Histotechnology: Fixation, Processing and Staining Quality
Histotechnology tests the physical chemistry of turning tissue into a slide: fixation, dehydration, clearing, embedding, sectioning and staining. Most exam questions are troubleshooting questions where an artefact on the slide points back to a specific step in the process.
Core rules
- Fixation: 10% neutral buffered formalin (about 4% formaldehyde, phosphate buffered to pH 7.0-7.4) is standard; the fixative-to-tissue volume ratio should be at least 10:1 and the recommended time is 8-24 hours for routine specimens. Buffering prevents formalin pigment and tissue acidification.
- Processing sequence: dehydration through graded alcohols, clearing in xylene (miscible with both alcohol and paraffin), infiltration with molten paraffin at 56-60 degrees C.
- H&E chemistry: haematoxylin is a basic dye requiring an aluminium mordant; in the presence of a mordant it stains nucleic acids blue-purple (basophilic). Eosin is an acidic dye staining proteins and cytoplasm pink (eosinophilic). Bluing (Scott tap water or lithium carbonate) restores the correct blue.
- Frozen sections are used for intraoperative diagnosis and for fat and enzyme studies that would be destroyed by processing; they cut at 4-6 micrometres and are stained rapidly.
Worked example
Prepare 2 L of 10% formalin working solution from a 37-40% formaldehyde stock. Reasoning: 10% formalin is 10% v/v of the saturated stock, so the dilution factor is 1 part stock to 9 parts diluent. Volume of stock = 2,000 mL / 10 = 200 mL, plus 1,800 mL of distilled water. If the stock is treated as 100% formalin for strength calculations instead, the result is identical because 10% formalin contains 3.7-4.0% formaldehyde by weight, which is the intended working concentration. The solution must then be buffered, since unbuffered formalin drifts acid and produces brown pigment.
Traps that cost marks
- Chatter or parallel lines on sectioning indicates a dull or damaged blade, not a processing problem; compression and wrinkles indicate a loose blade or too warm a block.
- Brown granular pigment can be formalin pigment, haemosiderin or melanin; haemosiderin is Prussian blue positive, melanin bleaches with potassium permanganate, and formalin pigment dissolves in saturated alcoholic picric acid.
- Under-differentiated haematoxylin leaves pale grey nuclei; over-differentiation in acid alcohol removes stain from nuclei entirely.
- Incomplete fixation shows as pale, smeary central areas in a block, especially in liver and lymph node.
- Decalcification with strong acids damages DNA and antigenicity; EDTA is gentler for immunohistochemistry.