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

Summary

Here, we present a protocol for harvesting and processing conjunctival biopsy to identify mucous membrane pemphigoid.

Abstract

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.

Introduction

Mucous membrane pemphigoid (MMP) is a chronic cicatrizing conjunctival disease of autoimmune etiology and affects mucosal membranes in the body^1,2. Chronic cicatrizing conjunctivitis has different etiologies, and differentiation of MMP from SJS, sarcoidosis, ocular squamous neoplasia, and pseudopemphigoid is necessary as the former requires systemic immunosuppression^3,4. Moreover, MMP is a potentially blinding disease where patients often present late and are misdiagnosed initially^1,2,

Protocol

The protocol follows the guidelines of the human research ethics committee of LV Prasad Eye Institute human research ethics committee.

1. Conjunctival biopsy technique

1. Plan biopsy from both eyes of the patient suspected of pemphigoid under topical or local anesthesia and operating microscope.
2. Take informed consent from the patient and list possible side effects of bleeding, scarring, and the need to start topical steroids following surgery.
3. Place 1-2 drops of 0.5% proparacaine in both eyes and wait 5 min before starting the procedure.
4. Ensure the patient does not feel pain when touch....

Results

The interpretation of DIF requires confirmation of the presence of the basement membrane in the tissue sections (Figure 1). In the PAS-stained slides, the basement membrane will appear as a bright pink membrane situated at the basal region of the conjunctival epithelium (Figure 1C). The basement membrane's continuity and architecture should also be commented upon. DIF positivity will be seen as bright green BM (Figure 1B), indicatin.......

Discussion

The technique of conjunctival biopsy described here uses OCT medium as a transfer medium, obviating the need for an additional transfer medium and avoiding the biopsy handling for transferring it to OCT medium for cryosectioning. A plastic cartridge mold filled with medium and biopsy is directly frozen to make tissue blocks and then sectioned. The advantages of OCT are that it freezes rapidly with minimal damage from crystal formation and encapsulates the tissue specimens. The most critical step in direct immunofluoresce.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 N hydrochloric acid	Finar  chemical Limited	7647-01-0
Acetone	Qualigens	Q33517
Albumin	DAKO	F0117
Basic fuchsin	Qualigens	Q39402
C3c	Diagnostic Biosystems	F003
C4c	Diagnostic Biosystems	F005
Cartridge	Polysciences	25376-500
Charcoal Activated	Sigma	C-5260
Cryostat	Leica	CM1860UV
Cryotome blades	Leica	818
DAPI	Abcam	Ab104139
Fibrinogen	Diagnostic Biosystems	F006
Fluorescence Microscope	Olympus	CX43
Forceps	Joja surgical	JS1528A
Humidity Chamber	Diagnostic Biosystems	SM365
IgA	Diagnostic Biosystems	F007
IgG	Diagnostic Biosystems	F008
IgM	Diagnostic Biosystems	F009
IHC Adhesive glass slides	Tomo	33140455
Lim's forceps	Speedway	S-7223
Michel's tansport medium	Fisher Scinitics	NC0253304
Microscopic cover glass	Blue star	https://www.bluestarslides.com
OCT Medium	Cryomatrix	6769006
PBS	DAKO	dm831
Periodic acid	Sigma	375810-25G
Potassium disulfite	Sigma	P2522-500G
Proparacaine 0.5%	Sunway Private	G/1118A
Westcott's scissors	Pillig Gurgical Intruments	423480