Eyeball, Bone Marrow Biopsy, and Calcified Bone Stain

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Introduction

  • Histopathology is the branch of laboratory science that studies tissues under a microscope to identify diseases and abnormalities.
  • Most tissue specimens can be processed using routine histological techniques; however, some tissues require special treatment because of their delicate or hard nature.
  • The eyeball is a highly delicate organ that requires careful fixation and processing to preserve its microscopic structures.
  • Bone marrow biopsy specimens contain both soft marrow and hard bone, making special processing and decalcification necessary.
  • Calcified bone tissues cannot be sectioned directly and must undergo decalcification before microscopic examination.

Eyeball Staining

Collection and Fixation

  • The eyeball should be collected immediately after surgical removal.
  • Excess handling and pressure should be avoided to prevent distortion of the globe.
  • The specimen should be properly labeled with patient details and orientation markings, if necessary.
  • The specimen should be transported to the laboratory without delay.

Fixatives

  • Immediate fixation is essential to prevent autolysis and tissue degeneration.
  • Davidson’s fixative is the preferred fixative for ocular specimens because it provides excellent preservation of retinal structures.
  • When Davidson’s fixative is unavailable, 10% Neutral Buffered Formalin can be used.
  • The eyeball should be completely immersed in an adequate volume of fixative (10–20 times the tissue volume).
  • Proper fixation helps maintain tissue morphology and improves staining quality.

Grossing Procedure

1. External Examination

  • Examine the shape, size, and color of the eyeball.
  • Note any abnormalities such as tumors, injuries, hemorrhage, or deformities.
  • Measure the anteroposterior, horizontal, and vertical diameters of the globe.

2. Examination of Cornea and Sclera

  • Observe the transparency and condition of the cornea.
  • Examine the sclera for any thickening, pigmentation, or lesions.

3. Examination of Optic Nerve

  • Measure the length and diameter of the optic nerve stump.
  • Check for any swelling, discoloration, or tumor involvement.

4. Orientation of the Specimen

  • Identify the superior, inferior, nasal, and temporal aspects of the globe.
  • Proper orientation is important for locating lesions accurately.

5. Sectioning of the Eyeball

  • The eyeball is usually cut along the pupil–optic nerve axis.
  • A sharp blade is used to obtain clean sections without damaging internal structures.
  • The globe is divided into two halves to expose the internal contents.

6. Internal Examination

  • Examine the lens, vitreous body, retina, choroid, and optic disc.
  • Note the presence of retinal detachment, tumors, hemorrhage, inflammation, or other pathological changes.

7. Tissue Sampling

  • Representative sections are taken from:
    • Cornea
    • Iris and ciliary body
    • Lens
    • Retina
    • Choroid
    • Optic nerve
    • Any abnormal lesion present

Periodic Acid-Schiff (PAS)

Principle

  • The Periodic Acid–Schiff (PAS) stain is used to demonstrate carbohydrates and carbohydrate-rich structures in tissues.
  • Periodic acid oxidizes glycol groups present in carbohydrates to form aldehydes.
  • These aldehyde groups react with Schiff’s reagent, producing a bright magenta or pink color.

In ocular tissues, PAS stain is particularly useful for demonstrating:

  • Corneal basement membrane
  • Descemet’s membrane
  • Lens capsule
  • Retinal basement membranes
  • Glycogen and mucopolysaccharides

Section

  • Paraffin-embedded tissue sections
  • Thickness: 4–5 µm
  • Sections should be properly deparaffinized and hydrated before staining

Staining Solutions Required

1. Periodic Acid Solution (0.5–1%)

  • Periodic acid – 0.5 g
  • Distilled water – 100 mL

2. Schiff’s Reagent

Contains:

  • Basic fuchsin
  • Hydrochloric acid
  • Sodium metabisulphite
  • Activated charcoal

3. Hematoxylin

  • Used as a counterstain.

4. Running Tap Water

  • For washing and color development.

Procedure

  1. Deparaffinize sections in xylene.
  2. Hydrate through descending grades of alcohol to water.
  3. Treat sections with periodic acid solution for 5–10 minutes.
  4. Wash thoroughly in distilled water.
  5. Immerse sections in Schiff’s reagent for 10–15 minutes.
  6. Wash in running tap water for 5–10 minutes until the pink color develops.
  7. Counterstain with hematoxylin for 1–2 minutes.
  8. Wash in water and blue the nuclei.
  9. Dehydrate through graded alcohols.
  10. Clear in xylene.
  11. Mount with DPX or suitable mounting medium.

Results

Tissue Component Colour
Basement membranes Bright magenta (pink-red)
Glycogen Magenta
Mucopolysaccharides Magenta
Lens capsule Magenta
Corneal structures Magenta
Nuclei (hematoxylin) Blue
Background tissue Pale pink

Masson’s Trichrome Stain 

Principle

  • Masson’s Trichrome stain is a special histochemical staining technique used to differentiate collagen fibers from muscle fibers and other tissue elements.
  • The stain employs three different dyes that selectively stain various tissue components based on their permeability and affinity for the dyes.

In eye tissue, Masson’s Trichrome stain is mainly used to demonstrate:

  • Collagen fibers in the cornea and sclera
  • Fibrosis and scar tissue
  • Connective tissue components of the eye

Section

  • Paraffin-embedded tissue sections
  • Thickness: 4–5 µm
  • Well-fixed tissue sections are preferred

Staining Solutions Required

1. Weigert’s Iron Hematoxylin

2. Biebrich Scarlet–Acid Fuchsin Solution

3. Phosphomolybdic/Phosphotungstic Acid Solution

4. Aniline Blue Solution

5. Acetic Acid (1%)

Procedure

  1. Deparaffinize sections and hydrate to water.
  2. Stain with Weigert’s Iron Hematoxylin for 10 minutes.
  3. Wash thoroughly in running water.
  4. Stain with Biebrich Scarlet–Acid Fuchsin solution for 10–15 minutes.
  5. Rinse in distilled water.
  6. Treat with phosphomolybdic/phosphotungstic acid solution for 10 minutes.
  7. Transfer directly to Aniline Blue solution for 5–10 minutes.
  8. Rinse briefly in distilled water.
  9. Differentiate in 1% acetic acid for 1–2 minutes.
  10. Dehydrate rapidly through alcohol.
  11. Clear in xylene and mount with DPX.

Results

Tissue Component Colour
Collagen fibers Blue
Muscle fibers Red
Cytoplasm Red
Keratin Red
Nuclei Black
Red blood cells Bright red

Congo Red Stain 

Principle

  • Congo Red is a special histochemical stain used for the demonstration of amyloid deposits in tissues.
  • Congo Red dye has a specific affinity for amyloid fibrils and binds to them, producing a characteristic red-orange color under ordinary light microscopy.
  • When viewed under polarized light, amyloid deposits show apple-green birefringence, which is considered diagnostic of amyloidosis.

In ocular pathology, Congo Red stain is used to detect amyloid deposits in the:

  • Conjunctiva
  • Cornea
  • Eyelid tissues
  • Lacrimal gland
  • Orbit

Section

  • Paraffin-embedded tissue sections
  • Thickness: 5–8 µm
  • Well-fixed tissue sections are preferred

Staining Solutions Required

1. Congo Red Solution

2. Harris or Mayer’s Hematoxylin

3. Alkaline Alcohol Solution

Procedure

  1. Deparaffinize the tissue sections in xylene.
  2. Hydrate through descending grades of alcohol to water.
  3. Stain sections with Congo Red solution for 20–30 minutes.
  4. Rinse briefly in distilled water.
  5. Differentiate in alkaline alcohol if required.
  6. Wash in running tap water.
  7. Counterstain with hematoxylin for 1–2 minutes.
  8. Wash and blue the nuclei.
  9. Dehydrate rapidly through alcohol.
  10. Clear in xylene and mount with DPX.

Results

Tissue Component Colour
Amyloid deposits Red to orange-red
Nuclei Blue
Background tissue Pale pink

Under Polarized Light

Structure Appearance
Amyloid deposits

Apple-green birefringence

Reticulin Stain

Principle

  • Reticulin stain is a special histochemical staining method used to demonstrate reticular fibers (Type III collagen) present in tissues.
  • The technique is based on silver impregnation, in which reticular fibers absorb silver salts and are subsequently reduced to visible black metallic silver deposits.
  • In ocular tissues, reticulin staining helps demonstrate the supporting connective tissue framework and basement membrane-associated structures.

Section

  • Paraffin-embedded tissue sections
  • Thickness: 4–5 µm
  • Well-fixed tissue sections are preferred

Staining Solutions Required

1. Potassium Permanganate Solution

2. Oxalic Acid Solution

3. Ferric Ammonium Sulfate (Sensitizer)

4. Ammoniacal Silver Solution

5. Formalin Solution

6. Gold Chloride Solution

7. Sodium Thiosulfate Solution

8. Nuclear Fast Red

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Oxidize with potassium permanganate for 2–5 minutes.
  3. Wash in water.
  4. Bleach with oxalic acid until sections become colorless.
  5. Wash thoroughly in distilled water.
  6. Sensitize with ferric ammonium sulfate.
  7. Wash and treat with ammoniacal silver solution.
  8. Reduce in formalin solution until reticulin fibers become visible.
  9. Wash and tone with gold chloride solution.
  10. Fix with sodium thiosulfate.
  11. Counterstain with Nuclear Fast Red.
  12. Dehydrate, clear, and mount.

Results

Tissue Component Colour
Reticulin fibers Black
Nuclei Red
Background tissue Pink to pale red

Bone Marrow Biopsy Staining

Collection of Specimen

Bone marrow biopsy is usually obtained from the posterior superior iliac spine using a special biopsy needle. The procedure provides a small cylindrical core of bone and marrow tissue for histopathological examination.

  • The biopsy specimen should be collected aseptically.
  • The tissue should be handled gently to avoid crushing artifacts.
  • The specimen should be placed immediately into a fixative after collection.
  • Delay in fixation may lead to autolysis and poor preservation of cellular details.
  • Proper labeling with patient identification and specimen details is essential.

Fixation

Fixation is necessary to preserve the cellular and structural components of the bone marrow.

Preferred Fixative

10% Neutral Buffered Formalin (NBF)

  • Most commonly used fixative for bone marrow biopsy specimens.
  • Provides good preservation of tissue architecture.
  • Suitable for routine histopathological examination.

Alternative Fixatives

  • Bouin’s Fixative
  • Zenker’s Fixative (less commonly used)
  • B-5 Fixative (used in some specialized laboratories)

Reticulin Stain (Silver Impregnation)

Principle

  • Reticulin stain is a silver impregnation technique used to demonstrate reticular fibers (Type III collagen) in tissues.
  • Reticular fibers have the ability to bind silver salts, which are then reduced to visible black metallic silver deposits.
  • This property is known as argyrophilia.

Section

  • Paraffin-embedded bone marrow biopsy sections
  • Thickness: 3–5 µm
  • Properly fixed and decalcified tissue sections

Reagents Required

1. Potassium Permanganate Solution

2. Oxalic Acid Solution

3. Ferric Ammonium Sulfate Solution

4. Ammoniacal Silver Solution

5. Formalin Solution

6. Gold Chloride Solution

7. Sodium Thiosulfate Solution

8. Nuclear Fast Red

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Oxidize with potassium permanganate for 2–5 minutes.
  3. Wash in distilled water.
  4. Bleach with oxalic acid until sections become colorless.
  5. Wash thoroughly in water.
  6. Sensitize with ferric ammonium sulfate solution.
  7. Wash and immerse in ammoniacal silver solution.
  8. Reduce the silver with formalin solution until reticulin fibers become visible.
  9. Tone with gold chloride solution.
  10. Fix with sodium thiosulfate solution.
  11. Counterstain with Nuclear Fast Red.
  12. Dehydrate, clear, and mount.

Results

Tissue Component Colour
Reticulin fibers Black
Nuclei Red
Background tissue Pink to pale red

 

Masson’s Trichrome stain

Principle

  • Masson’s Trichrome stain is a special histochemical staining technique used to differentiate collagen fibers from muscle fibers and other tissue components.
  • The stain uses three different dyes that selectively stain tissue elements based on their chemical properties.
  • In bone marrow biopsy specimens, Masson’s Trichrome stain is primarily used to detect and assess collagen fibrosis, which may occur in various bone marrow disorders.

Section

  • Paraffin-embedded bone marrow biopsy sections
  • Thickness: 3–5 µm
  • Properly fixed and decalcified tissue sections

Reagents Required

1. Weigert’s Iron Hematoxylin

2. Biebrich Scarlet–Acid Fuchsin Solution

3. Phosphomolybdic/Phosphotungstic Acid Solution

4. Aniline Blue Solution

5. Acetic Acid (1%)

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Stain with Weigert’s Iron Hematoxylin for 10 minutes.
  3. Wash thoroughly in running water.
  4. Stain with Biebrich Scarlet–Acid Fuchsin solution for 10–15 minutes.
  5. Rinse in distilled water.
  6. Treat with phosphomolybdic/phosphotungstic acid solution for 10 minutes.
  7. Transfer directly to Aniline Blue solution for 5–10 minutes.
  8. Rinse briefly in distilled water.
  9. Differentiate in 1% acetic acid for 1–2 minutes.
  10. Dehydrate rapidly through alcohol.
  11. Clear in xylene and mount with DPX.

Results

Tissue Component Colour
Collagen fibers Blue
Muscle fibers Red
Cytoplasm Red
Red blood cells Bright red
Nuclei Black

Prussian Blue Stain

Principle

  • Prussian Blue stain, also known as Perls’ Prussian Blue stain, is used to demonstrate ferric iron (Fe³⁺) in tissues.
  • Hydrochloric acid releases ferric iron from tissue proteins, and the free iron reacts with potassium ferrocyanide to form an insoluble blue-colored compound called Prussian Blue (ferric ferrocyanide).
  • In bone marrow biopsies, this stain is used to assess iron stores and diagnose disorders of iron metabolism.

Section

  • Paraffin-embedded bone marrow biopsy sections
  • Thickness: 3–5 µm
  • Properly fixed and decalcified sections

Reagents Required

1. 2% Potassium Ferrocyanide Solution

2. 2% Hydrochloric Acid Solution

3. Nuclear Fast Red

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Prepare freshly mixed equal parts of potassium ferrocyanide and hydrochloric acid.
  3. Immerse sections in the working solution for 20–30 minutes.
  4. Wash thoroughly in distilled water.
  5. Counterstain with Nuclear Fast Red for 5 minutes.
  6. Wash, dehydrate, clear, and mount.

Results

Tissue Component Colour
Ferric iron (hemosiderin) Blue
Nuclei Red
Background tissue Pink

 

PAS (Periodic Acid–Schiff) 

Principle

PAS stain is used to demonstrate glycogen, mucopolysaccharides, glycoproteins, and basement membrane substances. Periodic acid oxidizes carbohydrates to aldehydes, which react with Schiff’s reagent to produce a bright magenta color.

Section

  • Paraffin-embedded bone marrow biopsy sections
  • Thickness: 3–5 µm
  • Properly fixed and decalcified sections

Reagents Required

1. Periodic Acid Solution (1%)

2. Schiff’s Reagent

3. Hematoxylin

Used as a counterstain.

4. Distilled Water

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Treat with periodic acid solution for 5–10 minutes.
  3. Wash thoroughly in distilled water.
  4. Immerse in Schiff’s reagent for 10–15 minutes.
  5. Wash in running tap water for 5–10 minutes until the magenta color develops.
  6. Counterstain with hematoxylin.
  7. Wash, dehydrate, clear, and mount.

Results

Tissue Component Colour
Glycogen Magenta
Mucopolysaccharides Magenta
Basement membrane substances Magenta
Nuclei Blue
Background tissue Pale pink

Calcified Bone Tissue Staining

Fixation

Bone specimens are first fixed in:

10% Neutral Buffered Formalin

Duration:

    • 24–48 hours

Hematoxylin and Eosin (H&E) Stain

Principle

Hematoxylin and Eosin (H&E) stain is the most commonly used routine stain in histopathology. It provides excellent visualization of the general tissue architecture and cellular details.

  • Hematoxylin is a basic dye that stains acidic structures, particularly nuclei, blue to purple.
  • Eosin is an acidic dye that stains basic structures such as cytoplasm, muscle fibers, and connective tissue shades of pink.

In calcified bone tissue, H&E stain is used to study bone structure, bone marrow spaces, osteocytes, osteoblasts, and pathological changes after decalcification.

Section

  • Paraffin-embedded decalcified bone sections
  • Thickness: 4–5 µm
  • Properly fixed and decalcified tissue

Reagents Required

1. Harris or Mayer’s Hematoxylin

2. Acid Alcohol

3. Ammonia Water or Scott’s Tap Water

4. Eosin Solution (1%)

5. Graded Alcohols

6. Xylene

7. DPX Mounting Medium

Procedure

  1. Deparaffinize sections in xylene.
  2. Hydrate through descending grades of alcohol to water.
  3. Stain with hematoxylin for 5–10 minutes.
  4. Wash in running tap water.
  5. Differentiate in acid alcohol.
  6. Wash and blue in ammonia water or Scott’s tap water.
  7. Wash thoroughly in water.
  8. Counterstain with eosin for 1–2 minutes.
  9. Dehydrate through ascending grades of alcohol.
  10. Clear in xylene.
  11. Mount with DPX.

Results

Tissue Component Colour
Nuclei Blue to purple
Cytoplasm Pink
Collagen fibers Pink
Bone matrix Pink
Muscle fibers Pink to red
Red blood cells Bright red

 

Von Kossa Stain 

Principle

  • Von Kossa stain is a special histochemical stain used to demonstrate calcium deposits and mineralized bone tissue.
  • In this method, silver ions replace calcium salts present in the tissue.
  • Upon exposure to light, the silver is reduced to metallic silver, producing a black coloration.

Section

  • Paraffin-embedded bone sections
  • Thickness: 4–5 µm
  • Decalcified or undecalcified sections, depending on the purpose of the study

Reagents Required

1. Silver Nitrate Solution (5%)

2. Sodium Thiosulfate Solution (5%)

3. Nuclear Fast Red

Used as a counterstain.

4. Distilled Water

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Immerse sections in 5% silver nitrate solution.
  3. Expose to bright light or ultraviolet light for 30–60 minutes.
  4. Wash thoroughly in distilled water.
  5. Treat with 5% sodium thiosulfate for 2–5 minutes.
  6. Wash in running water.
  7. Counterstain with Nuclear Fast Red.
  8. Dehydrate, clear, and mount.

Results

Tissue Component Colour
Calcium deposits/mineralized areas Black
Nuclei Red
Background tissue Pink

Alizarin Red S Stain 

Principle

  • Alizarin Red S is a special histochemical stain used for the direct demonstration of calcium salts in tissues.
  • The dye reacts with calcium ions to form an orange-red to deep red complex, allowing easy visualization of calcium deposits.

Section

  • Paraffin-embedded bone sections
  • Thickness: 4–5 µm
  • Suitable for both decalcified and undecalcified tissue sections

Reagents Required

1. Alizarin Red S Solution (1–2%)

2. Acetone or Acetone-Xylene Mixture

3. Distilled Water

Procedure

  1. Deparaffinize and hydrate sections to water.
  2. Stain with Alizarin Red S solution for 2–5 minutes.
  3. Wash thoroughly in distilled water.
  4. Differentiate if required.
  5. Dehydrate rapidly through acetone.
  6. Clear and mount.

Results

Tissue Component Colour
Calcium deposits Orange-red to deep red
Nuclei Light blue (if counterstained)
Background tissue Pale pink

 

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