iron staining.

A brief of iron storage and iron staining.

1. IRON STAINING MODERATOR: DR ANEETA SINGH Presented by : Shikha Sharma

2. Introduction: Iron is an essential element for humans that is mainly absorbed from diet in the duodenal enterocytes and is transported in the plasma by an iron binding glycoprotein called transferrin , synthesised in liver. Transferrin delivers iron to the erythroid precursors, which possess high affinity transferrin receptors that mediate iron import through receptor mediated endocytosis . The major sites of iron storage are liver and mononuclear phagocytes .

3. Iron distribution in healthy young adults(mg) POOL MEN WOMEN Total 3450 2450 FUNCTIONAL (80%) Hemoglobin 2100 1750 Myoglobin 300 250 Enzymes 50 50 STORAGE (15-20%) Ferritin , hemosiderin 1000 400

4. STORAGE FORMS: FERRITIN: Ferritin is the primary iron storage form . It is water-soluble protein-iron complex and contains a spherical shell of apoprotein enclosing a core of hydrated ferric phosphate . A single ferritin molecule can hold upto 4500 iron atoms. Ferrous iron entering into ferritin is oxidised to ferric form by molecular oxygen , with apoferritin catalysing the oxidative process. Ferritin is present in circulation and serum ferritin levels are used as a marker of the total amount of iron stored in the body,hence serum ferritin is used as a diagnostic test for IDA and also to control chelation therapy in thalassemia major. Ferritin is found at highest levels in liver( 60%), and in muscles and reticuloendothelial cells(40%).

5. HEMOSIDERIN: Hemosiderin is a brown pigment that is present in reticuloendothelial cells of bone marrow, spleen and liver . HEMOSIDERIN is probably formed by partial degradation of aggregates of ferritin by lysosomes . It is a conglomeration of iron, ferritin proteins and other subcellular constituents. With normal iron stores ,only traces of hemosiderin are found in body and most of the iron is stored as ferritin but in iron overload states most iron is stored as hemosiderin .

6. Iron in hemosiderin is chemically reactive and turns blue-black when exposed to Potassium ferrocyanide , which forms the basis for the PRUSSIAN BLUE STAIN . Hemosiderin differs from ferritin as- 1.Unlike ferritin , hemosiderin is visible , water insoluble, crystalline protein-iron complex. 2.Hemosiderin has higher iron/protein ratio than ferritin . 3. Hemosiderin is more stable and less available form of storage iron than ferritin .

7. IRON WITHIN NORMOBLASTS 80-90% of iron that is taken up by erythroid precursor cells is ultimately taken up by mitochondria and incorporated into heme . NORMOBLASTS with prussian blue positive granules are called SIDEROBLASTS. These are normal erythroblasts that after prussian blue staining show a few blue granules scattered in cytoplasm, representing endosomes filled with excess iron not utilized for heme synthesis. If granules persist after enucleation,the mature cells are called SIDEROCYTES. Now if, a nucleated red cell precursor has 5 or more iron granules encircling more than 1/3 rd of the nuclear circumference, it is defined as a RINGED SIDEROBLAST. The abnormal deposition of iron is shown to be in mitochondria which align themselves around nuclei in a ringed fashion.

8. The most widely used stain for iron demonstration is prussian blue stain. Also known as PERLS’ PRUSSIAN BLUE after its inventor, a German Pathologist , Max Perls . IRON STAINING It is an extremely sensitive test, and can even detect single granules of iron in cells.

9. PRINCIPLE OF PRUSSIAN BLUE STAIN FOR FERRIC IRON IN HEMOSIDERIN: Hemosiderin forms the body’s iron stores. When the tissue is treated with an acid- ferrocyanide solution, it will result in the unmasking of ferric iron in hemosiderin , in the form of ferric hydroxide, Fe (OH)3 , by dilute hydrochloric acid. The ferric iron then reacts with a dilute potassium ferrocyanide solution to produce an insoluble blue compound ferric ferrocyanide (PRUSSIAN BLUE).

10. STAINING PROTOCOL OF IRON for bm samples FIXATIVE OF CHOICE: FORMOL-METHANOL (ratio 1:9). Methanol alone is also adequate. STAINING SOLUTION: Equal quantities of: 2% HCL and 4% potassium ferrocyanide . WORKING SOLUTION : Made by mixing 2 ml of each reagent. Also used is Safranin (0.1% nuclear fast red) for counterstaining.

11. Procedure: Fix the air dried bone marrow specimen in methanol for 15 min. Dry the slides in room temperature. Mix equal parts of HCL and potassium ferrocyanide prepared immediately before use. Immerse slides in this working solution for 15 min. in room temperature. Now wash the slides well with distilled water for 5 min. Counter stain with aqueous solution of safranin (0.1% nuclear fast red) for 10-15 sec. Wash with distilled water . Let the slides dry. RESULTS: FERRIC IRON : BLUE NUCLEI : RED CYTOPLASM : PINK

12. Notes to prevent FALSE POSITIVE RESULTs: Outdated reagents or improperly stored reagents may not be used. Iron contamination from glassware must be prevented. Avoid washing with tap water before placing into the working solution, as rust in the water or tap fixtures could cause false positive staining . Use distilled water. Wash well before staining with neutral red, as traces of iron can form a granular red deposit with neutral red.

13. GRADING OF IRON STOREs ON BMA (after Gale et al) none No iron granules seen. 1 Very slight Small granules in reticulum cells only under oil-immersion. 2 Slight Few small granules visible with low power lens. 3 Moderate Numerous small granules in all marrow particles. 4 Moderatel y heavy Large granules in small clumps. 5 Heavy Dense large clumps of granules. 6 Very heavy Very large deposits obscuring the marrow cells. INTERPRETATION OF PRELS’ STAIN ON BMA : GRADE 0 : Iron deficiency (a minimum of 7 BM particles must be available before concluding that hemosiderin is absent. GRADE 1,2,3 : n ormal iron stores. GRADE 4 to 6 : increased iron stores.

14. GRADING OF IRON STORES ON BMB GRADE 0 No hemosiderin granules. GRADE 1 Fine granules in every 3-4 HPF. GRADE 2 Heavier granules in every 2-3 HPF. GRADE 3 Granules in every HPF in one or more cells. GRADE 4 Massive ,abnormal deposits with clumps and heavy granules. INTERPRETATION OF IRON STORES ON BMB: GRADE 0 :Iron deficiency. GRADE 1-2 : Normal stores. GRADE 3-4 : Increased stores.

15. Grade 0: No iron granules seen.

16. Grade 2 iron stores in BMA With few small granules visible with low power lens.

17. BMA smear showing dense large clumps of iron .(BMA iron stores grade 5).

18. Thank you.