Histopathology: Tissue Sample Preparation

• Concerns the preparation for microscopic examination.
• This is achieved through submitting a total tissue sample or select part for examination to a series of steps.
• "FDD-CIET-SSML" includes: Fixation, Decalcification, Desiccation, Clearing, Infiltration, Embedding, Trimming, Sectioning, Staining, Slide Mounting and Labelling

Histopathologic Steps

• The first and most critical step is fixation, because it affects later procedures.
• Involves preservation.
• Removes calcium or lime salts from calcified tissues like bones and teeth.
• Desiccation removes intracellular and extracellular fluid/water.
• Dehydration removes alcohol.
• Clearing involves removing alcohol used in dehydration.
• Clearing is de-alcoholization.
• Infiltration/Impregnation is an identical process
• Embedding involves casting or blocking.
• Involves trimming to remove excess wax from tissue.
• Sectioning includes cutting tissue blocks into uniformly thin slices.
• The proper labelling involves appropriate and accurate identification
• Fresh and preserved tissues can both be used

Fresh Tissue Examination

• Examined in the living state to allow observation of protoplasmic activities like mitosis, motion, phagocytosis, and pinocytosis.
• Its use is limited because fresh tissues are not permanent and develop changes after death.

Primary and Secondary Signs of Death

• Primary signs occur during somatic death and include CNS, respiratory, and cardiovascular failure.
• Occurs after somatic death.

Secondary Signs of Death

• Algor mortis involves cooling of the body at a rate of 7°F/hr or 1-1.5°C/hr.
• This is the 1st demonstrable change.
• Rigor mortis includes stiffening of the skeletal muscles.
• Livor mortis involves post-mortem lividity or suggilation
• Appearance of purplish discoloration.
• Autolysis is the destruction of tissues by enzymes produced by the tissues, leading to liquefaction.
• Decomposition is the breakdown of organic matter under the influence of microorganisms, creating disagreeable odors.
• Degeneration is a retrograde pathologic process in cells where the cytoplasm deteriorates, yet the nucleus is preserved.

Methods of Fresh Tissue Examination

• Teasing or Dissociation involves dissecting/separating selected tissue in isotonic salt solution, then examining under a microscope
• Can be unstained with Bright-field microscope, or with differential dyes
• Can be stained with Phase Contrast Microscope.
• Squash preparation or crushing requires placing small tissue pieces no more than 1 mm in diameter on a slide, then compressing them with another slide or coverglass.
• Using capillary action injects the slide tissue sample with dyes.
• Frozen Section is used when rapid tissue diagnosis is needed.
• Frozen section is recommended when lipids/nervous tissue must be demonstrated.
• Smearing is useful for cytological examinations, especially for cancer.

Smearing Techniques

• Streaking involves a rapid and gentle application to obtain uniform distribution.

• Too thick or smears are unsuitable for examination

• Spreading is somewhat more tedious than streaking, but maintains intracellular relationships.

• It's especially recommended for fresh sputum, bronchial aspirates, and thick mucoid secretions.

• Pull-apart disperses material evenly over two slides.

• A single uninterrupted motion of pulling apart is necessary

• Useful for serous fluids and blood smears.

• Touch preparation / Impression smear is when the slide surface is in contact and pressed on the site.

• Cells may be examined without destroying their actual intercellular relationship and without separating them from their normal surroundings.

Steps for Processing Preserved Tissues

• This is essentially fixation or preservation
• The process constituents of the cells, and therefore of the tissues are fixed in a physical.
• Partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss.
• Involves maintaining minimum distortion or decomposition
• It is first and most important Step
• The primary aim is to preserve the morphologic and chemical integrity of the cell as life-like.
• Hardens and protects the tissue from trauma.
• Simplifies cutting during gross examination.
• Uses cross-linking between proteins to achieve protein stabilization.
• Leaving a tissue specimen in air can cause distortion of morphologic appearance
• When hypotonic, there is more water outside the cell and the solute concentration inside is high.
• In hypotonic conditions water enters the cell, causing swelling/ bursting.
• When tissue is in a strong salt water will move outside the cell and the cell shrinks..
• It's called lysis or cytolyis.

Mechanisms in Fixation

• Additive: The chemical constituent of the fixative is absorbed and becomes part of the tissue via cross-link formation or molecular complexes
• Non-additive: The fixing agent isn't incorporated, but it alters and stabilizes tissue through water removal.
• Cross-links are formed - This stops autolysis and decomposition.

Factors Involved in Fixation

• Ideal pH is between 6.0-8.0

• 7.0 pH is average

• Room temperature (18-30°C) is traditional.

• Tissue processors (Autotechnicon): 40-42°C

• EM & Histochemistry: 0-4°C

• Mast cells for EM: Room temperature

• Nucleic acids benefit from Rapid at higher temperatures (60°C)

• Urgent specimens at 100°C

• Use 1-2 mm² in Electron Microscopy

• 2cm² for Light Microscopy

• Thin Light Microscopy ≤0.4 cm (4mm) or as prescribed

• Large solid tissues such as uterus should be opened or sliced thinly to fix better

• Use Slightly hypertonic solutions (400-450 mOsm) for best results.

• Use Sucrose when adding to Osmium tetroxide

• Most formalin fixatives should be for 24 hours (washed out).

• Buffered formalin requires 2-6 hours up to 1 week.

• Fix for 3 hours for EM but new textbooks now say between 0-4 hours and average 2 hours

Considerations

• Tissues should be in the fixative as soon as it is removed from the body to prevent decomposition/autolysis.
• Fixation kills prevent growth in culture and drug/toxins
• 1mm/HOUR, the rate can be variable
• Volume: Traditionally 10-25x the volume is used
• Recently, 20x maximum known.
• Except Osmium tetroxide (5-10x) is used.

Ideal Tissue

• 2cm² in diameter.
• 4mm thick.
• Fix for 4-6 hours or 6-18 hours, according to new text books.
• Should be Cheap
• Should be Safe to handle
• Kills quickly
• Produces minimum shrinkage
• Properly Hardens
• Inhibits decomposition
• Permits rapid tissue penetration
• Makes cellular components insoluble
• Allows staining.

According to Action

• Microanatomical Fixatives are 10% Formol saline, 10% Neutral Buffered Formalin (NBF), Heidenhain's Susa, Formol sublimate (corrosive), Zenker's solution, Zenker-formol (Helly's), Bouin's solution, Brasil's solution
• Cytological Fixatives used for Preserving structural elements of a cell. It is specifically the nucleus
• B.F.N.C.H. Bouin’s fluid, Flemming’s fluid, Newcomer’s fluid , Carnoy’s fluid, Heidenhain’s Susa

Cytoplasmic Fixatives

• Helly's, Orth's, Regaud's, Flemming's fluid w/o Acetic acid (HAC), Formalin w/ post-chroming.

• HORFF never contain acetic acid.

• 10F-ANA; 10% Formol saline

• Absolute Ethyl alcohol

• Newcomer's fluid

• Acetone

Handling Precautions

• Autopsy materials should be fixed ASAP to prevent decomposition,
• Body placed in mortuary refrigerator (4°C) or arterial embalming if no toxicological testing
• Surgical specimens fixed as soon after removal or refrigerated to avoid tissue distortion.
• Fixative amount must be adequate.
• 20x volume but 5-10x in Osmium tetroxide
• For prolonged fixation it should not be less than 50-100 times that of the tissue
• Air-filled lungs float so cover with gauze.
• Eyes - Don't before fixed: inject Formol-alcohol first.
• Avoid water as can create crystals.
• Follow following factors for better results.
  • immediate examination, tissue structure are most important

Routine Fixatives

• Commercial Formaldehyde is approximately 35-40% formaldehyde gas by weight. (OR 37-40% weight in volume, according to Gregorios' textbook definition).
• Mixture - 10 mL formalin mixed water/saline Known solution
• Buffer with phosphate buffer/ Calcium-acetate
• Turbid in nature
• Formalin best

Disadvantages

• Irritating dermatitis to the dermis: troublesome
• Irritating fumes and asthma
  • Dark brown granules are from spleen
  • Add Saturated alcoholic picric acid or alcoholic 1% KOH when too many
• Prevents formalin, fat dispersal, and acid dissolution
  • Grade formaldehyde retards acid to form Paraformaldehyde

Stain Pigments

• Acidity Reduces quality
• Prevents Hemosiderium leaching.
• Formation from over fixations.

Removal of Pigments

• Lillie utilizes formaldehyde fixed specimens in acetone, 28% ammonia water and hydrogen peroxide.

• It utilizes 70 % alcohol in rinsing agent

• Kardasewitsch involves in removal

• Best for Microscopic, preservation on routine

Advantages of Saline

• Penetrate, preserving - Prevent shrinkage

• Disadvantage of the use

• 24 HOURS+ - it must changes

• How can be prevented

• Same - it removes

  • Best tissue to prevent precipitation

• Disadvantage of preparing

• Low penetration

Alcacohol Formaldehyde

• Can immuniprescious and dehydraate since coagulates

Metallic Formula

• Can protect cells
• Allows for brilliant metacromatic stain
• For tissue
• Damages the shells and corrosives