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CLINICAL MORPHOLOGY
FEATURES CYTOCHEMISTRY
ACUTE
LEUKEMIA
CYTOGENETICS
MOLECULAR
BIOLOGY
IMMUNOPHENOTYPING
ELECTRON MICROSCOPY
CYTOCHEMISTRY
Sudan Black
The sudan stains are lipid-soluble and can therefore detect cellular lipids.
Haemopoeitic cells contain finely dispersed cytoplasmic lipids and complex membrane
lipoproteins.
The sudan black stain produces a black color if lipids are present.
Positive reactions are associated with granulocytic cells (myeloid lineage); the staining intensity
increasing as the cell matures.
Monocytic cells have variable reactions but often have finely scattered granules.
Lymphoid cells are usually negative.
The sudan black stain is useful in identifying myeloid leukemia.
Auer rods are also positive – a score of > 3 % positive cells is considered positive.
Also, in rare cases of ALL the sudan black has been reported as positive. (ALL cells have many
vacuoles which may take up stain.)
In humans peroxidases are found in the liver, kidney and in the granules of myeloid and monocytic
cells.
Myeloperoxidase is located in the primary azurophilic granules of granulocytes.
Primitive myeloid cells (i.e. blasts) demonstrate myeloperoxidase positivity in areas such as the
endoplasmic reticulum and the golgi region.
Auer rods are usually positive. (Good stain to demonstrate auer rods.)
The intensity of staining increases as the cell matures and often parallels sudan black staining –
though not always.
Monocytes are often negative or weakly positive.
Lymphoid cells are always negative.
Positivity is indicated by a golden brown color (diabenzidine).
A count of > 3 % blasts is considered positive.
Esterases are a diverse group of enzymes. Their natural substrates are esters of carboxylic acids.
The esterases are capable of hydrolysing aliphatic and aromatic esters.
The esterases can be divided into 2 groups:
– 1. Those with low substrate specificity, i.e. the non-specific esterases.
– 2. Those with high substrate specificity, i.e. the chloro-acetate esterases or specific esterases.
(Positive in mature myeloid cells.)
The non-specific esterase (NSE) enzymes are used to recognize cells of monocytic lineage.
Two substrates can be used: ANA (Alpha-naphtyl acetate) and ANB (Alpha-naphtyl butyrate).
If cells are positive they give a dense brown color
Monocytic cells are sensitive to fluoride and lose their positivity after it is added.
The acetate esterase is strongly positive for monocytes and weakly positive for granulocytes.
The butyrate esterase is more specific for cells of the monocytic lineage.
The NSE stain can also be helpful in:
– T-cell leukemia: polar dot positivity.
– Megakaryoblastic leukemia (M7): ANAE gives strong positivity.
ANBE – weak to negative.
– Myelomonocytic leukemia: A combination of the acetate and butyrate stains (Combined stain)
can be useful in identifying cells which have both myeloid (blue) and monocytic (brown)
characteristics.
– Hairy cell leukemia: A distinct crescent pattern of positivity is seen in the cytoplasm of hairy
cells.
The phosphatase enzymes are widely distributed in haempoeitic cells and can be divided into two
groups:
IMMUNOPHENOTYPING
As different cell types display different antigens on their surface, immunophenotyping has become
an essential tool in the diagnosis of Leukemia.
Why do Immunophenotyping ?
– To establish lineage.
– To establish stage of maturation.
To identify bad or better prognostic groups; eg. very immature cells won't respond well to
chemo
– To differentiate between a malignant and reactive process.
.
To diagnose Acute leukemia we need to choose a panel of antibodies which will answer these
questions.
CD5 PRE-T-CELLS
Tdt +/_ HLA-DR
SUBCAPSULAR
CORTEX OF +/_ CD34
THYMUS
Cyt
CD3 CD7
CD5 EARLY
Tdt CD2 THYMOCYTES
CD7
Cyt CD2
CORTEX CD3 CD5
OF Tdt CD1
THYMUS CD4
CD8 COMMON
THYMOCYTES
+/_CD3
CORTEX Cyt CD7
OF CD3 CD2
THYMUS
CD5
Tdt
CD1
CD4
CD8
Cyt
CD3
MEDULLA Cyt CD7
OF CD3 CD7 CD2
THYMUS CD2
CD5 MATURE
CD5 +/_Tdt THYM0-
+/_Tdt CD3 CD3 CYTES
CD8 CD4
Note: CD7 called the pan-T marker (found on all T-cells from pre- to mature).
Markers like CD1 and CD3 indicate level of maturation.
Tdt is a nuclear enzyme (similar to DNA polymerase). Useful early marker for lymphoid leukemia.
HLA-DR HLA-DR
Tdt Tdt CD19
Cyt
CD22
H CHAIN
GERMLINE D-J ARRANGEMENT
GENES
L CHAIN
GERMLINE GERMLINE
GENES
LATE PRO B-CELL PRE B-CELL Cyt
Cyt CD22
CD22 Cyt µ
HLA-DR
HLA-DR
Tdt CD19 Tdt
CD19
CD10
CD10
B-CELL
SMIg
Y
HLA-DR
CD19
Tdt
Tdt
CD22
VDJ REARRANGED
IN HEAVY AND LIGHT CHAINS
(B-ALL -- BURKITTS)
Note: CD19 is a good marker for lineage – eg. B-cell not T-cell.
MYELOID MATURATION PATHWAY
CD34
PLURIPOTENTIAL
STEM CELL +/_ HLA-DR
CD34
PROGENITOR
CELL
HLA-DR
Cyt
MPO CD33
Cyt
CD13
Cyt
MPO
Cyt
CD13
CD41 Glycophorin HLA-DR
BLASTS CD42
CD33 CD
CD61 HLA-DR 34
CD71 CD33
(Transferrin receptor)
MEGAKARYOBLAST ERYTHROBLAST BLAST
CDIIb
HLA-DR
MONOBLAST MYELOBLAST
CD13 CD33
CD33
CDIIb CD13
+/_
CD14 (Monocytic CDIIb
marker)
HLA-DR
MONOCYTE PROMYELOCYTE
MATURE GRANULOCYTE
HLA-DR + -- +/--
Tdt + + --
CD34 + -- +
B-CELL MARKERS:
CD19 + -- --
CD10 (actually a maturation + -- --
marker but also for lineage)
T-CELL MARKERS:
CD2 -- + --
CD7 -- + --
B-CELL ALL T-CELL ALL AML
MYELOID MARKERS:
CD13 -- -- +
CD33 -- -- +
CD14 (More specific for -- -- + (in
monocytic line cells) M4 and M5)
MEGAKARYOBLAST:
CD41 -- -- + (M7)
CD42 -- -- + (M7)
CD61 -- -- + (M7)
ERYTHROID MARKERS:
Glycophorin A -- -- + (M6)
Transferrin receptor -- -- + (M6)
HYBRID LEUKAEMIA
Bi-phenotypic Leukemia :
3 types: Bi-phenotypic,
Bilineal,
Biclonal.
STEM CELLS
LEUKEMIC
CELLS
Lymphoid
Myeloid
IMMUNOPHENOTYPING AND PROGNOSIS:
Very controversial.
However some antigens can signify a bad response to treatment, e.g. CD7+ in Myeloid leukaemia,
CD34 in Myeloid leukemia (immature marker) and the P-glycoprotein which is known to cause
multi-drug resistance.
CYTOGENETICS
After staining the chromosomes with a Giemsa stain each chromosome has its own characteristic
pattern of banding. This makes it possible to locate abnormalities.
The short arm is called -p and the long arm -q.
The arms meet at the centromere.
Each arm is divided into regions which in turn are divided into bands.
The regions and bands are numbered from the centromere outwards.
The ends of the chromosome are telomeres.
Methodologies:
Traditional cytogenic techniques first use methods to induce the cells to divide so that metaphase
cells are obtained.
The chromosomes are then pretreated with trypsin or heat and stained with Giemsa. This allows the
various chromosome bands to be studied; called G-banding.
More recently (2002), specific DNA probes are used to identify genes or chromosome regions. The
probes are labelled with fluorochromes so that they can be detected. This technique is known as
Fluorescent in situ Hybridization (FISH).
Note: Molecular biology is a different field from Cytogenetics, using mainly Polymerase Chain
Reaction (PCR) to detect certain genes.
Notes from Cape Peninsula University of Technology (Fmr. Cape Technikon), 2002, Biomedical
Technology.