the peripheral blood monocyte count is > 5 X 109 /l
and is confirmed by cytochemistry or serum / urinary muraminidase.
Cytochemistry:
Sudan Black: Positive > 3 %
Myeloperoxidase: Positive > 3 %
Non-specific esterase: Positive.
A combined esterase with both chloroacetate and butyrate as substrates usually demonstrates the
presence of both myeloid and monocytic cells.
Electron Microscopy: Confirms presence of granulocytes.
Cytogenetics: Abnormalities of chromosome 16.
Immunophenotyping: Monocytic antigens CD11b and CD14.
M4-Eosinophilia
When M4 is associated with increased eosinophils.
Eosinophils are usually chloroacetate and PAS positive.
Cytogenetics: inversion 16.

5. M5: Acute Monocytic / Monoblastic Leukaemia


Clinical:
Fatigue, weight loss, bleeding from mouth and nose.
Gum-hyperplasia.
Skin-lesions.
CNS involvement.
Laboratory:
FBC: Anaemia and thrombocytopaenia.
White cells: High to very high (15 to +/_ 100 X 109 /l.).
Peripheral blood:
Increased blasts with monocytes and promonocytes constituting 25 75 % of cells. The blasts are:
Large with grey-blue cytoplasm.
Cytoplasm is irregular in outline with some tiny granules and can be vacuolated.
Nucleus is irregular with folded appearance, has a delicate chromatin pattern and contains nucleoli.
The cells are often described as having a fried egg appearance.
Bone Marrow:
FAB CRITERIA: M5
Blasts are > 30 % of non-erythroid cells.
Monocytic component: > 80 % of non-erythroid cells:
M5a-monoblastic
More immature. Monoblasts > 80 % of monocytic component.
M5b-monocytic
More mature. Monoblasts < 80 % of monocytic component.
Cytochemistry:
Myeloperoxidase: Often negatives.
Sudan Black: Can have granular positivity. (NB to describe the positivity.)
Chloroacetate esterase: Negative.
Non-specific esterase: Strongly positive and is inhibited by the addition of fluoride.
Acid phosphatase: Diffuse positivity.
Muraminidase: Increased.
Immunophenotyping: Monocytic markers CD14, CD11b.
6. M6: Acute Leukaemia with Predominant Erythroid Features:
Clinical:
Bleeding, enlarged liver and spleen.
Laboratory:
FBC: Variable counts.
Peripheral blood:
Consists of cells of both myeloid and erythroid origin.
Promyelocytes may also be present.
Bone Marrow:
FAB CRITERIA: M6
Erythroblasts: > 50 % of all nucleated cells.
Myeloblasts: > 30 % of non-erythroid cells.
Erythroid dysplasia is common and it is thought that M6 is the erythroleukaemic transformation of
MDS.

The erythroblasts are often megaloblastic: are large and are multinucleated.
The myeloid blasts may have auer rods and can be dysplastic.
Cytochemistry:
Sudan Black
Myeloperoxidase

Positive in myeloblasts.

PAS: Erythroblasts have diffuse or fine granular positivity with or without block positivity.
NSE: Can be positive.
Iron stain: Course siderotic granules; can have ringed sideroblasts.
Electron Microscopy:
Demonstrates iron laden mitochondria and ferritin granules.
Immunophenotyping:
Positive for transferrin receptor (CD71) and glycophorin A.
Myeloid antigens: CD13 and CD33.
7. M7: Acute Megakaryocytic Leukaemia
Clinical:
Fever, bleeding, enlarged spleen.
Laboratory:
FBC: Anaemia
White cells: Variable
Platelets: Variable.
Peripheral blood:
Occasionally may have increased blasts.
Blasts vary in size and are pleomorphic.
Cytoplasm is deeply basophilic and can have cytoplasmic budding or vacuoles.
Blasts may resemble myeloblasts or lymphoblasts.
There are often bizarre platelets, micromegakaryoblasts and bare nuclei.
Bone Marrow:
FAB CRITERIA: M7
Blasts: > 30 % of all nucleated BM cells.
Blasts must be demonstrated to be megakaryoblasts by immunophenotyping, EM or cytochemistry.
Bone marrow may be difficult to aspirate due to increased fibrosis.
Cytochemistry:
Myeloperoxidase: Negative
Sudan Black: Negative
Chloroacetate esterase: Negative
Non-specific Esterase: Butyrate: Weak to negative.
Acetate: Weak to strong.
(Monocytic leukaemias are strongly positive with both substrates.)
Acid Phosphatase: Positive
PAS: Positive in more mature cells.
Immunophenotyping: Platelet glycoproteins: Positive.
CD41, CD42, CD61.

Electron Microscopy: Platelet peroxidase can be demonstrated.


8. M0: Acute Leukaemia with Minimal Evidence of Differentiation
This category was added to the classification in 1990. Previously all cytochemical negative cases
were classified as lymphoid.
Clinical:
Fever, anaemia, bleeding.
Laboratory:
The blasts do not exhibit any typical features of myeloid or lymphoid cells.
They are sudan black and myeloperoxidase negative.
The blasts demonstrate evidence of myeloid differentiation by one of the following methods:
1. Electron Microscopic demonstration of myeloperoxidase.
2. Demonstration of myeloperoxidase by immunophenotyping.
3. Demonstration by immunophenotyping other myeloid antigens: CD13, CD33.
Bone Marrow:
FAB CRITERIA: M0
Blasts > 30 % of nucleated cells.
< 3 % of blasts are positive with sudan black and myeloperoxidase using Light Microscopy.
Blasts demonstrated to be myeloid by immunophenotyping or Electron Microscopy.
Q: Which two myeloid leukaemias are negative for Sudan black and Myeloperoxidase?
A: M7 and M0.
Lymphoid subtypes:
Acute Lymphoblastic Leukaemia
Acute lymphoblastic leukemia is predominantly seen in children between the ages of 2 and 10
years.
However, it is also seen in adults.
Clinical features:
Fatigue, fever, infection, nausea, vomiting.
Bone pain.
Haemorrhage.
Lymphadenopathy.
Hepatomegaly.
Blasts can infiltrate the cerebral spinal fluid and cause neurological problems.
Enlarged thymus in T-cell ALL.
THE FAB CLASSIFICATION
The FAB group have divided ALL into 3 groups: L1, L2 and L3.
The classification deals solely with morphology and therefore it is necessary to do further tests
Cytochemistry, Immunophenotyping and Cytogenetics.

1. FAB-Subtype: L1
Blasts are small (+/_ twice the diameter of small lymphocytes).
Nucleus: regular in shape,
can have occasional cleft or indentation.
Nucleoli: Inconspicuous.
Cytoplasm: Scanty (high nuclear-cytoplasmic ratio.),
intensely basophilic,
can contain occasional granules.
L1 may be of T- or B-cell lineage.
2. FAB-Subtype: L2
Blasts are large, heterogeneous (vary in size), and pleomorphic.
Nucleus: Irregular, often with folding and indentations.
Nucleoli: Often present and can be large.
Cytoplasm: Abundant (low N/C ratio.),
basophilic,
can be vacuolated and can have vacuoles.
L2 can be confused with myeloblasts.
Can be of T- or B-cell lineage.
The FAB group have divised a scoring system to distinguish the blasts of L1 from those of L2.
Based on morphology; not relevant anymore.
3. FAB-Subtype: L3
L3 is often referred to as Burkitts lymphoma as the lymph nodes of patients with Burkitts have
morphologically the same cells. L3 is the leukaemic form of Burkitts lymphoma.
Blasts are large and homogenous.
Nucleus: Regular in shape varying from round to oval.
Nucleolus: Sometimes prominent.
Cytoplasm: Low N/C ratio not as low as L2.
Strongly basophilic with variable but prominent vacuoles -- starry night appearance.
The cells of L3 have been shown to be mature B-cells as they have surface immunoglobulin.
Cytogenetics: t(8;14) is the most common finding.
Cytochemistry in ALL
Very little relationship exists between the FAB classification and cytochemical staining.
Sudan Black
Myeloperoxidase
Negative
Chloroacetate esterase
B-lineage ALL: PAS stain show block positivity.
T-lineage ALL: Strong polar-dot or localised positivity with the Acid phosphatase stain.

Non-specific esterase stain using both butyrate or acetate as substrate will show
polar-dot or localised positivity. (NB in stains to describe the result.)
L3: Vacuoles usually stain positive with Oil Red O stain.

PROBLEMS WITH THE FAB CLASSIFICATION OF ACUTE LEUKAEMIA


1. Some leukaemias have < 30 % blasts and may be wrongly classified as MDS (RAEBIT).
2. Not all cases fit into the FAB classification, especially those associated with a history of
chemotherapy.
3. The FAB classification does not allow for bi-lineage or bi-phenotypic leukemias (with
features of both myeloid and lymphoid cells.)
4. Important information such as immunophenotyping and cytogenetics have been excluded.
The MIC classification (morphology, immunophenotyping, cytogenetics) has been proposed
as an alternative; but (as at 2002) the FAB is still used in most laboratories.
TREATMENT OF LEUKAEMIA (as at 2002)
Treatment differs for myeloid and lymphoid leukaemia.
AML
Initial therapy is to induce a remission. This is usually achieved with a combination of drugs and
can vary from centrre to centre. They can consist of Cytosine arabinose, VP16, daunorubicin.
Acute Promyelocytic leukemia (APL-M3) is traeted with retinoic acid which induces maturation of
the cells.
Once remission is achieved consolidation therapy follows.
If the patient has a compatible donor a bone marrow transplant is recommended. If not, an
autologous transplant can be performed reinfusing patient's own remission marrow cells.
ALL
Childhood ALL has a very good prognosis and +/_ 80 % of patients expected to be cured. Drugs
used: Vincristine and Prednisone.
Consolidation is needed to completely irradicate the leukaemic clone.
Adult ALL does not have as good prognosis. 85 % will achieve remission but usually relapse. Bone
marrow transplant is the only hope of cure.

Revised Criteria for Classification of AML


M0
Large agranular blasts (resemble ALL L2, rarely L1).
Myeloperoxidase negative or < 3 % positive.
Myeloperoxidase positive with Immunochemistry or Electron Microscopy.
B- and T-lineage markers negative.
CD13 and/or CD33 positive.
TdT may be positive.

M1
Blast cells, agranular and granular types (types I and II) > 90 % of non-erythroid cells. At least 3 %
of these are positive for Peroxidase or Sudan black.
Remaining 10 % or less of cells are maturing granulocytes or monocytes.
M2
Sum of agranular and granular blasts (types I and II) is 30 89 % of non-erythroid cells.
Monocytic cells are < 20 %.
Granulocytes from promyelocytes to mature polymorphs are > 10 %.
M3
Majority of cells are abnormal promyelocytes with heavy granulation.
Characteristic cells containing bundles of Auer rods (faggots) invariably present.
Note: Microgranular variant also occurs.
M4
In the marrow, blasts > 30 % of non-erythroid cells.
Sum of myeloblasts, promyelocytes, myelocytes and later granulocytes is between 30 and 80 % of
non-erythroid cells.
> 20 % of non-erythroid cells are monocyte lineage.
If monocytic cells exceed 80 % diagnosis is M5.
Note: If marrow findings are as above and peripheral blood monocytic cells are > 5.0 X 10 9 /l,
diagnosis is M4.
If monocyte count is < 5.0 X 109 /l, M4 can be confirmed on basis of serum lysozyme,
combined esterase, etc.
Diagnosis of M4 is confirmed if > 20 % of marrow precursors are monocytic (confirmed by
special stains).
M4 with eosinophilia
Eosinophils > 5 % of non-erythroid cells in marrow.
Eosinophils are abnormal.
Eosinophils are chloroacetate and PAS positive.
M5
80 % of marrow non-erythroid cells are monoblasts, promonocytes or monocytes.
M5a : 80 % of monocytic cells are monoblasts.
M5b : < 80 % of monocytic cells are monoblasts, remainder are predominantly promonocytes and
monocytes.
M6
The erythroid component of the marrow exceeds 50 % of all nucleated cells.
30 % of the remaining non-erythroid cells are agranular or granular blasts (types I and II).
Note: If > 50 % erythroid cells but < 30 % blasts, diagnosis becomes myelodysplastic syndrome.
M7
30 % at least of nucleated cells are blasts.
Blasts identified by platelet peroxidase on Electron Microscopy, or by monoclonal antybodies.
Increased reticulin is common.

THE WHO CLASSIFICATION


# AML with Recurrent Cytogenetic Translocations

AML with t(8;21)(q22:q22) AML 1/ETO (includes some M2).


Acute Promyelocytic leukaemia with t(15;17)(q22:q11-12) and variants PML/RAR-alpha
(former M3).
AML with abnormal bone marrow eosinophils: inv 16 (p13q22) or t(16;16)(p13:q11).(FAB M4eosinophilia).
AML with 11q23 (MLL) abnormalities.
# AML with Multilineage Dysplasia

With prior myelodysplastic syndrome.


Without prior myelodysplastic syndrome.
# AML with MDS, Therapy related

Alkylating agent related.


Epipodophyllotoxin related.
Other types.
# AML not otherwise categorized

AML M0
AML M1
AML M2
AML M4 (except M4 Eosinophilia)
AML M5
AML M6
AML M7
Acute basophilic leukaemia
Acute panmyelosis with myelofibrosis
Acute biphenotypic leukaemia.

Notes from Cape Peninsula University of Technology (Fmr. Cape Technikon), 2002, Biomedical
Technology.
More course notes at: http://www.scribd.com/people/documents/2135965/folder/83622