Amyloid, pigments, and mineral deposits accumulate in tissues in a wide range of diseases. Their histochemical identification provides specific diagnoses that often cannot be made by any other technique.
Congo Red for Amyloid
Congo Red is the standard histochemical stain for amyloid. In alkaline solution, Congo Red binds to the beta-pleated sheet structure of amyloid fibrils via intercalation between adjacent beta-strands. Under brightfield microscopy, amyloid deposits stain salmon-pink to red. The diagnostic finding is apple-green birefringence under crossed polarizers — the result of Congo Red molecules aligned along the amyloid fibrils, which are themselves arranged in a highly ordered, birefringent array.
Amyloid types (AL, AA, ATTR, Aβ, etc.) cannot be distinguished by Congo Red alone — IHC for amyloid subtypes (kappa and lambda light chains, serum amyloid A, transthyretin, beta-amyloid) or mass spectrometry is required for typing. Congo Red sensitivity is high for large deposits but may miss early or small deposits — examine multiple sections under polarization. False-positive Congo Red binding can occur in elastic fibers, collagen, and hyaline — only apple-green birefringence confirms amyloid.
Thioflavin T
Thioflavin T is a fluorescent dye that binds amyloid and shows enhanced fluorescence with a characteristic emission shift. It is more sensitive than Congo Red for detecting small amyloid deposits but requires a fluorescence microscope and is less specific. Congo Red remains the gold standard for amyloid diagnosis.
Perls’ Prussian Blue for Iron
Perls’ Prussian Blue detects ferric iron (Fe3+) in tissue sections. Sections are treated with hydrochloric acid and potassium ferrocyanide; ferric ions react with ferrocyanide to form insoluble Prussian blue (ferric ferrocyanide). The reaction produces a blue precipitate at the site of iron. The background is typically counterstained with nuclear fast red.
Perls’ stain identifies hemosiderin (aggregated ferritin) in conditions of iron overload: hereditary hemochromatosis (hepatocyte iron), transfusion-related siderosis, alcoholic liver disease, and hemolytic anemias. In bone marrow, Perls’ grades stainable iron (0-6 scale). In lung, Perls’ demonstrates heart failure cells (hemosiderin-laden macrophages) in pulmonary congestion and confirms pulmonary hemosiderosis. In spleen and lymph nodes, iron deposition after hemorrhage or infarction is easily identified.
Von Kossa for Calcium
Von Kossa stain detects calcium deposits by substituting silver ions for calcium in insoluble calcium salts (phosphate, carbonate). Under ultraviolet light or bright light, the silver is reduced to metallic silver, appearing black or brown. The background is counterstained with nuclear fast red or van Gieson.
Von Kossa is used to demonstrate pathological calcification: dystrophic calcification in necrotic tissue, tumors (psammoma bodies in meningioma, papillary thyroid carcinoma, serous ovarian carcinoma), calcified heart valves, and calcific tendinitis. It also identifies metastatic calcification (calcium deposition in normal tissues due to hypercalcemia) in kidney, lung, and gastric mucosa. Von Kossa does not detect calcium oxalate — for oxalate, use Alizarin Red S at pH 4.0 or polarized light (oxalate crystals are birefringent).
Alizarin Red S for Calcium
Alizarin Red S forms an orange-red precipitate with calcium ions at alkaline pH (4.0-4.5). It is more specific for calcium than Von Kossa and detects both calcium phosphate and calcium oxalate. Alizarin Red is used for identifying calcium deposits in soft tissues, vascular calcification, and nephrocalcinosis. It is also the standard stain for identifying calcium in histologic sections of bone and teeth.
Bilirubin and Bile Stains
Fouchet’s stain (trichloroacetic acid and ferric chloride) oxidizes bilirubin to绿色的 biliverdin, then green/blue to the emerald-green color of bile. It is used to identify intrahepatic and extrahepatic cholestasis in liver biopsies and bile thrombi in canaliculi. Hall’s stain (Van Gieson with Fouchet’s) simultaneously stains bile (green/blue) and collagen (red). Bile pigment must be distinguished from lipofuscin (age pigment) and hemosiderin — Perls’ stains iron blue, and PAS stains lipofuscin variably.
Melanin Bleach
Melanin is an endogenous brown-black pigment that can obscure cellular detail and IHC interpretation. Melanin bleach (potassium permanganate followed by oxalic acid) removes melanin without damaging tissue morphology or antigenicity, allowing evaluation of cellular detail beneath heavy melanin deposits. Bleached sections can be used for IHC after protocol optimization.
Quality Control
Each special stain requires appropriate positive controls run concurrently: amyloid-containing tissue (Congo Red), iron-overloaded liver (Perls’), calcified artery (Von Kossa), and bile-stained liver (Fouchet’s). Control failure invalidates the run. Standard troubleshooting procedures apply — if controls are weak, check reagent freshness, pH, and incubation times.
