LEUKAEMIA

LEVEL 6
Contents
• Introduction to leukaemia
• Aetiology of leukaemia
• Pathophysiology of leukaemia
• classification of leukaemia
• Laboratory diagnostic test on Leukaemia
• Special stains on leukaemia
• Stain pattern used in leukaemia for diagnosis
INTRODUCTION
• The leukaemias are a group of disorders characterized by the
accumulation of malignant white cells in the bone marrow and
blood.Leukaemic cells do not function or mature normally.
• These abnormal cells cause symptoms because of: (i) bone marrow
failure (e.g. anaemia, neutropenia, thrombocytopenia); and
• (ii) infiltration of organs (e.g. liver, spleen, lymph nodes, meninges,
brain, skin or testes).
AETIOLOGY OF LEUKAEMIAS
1. ionizing radiation or exposure to chemicals such as benzene,
2. infection with certain viruses, HumanT cell leukaemia, virus type 1 and 11 in
adult T cell leukaemia and RNA retrovirus
3. treatment with cytotoxic drugs (e.g. alkylating agents)
4. Hereditary factors may also be involved, e.g. factors that activate oncogenes
(genes which are involved in producing malignant change) or chromosomal
abnormalities such as deletions, major translocations or additional
chromosomes. Some chromosomal changes have prognostic significance.
Pathophysiology of leukaemia
• The pathophysiology of leukaemia isn‘t completely understood, but
the initiating event is likely a mutation in the genetic material called
DNA that‘s present in the cells in the bone marrow.
• There is evidence that leukaemia in humans can be associated with
inappropriate expression of certain genes which appear to be
involved in regulating basic steps in cell proliferation.
Classification of Leukaemia
• The main classification is into four types: acute and chronic leukaemias, which are
further subdivided into lymphoid or myeloid.
• Acute leukaemias are usually aggressive diseases in which malignant
transformation occurs in the haemopoietic stem cell or early progenitors.
• Genetic damage is believed to involve several key biochemical steps resulting in (i)
an increased rate of proliferation, (ii) reduced apoptosis and (iii) a block in cellular
differentiation.
• Together these events cause accumulation in the bone marrow of early
haemopoietic cells known as blast cells .
Classification cont...

• The dominant clinical feature of acute leukaemia is usually bone

marrow failure caused by accumulation of blast cells although organ
infiltration also occurs.

• If untreated, acute leukaemias are usually rapidly fatal but,

paradoxically, they may be easier to cure than chronic leukaemias.
Classifaction cont...

• Based on whether the disease is acute (rapidly progressive)or chronic (slowly

progressive) and whether the leukaemic cells are of myeloid or lymphoid
origin, leukaemias can be broadly divided into:

1. Acute myeloid leukaemia (AML)

2. Acute lymphoblastic leukaemia (ALL)

3. Chronic myeloid leukaemia (CML)

4. Chronic lymphocytic leukaemia (CLL)

ACUTE & CHRONIC LEUKAEMIA.
• In acute leukaemias there is a clonal proliferation of blast cells (lymphoblasts in ALL, myeloblasts or
monoblasts, or rarely erythroblasts and megakaryocytes, in AML) which are difficult to differentiate in
Romanowsky stained bone marrow or blood films.

• Differentiation and treatment of acute leukaemias require specialist techniques including cytochemical-
staining (differentiates myeloid and lymphoid cells), and when possible, monoclonal antibody cell
marker studies (immunophenotyping) and chromosome studies (cytogenetics).

• In chronic leukaemias, the clone of tumour cells is sufficiently differentiated to be recognized in

Romanowsky stained preparations, i.e. lymphocytic cells in CLL and myeloid cells in CML.

• In most but not all forms of leukaemia the total leukocyte count is raised with very high counts being
found particularly in CML and CLL.
ACUTE MYELOID LEUKAEMIA(AML)
• AML is usually more common in adults. In tropical Africa, however,
AML and ALL are reported as having a similar frequency in children
below 15 y.
• In acute leukaemia the patient is anaemic, thrombocytopenic,
neutropenic, and blast cells predominate in the bone marrow and can
be seen in the blood film.
• In AML there is often inflammation of the gums,skin involvement, and
coagulation defects may occur in acute promyelocytic leukaemia.
AML Cont...

• The finding of Auer rods (needle or rod-like cytoplasmic inclusions) in

blast cells is diagnostic of AML, but these inclusions are not always
present in myeloblasts.
CLINICAL FEATURES IN AML
• Th e clinical features of AML are dominated by the pattern of bone
marrow failure caused by the accumulation of malignant cells within
marrow
CLINICAL FEATURES IN AML CONT..
• Infections are frequent and anaemia and thrombocytopenia are often
profound.
• A bleeding tendency caused by thrombocytopenia and disseminated
intravascular coagulation (DIC) is characteristic of the promyelocytic variant
of AML
• Tumour cells can infi ltrate a variety oftissues. Gum hypertrophy and infi
ltration (, skin involvement and CNS disease are characteristic of the
myelomonocytic and monocytic subtypes.
CLINICAL FEATURES IN AML CONT..
INVESTIGATION IN AML
• Haematological investigations reveal a normochromic normocytic anaemia
with thrombocytopenia in most cases.
• The total white cell count is usually increased and blood fi lm examination
typically shows a variable numbers of blast cells.
• The bone marrow is hypercellular and typically contains many leukaemic
blasts.
• Tests for DIC are often positive in patients with the promyelocytic variant of
AML
INVESTIGATION IN AML CONT..

• Biochemical tests are performed as a baseline before treatment

begins and may reveal raised uric acid or lactate dehydrogenase.
• The differential diagnosis includes acute lymphoid leukaemia (ALL) or
marrow infi ltration by other malignancies (e.g. carcinoma).
Morphological examples of acute myeloid leukaemia.
Classification of AML/ FAB
M1 = myeloblast without maturation
• Non granular cytoplasm or rare azurophilic granules
• Auer rods
• Mature myeloid cells not present
• Some blast cells myeloperoxidase positive
Classification of AML/ FAB cont...
M2 = Myeloblast wth maturation
•  Promyelocyte
•  More mature myeloid cells in addition to myeloblast
M3 = Hypergranular promyelocytic
•  Heavy cytoplasmic granulation and reniform nuclei
•  Multiple auer rods often in parallel bundle
•  Rare hypogranular type
M4 = Myelomonocytic (myelocyte + monocyte)
•  Immature myeloid
Classification of AML/ FAB cont..
M5 = Monocytic
•  Poorly differentiated type
•  Non specific esterase posiytive
•  Monoblast with non granular cytoplasm or rare azurophilic
granules/ auer rods
•  Promonocyte, monocyte in addition to monoblasts
Classification of AML/ FAB cont..
M6 = Erythroleukaemia (usually present in peripheral blood)
•  Erythroblast > 50% of marrorw nucleatyed cells
•  Myeloblast and promyelocyte increase
•  Morphological abnormalities
M7 = Megakaryoblastic
•  Megakaryoblasts
Acute lymphoblastic leukaemia
(ALL)
• ALL is most often found in children (males more often than females), AML
and ALL are reported as having a similar frequency in children below 15 y.
• cytopenic, neutropenic, and blast cells predominate in the bone marrow
and can be seen in the blood film.
• Bone pain, fever, bleeding, purpura, thrombosis and hepatosplenomegaly
are common and in ALL, also lymphadenopathy and central nervous
system involvement (late stages of disease).
ALL CONT... L1
L2
L3
L1
Clinical features in ALL
• Onset is usually abrupt
• Patient presents with symptom of anemia or hemorrhage
• Infective lesion of mouth and pharynx, fever headache and malaise
• Present sign of organ infiltration including
• Splenomegally
• Hepatomegally
• Lymphadenopathy
Laboratory diagnosis
• Blood picture:
• anemia, thrombocytopenia and moderate or marked increase in wbc;
majority blast cell
•  Anemia presenting more than 90%
•  RBC anisopoikilocytosis ++, Polichromasia + and occasional nRBCS
•  Platelets thrombocytopenia < 50x 109/l is common
•  WBCs subnormally to markedly elevated > 100x109/l, majority are
blast cells Neutropenia Lymphoblast Myeloblast, monoblast
Myeloblast contain auer rods
Cont... lab Diagnosis
Bone marrow
• Most cells are lymphoblast 50 – 100%
• Lymphoblasts contain round nucleus and 1—nucleoli and basophilic
cytoplasm without granules.
• Erythropoiesis – dyserythropoietic
• Megakaryocyte – reduced or absent
Classification of ALL according to
FAB classes/bone marrow
L1—Small Lymphoblast
• Homogenous
• Scanty Cytoplasm
• Regular Normal Nuclei
Classification of ALL according to
FAB classes/bone marrow
L2—Heterogenous Lymphoblast
• Variable Amount Of Cytoplasm Irregular Cleft Nuclei
• Large Nuclei
L3—Large Homogenous Lymphoblast
• Basophilic Cytoplasmround Nuclei
• Prominent Nucleoli
• Cytoplasm Vacuolation
• Other tests:
Cytochemical tests
• Myeloperoxidase & sudan black: AML Positive, ALL Negative
• Perioidic Acid Schiff: AML Negative, ALL Positive
• Non specific esterase stain: AML Positive ALL Negative
Chronic myeloid leukaemia (CML)
• In CML there is proliferation of myeloid cells. The abnormal
Philadelphia (Ph) chromosome* is present in the leukaemic cells of
more than 95% of patients with CML (also in 5–25% of patients with
ALL).

• *Formed by a translocation of the long arm from chromosome 22 to

the long arm of chromosome 9. It is associated with activation of the
BCR-ABL oncogene.
Clinical features of CML

• In the early stages of CML (chronic stage), patients usually have few
clinical symptoms.
• Splenomegaly, lethargy, and weight loss are common. CML develops
into AML which is preceded by an accelerated stage.
• The spleen and liver enlarge, and there is anaemia, thrombocytopenia
with bleeding tendency, fever, sweats and bone pain.
Clinical features of CML cont..
• CML is characterized by a greatly increased leukocyte count, up to 300
x10e9/l or more
Leukaemoid reactions

• In response to severe infections, malignancies, burns, and other

conditions, patients may have high white cell counts and blood films
which resemble those seen in chronic leukaemias.
• Leukaemoid reactions resembling CML can occur in tuberculosis,
meningococcal meningitis, septicaemia, severe megaloblastic
anaemia in pregnancy, eclampsia, acute hepatic necrosis, amoebic
liver abcess, burns and following severe haemorrahage.
Leukamoid reaction
• The main features which differentiate a leukaemoid reaction from
CML are as follows:
Leukamoid reaction CML
Total WBC Rarely more than 60 x 109/l 50–500 x109/l
Left shift Present,usually with toxic More prominent than
granules and neutrophill leukomid reaction No toxic
vaculation granulation. Blast
cells may be seen.
Eosinophil and Reduced or normal Increased
basophill
Neitrophil alkaline increased reduced
phosphate ratio
Leukamoid reaction

• Leukaemoid reactions resembling ALL can occur in miliary

tuberculosis, measles, whooping cough, chicken pox, syphilis,
infectious mononucleosis and malaria.
• A CLL-like blood picture can be seen with hyperactive malarial
splenomegaly.
Lab diagnosis of CML
Blood picture
. RBCs : anemia, Hb 8 – 10 g/dl, normocytic normochromic and occasional NRBCs
• WBCs : markedly elevated
• Count range: 50 – 500 x 109 with full spectrum of cells of granulocytic series
• Myelocyte > 20%
• Increased basophil is help to distinguish CGL from leucocytosis and leucomoid
reactions
•  Lymphocytopenia
•  Platelets: normal or increased
•  Myeloid erythroid ratio increased (25:1)
Diagnosis of CML cont..

• Bone marrow
• Marked granulocytic hyperplasia with predominance of
metamyelocyte, myelocytes, megakaryocyte increased
• Bone marrow cells usually look similar to the cell in the peripheral
blood
Chronic Lymphocytic Leukaemia
(CLL)
• In most forms of CLL there is a clonal proliferation of B lymphocytes.
Occasionally T lymphocyte CLL occurs. B cell CLL is rare in India and parts of
Asia.
• CLL may be misdiagnosed as hairy-cell leukaemia or prolymphocytic
leukaemia
• CLL is more often found in older persons (over 60 y) with more men affected
than women.
• In tropical Africa, CLL is also found in younger people. A lymphoproliferative
disorder may occur predominantly in young to middle aged women
associated with hyper-reactive malarial splenomegaly.
Clinical features of CLL
• Onset of disease is slow and in the early stages, asymptomatic.
• Most patients have weight loss, and develop lymphadenopathy and
hepatosplenomegaly (especially splenomegaly in malaria endemic
areas).
• In the later stages of CLL, neutropenia, thrombocytopenia and the
loss of normal functioning lymphocytes, lead to inadequate immune
responses resulting in bacterial, fungal, and viral infections.
Clinical features of CLL cont..
• develops, particularly due to hypersplenism and autoimmune
haemolysis and also thrombocytopenia as bone marrow failure
develops.
• CLL is staged from 0-IV based on the absolute lymphocyte count and
the presence of enlarged lymph nodes, liver or spleen, anaemia and
thrombocytopenia.
• A patient is classified as stage IV when all the features are present
and stage 0 when there is only an absolute lymphocytosis
(monoclonal lymphocytes).
Clinical features cont..
• Note: CLL does not usually develop into acute lymphoblastic
leukaemia.
• Patients die from immune deficiency, infection, bone marrow failure,
or infiltration of vital organs.
Lab diagnosis
Blood picture
• Hb: wide range from normal to depressed value .
• When anemia is due to hemolysis could be normochromic normocytic
• Rbcs: normochromic normocytic
• Reticulocytosis
• Direct Antiglobulin Test – POSITIVE
• Wbcs: Range between 20 – 150x 10x9/l occasionally > 1 million
• Cells are primary mature lymphocyte with B surface marker
• Some lymphocyte may be disrupted during the preparation of the film and such cells are
refered to smear cells
• Platelets: initially is normal or moderately reduced but later stage, marked
thrombocytopenia < 50 x 109/l is common
Diagnosis cont...
• Bone marrow
•  Lymphoblast
•  Decreased megakaryocytes
•  Granulocyte and erythroid precussors
Special stains used in the diagnosis
in leukaemia
• Cytochemistry is the study of chemical constituents of cells. These
elements may be enzymztic (e.g. peroxidase) or non-enzymatic (lipids
and glycogen).
• The cellular morphology of peripheral blood and bone marrow often
provides a provisional diagnosis, so cytochemical staining of cells has
provided a useful adjunct for differentiation of haemopoietic disease
especially acute leukaemia.
Special stains used in the diagnosis
in leukaemia cont.,.
• Differentiation between AML and ALL requires the facilities of a
specialist haematology laboratory to perform cytochemical staining
and immunophenotyping.
• An experienced haematologist is required to report bone marrow
preparations and classify ALL and AML.
Cytochemical staining

Lymphoblasts:
• Sudan black and myeloperoxidase negative and usually non-specific
esterase (NSE) negative.
• The periodic-acid (PAS) stain is often positive.
Myeloblasts:
• Sudan black and myeloperoxidase positive, PAS negative or diffusely
positive.
• Monoblasts are strongly positive for NSE and myeloblasts are diffusely
positive for NSE (resistant to inhibition by sodium fluoride). Monoblasts
may be weakly PAS positive.
Cytochemical stains cont,,,
• Details of cytochemical staining techniques, immunophenotyping
(monoclonal antibody cell marker studies) and cytogenetic studies
used to differentiate acute leukaemias can be found in Practical
Haematology and other haematology textbooks .
Cytochemical stains cont...
(Explanations)
• Myeloperoxidase: This stain is useful for differentiatind of blast of
acute myeloid leukaemia from those acut lymphoctic leukaemia.
• Esterases: Esterase reaction is used to differentiate myeloblast and
neutrophilic granulocyte from cells of monocyic origin.
Cytochemical stains cont...
(Explanations)
• Periodic Acid Schiff (PAS): Is helpful in the diagnosia of some acute
lymphocytic leukaemia and the erythroid type of acute myelogenous
leukaemia, as well as in the identfying abnormal erythroid precusors
in myelodysplastic syndromes.
• Leukocyte Alkaline Phosphatase: Used for differentiating chroni
myelogenous leukaemia from a leukaemoid reaction that may be
seen in sever infections.
• Sudan black: is useful technique for differentiation of AML from ALL
ASSIGNMENT
• Stain pattern used in leukaemia for diagnosis