Prussian blue stain

**Principle:** Tissue is treated with acidic potassium ferrocyanide (Perls reaction). The acid (HCl) releases ferric iron (Fe3+) from hemosiderin/protein complexes, and Fe3+ reacts with ferrocyanide to form ferric ferrocyanide (Prussian blue), an insoluble bright blue pigment. A nuclear fast red counterstain is often used. It stains only ferric iron in hemosiderin. It does not stain ferritin well in small amounts, heme iron within hemoglobin, or ferrous iron (Fe2+) without oxidation.

**Normal vs pathologic:** Hemosiderin is a golden-yellow/brown pigment on H&E and is a degradation product of ferritin (aggregated ferritin in lysosomes). It appears blue with Prussian blue stain. Normal bone marrow macrophages contain small amounts of stainable iron.

**Key clinical uses (high-yield):** - **Hereditary hemochromatosis** (HFE C282Y, autosomal recessive, chromosome 6): iron deposition in hepatocytes, pancreas, heart, skin, joints, and pituitary/testes. This causes cirrhosis, HCC risk, diabetes ("bronze diabetes"), dilated or restrictive cardiomyopathy, arthropathy (2nd/3rd MCP joints, chondrocalcinosis), and hypogonadism. Liver biopsy shows blue granules in hepatocytes (parenchymal pattern). Labs show increased ferritin, increased transferrin saturation, and increased serum iron, with decreased TIBC. Treatment is phlebotomy (or chelation, e.g., deferoxamine, deferasirox). - **Secondary hemosiderosis/iron overload** (e.g., repeated transfusions in thalassemia major or sickle cell disease): iron accumulates in reticuloendothelial cells (Kupffer cells, macrophages) first. - **Sideroblastic anemia:** bone marrow aspirate shows **ring sideroblasts**, which are erythroid precursors with iron-laden mitochondria that form a ring around the nucleus. Causes include X-linked ALAS2 mutation, myelodysplastic syndrome, alcohol, lead poisoning, isoniazid (B6 deficiency), and chloramphenicol. Pyridoxine (B6) is a treatment for some forms. - **Hemosiderin-laden macrophages:** "heart failure cells" in the lung alveoli (left heart failure with pulmonary congestion), diffuse alveolar hemorrhage (e.g., Goodpasture, GPA), and old bruises/hemorrhage (hemosiderin brown color progression). - **Bone marrow iron stores:** the gold standard for assessing iron stores. Absent stainable iron confirms **iron deficiency anemia**, whereas anemia of chronic disease shows normal or increased iron in macrophages with decreased iron in erythroblasts. - **Urine:** Prussian blue-staining hemosiderin in urinary sediment indicates chronic intravascular hemolysis (e.g., PNH, mechanical heart valve hemolysis), when renal tubular cells take up and shed hemosiderin. - **Brain:** Hemosiderin deposits in superficial siderosis of the CNS.

**Distinguishing from similar stains:** - **Prussian blue (Perls):** iron (blue) - **Congo red:** amyloid (apple-green birefringence under polarized light) - **PAS:** glycogen, fungi, and mucopolysaccharides (magenta) - **Fontana-Masson:** melanin (and argentaffin cells) - **Rhodanine/Orcein:** copper (Wilson disease) - **Von Kossa/Alizarin red:** calcium - **Ziehl-Neelsen:** acid-fast organisms

**Pearls:** Note that "Prussian blue" is also the name of an oral drug (ferric hexacyanoferrate) used to treat thallium and radioactive cesium (137Cs) poisoning by binding the ions in the gut and interrupting enterohepatic circulation. Do not confuse this with the stain.