Cytology Fixatives and Staining Techniques
Classified in Chemistry
Written at on English with a size of 3.84 KB.
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:
- Fixing: Send in a bottle and fix with 50% alcohol within 12 hours.
- Spin: Centrifuge the cell suspension in a cytospin for 10 minutes at 1500 rpm.
- Decanting: Remove the supernatant liquid.
- Homogenization, Extension, and Fixing: With 96% alcohol or Citospray.
- Staining: See below.
Staining Techniques
PAS Reaction (Periodic Acid-Schiff)
Demonstration of carbohydrates.
- Periodic acid (HIO4) oxidation reaction of the aldehyde groups.
- 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:
- Acetylation of 1,2-glycol groups.
- 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.