Wednesday, December 28, 2005

Examples of Embedding Instructions and Histochemical Stains

General comments: Difficult cases for embedding will be done by specific technicians and should be identified by name on the orders: e.g. Sandra to embed only!

Examples of embedding on cut edge: skin conjunctiva, cornea, iris, sometimes muscle.
Examples of embedding on flat face: muscle, soft tissue (fat, fibrous tissue, orbital.
Examples of embedding on cut end: temporal arteries, optic nerve.
Example embedding and sectioning instructions:

  • eyelid for seborrheic keratosis: bisect, embed on cut edge 1 ribbon or section stained
  • eye- Sandra to embed only! On cut face. Sections to include optic nerve. Stain with H&E and PAS.
  • cornea for bullous keratopathy: embed on edge, 1 section H&E and 1 section PAS
  • cornea for Salzmann's nodule: cornea, embed on cut edge 1 section H&E, 1 PAS, and one for oxidized aldehyde fuchsin stain
  • conjunctiva for pterygium: Sandra to embed only flatten at embedding station, trisect parallel to the long axis, embed slices on edge, cut 1 section stained with H&E and 1 section stained with PAS
  • temporal artery- initial sections: slide at 1 mm intervals and embed on cut end so that a cross section of the vessel is obtained, initial face and stain 1 section for H&E and one unstained.
  • Temporal artery if initial section is negative: Deeper q 20th through block. (depends on thickness of the sections q 30th is adequate if sections are 4-5 microns).
    Typical use of commonly employed special stains:
  • PAS- identify glycogen or basement membranes. Use for all corneas, conjunctiva, eyes.
  • Verhoeff Van Gieson (EVG)- stains elastic tissue. Use for temporal arteries and vascular tumors.
  • Infectious Package PAS, GMS, AFB and Gram stain. Use for suspected infections.

  • Shown are the Gram stain in the figure to the left with black arrow one pointing to gram positive cocci. Notice the size of the organism compared to the adjacent red nuclei and the clustered arrangement of bacteria. This is a control slide and will often show both gram positive and gram negative organisms.

The Gomori methenamine silver stain (GMS) highlights fungal hyphae. Branching of the hyphal structures are helpful to narrow the diagnostic possibilities. Here 45 degree dichotomous branching is seen at red number 1. It is also important to look for septae which are visible as thin lines splitting the hyphal structures (red arrow 2).

      The Ziehl- Neelsen stain is a carbol-fuchsin stain that is used for acid fast bacilli and show very small organisms that are red and elongated. Some are extracellular (lighted arrow 1) and some are intracellular (arrow 2). In this control slide the organisms are particularly abundant.

  • Dystrophy Package: Alcian Blue pH 0.4, Congo Red, Trichrome. Use for suspected BIGH3 dystrophies and macular dystrophy of the cornea.
  • Prussian Blue- stains iron. Use to stain basal epithelium in keratoconus or hemosiderin.
  • Giemsa- excellent for air dried cellular elements. Use for air dried cornea swabs for acanthameba, fine needle aspirations of suspected lymphomas.
  • Von Kossa- stains calcium black. Use for band keratopathy.
  • Masson’s trichrome stains muscle red, tendon blue. Use for rhabdomyosarcoma, slipped tendons, vascular tumors that have smooth muscle (e.g. cavernous hemangioma).
  • Mucicarmine stains neutral mucins pink. Use for suspected adenocarcinoma.
  • Oil red O- stains lipids red in frozen section. Not useful for fixed tissue. In our lab this stain is largely supplanted by osmication and thick sections for toluidine blue which give far better quality of staining and sectioning. (Rarely do the plastic surgeons send the tissue fresh anyway). Lipids stain bright green with this stain (see arrows below).

  • For other special stains see one of the many books in the lab such as the Laboratory Methods in Histotechnology, AFIP Manual, by Prophet, Mills Arrington and Sobin.


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