Histopathology and Laboratory Techniques
Specimen journey
Formal processing: fixation in 10% neutral buffered formalin (volume at least 10 times the tissue), dehydration in graded alcohols, clearing in xylene, embedding in paraffin, sectioning at 3–5 µm, staining with hematoxylin and eosin. Fixation time matters: underfixation causes poor morphology and false-negative immunohistochemistry; overfixation masks antigens. Frozen sections are for intraoperative decisions (margins, diagnosis) and are not permanent; they use cryostat sections and rapid H&E.
Special stains: PAS for glycogen, fungi, basement membrane; PAS with diastase distinguishes glycogen; Masson trichrome for collagen; reticulin for liver and bone marrow architecture; Congo red for amyloid (apple-green birefringence under polarized light); Prussian blue for iron; Fontana–Masson for melanin; Ziehl–Neelsen for acid-fast bacilli; Fite for leprosy; mucicarmine for Cryptococcus and mucin; Giemsa for blood and microorganisms; Grocott silver for fungi; alcian blue for mucin.
Quality control: daily stain checks, positive and negative controls, equipment maintenance, reagent logs, and documentation. Preanalytical errors (delays, wrong fixative) cause more problems than analytical ones.
Traps
Questions ask "best stain" for a substance, or confuse PAS with trichrome. Decalcification with strong acid can destroy DNA and antigens; EDTA is gentler.
Rule of thumb: name the substance first, then the stain - collagen = trichrome, iron = Prussian blue, amyloid = Congo red, acid-fast = ZN.
Worked example: a liver biopsy with suspected fibrosis - trichrome shows blue/green collagen and highlights bridging septa that H&E may miss.