Special Stains - PowerPoint by 2AT030jY

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									DEMONSTRATION OF IRON
(Perls's Prussian Blue reaction)
The majority of non-haem iron is stored as
haemosiderin (a ferric iron-protein complex), a
small amount as ferritin and the remainder is
contained in myoglobin and transferrin.
Haemosiderin is mostly concentrated in the RE
cells, particularly those in the bone marrow,
except in cases of haemachromatosis and
transfusion haemosiderosis.
Principle
Ferric iron in haemosiderin is released from protein by
treatment with dilute hydrochloric acid.
The free iron reacts with a dilute solution of potassium
ferrocyanide to produce an insoluble blue compound,
ferric ferrocyanide (Prussian Blue).
Ferritin is not demonstrated as it is water soluble and
is lost in the reaction.
Any contrasting, non-acidic counterstain may be used
but Neutral Red, safranin or eosin are the most
popular.
40% of developing
erythroblasts normally
contain a few haemosiderin
granules (siderotic granules)
and are classified as
sideroblasts.
These granules are seen in
Ramanowsky-stained blood
or bone marrow smears as
basophilic granules
(Pappenheimer bodies) and
give a positive reaction with
the Perls's reaction.
Increased numbers of sideroblasts are found
when haemoglobin synthesis is impaired.

When haem synthesis is at
fault (sideroblastic anaemia)
the granules accumulate in
mitochondria and form a ring
around the nucleus (ring
sideroblast).


The distribution of granules is normal when globin
synthesis is at fault (e.g. thalassaemia) or with iron
overload (haemachromatosis).
HEINZ BODIES
Heinz bodies represent denatured haemoglobin
precipitated in the red cell and attached to the
red cell membrane.
They are not visible with Ramanowsky stains
but show up with vital staining (crystal violet or
methyl violet) and phase microscopy.
Heinz bodies can be induced in great numbers
in susceptible individuals by incubating the
blood in acetylphenylhydrazine.
Heinz bodies appear as irregular, refractile,
purple inclusions, 1 to 3 μm in diameter and
located on the periphery of the cell.
A normal control subject will have at least one
Heinz body in each red cell.
A susceptible individual (G6PD deficiency, glutathione
reductase deficiency, or unstable haemoglobin) will have
5 or more Heinz bodies in >40% of the red cells.
DEMONSTRATION OF Hb H
Hb H inclusions develop in Hb Bart's hydrops
fetalis syndrome and in Hb H disease.
In Hb H disease many red cells develop
inclusions but in α-thalassaemia-2 only 1 in 1000
to 10 000 red cells develop inclusions.
In the presence of Hb H, incubation of red cells in 1.0%
brilliant cresyl blue stain (for one hour at 37°C) will
cause in vitro precipitation of Hb H.
Note: This is a supravital staining procedure.
The inclusions appear as multiple greenish-blue
bodies (golfball appearance) and should be
readily distinguished from the precipitated dots
and filaments in reticulocytes.

								
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