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^1,2,5. Only ocular involvement without systemic involvement can be seen in 27.4% of ocular MMP patients⁶. Diagnosis of MMP requires a conjunctival biopsy to show the presence of autoantibodies in the basement membrane^2,5. Direct immunofluorescence (DIF) is a pivotal diagnostic tool for identifying autoimmune diseases affecting the conjunctiva, such as MMP. DIF helps detect the in-situ deposition of immunoglobulins and complement components at the basement membrane zone (BMZ), a characteristic of MMP. The DIF positivity rates vary from 30% to 80%^1,5. However, some patients can have a negative biopsy, especially those with ocular-only MMP, but clinical documentation of disease progression is treated similarly to biopsy-positive ocular MMP cases^7,8. Hence, Dart et al. stressed the need for separate diagnostic criteria for ocular-only MMP⁸.

The proposed reasons for the low positivity rate across studies are advanced disease with extensive ocular surface scarring, inadequate tissue processing or staining techniques, and a biopsy-negative disease variant^8,9,10,11. A retrospective analysis of biopsies from 27 patients suspected of ocular MMP revealed only a 30% positivity rate, and 63.7% had only ocular involvement¹². DIF requires a specific protocol; not every diagnostic laboratory is attuned to that. There are differences regarding the biopsy site, whether to perform a biopsy from one or both eyes, and sample transfer solutions^13,14,15. In a retrospective analysis of the conjunctival biopsies at our center, the DIF positivity rate was 54.1%, with more positives when biopsied bilaterally (59% vs. 45%)⁸. Although a biopsy is critical, clinical signs and progressive ocular surface cicatrization help clinch the diagnosis of MMP. It is easier to diagnose when an extraocular site is involved, such as oral or skin, reported in 40% and 18% of patients, respectively⁵.

There are variations in the existing literature, and the protocol presented here can serve as a reference guide with modifications introduced to reduce the need for a unique transfer medium¹⁴. The biopsies are transferred in an optimal cutting temperature (OCT) medium and snap-frozen in the same transfer cartridge in the pathology lab, obviating the need for an additional transfer medium. The current paper outlines the methodology for DIF staining of conjunctival tissue, tissue harvesting, and sample transfer protocol.

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 touching the conjunctiva with fine forceps. If the patient still feels pain after applying topical proparacaine, inject 0.2 mL of subconjunctival 2% lidocaine with a 1 mL tuberculin syringe.
5. Place a speculum in the right or left eye, whichever is chosen first.
6. Examine the ocular surface for areas of scarring and keratinization. Select the biopsy area that is uninvolved and not scarred or keratinized.
   NOTE: In this study, the superior bulbar conjunctiva away from the limbus is preferred as the biopsy site, followed by the inferior bulbar conjunctiva.
7. Use Lim's forceps to lift the sclera off the conjunctiva and linearly excise with Westcott's scissors, measuring 4-5 mm long and 2-3 mm wide.
8. Transfer the whole biopsy immediately to an optimal cutting medium (OCT).
   NOTE: One can transfer biopsies in normal saline or a Michel transport medium.
9. Take a cartridge and a plastic tissue mould with a well in the centre, clean it with saline, and air-dry it.
10. Fill the cartridge with OCT medium separately for the right and left eye. Submerge the excised conjunctival tissue in the OCT medium and send it to pathology.
11. Repeat the similar steps for the contralateral side as well.

2. Transferring the tissue

1. Embed the biopsy specimens of the conjunctiva in an OCT medium to preserve tissue architecture and antigenicity if received in Michel's transport medium or normal saline.
2. Snap-freeze the embedded samples using liquid nitrogen or dry ice and store at -80 °C overnight.

3. Preparation and tissue sectioning

1. Section the frozen tissue into 4-6 µm slices (7 in number) using a cryostat at -20 °C to -25 °C.
2. Mount the sections on positively charged polylysine-coated microscope slides to ensure adhesion and store at -80 °C until staining in aluminum foil.

4. Immunofluorescent staining

1. Thaw slides at room temperature (RT) for 10 min and fix in cold acetone (100%) for 10 min to preserve antigens and prevent tissue damage.
2. Rinse with 1x PBS thrice, each time for 5 min to remove excess OCT medium and acetone.
3. Apply conjugated antibodies anti-IgG, anti-IgA, anti-IgM, and anti-C3 diluted (Fibrinogen-1:10 [positive control], IgG-1:10, IgA-1:10, IgM-1:10, C3-1:5, C4-1:10) over slides.
   NOTE: Antibodies are diluted in 1x PBS.
4. For negative controls, skip the application of conjugated antibodies and follow the remaining steps mentioned below.
5. Follow the manufacturer's guidelines for diluting the antibodies and standardize with known positive and negative controls.
6. Incubate the slides in a humidified chamber at 37 °C for 45-60 min.
7. Wash slides with PBS (3× for 5 min each) to remove unbound antibodies.
8. Counterstain with DAPI (4',6 -diamidino-2-phenylidole)-containing mounting medium for nuclear visualization.
9. Place a coverslip over the slides and allow them to cure for 20 min.
10. Examine slides using a fluorescence microscope equipped with appropriate filters for the fluorophores used (excitation green filter for 488 nm).
11. Document the findings with digital micrographs.

5. Rapid periodic acid - Schiff staining

1. Preparation of periodic acid Schiff (PAS)
   1. Dissolve 1 g of basic fuschin in 100 mL of hot distilled water.
   2. Add 10 mL of hydrochloric acid and 2 g of potassium disulfite to decolorize the dye.
   3. After cooling, add 0.5 g of activated charcoal, stir, and filter the solution to obtain clear periodic acid Schiff's reagent. Store in a dark bottle.
      NOTE: To test for Schiff's reagent, pour 10 mL of 37% formalin into a watch glass and add a few drops of Schiff's to be tested. A good Schiff's reagent will rapidly turn a red-purple color. A deteriorating Schiff's reagent will cause a delayed reaction, and the product will be a deep blue-purple.
2. PAS staining technique
   1. Hydrate the slides in water.
   2. Oxidize in 0.5% periodic acid solution for 5 min.
   3. Rinse in distilled water.
   4. Place in Schiff's reagent for 15 min (sections look light pink during this step)
   5. Wash in lukewarm tap water for 5 min (sections turn dark pink).
   6. Counterstain in Mayer's hematoxylin for 1 min.
   7. Wash in tap water for 5 min.
   8. Dehydrate the slides in the following solutions: One time in 80% ethyl alcohol for 1 min, two times in 90% ethyl alcohol for 1 min, two times in 100% ethyl alcohol for 1 min.
   9. Dehydrate the slides three times in xylene for 1 min each.
   10. Place a coverslip over the slides using a synthetic mounting medium (e.g., DPX mountant)
   11. Observe the PAS results under the bright field microscope.

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