Cytology Fixatives and Staining Techniques

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Cytology Fixatives

Solution: Cytology fixatives, formerly employing ether/alcohol 96 in equal parts, are now rarely used due to the hazardous nature of ether. The 96% alcohol is most often used. The procedure involves immersing the preparation in the fixative bath for a minimum of 10 to 15 minutes. Other alcohols, such as 100% methanol, 80% propanol, and 80% isopropanol, can also be used. Citospray is used in samples obtained by forced exfoliation.

Sample Types in a Cytology Laboratory

Samples that can reach the lab from samples obtained by:

  • Forced exfoliation: Rubbing or scraping with various instruments. This is applied to the skin and organs accessible from the outside.
  • Spontaneous exfoliation: Samples containing spontaneously exfoliating cells (urine, sputum).
  • Liquid-based cytology: Samples collected in a liquid transport medium.
  • Fine Needle Aspiration (FNA): Used for lesions in organs without direct access and no spontaneous secretion.

Urine Sample Processing in the Cytopathology Service

Outlines the procedure for a urine sample:

  1. Fixing: Send in a bottle and fix with 50% alcohol within 12 hours.
  2. Spin: Centrifuge the cell suspension in a cytospin for 10 minutes at 1500 rpm.
  3. Decanting: Remove the supernatant liquid.
  4. Homogenization, Extension, and Fixing: With 96% alcohol or Citospray.
  5. Staining: See below.

Staining Techniques

PAS Reaction (Periodic Acid-Schiff)

Demonstration of carbohydrates.

  1. Periodic acid (HIO4) oxidation reaction of the aldehyde groups.
  2. Formation of Schiff coloration (rojo-magenta).

PAS-Positive Substances:

  • Carbohydrates:
    • Simple carbohydrates (liver)
    • Neutral mucopolysaccharides (stomach, colon, bacterial capsule)
    • Mucoproteins (lungs, thyroid)
    • Glycoproteins
    • Glycolipids (CNS)
    • Pigments (ceroid, lipofuscin)

PAS-Negative Carbohydrates: Acid mucopolysaccharides.

Amylase Digestion

Amylases catalyze the hydrolysis of glycosidic bonds of starch and glycogen.

  • PAS-PASA-: Neutral mucopolysaccharides, glycoproteins.
  • PAS-PASA+: Non-glycogen carbohydrates.

Techniques to Identify False Positives

Based on blocking functional groups, and based on the negativity of the PAS reaction after:

  1. Acetylation of 1,2-glycol groups.
  2. Aldehyde Group Blocking: The existence of pre-existing aldehyde groups can lead to false positive results. In this case, a control tissue (not oxidized) and an oxidized tissue are compared.

Cationic Dye-Based Techniques for Acidic Carbohydrates

  • Toluidine Blue: Used in combination with PAS. Acidic GAGs stain blue, and neutral glycoproteins stain magenta.
  • Metachromasia (Toluidine Blue): Acidic chromotrope groups stain red, while the rest of the tissue stains blue.
  • Spicer Diamines: Oxidizing agent + (Cl3Fe). Stains sulfated GAGs. When combined with Alcian blue, it distinguishes neutral GAGs from acidic GAGs.
  • Colloidal Iron Method: Acidic groups in the tissue attract Fe3+, which is then demonstrated by the Prussian blue reaction.

Methenamine Silver Technique

Carbonyl radicals from the oxidation of carbohydrates reduce silver salts, producing a metallic silver precipitate on the structures. Positive staining occurs with PAS-positive glycoproteins.

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