Hemosiderin contains iron in the ferric form bound to a protein frame work. Hemosiderin is formed by partial degradation of aggregates of ferritin by lysosomes. It is present in reticuloendothelial cells of bone marrow, spleen and liver.
Difference between Hemosiderin and Ferritin
Unlike ferritin, hemosiderin is visible, water insoluble crystalline protein- iron complex
Hemosiderin has higher iron/protein ratio than ferritin
Hemosiderin is more stable and less available form of storage iron than Ferritin
Iron distribution in adults
Fixative containing acids should be avoided as the hemosiderin is soluble in strong acid solution
In some tissues iron is tightly bound to proteins and cannot be demonstrated . In such cases, if treated with hydrogen peroxide, iron is released and then demonstrated by using Perls Prussian blue reaction.
Different stains for iron
Perls Prussian blue reaction (Mallory method)
This test is used to demonstrate ferric iron
History –
It was discovered in 1706 and was first used as synthetic colour in paints by Diesbach in Berlin.
In 18th century, Prussian blue was uniform coat colour worn by infantry and artillery regiments of Prussian army and later by German soldiers.
In 1867, German Pathologist, Max Perls described it as histochemical stain and hence Known as “Perls Prussian Blue” or Berlin blue.
Principle
Tissue sections when treated with Hydrochloric acid, denatures the protein binding to hemosiderin molecules ,there by releasing Ferric (3+) ions. These Ferric ions combine with Potassium Ferrocyanide to form Ferric Ferrocyanide which is an insoluble bright blue pigment (Prussian blue)
Procedure
preparation of Ferrocyanide solution
2% Potassium Ferrocyanide – 25ml
2% Hydro chloric acid – 25ml
Take the section to water
Treat the sections with freshly prepared acid Ferrocyanide solution for 10 minutes to 15 minutes
wash well in distilled water
counter stain the nuclei with 0.5% aqueous neutral red or safranine for 1 minute
Wash rapidly in distilled water
Dehydrate, clear and mount the sections
Interpretation
Ferric iron – blue
Nuclei – Red
Erythrocytes yellow
Instead of HCl acid, 5% acetic acid can be used, as HCl may dissolve freshly formed deposits of iron
Counter stains which can be used in Perls staining method are
0.5% Aqueous neutral red
0.1% Safranine
0.5% Aqueous Eosin
0.1% Nuclear Fast red in 5% aluminium sulphate
0.5% Phloxine
0.5% Tartrazine
Uses of Perls stain – To demonstrate iron especially in tissues such as bone marrow, spleen and also to demonstrate iron deposits in liver (hemochromatosis) and lungs (Congestive heart failure)
Turnbull blue reaction
Purpose – Detect Ferrous iron in tissues
Principle – Tissue sections are treated with acidic solution of Potassium Ferricyanide. Any Ferrous iron present will react to form an Insoluble bright blue pigment called Turnbull blue (Ferrous ferricyanide)
Procedure –
Slides are placed in Potassium Ferricyanide solution (PotassiumFerricyanide – 0.4gm and HCl 0.006N – 40ml) for 1 hr.
Then wash in 1% acetic acid
Counter stain slides in Nuclear fast Red for 5 minutes
Rinse in distilled wayer
Dehydrate, clear and mount
Result –
Ferrous iron – blue
Back ground – Pink red
Difference between Prussian Blue and Turnbull blue
Prussian blue – Ferric ferrocyanide – Fe4[Fe(CN)6]3
Turnbull blue – Ferrous ferrocyanide – Fe3[Fe(CN)6]2
Quincke-Tirmann-Schmelzer method
Purpose – Detect both ferrous and ferric iron in tissues
Sections are treated with 10% of Aqueous Ammonium sulphide for 2 hours. Both Ferrous and Ferric iron is converted to black ferrous sulphide (Quincke reaction)
Treatment of Ferrous sulphide for 15 minutes with a freshly prepared mixture of equal parts of 1% HCl and 20% Potassium ferricyanide converts it to ferrous ferricyanide (Tirmann-Schmelzer reaction)
Hukill and Putts method
Purpose – Detect both Ferrous and Ferric form of iron
Procedure –
Sections are treated with Bathophenanthroline solution for 2 hrs at room temperature
Then rinse in distill water and counter stain with 0.5% aqueous Methylene blue for 2 minutes
Rinse in water, dehydrate, clear and mount the section
Result
Ferrous iron – red
Nucle – blue
Difference between sideroblast and ring sideroblast
Normoblasts with Prussian blue positive granules are called sideroblasts which are normal erythroblasts showing few blue granules scattered in the cytoplasm, representing endosomes filled with excess iron utilized for heme synthesis
Ringed sideroblast – Nucleated red cell precursor having 5 or more iron granules encircling more than 1/3rd of the nuclear circumference. Abnormal iron deposits are in mitochondria which align themselves around nuclei in ringed fashion
Siderocyte – if granules persist in cytoplasm even after enucleation, then the mature cells are called Siderocytes
Types of sideroblasts (WHO International working group on morphology of MDS)
Type 1- Less than 5 siderotic granules in cytoplasm
Type 2 – 5 or more siderotic granules in cytoplasm but not in perinuclear distribution
Type 3 or ringed sideroblasts – 5 or more siderotic granules in cytoplasm in perinuclear position or encompassing atleast 1/3rd of nuclear circumference
Type 1 and 2 – are seen in non siderotic anemia and Type 3 ia seen in sideroblastic anemia
Interpretation and grading of iron stores on Bone marrow aspiration
Interpretation and grading of iron stores on Bone marrow biopsy
Grade 0 – iron deficiency
Grade 1,2 – Normal iron stores
Grade 3, 4 – Increased iron stores
References
Christopher Layton, John D. Bancrofti, S Kim Suvarna. Fixation of Tissues. In: Theory and practice of histological techniques by John D. Bancrofti . 8th edition.
Histotecniques. In: Lynch Medical Laboratory technology by Mathew J. Lynch, Stanely S. Raphael. Saunders publication 1983
Internet sources
By –
Dr. V. Shanthi (Professor of Pathology, Narayana Medical College, Nellore)
Dr. N.Mohan Rao (Professor of Pathology, Narayana Medical College, Nellore)
Dr. B. Syam Sundara Rao (Professor of Pathology, Narayana Medical College, Nellore)
