Special Stains

1
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.
2
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.
3
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.
4
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).
5
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.
6
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.
7
A susceptible individual (G6PD deficiency,
glutathione reductase deficiency, or unstable
haemoglobin) will have 5 or more Heinz bodies in
gt40 of the red cells.
8
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 a-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
37C) will cause in vitro precipitation of Hb
H. Note This is a supravital staining procedure.
9
The inclusions appear as multiple greenish-blue
bodies (golfball appearance) and should be
readily distinguished from the precipitated dots
and filaments in reticulocytes.