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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