Histopathological and Cytologic Techniques - Tissue Processing

Overview and Steps of Tissue Processing

• Histology: The study of normal tissues.
• Histopathology: The study of abnormal tissues.
• Tissue Processing Outline: Tissue processing entails a series of sequential physical and chemical steps designed to prepare tissue specimens for microscopic examination.

Core Steps of Tissue Processing

1. Accessioning (Integral step depending on clinical condition/indication)
2. Fixation
3. Decalcification (Integral step depending on clinical condition/indication)
4. Dehydration
5. Clearing / Dealcoholization
6. Infiltration / Impregnation
7. Embedding / Casting / Blocking
8. Trimming
9. Sectioning / Microtomy / Cutting
10. Staining
11. Mounting
12. Labelling

Accessioning

• Specimen Coding Letters: Specific letters are assigned to specimens upon entry into the laboratory to designate the specimen type:
  • S: Surgical
  • A: Autopsy
  • C: Cytology
  • P: Pap smear
• Autopsy Examination:
  • An autopsy is a detailed examination of a deceased patient.
  • It is considered the gold standard for determining the patient's cause of death.
  • If the cause of death is unknown to the attending physician, an autopsy is performed to establish it.
• Separation of Gynecologic Cytology Specimens:
  • Pap smears fall under the category of cytology.
  • In the Philippines, Pap smears are handled separately from non-gynecological cytology specimens because Pap smears are gynecologic specimens containing normal vaginal flora.
  • Cross-Contamination Prevention: Separating these specimens prevents cross-contamination of sterile body fluid samples. For example, Cerebrospinal Fluid (CSF) is one of the most sterile specimens collected from the human body. If CSF is processed or stained using reagents intended for gynecological samples, the CSF may become contaminated with bacilli from normal vaginal flora.
• Labeling Guidelines:
  • Always use a pencil when writing specimen descriptions, labels, or accession numbers.
  • Warning: Never use ink-based markers or pens because the organic solvents and chemicals used in the histopathology laboratory will dissolve and erase ink labels.
• Specimen Dimensions for Processing:
  • Standard specimen processing size: $3 \times 2\,\text{cm}$ in surface area and $3-5\,\text{mm}$ in thickness.
  • During gross examination, the pathologist selects representative defective or pathological sections to fit properly inside standard tissue cassettes.

Fixation: Definition, Aims, and Mechanisms

• Definition: Fixation is the primary and most critical step in tissue processing. It is the process of preserving cells and tissue constituents in a condition as close to life as possible.
• Overall Purpose: To prevent cell degeneration, autolysis, putrefaction, decomposition, and structural distortion.

Primary Aim of Fixation

• To preserve the morphological and chemical integrity of the cell in a life-like manner.
• Preserves cellular architecture and biochemical constituents (especially proteins).
• Enables histochemical and staining techniques. For example, Alkaline Phosphatase (ALP) can be detected qualitatively in histopathology (presence vs. absence), whereas clinical chemistry measures ALP quantitatively.

Secondary Aim of Fixation

• To harden and protect the tissue from the mechanical trauma of subsequent handling and processing.
• Tissue Consistency Changes: Soft tissue becomes firm or rubbery following fixation with formalin.
• Fruit Handling Analogy: Soft tissue subjected to handling without prior fixation suffers mechanical trauma (similar to fruit becoming bruised or "malamog" when bumped or dropped). Fixation hardens tissue to prevent trauma, but it must not become excessively hard (like stone).

Questions & Discussion on Fixation

• Question #1: How does fixation preserve tissues?
  • Answer: Through the preservation of proteins.
  • Explanation: Protein is a major structural constituent of human tissue. Preserving tissue architecture requires maintaining its internal biochemical protein framework.
• Question #2: What is the mechanism of preservation?
  • Answer: Cross-linking of proteins.
  • Explanation: When proteins are exposed to heat or chemical fixatives, they cross-link and aggregate together, forming a structurally stable, insoluble matrix.
• Question #3: When should fixation be done?
  • Answer: Immediately.
  • Explanation: Fixation must be executed immediately upon removal of the tissue or organ from the living body. Once isolated from its blood supply, tissue immediately begins to decline due to autolysis.
  • Consequences of Delay: Delays lead to degeneration, putrefaction, decomposition, and distortion.
  • Operating Room (OR) Protocol: Surgeries can take prolonged periods. Containers filled with fixative must be brought directly into the operating room so specimens can be submerged immediately after excision, rather than waiting for surgery completion or transport.
  • Verification Warning: Never assume an unlabeled liquid in a specimen container is a fixative. Always verify. If a specimen is found submerged only in normal saline, replace it with appropriate fixative immediately.

Methods of Fixation

1. Heat Fixation:
   • Example: Bacterial smear preparation in microbiology (passing the slide through a flame).
   • Effect: Coagulates cellular proteins to preserve cell structure without causing bursting, keeping cells intact during staining.
2. Chemical Fixation:
   • Example: Submerging tissues in liquid chemical fixatives (e.g., $10\%$ Formalin) to induce cross-linking of cellular proteins.

Gross Examination

• Gross examination involves inspecting the specimen visually and tactilley before sectioning.
• Parameters Recorded:
  1. External appearance
  2. Size
  3. Shape
  4. Consistency
  5. Number of tissue pieces (e.g., multiple skin moles)
  6. Color
  7. Three-dimensional measurements in centimeters ($\text{cm}$): Length $\times$ Width $\times$ Height
• Grossing Procedure Example: When receiving a large specimen like a Total Abdominal Hysterectomy, the organ is not processed whole. The pathologist examines the gross appearance, slices the organ into smaller sections, selects defective or suspicious regions, cuts sections to approximately $3-5\,\text{mm}$ thickness, and places them inside labeled tissue cassettes.

Effects and Characteristics of Fixatives

General Effects of Fixatives

• Hardens soft and friable tissues to protect against trauma.
• Makes tissue resistant to damage and osmotic distortion.
• Inhibits autolysis and bacterial decomposition due to antimicrobial properties.
• Increases optical differentiation and staining contrast of cellular constituents by cross-linking proteins.
• Acts as a mordant or accentuator (intensifies stain action, similar to iodine in Gram staining).
• Reduces infectious hazards from pathogenic tissues.

Characteristics of an Ideal Fixative

• Cheap and readily available.
• Stable during storage.
• Safe to handle and non-toxic.
• Prevents tissue distortion and minimizes shrinkage.
• Inhibits bacterial decomposition and autolysis.
• Penetrates tissue rapidly.
• Hardens tissue appropriately.
• Isotonic with minimal osmotic effect on cells.
• Permits subsequent staining procedures.
• Karnovsky's Fixative Exception: Karnovsky's fixative fulfills almost all ideal characteristics, but fails on cost—it is extremely expensive.

Mechanism of Action: Additive vs. Non-Additive

• Additive Fixation:
  • The fixative chemically reacts with and becomes a permanent part of the tissue by forming cross-links or molecular complexes.
  • Examples: Formalin, Mercuric chloride, Osmium tetroxide.
• Non-Additive Fixation:
  • The fixative does not combine chemically with the tissue. Instead, it stabilizes tissue structures by removing bound water, precipitating proteins, and dehydrating the tissue.
  • Examples: Alcoholic fixatives.
  • Decomposition Example: Comparing two bodies—one found in a river and one on a road—the body from the river decomposes and becomes malodorous much faster because water accelerates decomposition. Removing bound water stabilizes tissue against autolytic decay.

Factors Involved in Fixation

• pH:
  • Optimal range: $6.0 - 8.0$
  • Fixation must be maintained near neutral pH. Extreme acidity or alkalinity damages cellular structures and causes pigment artifacts.
• Temperature:
  • Room Temperature (RT): Standard traditional fixation temperature.
  • $40^\circ\text{C}$: Used in automated tissue processors.
  • $0 - 4^\circ\text{C}$: Mandatory for electron microscopy and histochemistry to preserve delicate chemical constituents and enzymes.
  • $60^\circ\text{C}$: Used for rapid diagnostic examination (e.g., skin fixation).
  • $100^\circ\text{C}$: Used for fixing tissues infected with Tuberculosis (TB). Calcified lung tissue from TB is extremely dense and hard; boiling accelerates fixative penetration.
  • Thermal Acceleration & Risks: Heating speeds up fixation, but excessively high temperatures cook and ruin tissue morphology.
• Time Duration:
  • Primary Fixation: $2 - 6\,\text{hours}$.
  • Electron Microscopy: $3\,\text{hours}$.
• Thickness:
  • Light Microscopy: Maximum $2\,\text{cm}^2$ surface area, no thicker than $0.4\,\text{cm}$ ($4\,\text{mm}$).
  • Electron Microscopy: $1 - 2\,\text{mm}^2$
  • Whole Brain Specimen: Suspended whole for $2 - 3\,\text{weeks}$ by a cord tied around the Circle of Willis.
  • Large Solid Organs: Must be sliced thinly or opened to allow fixative penetration.
• Concentration:
  • Higher concentrations are not necessarily better; excessive chemical concentration causes severe tissue damage.
  • $10\%$ Formalin: Standard routine concentration.
  • $3\%$ Glutaraldehyde: Standard routine concentration.
  • $0.25\%$ Glutaraldehyde: Used for electron microscopy.
• Osmolality:
  • Slightly hypertonic solutions ($400 - 450\,\text{mOsm}$) yield optimal structural preservation.

Practical Considerations and Other Factors Affecting Fixation

• Speed: Fixation must begin immediately upon excision.
• Rate of Penetration:
  • Formalin penetrates tissue at a rate of approximately $1\,\text{mm/hour}$.
  • A $2\,\text{mm}$ thick tissue section requires $2\,\text{hours}$ for complete penetration.
• Volume Ratio:
  • $20:1$ Ratio: Fixative volume to tissue volume ratio of $20:1$ provides maximum effectiveness and is the standard rule.
  • $5 - 10:1$ Ratio: Used for Osmium Tetroxide (due to high cost and toxicity).
  • $50 - 100:1$ Ratio: Used for prolonged fixation and museum preparation specimens.
  • Minimum Requirement: The tissue must be completely submerged in the fixative.
• Factors Retarding Penetration:
  • Presence of organic contaminants: Mucus, fat, and blood retard fixative penetration.
  • Laboratory Protocol: Wash blood-soaked tissues with Normal Saline Solution (NSS) prior to immersion in fixative.
  • Cold temperatures reduce penetration rates.
• Factors Accelerating Fixation:
  • Agitation: Mechanical agitation (rotary or vertical oscillation, or pressurized fluid exchange in automated processors) accelerates fluid exchange. Efficient agitation reduces overall processing time by up to $30\%$.
  • Moderate Heat: Warm temperatures ($37 - 56^\circ\text{C}$) accelerate fixation rate.
  • Vacuum application.

Classification and Systematics of Fixatives

Classification According to Composition

1. Simple Fixatives: Contain only one chemical constituent in the solution.
   • Examples: Formaldehyde, Glutaraldehyde, Mercuric chloride, Chromic acid, Picric acid, Glacial acetic acid, Acetone, Alcohol, Osmium tetroxide.
2. Compound Fixatives: Contain two or more chemical constituents combined to obtain optimal balanced effects.

Classification According to Action

1. Microanatomical Fixatives: Preserve overall cellular and tissue architecture without altering spatial and intercellular relationships.
   • Examples: Bouin's solution, Brasil's solution, Zenker's solution, Zenker's formol (Kelly's solution), $10\%$ Neutral Buffered Formalin (NBF), $10\%$ Formol saline, Formol sublimate (Formol corrosive), Heidenhain's SuSa.
2. Cytological Fixatives: Preserve specific intracellular organelles and inclusions.
   • Nuclear Fixatives: Contain Glacial Acetic Acid (pH is acidic; enhances nuclear detail and chromatin visibility).
     • Mnemonic: BFNCH ("Bench")
     • Bouin's fluid
     • Flemming's fluid
     • Newcomer's fluid
     • Carnoy's fluid
     • Heidenhain's SuSa
   • Cytoplasmic Fixatives: Must NEVER contain glacial acetic acid (acetic acid dissolves cytoplasmic structures and mitochondria).
     • Examples: Helly's fluid, Orth's fluid, Regaud's fluid, Flemming's fluid without acetic acid, Formalin with post-chroming.
3. Histochemical Fixatives: Preserve biochemical constituents (enzymes, proteins, carbohydrates, lipids) for histochemical analysis.
   • Mnemonic: FANA
   • Formol saline ($10\%$)
   • Absolute Ethyl Alcohol
   • Newcomer's fluid
   • Acetone

Specific Tissue Element Preservation Guidelines

• Lipid Fixation: Cryostat sections, Formol calcium, Osmium tetroxide, Acid dichromate fixatives.
• Carbohydrate (Glycogen) Fixation: Alcoholic fixatives (Rossman's fluid, Brasil's fluid, Cold absolute alcohol). Warning: Never use water-based fixatives or $10\%$ Formalin for glycogen preservation; water dissolves glycogen.
• Protein Fixation: $10\%$ Neutral Buffered Formalin (NBF), Neutral buffered formaldehyde.

Specific Chemical Fixatives

Aldehyde Fixatives

1. Formaldehyde (Formalin):
   • Gas produced by the oxidation of methyl alcohol.
   • Concentration: Pure stock solution is $37 - 40\%$ formaldehyde gas in water. The working concentration for tissue fixation is $10\%$ formalin (which equals $3.7 - 4.0\%$ free formaldehyde gas).
   • Handling Warning: Concentrated solutions must never be neutralized without precaution, as this can trigger explosions.
   • Formalin Pigment Artifacts:
     • Paraformaldehyde: White crystalline precipitate formed on prolonged standing or cold storage. Removed by adding $10\%$ methanol or by filtration.
     • Acid Formaldehyde Hematin: Dark brown/black granular pigment formed when unbuffered acidic formalin reacts with hemoglobin in blood-rich tissues. Obscures tissue details. Removed using saturated picric acid, alcoholic potassium hydroxide (KOH), Kardasewitsch method, or Lillie's method.
2. $10\%$ Neutral Buffered Formalin (NBF) / $\text{PO}_4$ Buffered Formalin:
   • Best general tissue fixative.
   • Best fixative for tissues containing iron granules (demonstrated via Prussian blue stain).
   • Contains a double phosphate buffer to maintain neutral pH ($pH\,6.8 - 7.2$), preventing acid formaldehyde hematin pigment formation.
   • Rate of penetration: $1\,\text{mm/hour}$.
3. $10\%$ Formol Saline:
   • Formalin diluted in $0.9\%$ / $10\%$ NaCl.
   • Recommended for Central Nervous System (CNS) specimens, lipids, and routine post-mortem autopsy materials.
4. Formol Calcium:
   • Formalin solution containing calcium chloride.
   • Recommended for preserving lipids in frozen sections.
5. Formol Corrosive (Formol Sublimate):
   • Formalin combined with mercuric chloride (sublimate).
6. Glutaraldehyde:
   • Standard fixative for electron microscopy ($3\%$ solution) and electron histochemistry/immunocytochemistry ($0.25\%$ solution).
7. Karnovsky's Paraformaldehyde-Glutaraldehyde:
   • Combination of paraformaldehyde and glutaraldehyde.
   • One of the absolute best fixatives for electron microscopy and immunohistochemistry; drawback is that it is extremely expensive.
8. Acrolein:
   • Rapidly penetrating aldehyde used for immunohistochemistry and immunocytochemistry.

Metallic Fixatives

1. Mercuric Chloride:
   • Heavy metal fixative that penetrates rapidly and enhances nuclear detail.
   • Disadvantage: Forms dense black mercuric chloride pigments in tissue and causes tissue shrinkage.
   • Mercuric Fixative Mnemonic: BOSCHZ
     • B5 fixative: Excellent for Bone Marrow (BM) biopsies (Fixation time: $1.5 - 2\,\text{hours}$).
     • Ohlmacher's fluid
     • Schaudinn's fluid
     • Carnoy-Lebrun fluid
     • Heidenhain's SuSa: "Su" = sublimate (mercuric chloride), "Sa" = saure (acid; contains acetic acid). Excellent for tumor biopsies (skin) (Fixation time: $3 - 12\,\text{hours}$).
     • Zenker's fluid: Contains mercuric chloride and potassium dichromate. Used for small pieces of liver, spleen, connective tissue fibers, and nuclei (Fixation time: $12 - 24\,\text{hours}$).
     • Zenker's Formol (Helly's Solution): Zenker solution with formalin added. Fixative for pituitary gland, bone marrow, and blood-containing organs.
   • Dezenkerization Process: The mandatory step to remove black mercuric chloride artifacts from sections fixed in mercuric fixatives:         $\text{Water} \rightarrow 0.5\%\,\text{Iodine in } 70\%\,\text{Ethanol} \rightarrow \text{Water} \rightarrow \text{Sodium Thiosulfate} \rightarrow \text{Water}$
     • Mercuric chloride binds with iodine to form mercuric iodide. Sodium thiosulfate then removes the excess iodine and renders the compound soluble in water, dissolving black deposits.
2. Lead Fixatives:
   • Fixes connective tissue mucin and is recommended for acid mucopolysaccharides.
3. Chromate Fixatives:
   • Chromic Acid: Used in $1 - 2\%$ aqueous solution. Precipitates all proteins and preserves carbohydrates. Strong oxidizing agent; must be mixed with reducing agents (like formalin) immediately before use to prevent decomposition.
   • Regaud's Fluid (Moller's / Molliflex): Demonstrates chromatin, mitochondria, mitotic figures, Golgi bodies, and RBCs.
   • Orth's Fluid: Used to study early degenerative processes, tissue necrosis, and myelin preservation.
   • Potassium Dichromate ($\text{K}_2\text{Cr}_2\text{O}_7$): Preserves lipids and mitochondria ($pH\,4.5 - 5.2$).

Picric Acid Fixatives

• Properties: Excellent for glycogen. Precipitates all proteins by forming crystalline picrates with amino acids and reacting with histones.
• Warnings:
  • Picric acid is highly explosive when dry.
  • Never wash tissues in water prior to dehydration when using picric acid fixatives.
  • Imparts an intense yellow stain to tissues.
• Removal of Yellow Pigment: Excess picric acid pigment is removed by washing tissue blocks in $70\%$ ethanol.
• Formulations:
  1. Bouin's Fluid / Solution:
     • Recommended for fixation of embryos and pituitary biopsies.
     • Standard fixation time: $24\,\text{hours}$. Prolonged storage in Bouin's causes severe acid hydrolysis and loss of stainable DNA and RNA.
  2. Brasil's Alcoholic Picroformol Fixative (Brasil's Fluid):
     • Best fixative for glycogen.

Glacial Acetic Acid

• Water-free (anhydrous) acetic acid that solidifies and freezes at $16 - 17^\circ\text{C}$.
• Not used alone due to severe tissue swelling properties; incorporated into compound fixatives (typically at $5\%$ concentration) to counteract tissue shrinkage caused by other reagents.
• Fixes and precipitates nucleoproteins, chromosomes, and chromatin material (nuclear fixative).

Alcohol Fixatives

• Protein denaturants; act as non-additive fixatives by removing bound water.
• Disadvantage: Cause excessive tissue brittleness and hardness if used for routine fixation.
• Types:
  1. Methanol (Methyl Alcohol): Used for fixing dry and wet blood smears (Peripheral Blood Smears - PBS) and bone marrow smears. Highly toxic to optic nerves (inhalation/ingestion causes blindness).
  2. Ethanol (Ethyl Alcohol): Preserves but does not fix glycogen. Avoid water-containing fixatives when preserving glycogen. Causes polarization artifact (glycogen is pushed toward the cell periphery).
  3. Isopropanol (Isopropyl Alcohol): Used for fixing touch preparations.
  4. Carnoy's Fluid: The most rapid fixative. Fixation time is $1 - 3\,\text{hours}$. Used for fixing chromosomes, lymph glands, and urgent intraoperative biopsies.
  5. Alcoholic Formalin (Gendre's Fixative): Used to preserve sputum specimens for direct sputum microscopy.
  6. Newcomer's Fluid: Used for mucopolysaccharides and nuclear proteins; gives superior results in Feulgen stain (DNA demonstration) compared to Carnoy's.

Questions & Discussion on Specific Fixatives

• Question #4: What is the classification of a fixative whose name contains the term "Zenker"?
  • Answer: Mercuric chloride fixative (heavy metal).
• Question #5: Is aldehyde fixative an additive or non-additive fixative?
  • Answer: Additive fixative.
• Question #6: Is metallic fixative an additive or non-additive fixative?
  • Answer: Additive fixative.
• Question #7: Is alcohol fixative an additive or non-additive fixative?
  • Answer: Non-additive fixative. Alcohol removes bound water, inhibiting decomposition.
• Question #8: What is the most rapid fixative called?
  • Answer: Carnoy's Fluid.
• Question #9: What is the best general fixative?
  • Answer: Neutral Buffered Formalin (NBF).
• Question #10: What is the best fixative used in preserving glycogen?
  • Answer: Brasil's Fluid.

Other Fixative Agents

1. Osmium Tetroxide:
   • Pale yellow powder that dissolves in water up to $6\%$ at $20^\circ\text{C}$ to form a strong oxidizing solution.
   • Inhibits Hematoxylin staining (cannot be used for H&E stain).
   • Forms black precipitate crystals (osmium oxide) in lipids. Used for electron microscopy and lipid staining.
   • Flemming's Solution: Chrome-osmium-acetic acid mixture. Fixes fat and nuclear detail permanently.
   • Flemming's Solution without Acetic Acid: Preserves cytoplasmic details and mitochondria.
2. Trichloroacetic Acid (TCA):
   • Precipitates proteins ($10\%\,\text{w/v}$ concentration). Functions as both a fixative and a decalcifying agent. Swelling effect counteracts tissue shrinkage.
3. Acetone:
   • Used at ice-cold temperatures ($-5^\circ\text{C} \text{ to } 4^\circ\text{C}$).
   • Used for preserving diffusible enzymes (phosphatases and lipases).
   • Used for fixing brain tissue for Rabies diagnosis (detection of Negri bodies under the microscope).
   • Used in enzyme histochemistry.
4. Heat Fixation / Microwave Fixation:
   • Causes thermal coagulation of proteins. Used for bacteriologic smears.
   • Optimal microwave temperature: $45 - 55^\circ\text{C}$.
   • Underheating: Leads to poor sectioning quality.
   • Overheating ($>65^\circ\text{C}$): Causes cellular vacuolation, cytoplasmic overstaining, and pyknotic nuclei.

Fixation Terminologies, Principles, Precautions, and Difficulties

Fixation Terminologies

• Secondary Fixation: Placing an already fixed tissue into a second, completely different fixative solution to achieve special staining effects or further firm soft tissues.
• Post-Chromatization: A specific form of secondary fixation where primarily fixed tissue is submerged in an aqueous solution of $2.5 - 3\%$ Potassium Dichromate.
• Washing Out: The process of removing excess fixative, pigments, or chemical artifacts from tissue sections using appropriate solvents.

Fixation Pigments and Washing Out Summary

17 Principles and Precautions in Fixation

1. Surgical and autopsy materials must be fixed immediately after removal.
2. All tissue containers and cassettes must be properly labeled with pencil.
3. If tissues are refrigerated, avoid $0^\circ\text{C}$ (freezing causes ice crystal artifacts and repeated freeze-thaw cycles destroy tissue architecture).
4. Tissue sections should not exceed $5\,\text{mm}$ thickness (except in lung edema, where $1 - 2\,\text{cm}$ thickness is acceptable).
5. Purulent exudates, transudates, or suspicious infectious fluids must be saved for microbiological culture before placing tissue in fixatives.
6. Fixative volume must be adequate ($20:1$ ratio).
7. Prevent specimen contamination.
8. Wash tissues thoroughly before staining.
9. Solid parenchymal organs must be injected with fixative directly into their structure.
10. Hollow organs (e.g., stomach, intestines) should be opened or packed with cotton soaked in fixative (cotton acts as a weight to submerge organs).
11. Air-filled lungs naturally float on liquid fixatives. To keep them submerged, cover the lungs with several layers of gauze pads soaked in fixative. (If lung tissue sinks without gauze, it indicates fluid accumulation or potential drowning as cause of death).
12. Human brain specimens should undergo intravascular perfusion or be suspended whole by a cord tied around the Circle of Willis for $2\,\text{weeks}$.
13. Eyes must NEVER be dissected before fixation. Eyes are fixed whole in containers to prevent loss of vitreous humor.
14. Frozen sections may develop ice crystal artifacts; touching the tissue block briefly with a warm finger softens tissue for cutting.
15. Muscle tissues must be stretched on a card for $30\,\text{minutes}$ prior to fixation to prevent curling and rigor distortion.
16. Water must NEVER be used on tissues intended for glycogen demonstration (glycogen is water-soluble).
17. Excessively hard tissues can be washed overnight in running water and submerged in $4\%$ aqueous phenol for $1 - 3\,\text{days}$ (Lendrum's Method) to soften.

Difficulties Encountered in Fixation

Decalcification

• Definition: The process of removing calcium or lime salts from bones, teeth, and calcified tissues following fixation.
• Indication: An optional step performed only when tissues contain hard calcium deposits.
• General Rules:
  • Fixation must precede decalcification.
  • Recommended fluid to tissue volume ratio: $20:1$.
  • Optimum temperature: Room temperature ($18 - 30^\circ\text{C}$).
  • Standard duration: $24 - 48\,\text{hours}$ (dense cortical bone requires up to $14\,\text{days}$ or longer).
  • Effect of Heat: Heat accelerates decalcification but severely damages tissue. At $37^\circ\text{C}$, nuclear staining with Van Gieson's stain is impaired. At $55^\circ\text{C}$, tissues undergo complete digestion within $24 - 48\,\text{hours}$.

Types of Decalcifying Agents

1. Acids:
   • Nitric Acid ($5 - 10\%$): Most common rapid decalcifier. Used for routine and urgent biopsies.
     • Formol Nitric Acid: Rapid acting; produces less destruction than $10\%$ aqueous nitric acid.
     • Perenyi's Fluid: Acts as both a tissue softener and a decalcifying agent. Imparts a yellow color due to nitrous acid formation, which is prevented/removed by neutralizing with $5\%$ sodium sulfate and washing in running tap water for $12\,\text{hours}$.
     • Phloroglucin Nitric Acid: The most rapid decalcifying agent.
   • Hydrochloric Acid (HCl): Inferior to nitric acid due to slower action and greater tissue distortion.
     • Von Ebner's Solution: Contains HCl; used for small pieces of bone and surface decalcification.
   • Formic Acid ($5\%$): Best general decalcifying agent (counterpart to NBF in fixation). Functions as both a fixative and decalcifying agent.
   • Trichloroacetic Acid ($5\%$ TCA): Functions as a protein precipitant, fixative, and decalcifier.
   • Sulfurous Acid: Very weak solution suitable only for minute bone spicules.
   • Chromic Acid (Flemming's Fluid): Fixative and decalcifier for minute bone spicules; forms bottom precipitates requiring frequent fluid changes.
2. Chelating Agents:
   • Reagents that bind calcium ions (sequestration).
   • EDTA (Ethylene Diamine Tetraacetic Acid / Versene): Versene is the powder preparation of EDTA. Excellent for electron microscopy, immunohistochemistry, and enzyme staining.
3. Ion Exchange Resins:
   • Resins that accelerate decalcification when using formic acid solutions. Not recommended for mineral acids (HCl or $\text{HNO}_3$).
4. Electrophoresis:
   • Employs electric current to move positively charged calcium ions ($\text{Ca}^{2+}$) toward the negative electrode (cathode).
   • Question #11: Where will calcium go during electrophoretic decalcification?
     • Answer: It migrates towards the cathode.

Measuring the Extent of Decalcification

1. Physical or Mechanical Method:
   • Probing or gently bending the tissue with a needle/stick. Crude and unreliable; can introduce mechanical damage artifacts.
2. X-Ray Method:
   • Most ideal, most sensitive, and most reliable method (though expensive). Tissues are fully decalcified when bone structures are no longer visible on an X-ray image.
3. Chemical Method (Ammonium Oxalate Test):
   • The decalcifying fluid is mixed with ammonium oxalate.
   • White precipitate formation: Indicates calcium oxalate presence (decalcification is incomplete).
   • Solution remains clear: Indicates absence of calcium (decalcification is complete).

Tissue Softeners

• Used to soften excessively hard tissues (chitin, hair, nails, dense fibrous tissue):
  1. Lendrum's Method: Submerging tissue in $4\%$ Phenol.
  2. Molliflex: Softening agent (disadvantage: tissues appear soapy and swollen).
  3. $2\%$ Hydrochloric Acid.
  4. $1\%$ Hydrochloric Acid in $70\%$ Alcohol.

Post-Decalcification Process

• Purpose: Mandatory neutralization step to remove and neutralize residual acid bound to tissue, preventing continued tissue destruction.
• Methods:
  1. Immersing tissue in saturated Lithium Carbonate or $5 - 10\%$ Sodium Bicarbonate.
  2. Rinsing in running tap water.
  3. Storing in formol saline with $15\%$ sucrose or Phosphate-Buffered Saline (PBS) with $15 - 20\%$ sucrose at $4^\circ\text{C}$.

Dehydration

• Aim: To remove all water and fixative from the tissue block and replace it with dehydrating fluid in preparation for impregnation.
• Principle: Tissues are passed through increasing strengths of alcohol (e.g., $65/70\% \rightarrow 95\% \rightarrow 100\%$ absolute alcohol) to prevent cell distortion caused by sudden osmotic changes.
• Delicate / Embryonic Tissues: Must begin dehydration in a lower concentration ($30\%$ ethyl alcohol).
• Rehydration: Passing tissues through decreasing concentrations of alcohol to reintroduce water.

Characteristics of an Ideal Dehydrating Solution

• Dehydrates rapidly without tissue shrinkage or distortion.
• Does not evaporate rapidly.
• Dehydrates fatty tissues.
• Does not harden tissues excessively.
• Does not remove stains.
• Non-toxic and non-flammable (Note: alcohol fail on flammability, as all dehydrating alcohols are highly flammable).

Dehydrating Agents

1. Alcohol:
   • Ethanol (Ethyl Alcohol): Best and most common dehydrating agent for routine tissue processing.
   • Methyl Alcohol (Methanol): Used for dehydrating blood films and Peripheral Blood Smears (PBS).
   • Butyl Alcohol (Butanol): Utilized in plant and animal microtechniques.
   • Industrial Methylated Spirit (Denatured Alcohol): Ethanol plus a small percentage of methanol.
   • Isopropyl Alcohol (Isopropanol): Excellent substitute for ethanol; widely used in microwave processing schedules.
2. Acetone: Rapid dehydrating agent and fixative; recommended for urgent biopsies.
3. Diethylene Dioxide (Dioxane): Dual-purpose reagent (acts as both dehydrating agent and clearing agent).
4. Ethylene Glycol Monoethyl Ether (Cellosolve): Dual-purpose dehydrating and clearing reagent.
5. Tetrahydrofuran (THF): Dual-purpose dehydrating and clearing reagent.

Note on Dual-Purpose Reagents: Dioxane, Cellosolve, and THF allow tissues to proceed directly from dehydration to paraffin infiltration. However, they are expensive and toxic.

Additives and Indicators in Dehydration

• $4\%$ Phenol in $95\%$ Ethanol: Added to dehydrating baths as a tissue softener for hard, dense fibrous tissues, tendons, or nails.
• Anhydrous Copper Sulfate:
  • Added to the final absolute alcohol dehydrating bath as a water indicator and dehydrant.
  • White powder preparation: Indicates complete dehydration (no water present).
  • Turns Blue: Indicates presence of water (incomplete dehydration).
• Xylene Incomplete Dehydration Test:
  • When tissues are transferred into xylene, if the xylene turns milky/cloudy, residual water is present (incomplete dehydration).
  • If xylene remains clear, no water is present (complete dehydration).

Clearing / Dealcoholization

• Aim: To remove the dehydrating agent (alcohol) from the tissue and replace it with a fluid miscible with both alcohol and paraffin wax.
• Term Origin: Called clearing because reagents have a high refractive index, making tissue sections transparent or clear.
• Ideal Characteristics: Miscible with alcohol, paraffin wax, and mounting media (Canada balsam); high refractive index; low viscosity.
• Viscosity Factor: High viscosity retards penetration rate.
• Exposure Hazard: Excessive exposure to clearing agents renders tissue hard and brittle.

Clearing Agents

1. Xylene (Xylol):
   • Most commonly used routine clearing agent.
   • Clearing time: $30 - 60\,\text{minutes}$.
   • Advantage: Rapid and cheap; used for embedding and mounting.
   • Disadvantage: Exposure exceeding $3\,\text{hours}$ makes tissue excessively hard and brittle.
   • Water Indicator: Turns milky when water is present.
2. Toluene:
   • Clearing time: $1 - 2\,\text{hours}$.
   • Advantage: Tissues do not become hard or brittle even after $24\,\text{hours}$ immersion. Not carcinogenic.
   • Disadvantage: Slower penetration; expensive and toxic.
3. Benzene:
   • Clearing time: $15 - 60\,\text{minutes}$.
   • Advantage: Rapid clearing for urgent biopsies; does not harden tissue.
   • Disadvantage: Carcinogenic. Causes severe bone marrow damage and aplastic anemia.
4. Chloroform:
   • Clearing time: $6 - 24\,\text{hours}$ (slowest clearing agent).
   • Advantage: Recommended for tough tissues, nervous tissue, lymph nodes, embryos, and large tissue specimens. Does not cause brittleness.
   • Disadvantage: Causes hepatotoxicity (liver damage). Tissues tend to float in chloroform (Remedy: wrap tissue in cotton gauze to sink).
5. Cedarwood Oil:
   • Recommended for CNS tissues, cytological specimens, and delicate histology. No tissue distortion even upon prolonged storage, but extremely expensive and slow.
6. Aniline Oil: Recommended for embryos, insects, and delicate tissues.
7. Clove Oil: Causes minimal shrinkage, but deteriorates and becomes adulterated over time.
8. Carbon Tetrachloride: Similar to chloroform; used for double embedding.
9. Glycerine / Gum Syrup: Clears tissues without dealcoholization (used in frozen sections).

Comparative Summary of Clearing Agents

Impregnation / Infiltration

• Aim: To fill up all tissue spaces, cavities, and cellular voids with a solid supporting medium (such as paraffin wax), giving tissue sufficient rigidity to permit sectioning into thin ribbons without crumbling.

Paraffin Wax Impregnation

• Properties: Most common, simplest, and best embedding medium. Rapid processing within $24\,\text{hours}$.
• Melting Point (MP): Standard melting point is $56^\circ\text{C}$.
  • Laboratory Temperature Adjustments:
    • If lab temperature is $20 - 24^\circ\text{C}$: Set wax MP to $54 - 58^\circ\text{C}$.
    • If lab temperature is $15 - 18^\circ\text{C}$: Set wax MP to $50 - 54^\circ\text{C}$.
• Paraffin Oven Temperature: Regulated at $55 - 60^\circ\text{C}$ ($2 - 5^\circ\text{C}$ above the melting point of the wax).
  • Warning: Overheating paraffin causes tissues to become hard, cooked, and brittle.
• Water Removal from Reused Wax: Heating used wax to $100 - 105^\circ\text{C}$ evaporates accumulated water. Used wax should be filtered (Greens No. 904 paper) and reused a maximum of twice.
• Storage Advantage: Formalin-fixed, paraffin-embedded (FFPE) tissue blocks can be stored indefinitely at room temperature for historical studies and nucleic acid recovery.

Three Methods of Paraffin Wax Impregnation

1. Manual Processing:
   • Tissues are manually transferred through at least 4 changes of melted paraffin wax at $15\,\text{minute}$ intervals, followed by a final fresh paraffin immersion for $3\,\text{hours}$.
2. Automatic Tissue Processor (AutoTechnicon):

*   Employs an automated machine that moves tissue cassettes sequentially through fixative, dehydrating, clearing, and impregnating stations.
*   *Features*: Standard 12 individual processing steps/jars; transfer arm; electrical clock timer; thermostat-controlled paraffin baths set at $3^\circ\text{C}$ above wax MP.
*   *Agitation*: Continuous vertical or rotary oscillation accelerates fluid exchange.
*   *Maintenance Warning*: If paraffin smells like xylene or clearing agent, the wax is polluted and must be completely replaced.

3. Vacuum Embedding:

*   Involves wax impregnation under negative atmospheric pressure inside a sealed vacuum oven.
*   *Equipments*: Heavy brass chamber, airtight rubber valve glass lid, manometer, and thermostatically controlled water jacket maintained at $2 - 4^\circ\text{C}$ above wax MP.
*   *Pressure Limit*: Vacuum negative pressure maintained at **$400 - 500\,\text{mmHg}$**.
*   *Advantage*: Reduces impregnation time by $25 - 75\%$. Removes trapped air bubbles from porous organs (e.g., lungs, brain, decalcified bone, eyes).

Paraffin Wax Substitutes

• Paraplast: Highly purified paraffin mixed with synthetic plastic polymers. MP $56 - 57^\circ\text{C}$. More elastic and resilient; produces uniform serial ribbons for dense tissues (bone, brain).
• Embeddol: MP $56 - 58^\circ\text{C}$. Less brittle and less compressible than paraffin.
• Bioloid: Synthetic wax specially recommended for embedding whole eye sections.
• Tissue Mat: Paraffin mixture containing rubber.
• Ester Wax: Harder wax, MP $46 - 48^\circ\text{C}$. Soluble in $95\%$ alcohol; enables impregnation without prior clearing. Cut using sliding microtomes.
• Water-Soluble Wax (Carbowax):
  • Polyethylene Glycol (PEG) polymers, MP $38 - 42^\circ\text{C}$ or $45 - 56^\circ\text{C}$.
  • Miscible directly with water. Does not require dehydration or clearing (tissue goes directly from fixation/washing into melted Carbowax).
  • Ideal for histochemical enzyme studies because exposure to alcohol and xylene is avoided.

Non-Paraffin Impregnating Media

1. Celloidin (Colloidion / Colloidin):
   • Purified nitrocellulose soluble in ether and alcohol.
   • Used for large hollow cavities, hard dense bones/teeth, and whole brain/embryo sections. Does not require heat (zero thermal shrinkage).
   • Processing Time: Extremely slow (takes days to weeks).
   • Wet Celloidin: Tissue blocks stored in $70 - 80\%$ alcohol until cutting. Preferred for whole eye sections.
   • Dry Celloidin: Stored in Gilson's mixture (equal parts chloroform and cedarwood oil).
   • Low Viscosity Nitrocellulose (LVN): Soluble form used at higher concentrations for faster penetration.
2. Gelatin:
   • Water-soluble; does not require dehydration or clearing.
   • Used for delicate histochemical/enzyme sections and frozen sections to prevent fragmentation of friable tissue. Tissue thickness must be $\le 2 - 3\,\text{mm}$. Contains $1\%$ phenol to prevent mold growth.
3. Plastics / Resins:
   • Used for electron microscopy and thin plastic embedding.
   • Classifications:
     • Epoxy: Bisphenol A (Araldite), Glycerol (Epon), Cyclohexene dioxide (Spurr).
     • Polyester
     • Acrylic

Embedding / Casting / Blocking

• Definition: The process of placing the infiltrated tissue in a precisely oriented position inside a mold filled with liquid embedding medium, which is then allowed to solidify.
• Melted Paraffin Temperature for Embedding: Maintained at $5 - 10^\circ\text{C}$ above the melting point of the wax to ensure a liquid state during manipulation.

Orientation

• Definition: Correct spatial alignment of the tissue section within the mold, on the microtome, and on the glass slide.
• Critical Importance: Proper orientation ensures diagnostically vital tissue structures are exposed during cutting. Incorrect placement leads to missing or destroying critical pathological elements.

Double Embedding

• Definition: Process where tissue is infiltrated and embedded first in a primary supporting medium (e.g., celloidin or agar), and then infiltrated and embedded a second time in paraffin wax.

Types of Embedding Molds

• Leuckhart's Embedding Molds:

*   Consists of two L-shaped heavy brass or metal strips resting on a flat metal plate. Adjustable size for large specimens.

• Compound Embedding Unit:

*   Series of interlocking metal plates forming multiple compartments to embed multiple tissue blocks simultaneously.

• Plastic Embedding Rings:

*   Ring placed over specimen, filled with wax, and attached to a block holder.

• Metal Base Molds:

*   Stainless steel base molds fitted with plastic embedding rings that serve as block holders during microtomy.

• Disposable Molds:
  • Peel Away: Thin plastic molds peeled off once wax solidifies.

*   *Plastic Ice Trays*: Multi-compartment trays.

*   *Paper Boats*: Folded paper molds; cheap, customizable size.

Trimming

• Definition: The removal of excess paraffin wax from the block edges to expose the tissue face.
• Ideal Block Geometry: Four-sided prism or truncated pyramid.
• Note: Molds like Peel Away yield pre-shaped blocks that minimize trimming.

Sectioning / Microtomy / Cutting

• Definition: Microtomy is the process by which embedded tissue blocks are cut into ultra-thin slices (tissue ribbons/sections) using a precision mechanical instrument called a microtome.

Section Thickness Specifications

• Histopathology (Light Microscopy):
  • General Thickness Range: $4 - 6\,\mu\text{m}$
  • Routine Section Thickness: $3 - 5\,\mu\text{m}$
  • Frozen Section Thickness: $10 - 15\,\mu\text{m}$ (thicker because tissue is unfixed/unprocessed)
• Electron Microscopy:
  • General Thickness: $0.5\,\mu\text{m}$
  • Very Thin Sections: $60 - 100\,\text{nm}$
  • Semi-Thin Sections: $0.5 - 1\,\mu\text{m}$

Microtome Knife Profiles

• Plane-Concave Knife ($25\,\text{mm}$ length, Profile A):

*   One side flat, one side concave. Concave side used for paraffin sections; flat side used for celloidin sections.

• Biconcave Knife ($120\,\text{mm}$ length, Profile B):

*   Both sides concave. Used for cutting paraffin sections on rotary microtomes.

• Plane-Wedge Knife ($100\,\text{mm}$ length, Profile C):

*   Both sides straight wedge-shaped. Used for frozen sections or extremely hard/tough specimens on sledge/sliding microtomes.

Knife Materials

• Disposable blades (standard modern practice).
• Stainless steel, Tungsten carbide, Steel.
• Glass knives (Ralph knives for ultramicrotomy).
• Diamond knives (used for cutting plastic/resin sections for electron microscopy).

Microtome Knife Angles

• Bevel Angle: Angle formed between the cutting facets. Standard: $27^\circ - 32^\circ$.
• Clearance Angle: Angle between the block face and the cutting facet of the knife. Standard: $0 - 15^\circ$ (prevents friction between block and knife).
• Cutting Angle: Angle formed between the line of cut and the knife facets. Standard: $15^\circ$.

Knife Sharpening Stages: Honing vs. Stropping

Types of Microtomes

1. Rocking Microtome (Cambridge):

*   Invented by Paldwell Trefall in 1881. Simplest microtome. Cuts sections in a curved plane by up-and-down rocking. Disadvantage: difficulty reorienting block.

2. Sliding Microtome:

*   Invented by Adams in 1789.
*   *Base Sledge*: Block moves on a track; knife remains stationary.
*   *Standard Sliding*: Block remains stationary; knife slides horizontally across the block. **Warning**: Dangerous due to exposed sliding knife (acting like a guillotine/garrote).

3. Rotary Microtome (Minot):
   • Invented by Minot in 1885-1886.
   • Most common microtome used today for paraffin-embedded sections.
   • Operating Principle: Rotation of the handwheel operates a synchronized feed mechanism. 3 essential parts: Block Holder (moves vertically), Knife Carrier (advances horizontally based on thickness setting), and Pawl/Ratchet Feed Wheel/Adjustment Screws.
4. Vibrotome: Cuts unfixed, unfrozen tissue sections submerged in buffer for histochemical enzyme demonstrations.
5. Ultrathin Microtome (Ultramicrotome):

*   Equipped with a viewing microscope eyepiece and diamond knife. Cuts ultrathin sections ($60 - 100\,\text{nm}$) for electron microscopy.

6. Freezing Microtome / Cryostat:

*   Invented by Queckett in 1848. Uses cold microtomy inside a refrigerated cabinet ($-10 \text{ to } -30^\circ\text{C}$) for intraoperative frozen sections.
*   *Clinical Purpose*: Rapid diagnostic evaluation during ongoing surgery (turnaround time within minutes). Helps surgeons determine malignancy and surgical margins immediately (e.g., expanding from breast lump excision to full mastectomy if aggressive cancer is identified).

Types of Tissue Sections and Corresponding Microtomes

• Paraffin Section: Cut using Rocking or Rotary Microtomes.
• Celloidin Section: Cut using Sliding Microtomes.
• Frozen Section: Cut inside Cryostat / Freezing Microtome.

Auxiliary Equipment in Microtomy

• Flotation Water Bath:
  • Used to float, stretch, and flatten paraffin tissue ribbons, removing wrinkles and folds.
  • Temperature Setting: Set $10^\circ\text{C}$ below the melting point of the paraffin wax used.
  • Example: If wax MP is $56^\circ\text{C}$, the flotation bath temperature is set to $46^\circ\text{C}$.
• Drying Oven / Slide Warmer:
  • Used to melt wax and dry slides after fishing sections out of the flotation bath.
  • Temperature Setting: Set $5^\circ\text{C}$ above the melting point of the wax.
• Forceps: Fine-tipped serrated forceps used to manipulate floating ribbons.
• Clean Slides: Clean, grease-free glass slides used to pick up sections.

Staining

• Definition: Staining imparts artificial color to tissue components to increase optical contrast and differentiation under the microscope.

Groups of Tissue Staining

1. Histologic Staining: Direct interaction between dyes and cellular structural constituents.
2. Histochemical Staining: Coloration produced by chemical reactions targeting specific biochemical constituents (e.g., glycogen, enzymes) rather than general tissue structures.
3. Immunochemical (Immunohistochemical) Staining: Uses labeled antigen-antibody interactions to detect specific phenotypic markers.

Methods of Staining

• According to Mordant/Accentuator:
  • Direct Staining: Application of aqueous or alcoholic dye solutions directly to tissue (e.g., Methylene blue, Eosin).
  • Indirect Staining: Uses an intermediate agent—a mordant or accentuator—to link the dye to tissue or intensify dye action. Examples: Ehrlich's hematoxylin (potassium alum mordant), Weigert's hematoxylin (iron mordant), Loeffler's methylene blue (potassium hydroxide accentuator), Carbol fuchsin (phenol accentuator).
• According to Differentiator:
  • Progressive Staining: Staining is stopped once desired color intensity is reached. No differentiator (decolorizer) is applied. Examples: Carazzi's hematoxylin, Mayer's hematoxylin.
  • Regressive Staining: Tissue is intentionally overstained, and excess stain is selectively removed using a differentiator/decolorizer (e.g., acid alcohol). Example: Harris' hematoxylin.
• According to Color Result:
  • Orthochromatic Staining: Tissue elements stain the exact same color as the dye solution used (e.g., Methylene blue staining structures blue).
  • Metachromatic Staining: Tissue elements stain a color distinctly different from the original dye solution color (e.g., Methyl violet, Bismarck brown, Toluidine blue staining structures reddish-purple).
• Vital Staining:
  • Intravital Staining: Injecting dye into a living animal body (e.g., Lithium, Carmine, India ink).
  • Supravital Staining: Staining living cells immediately after removal from the body (e.g., Neutral red [best vital stain], Janus green for mitochondria, Trypan blue, Reticulocyte count in hematology).

Categories of Stains

1. Natural Stains: Derived from natural plant or animal sources.
   • Hematoxylin: Extracted from the heartwood of Hematoxylon campechianum.
   • Cochineal Dyes: Extracted from female Coccus cacti scale insects. Yields Carmine dye. Best's Carmine is used for glycogen demonstration.
   • Orcein: Extracted from lichens; used for staining elastic fibers.
   • Saffron
2. Synthetic Stains (Aniline / Coal Tar Dyes):
   • Derived from hydrocarbon benzene.
   • Chemical Formulation: Chromogen (chromophore imparting visible color) + Auxochrome (salt-forming group attaching dye to tissue) = Dye.

Principles and Questions on Staining Chemistry

• Question #12: How long do we keep tissue blocks and glass slides in histopathology specimen retention?
  • Answer: Indefinitely.
• Question #13: What is the pH of the cell nucleus—acidic or basic?
  • Answer: Acidic (due to Deoxyribonucleic Acid - DNA).
• Rules of Chemical Affinity: Opposite charges attract:
  • Acidic tissue structures (e.g., DNA in nucleus) are basophilic; they attract basic dyes (e.g., Hematoxylin, Methylene blue).
  • Basic tissue structures (e.g., proteins in cytoplasm) are acidophilic; they attract acidic dyes (e.g., Eosin, Picric acid).
  • Neutral Dyes: Formed by combining aqueous acid and basic dyes (e.g., Romanowsky dyes); stain nucleus and cytoplasm simultaneously.

Common Staining Solutions

Ripening of Hematoxylin

• Hematoxylin itself is non-staining and must be oxidized (ripened) into Hematin.
• Natural Ripening: Exposure to air and sunlight (takes $3 - 4\,\text{months}$). Examples: Ehrlich's and Delafield's hematoxylin.
• Artificial Ripening: Chemical oxidation using chemical agents ($\text{H}_2\text{O}_2$, Mercuric oxide, Potassium permanganate, Sodium iodate). Examples: Mayer's and Harris' hematoxylin.

Specific Hematoxylin Formulations

1. Aluminum Hematoxylins (Alum Mordant): Stains nuclei red initially, then turns blue (blueing step). Routinely used in H&E.
   • Harris Hematoxylin: Artificially ripened with mercuric oxide. Used in routine H&E, cytology (Pap smears), and sex chromosome staining.
   • Ehrlich's Hematoxylin: Naturally ripened. Glycerin added to prolong shelf life. Recommended for bone and cartilage.
   • Delafield's Hematoxylin: Naturally ripened.
   • Mayer's Hematoxylin: Artificially ripened with sodium iodate. Progressive stain.
   • Cole's Hematoxylin: Ripened with alcoholic iodine.
   • Carazzi's Hematoxylin: Ripened with potassium iodate. Used for frozen sections.
2. Iron Hematoxylins (Iron Mordant/Oxidizer): Regressive stains.
   • Weigert's Hematoxylin: Uses ferric ammonium chloride. Standard iron hematoxylin for muscle fibers and connective tissue.
   • Heidenhain's Hematoxylin: Uses iron alum. Used for nuclear and cytoplasmic inclusions.
   • Loyez Hematoxylin: Used for frozen sections.
   • Verhoeff Hematoxylin: Stains elastic fibers black.
3. Tungsten Hematoxylin: Phosphotungstic Acid Hematoxylin (PTAH). Uses $1\%$ phosphotungstic acid; progressive stain for CNS and general tissue.
4. Lead Hematoxylin: Solcia hematoxylin; demonstrates endocrine cells.
5. Molybdenum Hematoxylin: Thomas hematoxylin; demonstrates collagen and endocrine granules.

Eosin Counterstains

• Red acidic xanthene dye used as a counterstain for cytoplasm and connective tissue.
• Yellow (Eosin Y): Most common; water-soluble; gives green-yellow fluorescence.
• Erythrosin B (Eosin B)
• Ethyl Eosin (Eosin S): Alcohol-soluble.

Alphabetical Reference of Other Key Stains

• Acid Fuchsin - Picric Acid (Van Gieson's Stain): Demonstrates collagen/connective tissues.
• Acridine Orange: Basic fluorochrome. Discriminates living vs. dead cells (DNA = green fluorescence; RNA = red fluorescence).
• Acridine Red 3B: Demonstrates calcium salts and phosphatase.
• Alcian Blue: Water-soluble dye; stains acid mucopolysaccharides blue.
• Aniline Blue: Cytoplasmic counterstain for epithelial sections.
• Basic Fuchsin: Plasma stain for acid-fast organisms and mitochondria. Includes Carbol fuchsin, Schiff's reagent, Aldehyde fuchsin (Gomori's).
• Benzidine: Stains hemoglobin.
• Bismarck Brown: Counterstain for Gram's stain, Acid-Fast stain, and Pap smear (EA 50).
• Carmine: Chromatin stain; combined with aluminum chloride (Best's Carmine) for glycogen.
• Celestine Blue: Good nuclear stain resistant to strong acids.
• Crystal Violet: Stains nuclear chromatin, amyloid in frozen sections, and platelets.
• Giemsa Stain: Differentiates leukocytes in blood smears.
• Gold Sublimate: Gold chloride + mercuric chloride for metallic neuroglia impregnation.
• Iodine: Oldest stain. Gram's iodine (microorganisms/fibrin); Lugol's iodine (amyloid, glycogen).
• Janus Green: Supravital stain for mitochondria.
• Malachite Green: Counterstain for Ascaris eggs, RBCs, and bacterial spores (Schaeffer-Fulton).
• Methylene Blue: Basic nuclear stain.
• Neutral Red: Best vital stain for cell granules and phagocytic vacuoles.
• Orcein: Stains fine elastic fibers in skin.
• Osmium Tetroxide: Stains lipids black.
• Picric Acid: Fixative, yellow pigment, and counterstain in Van Gieson.
• Prussian Blue: Stains iron granules blue.
• Rhodamine B: Used with osmic acid for blood and glandular tissues.
• Silver Nitrate: Demonstrates spirochetes, reticulum, and nerve fibers.
• Toluidine Blue: Stains Nissl granules and chromophilic bodies.
• Victoria Blue: Demonstrates neuroglia in frozen sections.

Tissue Adhesives

• Substances applied to slides to prevent tissue detachment. Modern laboratories avoid routine adhesives because adhesives stain non-specifically and create background artifacts.

1. Mayer's Egg Albumin: Egg white (adhesive) + Glycerol (increases viscosity) + Thymol (prevents molds). Disadvantage: background staining.
2. $1\%$ Gelatin: Added to flotation bath.
3. Cellulose ($1\%$ Methyl Cellulose): Non-staining.
4. Poly-L-Lysine: Widely used in Immunohistochemistry (IHC). Superior attachment with zero background staining.
5. Sodium Salicylate: Commercial syrup diluted $1:10$.
6. Resin (Araldite): Epoxy resin diluted $1:10$ with acetone.

Hematoxylin and Eosin (H&E) Staining Protocol

Staining Equipment

• Coplin Jar: Holds $5 - 9$ slides.

• Slotted Staining Dishes: Holds $5 - 19$ slides.

• Metal/Glass Staining Racks/Carriers: Holds $10 - 30$ slides.

12 Sequential Steps in Routine H&E Staining

1. Xylene: Deparaffinization (removes paraffin wax).
2. Descending grades of alcohol: Rehydration (reintroduces water).
3. Water: Rinse.
4. Washing out: Removes fixative artifact pigments (e.g., cold water for osmium tetroxide).
5. Hematoxylin: Stains acidic nuclear structures light blue.
6. Acid Alcohol: Differentiator (selective decolorization of excess stain).
7. Ammonia Water: Blueing agent (converts reddish nuclear hematoxylin to intense blue lake). Alternatives: Lithium carbonate ($\text{Li}_2\text{CO}_3$), Scott's tap water, tap water.
8. Water: Rinse.
9. Eosin Y: Counterstain (stains cytoplasm pale pink).
10. Ascending grades of alcohol: Dehydration.
11. Xylene: Dealcoholization / Clearing.
12. Mount and Label.

Color Transitions During Key H&E Stages

Mounting, Ringing, and Labelling

Mounting

• Definition: Placing a syrup-like mounting medium and glass coverslip over the tissue section to protect it from mechanical scratching, air oxidation, and fading, and to enable microscopic examination.
• Hardening: Slides are incubated at $37^\circ\text{C}$ for $12 - 24\,\text{hours}$ after mounting.
• Refractive Index (RI): The mounting medium must have a refractive index close to glass ($\text{RI} = 1.518$) to prevent light refraction.

Coverslip Specifications

• Coverslip # 1.5: Thickness is $180\,\text{nm}$. Recommended for photomicrography and research.
• Coverslip # 1: Thickness is $150\,\text{nm}$. Standard coverslip routinely used in diagnostic laboratories.
• Standard Dimensions: $18 \times 18\,\text{mm}$, $20 \times 20\,\text{mm}$, $22 \times 22\,\text{mm}$, $24 \times 24\,\text{mm}$.

Kinds of Mounting Media

1. Aqueous Mounting Media:
   • Used for mounting water-miscible preparations, fat stains, and immunofluorescent sections.
   • General Composition: Gelatin/Gum Arabic (solidifies medium), Glycerol (prevents drying/cracking), Sugar (increases RI), Preservative (Merthiolate).
   • Glycerin: $\text{RI} = 1.46$.
   • Farrant's Medium (Gum Arabic): $\text{RI} = 1.44$. Standard semi-permanent medium for fat stains.
   • Apathy's Medium: Recommended for methylene blue stained nerve preparations.
   • Brun's Fluid: Used for mounting frozen sections from water.
2. Resinous Mounting Media:
   • Used for routine H&E sections.
   • Canada Balsam: Natural resin extracted from the tree Abies balsamea. $\text{RI} = 1.524$. Diluted with xylene or toluene. Disadvantage: Darkens, oxidizes, and turns yellow over time.
   • Synthetic Resins: XAM, Clarite, Permount, HSR, Clearmount, Eukitt. Resinous synthetic media that do not yellow rapidly.

Ringing

• Definition: The process of applying an airtight seal (e.g., black cement) around the outer margins of the coverslip.
• Purpose: Prevents evaporation of mounting media, prevents fluid leakage, protects against oxidation, and prevents coverslips from detaching during alcohol slide cleaning.
• Cleaning Warning: Never clean mounted slides with xylene, as xylene dissolves resinous mounting media and detaches coverslips.
• Ringing Media: Kronig Cement, Durofix.

Labelling

• Definition: Permanent identification applied to one end of the glass slide.
• Information Recorded: Specimen accession number and year (e.g., S-25-1234).
• Rule: Use accession numbers rather than patient names to ensure privacy, standardized filing, and indefinite archival tracking.