HISTOLOGY
LECTURE # 30
INTRODUCTION TO SPECIAL STAINS TECHNIQUES: PIGMENTS & MINERALS STAINING Rationale: Special stain techniques are one of the major processes done in the histology laboratory. These techniques are performed to be evaluated with the diagnostics slides from H&E’s. These stains are used as an aid and a diagnostic tool for a final diagnosis.
Objective: Once completed this lecture, the student should be able to: a)
Describe the different types of pigment & mineral staining.
b)
Learn the various methods of stain demonstration.
c)
Learn the classification for iron, urate crystals, copper and other granule substances staining.
d)
Learn the procedures and diagnostic tools for each stain.
PIGMENT & MINERALS Types of Pigments • • • • • •
Endogenous Pigment Exogenous Pigment Hematogeneous pigment Anthracotic pigment endogenous, non-hematogeneous pigment Mineral
Definitions: Pigment - coloring agents deposited, frequently as cytoplasmic inclusions or granules in cells and tissue. Three majors groups: • Artifact Pigments • Exogenous pigments • Endogenous Hematogeneous pigments I. Artifact Pigments These pigments are deposited in the tissue as a result of a chemical action during processing, and most commonly result from fixation. They normally lie on top of the tissue and not within the cell. Examples: 1. Mercury - any mercury containing fixative 2. Formalin - acidic formaldehyde solution 3. Chrome pigments - potassium dichromate solutions II. Removal of Pigments 1. Mercury Pigments - Iodine solution followed by a Sodium Thiosulfate solution 2. Chrome Pigments - Washed thoroughly in water before dehydration 3. Formalin Pigments - alcoholic picric acid or alkalis, resistant to strong acids III. Exogenous Pigments – These pigments are formed externally and then taken into the body by various routes. A. Carbon (Anthracotic pigment) - black pigment seen in lung and lymph nodes. It resists bleach and extraction procedures. These are demonstrated with silver stains. B. Asbestos fibers - birefringement fibers of magnesium silicate that may be found in people exposed to large quantities of asbestos. This can lead to mesothelioma. After inhalation the fibers become coated with an iron-containing protein and are known as asbestos bodies. These are demonstrated with the Prussian Blue reaction for iron. C. Tattoo pigments - found in tattooed skin, but occasionally found in associated lymph nodes. They include a series of organic and inorganic pigments. IV. Endogenous Hematogeneous Pigments ¾ These pigments are formed within the blood in the body. ¾ They are classified as Hematogeneous (from blood) or non-Hematogeneous. A. HEMATOGENEOUS 1. Hemoglobin - is a conjugated protein that is found normally in red blood cells and that is responsible for transporting oxygen from the lungs to other parts of the body. a) Staining: 1) Eosin staining 2) Okajima Stain 2. Hemosiderin - large amount are found only in pathologic conditions. If the production and destruction of RBC are not balanced, there may be increased deposition of hemosiderin in tissue • Hemochromatosis - disease caused by excessive absorption of dietary iron, is also characterized by excessive hemosiderin deposits. a) Staining: 1) Prussian Blue Reaction
3. Bile pigments - Biliverdin also results from destruction of RBC’s and further breakdown of the heme portion of hemoglobin. a) Biliverdin - is transported to the liver, where it undergoes reduction to bilirubin, which is not normally deposited in tissue but is removed from circulation by the liver and the secreted as one of the components of bile. b) Jaundice - caused by the obstruction of normal bile flow. c) Staining: 1) Hall’s Stain for Bile 4.
Hematoidin - similar to bilirubin and oxidized to biliverdin by bile demonstrating techniques. It’s formed in tissue as a result of hemorrhage and reduced oxygen tension.
Endogeneous Pigments Non-Hematogeneous
Hematogeneous Lipidic
Hemoglobin
Lipofuchsin Globin
Ceroid
Non- Lipidic Melanin
Heme (pigment part)
(protein part) Amino acid pool
Bile pigments Iron Reused in new hemoglobin synthesis
Deposited in tissue as hemosiderin
Biliverdin Bilirubin further breakdown products
V. Endogenous Non-hematogeneous Pigments ¾ These pigments are not from blood. ¾ NON-HEMATOGENEOUS Non lipidic pigments 1. Melanin - mixture of substances bound to proteins. Brown to black pigment present normally in the hair, skin, retina, iris, and certain parts of the central nervous system. Synthesis of Melanin Oxidation by tyrosine (amino acid) dopa
tyrosine (enzyme)
DOPA (3,4-dihydroxy phenylalanine)
further oxidation
melanin
through several steps
2. This reaction allows the histochemical demonstration of melanocytes, the cells responsible for the formation of melanin pigments. a) Melanin bleaching = when melanin is present in large amounts, cell details may be obscured. An oxidizing agent is used in order to remove the excess melanin. » 10% Potassium permanganate followed by Oxalic acid. lipidic pigments 1. Lipofuchsin - referred to as “wear and tear” pigment, collects in the more permanent cells: heart, liver, neurons. It is a yellow-brown pigment that stains in Oil red O, Sudan Black B, and PAS reaction. 2. Ceroid - is a brownish-yellow pigment described in hepatocytes and macrophages of rats with experimentally induced cirrhosis of the liver. Rarely seen in humans. 1) Stains: a) Oil Red O b) Sudan Black B
VI. Endogenous Deposits A. Urates, are deposited in tissue (gouty tophi) or around joints in person suffering from gout. B. Gout - a disorder in uric acid metabolism 1. Sodium urate are very soluble in water 2. dissolves if fixed in routine aqueous fixatives 3. alcoholic fixatives are recommended 4. Birefringent and identified by polarizing microscope a) Stain: Argentaffin reaction - Methenamine silver VII. Minerals ¾ Metallic and non metallic ions necessary for growth and other bodily functions. A. Ions demonstrated with special stains are: • Calcium • Ferrous/Ferric • Cupric • Phosphate • Carbonate B. Metallic elements are pathologically in tissue: Silver Lead Copper Gold C. Microincineration - method employed to study inorganic substances found in tissue. Process: Slides are cut at 4 µm and mounted on high temperature glass, the slides are incinerated up to 650oC, once completed slides are allowed to cool, then removed from the oven, coverslipped with glycerol mounting medium and microscopically examined. VIII. Types of Cytoplasmic Granules • Adrenal chromaffin cells • Pancreatic endocrine cells • gastrointestinal enterochromaffin cells • “C” cells of the thyroid • Pituitary cells • Neuroendocrine cells Demonstration: ¾ Argyrophilic - have the ability to be impregnated by silver, but do not have the ability to reduce the silver to a metallic form without the help of a reducing agent. (NEED A REDUCER) ¾ Argentaffin - They have the ability to be impregnated by silver and the ability to reduce the silver to a metallic form without the need of a reducing agent. (NO REDUCER NEEDED) ***Argentaffin cells will give a positive reaction with argyrophil techniques, but argyrophil cells will not yield positive results with argentaffin techniques ***
Special Staining Techniques Prussian Blue Stain for Ferric Ion (Fe+3) Purpose: The detection of ferric iron in tissue. Found normally in spleen and bone marrow. Principle: Detects the loosely bound protein complexes (As in hemosiderin). Strong bound iron will not react. Sections are treated with an acidic solution of potassium ferrocyanide and any ferric ion present will react in a bright blue pigment called Prussian Blue. Fixative: Alcohol or 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections. Control: a section containing ferric iron must be used. Reagents: 2% Potassium Ferrocyanide: Potassium ferrocyanide & distilled water 2% Hydrochloric acid Solution: Concentrated hydrochloric acid & distilled water Nuclear Fast Red solution: Nuclear fast Red, Aluminum sulfate, distilled water & Thymol Procedure: 1. Deparaffinize and hydrate slides to water 2. Place slides in a freshly prepared solution of a 50:50 mixture of 2% potassium ferrocyanide & 2% hydrochloric acid for 20 minutes at 60oC. 3. Wash slides thoroughly in water. 4. Counterstain slides in Nuclear fast Red solution for 5 minutes. 5. Rinse slides in running tap water for 1 minute. 6. Dehydrate sections in 95% alcohol and two changes of absolute alcohol. 7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Nuclei & hemofuchsin …………………………………………………...…Bright Red Hemosiderin (iron)……………………………………………………….….Blue Background…………………………………………………………….…….Pink
Turnbull’s Blue Stain for Ferrous Iron (Fe+2) Purpose: The detection of ferrous iron in tissue. Principle: Detects the ferrous iron in tissue. Sections are treated with an acidic solution of potassium ferricyanide and any ferrous iron present will react to form an insoluble bright blue pigment called Turnbull’s blue (ferrous ferricyanide). Fixative: Alcohol or 10% Neutral buffered formalin Technique: 4 to 5 µm paraffin sections Control: a section containing ferrous iron must be used. Reagents: 0.06N Hydrochloric acid: Hydrochloric acid & Distilled water Potassium Ferricyanide Staining Solution:: Potassium ferricyanide, 0.06N Hydrochloric acid Prepare fresh before use. 1% Acetic acid: glacial Acetic acid & distilled water Nuclear Fast Red solution: Nuclear fast Red, Aluminum sulfate, distilled water & Thymol Procedure: 1. Deparaffinize and hydrate slides to water 2. Place slides in a freshly prepared ferricyanide staining solution and stain for 1 hour at Room temperature. 3. Wash slides in 1% acetic acid thoroughly in water. 4. Counterstain slides in Nuclear fast Red solution for 5 minutes. 5. Rinse slides in running tap water for 1 minute. 6. Dehydrate sections in 95% alcohol and two changes of absolute alcohol. 7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Ferrous iron …………………………..……...………………………………………………Bright Red Background…………………………..……………………………………………………….Pink
ASBESTOS FIBERS
ASBESTOS FIBERS
Schmorl’s Technique for Reducing Substances Purpose: The detection of ferrous iron in tissue. Principle: Detects the ferrous iron in tissue. Sections are treated with an acidic solution of potassium ferricyanide and any ferrous iron present will react to form an insoluble bright blue pigment called Turnbull’s blue (ferrous ferricyanide). Fixative: 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections. Control: a section containing melanin or argentaffin granules. Reagents: 1% Stock ferric chloride: Ferric chloride & distilled water 0.1% Potassium Ferricyanide: Potassium ferricyanide & distilled water Ferric chloride-Potassium ferrocyanide: 1% ferric chloride & 0.1% Potassium ferricyanide, pH to 2.4 with 1N hydrochloric acid Mayer’s Mucicarmine Solution Metanil Yellow: Metanil yellow, Distilled water & glacial acetic acid Procedure: 1. Deparaffinize and hydrate slides to water. 2. Place slides in a freshly prepared ferric chloride - potassium ferricyanide solution and stain for 3 changes of 7 minutes each. 3. Rinse sections in distilled water. 4. Stain slides in working Mayer’s Mucicarmine for 1 hour. 5. Rinse slides rapidly in distilled water. 6. Counterstain slides in Metanil yellow for a few seconds. 7. Rinse slides rapidly in distilled water. 8. Dehydrate sections in 95% alcohol and two changes of absolute alcohol. 9. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Reducing Substances.………………………………………..………………Blue-green Goblet Cells, mucin…………………………………………………….……Rose Background………………………………………………………………….Yellow-green
Fontana Masson for Melanin and Argentaffin granules Purpose: Demonstration of argentaffin substances such as melanin, argentaffin granules of carcinoid tumors, and some neurosecretory granules. Principle: Some processes are argentaffin meaning they have the ability to bind silver from a silver solution and to reduce it to the visible metallic silver without the need for a separate reducing agent. Fixative: 10% Neutral buffered formalin, alcoholic fixative should be avoided since it tends to dissolves the argentaffin granules. Technique: 4 to 5 µm paraffin sections. Control: a section of skin for melanin, appendix or small intestine for argentaffin granules. Reagents: 10% Silver Nitrate Fontana Silver Solution: 10% Silver Nitrate & Ammonium hydroxide Gold Chloride Sodium Thiosulfate Nuclear Fast Red Procedure: 1. Deparaffinize and hydrate slides to water. 2. Place slides in silver nitrate solution in water bath at 56 degrees. For 1 hour. 3. Rinse sections in distilled water. 4. Immerse slides in gold chloride for 10 minutes. 5. Rinse slides rapidly in distilled water. 6. Place slides in Sodium thiosulfate for 5 minutes. 7. Rinse slides in distilled water. 8. Counterstain slides in Nuclear fast Red for 5 minutes. 9. Wash slides for at least 1 minute in distilled water. 10. Dehydrate sections in 95% alcohol and two changes of absolute alcohol 11. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Melanin .………………………….………………………….............................…Black Argentaffin granules………………………………………………………………Black Nuclei……………………………….................………………………………….Pink
Grimelius’ Argyrophil Stain Purpose: Demonstration of argyrophil granules in neurosecretory tumors. Principle: Some processes are argyrophilic meaning they need a reducing agent in order to bring the silver to a visible metallic form. Fixative: 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections Control: an argyrophilic-positive carcinoid tumor is preferred, but a section of small intestine can be used. Glassware should be chemically cleaned. Reagents: 1% Silver Nitrate: Silver nitrate & Distilled water 0.2M Acetic acid: Glacial Acetic acid & Distilled water 0.2M Sodium Acetate: Sodium acetate & Distilled water Working Silver Solution: Acetic acid-acetate Buffer, 1% Silver nitrate & Distilled water Reducing Solution: Hydroquinone, Sodium sulfite & Distilled water Nuclear Fast Red: Nuclear Fast Red, Distilled water & Thymol Procedure: 1. Deparaffinize and hydrate slides to water. 2. Place slides in working silver nitrate solution in water bath at 60 degrees for 1 hour. 3. Drain slides briefly. 4. Place slides in freshly prepared reducing solution preheated to 40 - 50 degrees Celsius for 1 minute. 5. Rinse slides well in distilled water. 6. Repeat step 2 for 10 minutes. 7. Drain slides briefly and place in reducing solution again for 1 minute. 8. Rinse slides in distilled water for 2 minutes. 9. Counterstain slides in Nuclear Fast Red solution for 5 minutes. 10. Dehydrate sections in 95% alcohol and two changes of absolute alcohol 11. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Argyrophil granules.……………………..........................................................…Dark brown to black Argentaffin granules…………………………………………………………..…Dark brown to black Nuclei……………………………...............…………………………………… Red Background……………………………………………………...………………Pale yellow-brown
Churukian-Schenk Method for Argyrophil Granules Purpose: Demonstration of argyrophil granules in neurosecretory tumors. Principle: Some processes are argyrophilic meaning they need a reducing agent in order to bring the silver to a visible metallic form. Fixative: 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections Control: an argyrophilic-positive carcinoid tumor is preferred, but a section of small intestine can be used. Glassware should be chemically cleaned. Reagents: 0.3% Citric acid: Citric acid & distilled water Acidified water: Distilled water and enough 0.3% Citric acid solution. 0.5% Silver Nitrate: Silver nitrate & Distilled water Reducing Solution: Hydroquinone, Sodium sulfite & Distilled water (Prepare just before use) Nuclear Fast Red: Nuclear Fast Red, Distilled water & Thymol Procedure: 1. Deparaffinize slides through xylene and alcohol's to acidified water, pH 4.0 to 4.2. 2. Place slides in 0.5% Silver nitrate solution in a 43 degrees water bath for 2 minutes. Transfer to a 58 degrees water bath for 2 hours. 3. Rinse slides in three changes of distilled water. 4. Transfer slides to reducing solution already heated in the 58 degrees water bath for 30 to 60 minutes. 5. Rinse sections in three changes of distilled water. 6. Return sections to the same silver nitrate of step 2 at 58 degrees for 10 minutes. 7. Rinse slides in three changes of distilled water. 8. Place slides in the same reducing solution of step 4 at 58 degrees for 5 minutes. 9. Rinse slides in three changes of distilled water. 10. Counterstain slides in Nuclear Fast Red solution for 3 minute. 11. Rinse slides in three changes of distilled water. 12. Dehydrate sections in two changes of 95% alcohol and two changes of absolute alcohol. 13. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Argyrophil granules.……….……………………….……………........................…Black Argentaffin granules……………………………………………………………..…Black Nuclei………………………………………...............…………………………… Red Background………………………………….……………………...………………Yellow-brown
Gomori’s Methenamine Silver Method for Urates Purpose: Demonstration of urates in tissue. Principle: Urates are stained black by this method, it is presumed that it reacts with the silver, which is then reduced to its metallic form. Fixative: Absolute alcohol is required. Technique: 4 to 5 µm paraffin sections Control: a section containing urate crystals. Reagents: 5% Silver Nitrate: Silver nitrate & Distilled water 3% Methenamine Solution: Methenamine & distilled water Stock Methenamine-Silver Solution: 5% silver Nitrate & 3% Methenamine solution. 5% Sodium Borate (Borax): Sodium borate & distilled water 3% Sodium thiosulfate: sodium thiosulfate & distilled water Stock light green solution: Light green, distilled water & glacial acetic acid Working Light green: Stock light green and distilled water. Procedure: 1. Deparaffinize slides and rinse in several changes of absolute alcohol. Do not hydrate slides. 2. Place sections in working methenamine-silver solution which has been preheated at 60 degrees. 3. Incubate slides for 30 minutes at 60 degrees. The urate crystal should turn black. 4. Rinse slides in distilled water. 5. Place sections in 3% sodium thiosulfate (hypo) for 5 minutes. 6. Wash slides in running tap water for 2 to3 minutes. 7. Rinse slides in several changes of distilled water. 8. Counterstain slides in working light green solution for 1 1/2 to 2 minutes. 9. Dehydrate sections in two changes of 95% alcohol and two changes of absolute alcohol. 10. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Urates……………………………………………...……………………........................…Black Background……………………………………...………………………...………………Green
Bile Stain (Hall) Purpose: Demonstration of bilirubin in tissue. Principle: Bilirubin is oxidized to biliverdin in an acid medium. This oxidation reaction is rapidly accomplished by ferric chloride in trichloroacetic acid medium. Fixative: 10% Neutral buffered formalin Technique: 4 to 5 µm paraffin sections Control: a section containing bile must be used as a control. Reagents: 10% Ferric chloride: Ferric chloride & Distilled water Fouchet’s Reagent: Trichloroacetic acid, 10% Ferric chloride & distilled water. Van Gieson’s solution: 1% acid fuchsin & Saturated Picric acid. Procedure: 1. Deparaffinize and hydrate slides to distilled water. 2. Wash sections well in distilled water. 3. Stain sections in freshly prepared Fouchet’s solution for 5 minutes. 4. Wash slides in tap water, then rinse in distilled water. 5. Stain sections in Van Gieson’s solution for 5 minutes. 6. Place slides directly into 95% alcohol and rinse well. 7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Bile or Bilirubin……………………………………………...…….…Emerald green to olive drab Background…………………………………………...………………Yellow
Von Kossa Calcium Stain Purpose: To identify the presence of calcium in tissue.. Principle: This is a chemical reaction, even though is going in an indirect way of detecting calcium. The silver reacts with the anions, primarily carbonate and phosphate, of the calcium salts. Bright light reduces the silver salt to metallic silver and unreduced silver is removed with sodium thiosulfate. Fixative: 10% Neutral buffered formalin. Alcohols are preferred. Technique: 4 to 5 µm paraffin sections Control: a section containing calcium must be used as a control. Reagents: 5% Silver nitrate: Silver nitrate & distilled water 5% Sodium thiosulfate: Sodium thiosulfate & distilled water Nuclear Fast Red: Nuclear fast Red, distilled water & Thymol Procedure: 1. Deparaffinize and hydrate slides to distilled water. 2. Place sections in silver nitrate solution and expose to bright sunlight for 10 to 20 minutes. If the day is overcast, longer incubation will be necessary. Check the slides periodically and stop the reaction when the calcium salts are brown-black. 3. Wash sections well in distilled water. 4. Place slides in Sodium thiosulfate for 2 to 3 minutes. 5. Wash slides well in distilled water. 6. Counterstain slides in Nuclear-fast red for 5 minutes. 7. Wash sections well in distilled water. 8. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Calcium salts……………………………………………....…….…Black Background…………………………………………...………………Red
Alizarin Red S Calcium Stain Purpose: To identify the presence of calcium in tissue.. Principle: Alizarin red S will react with the cations calcium, magnesium, manganese, barium, and strontium, but normally only calcium is present in tissue in sufficient quantities for demonstration. Calcium forms an alizarin red s-calcium complex in a chelation process. Fixative: Alcoholic fixatives or 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections Control: a section containing calcium must be used as a control. Reagents: Alizarin Red S Staining solution: Alizarin Red S & Distilled water; Mix the solution and pH to 4.1 to 4.3 with 0.5% ammonium hydroxide. The pH is critical. Procedure: 1. Deparaffinize and hydrate slides to 50% alcohol. 2. Rinse slides rapidly in distilled water. 3. Place sections in alizarin red S staining solution. Check the reaction microscopically and remove slides when an orange-red lake forms (30 seconds to 5 minutes). 4. Shake off excess dye and carefully blot the sections. 5. Dehydrate in acetone (10 to 20 seconds) and in acetone-xylene mixture (10 to 20 seconds). 6. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Calcium deposits……………………………….………………………....…….…Orange-red
Paraffin Section
Wet (Cytology) Section
Rhodanine Method for Copper Purpose: To detect copper in tissue, especially in liver in Wilson’s Disease. Principle: This procedure is more sensitive than the rubeanic acid method; however, it has suggested that Rhodanine demonstrates the protein to which the copper binds rather than the copper itself. Therefore, although considered more sensitive, it may be less specific than the rubeanic acid methods and falsepositive results may be obtained. Fixative: 10% Neutral buffered formalin. Technique: 4 to 5 µm paraffin sections Control: a section containing copper must be used as a control. Reagents: Saturated Rhodanine solution: Rhodanine & absolute alcohol Working Rhodanine solution: Saturated Rhodanine solution & distilled water. Filter before use. Diluted Mayer’s Hematoxylin: Mayer’s hematoxylin & distilled water. 0.5% Sodium borate: Sodium borate & distilled water. Procedure: 1. Deparaffinize and hydrate slides to distilled water. 2. Using plastic coplin jars, place slides in 50 mL of working Rhodanine solution. Leave 18 hours at 37 degrees. 3. Rinse slides well in distilled water. 4. Stain slides in Mayer’s hematoxylin for 10 minutes. 5. Rinse slides in distilled water. 6. Rinse slides quickly in 0.5% sodium borate solution. 7. Clear slides in three changes of xylene and mount in a synthetic resin mounting media. Results: Copper………………………….………………………………....…….…Bright red to red yellow Nuclei………………………..…………………………………………….Light blue
