Chapter-1 Histotechniques: Tissue Processing Step by Step

TISSUE PROCESSING

When studying tissues under a microscope, proper tissue processing is crucial to obtain accurate and high-quality results. Histotechniques, also known as histology techniques, involve following series of steps Fixation, Dehydration, Clearing, Infiltration, Embedding, Trimming, Section cutting, staining, mounting and labeling etc. to prepare tissues for microscopic examination.

FIXATION AND FIXATIVES

Primary aim: preserve the morphological and chemical integrity of the cell in as life-like manner. – Shape, structure, intercellular relationship and chemical constituents of tissues are preserved. – Prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body.

Secondary goal: harden and protect the tissue from the trauma of further handling

MAIN FACTORS INVOLVED IN FIXATION:

1. Hydrogen Ion Concentration – pH 6 and 8 .
2. Temperature – Formalin heated at 60C
3. Thickness of section – 2cm2 for light microscopy
4. Osmolality – slightly hypertonic
5. Concentration – low conc. of glutaraldehyde
6. Duration of fixation – 2-6 h in buffered formalin

EFFECT OF FIXATIVES

•  harden soft and friable tissues
• make the cells resistant to damage and distortion
• inhibit bacterial decomposition
• increase optical differentiation of cells and tissues
• act as mordants or accentuators
• reduce the risk of infection

CHARACTERISTICS OF A GOOD FIXATIVE

1. Cheap
2. Stable
3. Safe to handle
4. Kills the cell quickly producing minimum distortion of cell constituents.
5. Inhibit bacterial decomposition
6. Produce minimum shrinkage of tissues
7. Harden tissues making cutting sections easier
8. Isotonic, causing minimal physical and chemical alteration of the cells and their constituents.
9. Make cellular components insoluble to hypotonic solutions

TYPES OF FIXATIVES :

1. According to composition

• Simple Fixative – made up of only one component substance such as- Formaldehyde ( Most used fixative), Glutaraldehyde, Mercuric Chloride, Potassium dichromate, Chromic acid , Picric Acid, Acetic Acid, Acetone ,Alcohol, Osmium tetra oxide etc.
• Compound Fixative – made up of two or more fixatives such as Zenker’s solution, Bouins Fluid etc.

2. According to Action

• Microanatomical Fixatives –  permits the general microscopic study of tissue structures such as
  1. 10% Formol Saline
  2. 10% Neutral Bufered Formalin
  3. Heidenhain’s Susa
  4. Formol sublimate
  5. Zenker’s solution
  6. Zenker formol
  7. Ouin’s solution
  8. Brasil’s solution

•  Nuclear Fixative – Preserve nuclear structures such as,

1. 1. Flemming’s fluid
   2. Carnoy’s fluid
   3. Bouin’s fluid
   4. Newcomer’s fluid
   5. Heidenhain’s Susa

• Cytological Fixatives – preserves cytoplasmic structures such as,

1. 1. Flemming’s fluid without acetic acid
   2. Kelly’s fluid Formalin with “post-chroming”
   3. Regaud’s fluid (Muller’s fluid)
   4. Orth’s fluid

•  Histochemical Fixatives – preserve chemical contents of cells and tissues such as

1. 1. LIPID FIXATIVE – Mercuric chloride and Potassium dichromate
   2. PHOSPHOLIPIDS FIXATIVE – Baker’s formal calcium
   3. CARBOHYDRATE FIXATIVE – Alcoholic formaldehyde
   4. PROTEIN FIXATIVE – Neutral buffered formal saline or formaldehyde
   5. GLYCOGEN FIXATIVE – Rossman’s fluid or absolute alcohol

  Composition, Advantage, Disadvantage & Use of Fixative

1. Formaldehyde –

 A. 10% formalin: widely used (10% formalin)

Disadvantage –

1. fumes are irritating to the nose and eyes
2. prolonged storage may induce precipitation of white paraformaldehyde

Notes –    Removal of precipitate is addition of 10% methanol

B. 10% formal – Saline –

1. – 40% Formaldehyde + NaCl + Distilled water –
2. fixation of CNS Tissues and General post-mortem tissues –
3. preserves enzymes and proteins

C. 10% Neutral Buffered Formalin/Phosphate-Buffered Formalin –

1. Sodium dihydrogen phosphate + Disodium hydrogen phosphate + 40%Formaldehyde + Distilled water
2. Preservation of surgical, post-mortem and research specimens
3. Best fixative for iron-containing tissues

D. Formal-Corrosive (Formal Sublimate)

1. Aq. Mercuric Chloride + 40% Formaldehyde
2. Routine post-mortem tissues
3. Excellent in silver reticulum methods
4. Fixes lipids, especially neutral fats and phospholipids

E. Alcoholic Formalin (Gendre’s Fixative)

1. 95% Ethyl Alcohol saturated with picric acid + Strong formaldehyde solution + glacial acetic acid.
2. Immunoperoxidase studies on tissues
3. Used for rapid diagnosis
4. Good for preservation of glycogen and for micro-incineration
5. Used to fix sputum, since it coagulate mucus

F. Glutaraldehyde

1. two formaldehyde residues linked by 3C chains
2. used for enzyme histochemistry and electron microscopy
3. preserves plasma proteins

3. METALLIC FIXATIVES

• MERCURIC CHLORIDE

1. 1. Mercuric Chloride + Potassium Dichromate + Sodium Sulfate + Distilled Water
   2. most common metallic fixative
   3. Tissues fixed with mixtures containing mercuric chloride (except Susa) contain black precipitates of mercury.
   4. Routine fixative of choice for preservation of cell detail in tissue photography.
   5. Renal tissues, Fibrin, Connective tissues and muscles
   6. Black deposits may be removed by adding saturated iodine solution in 96% alcohol, the iodine being decolorized with absolute alcohol in the subsequent stages of dehydration.

• Zenker’s Fluid

1. 1. Mercuric Chloride + Glacial Acetic Acid
   2. fixing small pieces of liver, spleen, connective tissue and nuclei
   3. may act as mordant
   4. Mercuric deposits may removed by immersing tissues in alcoholic iodine solution. “de-zenkerization”

• Zenker-formol (Helly’s solution)

1. 1. Mercuric chloride + Potassium dichromate + Sodium sulphate + Distilled water + Strong formaldehyde (40%)
   2. Fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver.
   3. Preserves cytoplasmic granules
   4. Brown pigments are produced if tissues are allowed to stay for more than 24 hours.
   5. Pigments can be removed by immersing the tissue in saturated alcoholic pricric acid or sodium hydroxide

• Heidenhain’s Susa Solution
• 1. Mercuric chloride + Sodium chloride + Trichloroacetic acid + Glacial Acetic Acid + Formaldehyde (40%) + Distilled water
  2. tumor biopsies especially of the skin
  3. Excellent cytological fixative
  4. Mercuric chloride deposits may be removed by immersion on alcoholic iodine solution
  5. the tissue should be transferred directly to a high-grade alcohol, to avoid undue swelling of tissues caused by treatment with low-grade alcohol or water.

• B-5 Fixative
  1. Distilled water + Mercuric Chloride + Sodium acetate
  2. Commonly used for bone marrow biopsies

4. CHROMATE FIXATIVES

• Chromic Acid

1. 1. used in 1-2% aqueous solution
   2. Precipitates all proteins and adequately preserves carbohydrates.
   3. Strong oxidizing agent, strong reducing agent must be added.

• Potassium Dichromate

1. 1. Used in 3% aqueous solution
   2. Preserves lipids
   3. Preserves mitochondria

• Regard’s (Muller’s) Fluid

1. 1. Potassium dichromate + Strong formaldehyde 40%
   2. Demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues
   3. Prolonged fixation gives out black deposits and can be removed by running tap water.

• Orth’s Fluid

1. 1. Study of early degenerative proceses and tissue necrosis
   2. Demonstrates rickettsiae and other bacteria
   3. Preserves myelin

5. LEAD FIXATIVES

1. used in 4% aqueous solution
2. recommended for acid mucopolysaccharides
3. fixes connective tissue mucin
4. forms insoluble lead carbonate due to prolonged standing which can be removed by filtration or adding acetic acid drop by drop

6.PICRIC ACID FIXATIVES

1. Yellow color may be removed by treatment with another acid dye or lithium carbonate
2. Excellent fixative for glycogen demonstration
3. Suitable for Aniline stains

• Bouin’s Solution

1. 1. Solution of picric acid + Strong formaldehyde 40% + Glacial acetic acid
   2. Fixation of embryos and pituitary biopsies
   3. Preserving soft and delicate structures (endometrial curettings)
   4. Yellow stain is useful for handling fragmentary biopsies.
   5. Collagen, elastic connective tissues

• Brasil’s Alcoholic Picroformal Fixative

1. 1. Formaldehyde + Picric Acid + Ethanol/Isopropyl Alcohol + Tricbloroacetic acid
   2. Fixative for glycogen
   3. Less messy than Bouin’s solution

7. GLACIAL ACETIC ACID

1. Precipitates chromosomes and chromatin materials
2. Essential constituent of most common compound nuclear fixatives

8. ALCOHOLIC FIXATIVES

1. Ideal for small tissue fragments
2. Used as a fixative and dehydrating agent

• Methyl Alcohol: fixing dry and wet smears, blood smears and bone marrow tissue
• Isopropyl Alcohol 95% – fixing touch preparations
• Ethyl Alcohol: Blood, tissue films and smears
• Carnoy’s Fluid

1. 1. Absolute alcohol + Chloroform + Glacial acetic acid
   2. Fixing chromosomes, lymph node glands and urgent biopsies
   3. Fix brain tissue for diagnosis of rabies

• Newcomer’s Fluid
  1. Isopropyl alcohol + Propionic acid + Petroleum ether + Acetone + Dioxane
  2. fixing mucopolysaccharides and other proteins

9. OSMIUM TETRAOXIDE (OSMIC ACID)

1. 1. Pale yellow powder which dissolves in water to form strong oxidizing solution.
   2. fixes conjugated-fats and lipids permanently by making them insoluble during subsequent treatment with alcohol and xylene
   3. helps preserve cytoplasmic structure
   4. fixes myelin and peripheral nerves well
   5. fixes materials for ultrathin sectioning in electron microscopy, since it rapidly fixes small pieces of tissues and aids in their staining
   6. black osmic oxide crystals may be dissolved in cold water
   7. Osmic acid-fixed tissues must be washed in running water for at least 24 hours to prevent formation of artefacts

• Flemming’s Solution

1. 1. common chrome-osmium acetic acid fixative used
   2. Recommended for nuclear preparation of such sections
   3. Aqueous chromic acid 1% + Aqueous osmium tetraoxide 2% + Glacial acetic acid
   4. An excellent fixative for nuclear structures -permanently fixes fat

• Flemming’s solution without acetic acid

1. 1. Made up only of chromic and osmic acid
   2. Removal of acetic acid from the formula serves to improve the cytoplasmic detail of the cell

10. TRICHLOROACETIC ACID

1. incorporated into compound fixatives
2. precipitates proteins
3. marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives
4. may be used as a weak decalcifying agent

11. ACETONE

1. Used at a cold temperature ranging from 5*C to 4*C
2. Recommended for the study of water diffusible enzymes especially phosphatases and lipases
3. Used in fixing brain tissues for diagnosis of rabies

Factors that affect Fixation of the Tissues:-

• RETARDED BY:

1. 1. Size and thickness of the tissue specimen-largest tissues require more fixatives and longer fixation time
   2. Presence of Mucus-prevents complete penetration of fixative; hence, tissues that contain mucus are fixed slowly and poorly. Excess mucus may be washed away with normal saline solution.
   3. Presence of fat- fatty tissues should be cut in thin sections and fixed longer.
   4. Presence of blood- tissues containing large amount of blood should be flushed out with saline before fixing
   5. Cold temperature- inactivates enzymes

• ENHANCED BY:

1. 1. Size and thickness of tissues- smaller and thinner tissues requires less fixative and shorter fixation time
   2. Agitation- fixation is accelerated when automatic or mechanical tissue processing is used.

Important Notes:-

1. SECONDARY FIXATION : Process of replacing an already fixed tissue in a second fixative order Usually with 10% formalin or 10% formal saline as primary fixative

2. POST CHROMATIZATION: form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5- 3 % potassium dichromate for 24 hours to act as mordant for better staining effects

3. WASHING-OUT: Process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues

1. 1. 1. Tap water: Removes excess chromates from tissues fixed in Kelly’s, Sinker’s, and Fleming’s solutions -excess formalin -excess osmic acid
      2. 50-70% alcohol: Used to wash out excess amount of picric acid (Bouin’s solution)
      3. Alcoholic iodine: Used to remove excessive mercuric fixatives

“DEHYDRATION”

Process of removing intercellular and extracellular water from the tissue after fixation and prior to wax impregnation is known as dehydration. Whatever the dehydrating agent is used, the amount in each stage should not be less than 10 times the volume of the tissue in order to ensure complete penetration of the tissue by the dehydrating solution.

Characteristics of Ideal Dehydrating Agent

1. Should dehydrate rapidly
2. Should not evaporate very fast
3. It should be able to dehydrate even fatty tissues
4. It should not harden tissues excessively
5. It should not remove stains
6. It should not be toxic to the body
7. It should not be a fire hazard

Commonly used Dehydrative Agents : –

1. Alcohol (most common)- For Routine dehydration of tissues
2. Acetone – For Urgent biopsies
3. Dioxane 4 Cellosolve
4. Triethyl phosphate
5. Tetrahydrofuran – For dehydrates and clears tissue

 Clearing (De-Alcoholization)

Clearing: Alcohol or dehydrating agent is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is to be impregnated

CHARACTERISTICS OF A GOOD CLEARING AGENT: –

1. Should be miscible with alcohol.
2. Should be miscible with, and easily removed by melted paraffin wax
3. Should not produce excessive shrinkage
4. Should not dissolve out aniline dyes
5. It should not evaporate quickly in a water bath.
6. Should make tissues transparent.

Commonly used Clearing Agents: –

1. Xylene (most common) – Routine histologic processing | Urgent biopsies which it clears within 15-30 minutes | Becomes milky when an incompletely dehydrated tissue is immersed in it.

2. Toluene :- Substitute for xylene or benzene  | Miscible with both absolute alcohol and paraffin  |  Much more expensive than Xylene

3. Benzene :- Urgent biopsies and routing purposes | Excessive exposure to benzene may be extremely toxic to man and may become carcinogenic or it may damage the bone marrow resulting in aplastic anemia.

4. Chloroform :- Slower action than xylene  |  Recommended for tough tissues, for nervous tissues, lymph nodes and embryos because it causes minimum shrinkage and hardening of tissues

5. Cedarwood Oil :- Used to clear both paraffin and celloidin sections | Recommended for CNS tissues and cytological studies, particularly smooth muscles and skin. | Becomes milky upon prolonged storage and should not be filtered before use.

6. Aniline Oil :- Clearing embryos, insects and very delicate specimens due to its ability to clear 70% alcohol without excessive tissue shrinkage

7. Clove Oil :- Tissues become brittle, aniline dyes re not removed and celloidin is dissolved

8. Carbon tetrachloride :- Similar to chloroform except it’s a lot cheaper

Also Read

1. Bile Salt (Hay^’s Sulphur Method)

2. Stool Examination: What You Need to Know

3. Estimation of occult blood in stool by Benzidine method.

4. “Unlocking the Secrets of Urine: A Comprehensive Guide to Urine Examination”

5. Glucosuria (Benedict Method)
6. Ketone Body (Rotheras, Gerhardt’s and Strip Method)
7. Proteinuria (Heat and acetic acid, Sulphur Salicylic Acid, Hellers or Nitric acid, and Esbach albuminometer Method)
8. Bence Jones Proteinuria (HCl Method)
9. Urobilinogen (Ehrlich Method)
10. Bile Pigment (Fouchets Method)
11. Occult Blood (Orthotoluidine and Benzidine Method).
12. Sedimentation Preparation
13. Slide Preparation
14. Microscopic Examination.