Cytology Fixatives and Staining Techniques

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.