Technique of Conjunctival Biopsy and Direct Immunofluorescence for Diagnosing Mucous Membrane Pemphigoid

Ocular mucous membrane pemphigoid (MMP) presents as chronic cicatrizing conjunctivitis that, if left untreated, can cause blindness. Timely diagnosis and immunosuppressive therapy prevent disease progression. Direct immunofluorescence staining (DIF) of the conjunctival biopsy for immune complex deposition in the basement membrane is diagnostic of MMP. The biopsy positivity rate depends on biopsy characteristics, laterality, old age, and disease activity. The current methods paper describes the technique of harvesting the conjunctival biopsy and using optimal cutting temperature medium-filled cartridges for sample transfer and block preparation. A biopsy is performed from a non-scarred, uninvolved, or relatively less-involved region of the conjunctiva in both eyes. The usual biopsy size is 4-5 mm long, 2-3 mm wide, and should have tiny underlying subconjunctival tissue so that the basement membrane is not missed. We recommend performing periodic acid Schiff staining to confirm the presence of the basement membrane. Using a specified protocol, the cryosections from biopsies are stained with fluorescent-conjugated antibodies IgG, IgM, IgA, and complement factors C3c and C4c. A positive biopsy shows the green positive fluorescence in the basement membrane, and even if one of the antibodies is positive, it is MMP positive. A negative biopsy does not rule out pemphigoid; however, a positive one confirms the MMP and differentiates it from drug-induced cicatrizing conjunctivitis or pseudopemphigoid.