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Direct Immunofluorescent (DIF) Services

Specimen requirements:

  • Punch biopsy (3 or 4 mm) of perilesional tissue submerged in Michel’s transport medium. The specimen should be shipped or couriered overnight.

  • “Perilesional” means within 2 cm of normal perilesional skin. The erythema surrounding the vesicle can be included in the biopsy if necessary due to anatomical limitations. 

  • Diagnostic Performance of Direct Immunofluorescence Microscopy Studies by Biopsy Sites in Autoimmune Subepidermal Blistering Dermatoses: A Prospective Study. Haefliger S, Sitaru S, et al: Br J Dermatol; 2020;183 (November): 970-972.

  • Method:

    • The specimen is transported in Michel’s medium and cut into 4-micron-thick slices using a cryomicrotome. Fluorescein-conjugated antibodies specific for immunoglobulin IgG, IgM, and IgA, and complements are used to stain ultra-thin sections. The slides are then evaluated under an immunofluorescent microscope.

  • Transport Medium

    • Mitchel’s solution (provided by Benchmark Diagnostics)

    • Mitchel’s solution bottles are better stored at room temperature and are okay to use for one year.

    • It should not be used after the expiration date.

    • If Michel’s medium is unavailable or expired, normal saline can be used as a transport medium; however, it should be available for testing in the laboratory within 24 hours of the biopsy. In that case, the biopsy should not be done on Fridays as the specimen may remain in saline for over 48 hours which may cause cellular autolysis.

    • According to one study, honey can also be used as a transport medium. The tissue remains optimum for use until one week.

    • Formalin should NOT be used for fixation or transportation.


Indication for DIF testing:

Vesiculobullous disorders

  • Subepidermal blisters (Damage dermal-epidermal junction adhesion molecules)

    • Pemphigoid group

    • Dermatitis herpetiformis

    • Linear IgA disease

    • Herpes (pemphigoid) gestationis

    • Epidermolysis bullosa acquisita

    • Porphyria

  • Acantholytic disorders (Damage to intercellular adhesion molecules)

    • Pemphigus group

    • Vasculitis and vasculopathic diseases

      • IgA vasculitis/Henoch Schonlein Purpura

      • Mixed cryoglobulinemia

      • ANCA positive vasculitides

      • Cocaine-induced retiform purpura

    • Collagen vascular and autoimmune disease

      • Lupus erythematosus

      • Mixed connective tissue disease

      • Epidermolysis bullosa acquisita

    • Deposition disorder

      • Porphyria cutanea tarda


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