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