PRINCIPLE:

This test stains haemosiderin, a storage form of iron. Fixed blood films or bone marrow preparations are incubated in a solution of potassium ferrocyanide in dilute hydrochloric acid.

The acid liberates loosely bound iron, and the ferric ions then react with the potassium ferrocyanide to form ferric ferrocyanide. This insoluble blue compound, Prussian blue, is located at the site of the reaction.

SPECIMEN:

Air dried blood films or bone marrow squashes.
Bone marrow squashes must contain fragments as the iron is present in and around the fragments

INTERPRETATION:

Haemosiderin stains as blue-black granules.
This test is performed so that iron stores in bone marrow may be assessed and to detect the presence of abnormal siderotic granules in erythrocytes and erythroblasts i.e siderocytes (Pappenheimer bodies in Leishman?s stain) and sideroblasts.

Siderocytes

These are red cells containing one or more granules of haemosiderin that vary in size from 1.5um down to the limits of visibility. They are not seen in peripheral red blood cells in health. Increased numbers are seen in some haemolytic anaemias, following splenectomy, in hyposplenic states, megaloblastic anaemia, haemochromatosis and haemosiderosis. Also, when marrow is stressed and reticulocytes do not complete their maturation in the marrow, siderocytes appear in the peripheral blood cells.

Sideroblasts

These are normoblasts that contain siderotic granules. They are more numerous
and larger in marrow normoblasts where haem synthesis is disturbed.

Ringed Sideroblasts

Are sideroblasts in which the haemosiderin is arranged in a collar encircling the nucleus of the cell. They are found in increased numbers in the erythroid cells of marrow in sideroblastic anaemia.
Assessment of Marrow Iron Stores
Haemosiderin appears as accumulations of small granules lying free, or in the phagocytic cells in marrow fragments.

Iron stores in bone marrow aspirates are graded as:

0 No iron granules observed
1+ Small granules in reticulum cells seen only with oil immersion lens.
2+ Few small granules visible with low power lens.
3+ Numerous small granules in all marrow particles.
4+ Large granules in small clumps.
5+ Dense large clumps of granules.
6+ Very large deposits obscuring marrow detail.