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

We are trying to increase the adoption of conjunctival biopsy for the diagnosis of mucous membrane pemphigoid, which is a chronic cicatrizing conjunctivitis, and if left untreated, it can cause blindness. Direct immunofluorescent staining of the conjunctival biopsy for immune complex deposition in the patient membrane is diagnostic of MMP, but is not performed at many centers across the world. The current methods paper describe the techniques of the harvesting conjunctival biopsy and using optimal cutting temperature medium filled with cartridges for sample transfer and blood preparation, which reduce the sample handling.

To begin, plan a biopsy from both eyes of the patient suspected of pemphigoid under topical or local anesthesia using an operating microscope. Take informed consent from the patient and clearly explain the possible side effects, including bleeding, scarring, and the potential need to start topical steroids after surgery. Before taking a biopsy, prepare the cartridges by cleaning them using normal saline and filling with optimal cutting temperature, or OCT medium separately for each eye.

Now place a speculum into the selected eye, either right or left. After applying topical proparacaine, if the patient still experiences pain, inject 0.2 milliliters of 2% lidocaine subconjunctivally using a 1 milliliter to tuberculin syringe. After lifting the conjunctiva off the sclera using limbs forceps, perform a linear excision measuring approximately 4-5 millimeters long and 2-3 millimeters wide with a pair of Westcott scissors.

Immediately transfer the entire biopsy into an optimal cutting temperature medium. Snap freeze the embedded samples using liquid nitrogen or dry ice. Then store them at -80 degrees Celsius overnight.

Section the frozen tissue into 4-6 micrometer thick slices using a cryostat maintained at -20 to -25 degrees Celsius. Mount the sections onto positively charged polylysine-coated microscope slides to ensure adhesion. Then wrap the slides in aluminum foil before storing at -80 degrees Celsius until staining.

Thaw the slides at room temperature for 10 minutes. Then fix the tissue in 100%cold acetone for another 10 minutes to preserve antigens and minimize tissue damage. Now, rinse the slides with OBS three times, each for five minutes to eliminate residual OCT medium and acetone.

Apply diluted conjugated antibodies, anti-IgG, anti-IgA, anti-IgM, and anti-IgC3 over the slides. Do not apply the antibodies for the negative control slide. Now incubate the slides in a humidified chamber at 37 degrees Celsius for 45-60 minutes.

Then wash the slides with PBS three times for five minutes each to remove unbound antibodies. Counterstain with a mounting medium containing DAPI for nuclear visualization. Lastly, place a cover slip over each slide and allow them to cure for 20 minutes.

Examine the slides using a fluorescence microscope equipped with the appropriate filters, including a green excitation filter at 488 nanometers for fluorophore detection. The basement membrane was confirmed in conjunctival biopsies using PAS staining, appearing as a continuous bright pink band at the base of the epithelium. Direct immunofluorescence positivity was visualized as bright green fluorescence along the basement membrane.

Direct immunofluorescence positive biopsies showed fluorescence for immunoglobulins IgM, IgA and IgG, while negative biopsies lacked fluorescence. The positive control showed strong green fluorescence at the basement membrane.