Specimen Handling for Direct Immunofluorescence (DIF)

In order to help us process and diagnose your tissue samples, we offer the following guidelines.

  • Specimens taken for DIF of bullous/desquamative lesions must be accompanied by another specimen for routine histopathology.
  • Either two biopsies can be taken, or a single elliptical wedge can be procured and then hemisected.
  • One specimen goes into formalin, the other into special DIF transport media supplied on request from our lab.

The diagnosis of bullous/desquamative lesions requires an assessment of where the epithelium is splitting. Clinically, obtaining these biopsies can be problematical due to the tendency for the epithelial layer to slough. It is best to seek a site in the anterior portion of the mouth and take care not to grasp the specimen with forceps. Eight-millimeter punch biopsies work quite well, and one should always biopsy the edge of the lesion to include both pathologic and normal appearing tissues.

How does DIF work?

Immunoglobulins from rabbits, goats or mice are used as reagents. These immunoglobulins are directed to antigens in the host tissue and many of these antigens are immunoglobulins or autoantibodies (i.e. an antibody to an antibody). Many antigens are damaged by formalin fixation and tissue processing; therefore a special medium is used and the sections are cut from frozen tissue samples.

The anti-human immunoglobulins are tagged with a fluorescent dye that can only be visualized with an ultraviolet light source (fluorescent) microscope.

How are results interpreted?
  • Localization of fluorescent antibodies
  • Basement membrane fibrinogen: lichen planus, lichenoid lesions
  • Basement membrane C-3, IgG, IgM and/or IgA: mucous membrane pemphigoid
  • Basement membrane IgM: lupus erythematosus
  • Intercellular (desmosomal region) C-3, IgG, IgM and/or IgA: Pemphigus vulgaris
  • Vascular wall C-3 and IgG: Erythema multiform

For the most part DIF is a useful adjunct for erosive diseases such as lichen planus, pemphigoid, pemphigus and erythema multiforme.