Introduction
- Identification of mineral pigment stains is inorganic substances that accumulate in tissues due to various physiological, pathological, or environmental processes.
- In histopathology, the identification and demonstration of these pigments are essential for diagnosing a range of conditions, such as hemorrhage, aging, metabolic disorders, and environmental exposure.
- The presence of mineral pigments can offer significant insight into the underlying pathology of a disease, revealing the presence of tissue damage, abnormal metabolism, or cellular degradation.
- Common mineral pigments found in tissues include hemosiderin (iron pigment), calcium (as calcifications), melanin, and various metal-based pigments (such as lead).
- These pigments have distinct staining characteristics, making them identifiable through specific histological staining techniques.
- These stains help to highlight the pigments’ presence and distribution within tissues, aiding pathologists in their diagnostic assessment.
Histopathological stain
Perls’ Prussian blue reaction stain for ferric iron
- This method is considered to be the first classical histochemical reaction.
- Treatment with an acid ferrocyanide solution will result in unmasking ferric iron in the form of the hydroxide, Fe(OH)3.
- The ferric iron then reacts with a dilute potassium ferrocyanide solution to produce an insoluble blue compound, ferric ferrocyanide (Prussian blue).
Fixation: – Avoid the use of acid fixatives. Chromates will also interfere with the preservation of iron.
Sections: – Works well on all types of sections, including resin.
Acid ferrocyanide solution
1% aqueous potassium ferrocyanide 20 ml
2% aqueous hydrochloric acid 20 ml
Preferably freshly prepared, mix just before use.
Method
1. Take a test and control section to water.
2. Treat sections with the freshly prepared acid ferrocyanide solution for 10–30 minutes.
3. Wash well in distilled water.
4. Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red.
5. Wash rapidly in distilled water.
6. Dehydrate, clear and mount in synthetic resin.
Results
Ferric iron – Blue
Nuclei – Red
Lillie’s method for ferric and ferrous iron
Fixation: – Avoid the use of acid fixatives. Chromates will also interfere with the preservation of iron.
Sections: – Paraffin wax, frozen and resin.
Method
1. Take test and control sections to distilled water.
2. Dissolve 400 mg of potassium ferrocyanide in 40 ml of 0.5% hydrochloric acid when testing for ferric iron.
-
- For testing ferrous iron, substitute 400 mg of potassium ferricyanide.
- Prepare just before use. Expose sections for 30 minutes.
3. Wash well in distilled water.
4. Stain nuclei with 0.1% aqueous nuclear fast red for 5 minutes.
5. Rinse in distilled water.
6. Dehydrate, clear, and mount in synthetic resin.
Results
Ferric iron dark Prussian – Blue
Ferrous iron dark Turnbull’s – Blue
Nuclei – Red
Leuco patent blue V method for haemoglobin
Fixation: – Formalin is best; Drury and Wallington have noted poor preservation with Heidenhain’s Susa (1980).
Solutions
Stock solution
1% aqueous patent blue V (CI 42045) 25 ml
Powdered zinc 2.5 g
Glacial acetic acid 0.5 ml
Mix well on a magnetic stirrer. The solution will become pale green-blue. Filter and store in a refrigerator at 3–6°C. The solution is stable for about 1 week.
Working solution
Stock solution 10 ml
Glacial acetic acid 2 ml
3% hydrogen peroxide 1 ml
Prepare immediately before use.
Method
1. Take test and control sections to distilled water.
2. Stain in patent blue solution for 5 minutes at room temperature.
3. Rinse in distilled water.
4. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 1 minute.
5. Rinse in distilled water.
6. Dehydrate, clear, and mount in synthetic resin.
Results
Hemoglobin peroxidase (red blood cells and neutrophils)
Dark – Blue
Nuclei – Red
Modified Fouchet’s technique for liver bile pigments
Fixation:- Any fixative appears suitable.
Sections:- Any.
Solutions
Fouchet’s solution
25% aqueous trichloroacetic acid 36 ml
10% aqueous ferric chloride 4 ml
Freshly prepared before use.
van Gieson stain
Dissolve 100 mg of acid fuchsin in 100 ml
of saturated aqueous picric acid.
Method
1. Take test and control sections to distilled water.
2. Treat with the freshly prepared Fouchet’s solution for 10 minutes.
3. Wash well in running tap water for 1 minute.
4. Rinse in distilled water.
5. Counterstain with van Gieson solution for 2 minutes.
6. Dehydrate, clear and mount in synthetic resin.
Results
Bile pigments emerald – Blue-green
Muscle – Yellow
Collagen – Red
Gmelin technique for bile pigments
- This technique is the only method that shows an identical result with liver bile, gallbladder bile and hematoidin.
- The method tends to be messy, capricious, and gives impermanent results.
- Deparaffinized tissue sections containing bile pigments are treated with nitric acid, and a changing color spectrum is produced.
- It can be unreliable, so it is advisable to repeat the test at least three times before a negative result is acceptable.
- A popular modification of this technique is that of Lillie and Pizzolato (1967), in which bromine in carbon tetrachloride is used as an
oxidant.
Sections:- Paraffin wax embedded.
Method
1. Take sections to distilled water and mount in distilled water.
2. Place the mounted section under the microscope using an objective with a reasonable working distance.
3. Place 2–3 drops of concentrated nitric acid on one side of the coverglass and draw under the coverglass using a piece of blotting paper on the opposite side.
4. Remove excess solution and observe the pigment for color changes.
Results:- Bile pigments will gradually produce the following spectrum of color change: yellow-green-blue-purple-red.
Masson-Fontana method for melanin
Fixation:- Formalin is best; chromate and mercuric chloride should be avoided.
Sections:- Works on all types of sections, although some adjustment may be necessary for resin sections.
Preparation of silver solution (after Fontana)
- Place 20 ml of a 10% aqueous silver nitrate solution in a glass flask.
- Using a fine-pointed dropper pipette, add concentrated ammonia drop by drop, constantly agitating the flask until the formed precipitate almost dissolves.
- This titration is critical if the method is to work consistently well.
- The endpoint of the titration is seen when a faint opalescence is present, and is best viewed using reflected light against a black background.
- If too much ammonia is inadvertently added, adding 10% silver nitrate drops will restore the opalescence.
- Add 20 ml of triple-distilled water to this correctly titrated solution and then filter into a dark bottle.
- Store the solution in the refrigerator and use within 4 weeks, but note that ammoniacal silver solutions are potentially explosive if stored incorrectly.
Method
1. Take test and control sections to distilled water.
2. Treat with the ammoniacal silver solution in a Coplin jar covered with aluminium foil, for 30–40 minutes at 56°C or overnight at room temperature.
3. Wash well in several changes of distilled water.
4. Treat sections with 5% aqueous sodium thiosulfate (hypo) for 1 minute.
5. Wash well in running tap water for 2–3 minutes.
6. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 5 minutes.
7. Rinse in distilled water.
8. Dehydrate, clear and mount in a synthetic resin.
Results
Melanin, argentaffin, chromaffin and some lipofuscins – Black
Nuclei – Red
Microwave ammoniacal silver method for argentaffin and melanin
Fixation:- 10% buffered neutral formalin.
Sections:- Paraffin wax embedded.
Preparation of solutions
- To 10 ml of 2% silver nitrate, add 5 ml of 0.8% lithium hydroxide monohydrate.
- Then add 28% ammonium hydroxide, drop by drop with constant shaking, until the precipitate almost dissolves.
- Make the solution 200 ml with distilled water and store in a refrigerator at 3–6°C.
- The solution is stable for at least 1 month. 0.2% aqueous gold chloride 2% aqueous sodium thiosulfate.
Method
1. Take slides to distilled water.
2. Place slides in 40 ml of refrigerated cold ammoniacal silver solution in a plastic Coplin jar and microwave at a power setting of 360 W
for 35 seconds.
-
- Gently agitate the Coplin jar for about 15 seconds.
- Microwave again at the same power for 35 seconds.
- Gently agitate the Coplin jar for about 15 seconds.
- Allow the slides to remain in the hot solution (approximately 80°C) for 2–3 minutes or until the sections appear a light brown.
3. Rinse in four changes of distilled water.
4. Place slides in 0.2% aqueous gold chloride for 1 minute.
5. Rinse in two changes of distilled water.
6. Place slides in 2% aqueous sodium thiosulfate for 1 minute.
7. Rinse in four changes of distilled water.
8. Counterstain with 0.1% aqueous nuclear fast red for 3 minutes.
9. Rinse in three changes of distilled water.
10. Dehydrate, clear, and mount in synthetic resin.
Results
Melanin, argentaffin, chromaffin, lipofuscin and other silver reducing substances – Black
Nuclei – Red
Schmorl’s reaction
Fixation:- 10% buffered neutral formalin.
Ferric-ferricyanide solution
Freshly prepared 0.4% aqueous potassium ferricyanide 4 ml
Freshly prepared 1% aqueous ferric chloride (or 1% ferric sulfate) 30 ml
N.B. Use this solution soon after mixing.
Method
1. Take test and control sections to distilled water.
2. Treat sections with the ferric-ferricyanide solution for 5–10 minutes.
3. Wash well in running tap water for several minutes to remove all residual ferricyanide from the section.
4. Lightly counterstain with 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 5 minutes.
5. Dehydrate, clear and mount in synthetic resin.
Results
Melanin, argentaffin cells, chromaffin, some lipofuscins, thyroid colloid and bile – Dark blue
Nuclei – Red
Bleaching melanin pigment using hydrogen peroxide
Fixation:- 10% neutral buffered formalin.
Solution
40% H2O 25 ml
Phosphate buffered saline (pH 7.6) 45 ml
(Dissolve PBS tablets according to instructions in 200 ml of distilled water per tablet.)
Make the solution fresh and place it in a 50 ml Coplin jar.
Method
1. Take test and control sections to distilled water.
2. Prepare the incubating solution fresh and place it in a water bath or oven at 60°C for 10 minutes.
3. Place slides into the incubating solution and ensure the Coplin jar lid is securely placed over the jar.
4. Incubate in the jar for 1 hour.
5. Remove slides and wash in running tap water for 3 minutes.
6. Continue with immunohistochemical procedures
(heat-mediated or enzyme digestion antigen retrieval techniques)
Ferrous ion uptake reaction for melanin
Fixation:- Formalin is best; avoid all chromate fixatives.
Sections:- Paraffin wax embedded.
Solutions
2.5% ferrous sulfate
1% potassium ferricyanide in 1% acetic acid
Method
1. Take test and control sections to distilled water.
2. Place in 2.5% ferrous sulfate for 1 hour.
3. Wash with six changes of distilled water.
4. Place 1% potassium ferricyanide in 1% acetic acid for 30 minutes.
5. Wash with four changes of distilled water.
6. Counterstain with 0.5% aqueous neutral red or 0.1% nuclear fast red.
7. Rinse in two changes of distilled water.
8. Dehydrate, clear and mount in synthetic resin.
Results
Melanins and neuromelanin – Dark green
Nuclei – Red
Nile blue method for melanin and lipofuscin
Fixation:- 10% buffered neutral formalin.
Sections:- Paraffin wax embedded.
Nile blue solution
Nile blue (CI 51180) 0.05 g
Distilled water 99 ml
Sulfuric acid 1.0 ml
Dissolve Nile blue in distilled water, then add sulfuric acid.
Method
1. Take test and control sections to distilled water.
2. Place in Nile blue solution for 20 minutes.
3. Wash with four changes of distilled water.
4. Mount in an aqueous mountant (e.g. glycerin jelly).
Results
Melanin – Dark blue
Lipofuscin – Dark blue
Nuclei – Blue or unstained
Long Ziehl-Neelsen method
Fixation:- Any routine fixative.
Sections:- Works well on all types of tissue sections.
Method
1. Take slides to distilled water.
2. Stain in a Coplin jar with filtered carbol fuchsin using a 60°C water bath for 3 hours, or overnight at room temperature.
3. Wash well in running water.
4. Differentiate in 1% acid alcohol until the background staining is removed.
5. Wash well in running tap water.
6. Counterstain nuclei with 0.25% aqueous methylene blue in 1% aqueous acetic acid for 1 minute.
7. Dehydrate, clear and mount in synthetic resin.
Results
Lipofuscin – Magenta
Ceroid – Magenta
Nuclei – Blue
Background pale magenta to pale blue
Aldehyde fuchsin technique
Fixative:- 10% buffered neutral formalin.
Sections:- Paraffin wax embedded.
Solutions
Acidified potassium permanganate solution (0.25% aqueous potassium permanganate in 0.1% sulfuric acid)
2% aqueous oxalic acid
Aldehyde fuchsin
-
- Dissolve 1 g pararosanilin (CI 42500) in 100 ml aqueous 70% ethanol.
- Add 1 ml concentrated hydrochloric acid and 2 ml paraldehyde or acetyl aldehyde, shaking the mixture thoroughly.
- Stand for 3–5 days at room temperature or preferably longer, near natural light, to allow the solution to blue.
- Store the solution at 4°C. The solution will remain viable for approximately 2 months.
- Any increase in the background staining will indicate deterioration of the staining solution.
Method
1. Take sections in distilled water.
2. Treat with acidified potassium permanganate solution for 5 minutes.
3. Wash well in distilled water and treat for 2 minutes with oxalic acid solution to bleach the section.
4. Wash well in distilled water.
5. Rinse in 70% ethyl alcohol.
6. Stain section in aldehyde fuchsin for 5 minutes. Longer staining times will be needed as the solution ages.
7. Rinse in 70% ethyl alcohol, followed by a rinse in three changes of distilled water.
8. Dehydrate, clear and mount in synthetic resin.
Results
Lipofuscin – Purple
Elastic fibers – Purple
Alizarin red S method for calcium
Fixation:- Buffered neutral formalin, formal alcohol and alcohol.
Sections:- Paraffin wax or frozen.
Solutions
1% aqueous alizarin red S (CI 58005) adjusted to pH 4.2 or pH 6.3–6.5 with 10% ammonium hydroxide.
0.05% fast green FCF (CI 42053) in 0.2% acetic acid.
Method
1. Take sections to 95% alcohol.
2. Stand slides on end and thoroughly air dry.
3. Place sections in a Coplin jar filled with the alizarin red S solution for 5 minutes.
4. Rinse quickly in distilled water.
5. Counterstain with fast green for 1 minute.
6. Rinse in three changes of distilled water.
7. Dehydrate, clear and mount in synthetic resin.
Results
Calcium deposits – Orange-red
Background – Green
Rubeanic acid method for copper
Fixative:- 10% buffered neutral formalin.
Rubeanic acid solution 0.1% rubeanic acid (dithiooxamide) in absolute ethyl alcohol 5 ml
10% aqueous sodium acetate 100 ml
Prepare fresh before use.
Method
1. Take the test section and a known positive control section to distilled water.
2. Place sections in a Coplin jar filled with rubeanic acetate solution for at least 16 hours at 37°C. Times may need to be extended, and the method is best carried out in a water bath.
3. Wash in 70% ethyl alcohol.
4. Rinse briefly in distilled water.
5. Drain section and blot dry.
6. Lightly counterstain with 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 1 minute.
7. Rinse in distilled water.
8. Dehydrate, clear, and mount in synthetic resin.
Results
Copper greenish – Black
Nuclei – Pale red
Rhodizonate method for lead salts
Fixation
- Avoid the use of mercury-containing fixatives.
- Bones containing lead salts can be decalcified in 5–10% sulfuric acid containing 5–10% sodium sulfate.
- This procedure should convert lead deposits into insoluble lead sulfate.
Sections:- Paraffin wax embedded.
Solutions
Rhodizonate solution Sodium rhodizonate 100 mg
Distilled water 50 ml
Glacial acetic acid 0.5 ml
0.05% fast green FCF (42053) in 0.02% acetic acid
Method
1. Take sections in distilled water.
2. Place in rhodizonate solution for 1 hour.
3. Rinse well in distilled water.
4. Counterstain in 0.05% aqueous fast green in 0.2% acetic acid for 1 minute.
5. Rinse in three changes of distilled water.
6. Dehydrate, clear and mount in synthetic resin.
Results
Lead salts – Black
Background – Green
Solochrome azurine method for beryllium and aluminum
Fixation:- Not critical.
Sections:- Paraffin wax or frozen.
Solutions
a. 0.2% solochrome azurine (Pure blue B)
b. 0.2% solochrome azurine in normal sodium hydroxide
Method
1. Take two test sections to distilled water.
2. Stain one section in solution a and one in solution b for 20 minutes.
3. Wash in distilled water.
4. Lightly counterstain in 0.5% aqueous neutral red or 0.1% aqueous nuclear fast red for 5 minutes.
5. Wash in distilled water and mount in synthetic resin.
Results
Solution a: aluminum and beryllium – Blue
Solution b: beryllium only – Blue-black
Nuclei – Red
Rhodanine method for silver
Fixation:- Not critical, but avoid the use of mercury-containing fixatives.
Sections:- Paraffin wax; frozen.
Incubating solution
P-Dimethylaminobenzylidene-rhodanine (saturated solution in 90%) 3.5 ml
M nitric acid 3 ml
Distilled water 93.5 ml
Method
1. Take paraffin wax sections to distilled water.
2. Incubate sections in rhodanine solution at 37°C for 24 hours.
3. Wash well in distilled water.
4. Mount in glycerin jelly.
Results
Silver deposits – Red-brown