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HAEMATOLOGY

THE DIAGNOSIS OF ACUTE LEUKAEMIA

CLINICAL MORPHOLOGY
FEATURES CYTOCHEMISTRY

ACUTE
LEUKEMIA
CYTOGENETICS

MOLECULAR
BIOLOGY

IMMUNOPHENOTYPING

ELECTRON MICROSCOPY
CYTOCHEMISTRY

Cytochemical stains are an essential part of the diagnosis of Acute Leukemia.


The stains reflect the chemical composition of the cells through color reactions.
The reactions do not damage the cell so that it can still be recognised.
Each cell lineage contains different enzymes and compounds and therefore cytochemical stains can
contribute to the subtyping of leukemia.

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.)

The Myeloperoxidase 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.

Periodic Acid Schiffs

The PAS stain identifies the glucose polymer glycogen.


Mature neutrophils have high levels of cytoplasmic glycogen.
Myeloblasts are usually negative.
The usefulness of the PAS reaction lies in the following conditions:
– Erythroleukemia (M6): erythroid cells can have a distinct block positivity.
– Megakaryoblastic (M7): positive in the more mature cells.
– B-lineage leukemia: can also have block positivity. (ALL of B-lymphocytes.)
– M14 eosinophilia: the granules of the eosinophils are usually positive.

A positive PAS is usually characterized by a deep magenta color.


The Esterase Stains

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 chloroacetate esterase is positive in granulocytic cells.


It is usually positive in the more mature cells and usually negative in blasts.
A positive reaction is identified by a red granular or blue granular color, depending on the type of
substrate used.

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 Stains

The phosphatase enzymes are widely distributed in haempoeitic cells and can be divided into two
groups:

● The alkaline phosphatases


Alkaline phosphatases are seen in cells of granulocytic origin. It is decreased or absent in
patients with CML.
● The acid phosphatases
This enzyme has been demonstrated in lysosomes where it partakes in digestive processes of
the cell.
A positive Acid Phosphatase reaction is indicated by a bright red color. Most leucocytes
have a positive reaction. Monocytes are often very positive.
Usefulness of the stain:
T-cells have distinct localised focal or polar-dot positivity which is located in the golgi-zone
of the cell.
“Hairy cells”: Most cells lose their positivity when tartrate is added to the substrate. Hairy
cells are resistant and retain their positivity = tartrate resistant (TRAP positive).
Megakaryoblasts are often positive and therefore the stain can be useful in diagnosis of M7.
Other stains

Oil Red O: Useful in diagnosis of L3 or Burkitts Lymphoma.


Feulgen: Demonstrates DNA and can be useful in identifying nucleoli.

IMMUNOPHENOTYPING

As different cell types display different antigens on their surface, immunophenotyping has become
an essential tool in the diagnosis of Leukemia.

Cluster Designation or CD number


Many antibodies are clustered into groups which are specific for a particular antigen. Each cluster
has been given a cluster designation (CD) number, eg. CD2, CD3, CD4, CD8 etc.
For example the antibodies WT1, 3A1 and Leu7 are all specific for a T-cell antigen. This antigen
has been given the number CD7.

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.

Maturation sequences of cell lineages:

MATURATION PATH OF T-CELLS


CD7

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

T-cytotoxic cell T-helper cell

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.

MATURATION PATHWAY OF B-CELLS

STEM CELL EARLY PRO B-CELL


CD34 +/_CD34

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

H CHAIN V-DJ ARRANGEMENT VDJ REARRANGED


GENES

L CHAIN GERMLINE VDJ REARRANGED


GENES

(COMMON ALL) (PRE B- ALL)

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

Monocytic line Myeloid line (granulocytes)


platelet line RBC line
HLA-DR
CDIIb +/_CD
CD14 34
CD13 CD33
CD
CD33 13

CDIIb
HLA-DR

MONOBLAST MYELOBLAST
CD13 CD33
CD33
CDIIb CD13

+/_
CD14 (Monocytic CDIIb
marker)
HLA-DR

MONOCYTE PROMYELOCYTE

MATURE GRANULOCYTE

Note: Cyt MPO = Cytoplasmic myeloperoxidase

IMMUNOLOGICAL MARKERS IN ACUTE LEUKAEMIA

B-CELL ALL T-CELL ALL AML

STEM CELL MARKERS:

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)

IMMUNOGLOBULIN GENES rearranged germ line or germ line


rearranged

T-CELL RECEPTOR GENES germ line or rearranged germ line


rearranged

HYBRID LEUKAEMIA

Bi-phenotypic Leukemia :

Some leukemias can express both Myeloid and Lymphoid markers.


These leukemias have a very bad prognosis.
Dual labelling with both a myeloid and lymphoid antibody is useful in making the diagnosis.
Also note that +/_ 30 % of leukemias can express one marker of a different lineage.
This is known as aberrant marker expression.

Scoring system for bi-phenotypic leukemia


POINTS B-LINEAGE T-LINEAGE MYELOID

2 cCD22 cCD3 (not surface CD3) Myeloperoxidase


(markers highly cIgM anti-TCR αβ (any method)
lineage-specific, CD79a anti-TCR γδ anti-lysozyme
so carry more
points)
1 CD10, CD19, CD20 CD2, CD5, TCR-rarrangement CD33, CD13, CD14,
Sudan black or other
myeloid lineage stain.

0.5 Tdt, CD24 Tdt, CD7, CD1a CD14, CD15, CD64,


(least specific for CD117
a lineage)

A score of > 2 in two or more lineages is required to diagnose bi-phenotypic leukaemia.

Definition of Hybrid Leukemia:


Acute leukemia which demonstrates the malignant transformation of two lineages.

3 types: Bi-phenotypic,
Bilineal,
Biclonal.

2 explanations: Lineage infedelity (McCullock)


Lineage promiscuity (Greaves).

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.

Also see: http://en.wikipedia.org/wiki/Immunophenotyping

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.

The cells of most haematological malignancies will have chromosomal abnormalities.


In some disorders consistent defects have been identified and contribute to the diagnosis.
Some malignancies have a spread of abnormalities which signifies the development of sub-clones.

Some of the more important diagnostic abnormalities:

Disorder Chromosomal abnormality Gene

1. CML t(9:22)(q34;q11) BCR/ABL


2. AML M2 t(8:21)(q22;q22) ETO/AML 1
3. AML M3 t(15:17)(q22;q12) PML/RARA
4. AML M4-eo inv 16 myH11-CBFB
5. ALL L3 t(8:14) immunoglobulin

Chromosomal abnormalities may also provide prognostic information:


1. inv 16: good prognosis.
2. Abnormalities associated with chromosome 7: bad prognosis.

Notes from Cape Peninsula University of Technology (Fmr. Cape Technikon), 2002, Biomedical
Technology.

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