Mucous membrane pemphigoid (MMP) is a chronic autoimmune subepithelial blistering disorder characterized by autoantibody-mediated injury to components of the epithelial basement membrane zone. It predominantly affects mucosal surfaces, including the oral cavity, ocular surface, nasopharynx, esophagus, and anogenital mucosa. ^, Monosite ocular involvement is termed ocular MMP (oMMP) or ocular cicatricial pemphigoid and remains one of the most diagnostically challenging causes of chronic cicatrizing conjunctivitis. Early oMMP can present with subtle, nonspecific symptoms such as recurrent conjunctivitis that may respond transiently to topical therapy, delaying disease recognition. More commonly, patients present with variable and sometimes minimal ocular surface inflammation and ocular surface and progressive conjunctival fibrosis that become clinically apparent only at more advanced stages. Diagnostic difficulty is further complicated by the overlap between oMMP and other inflammatory or cicatrizing conjunctival disorders, in which conjunctival and extraocular biopsies with adjunctive histopathology can aid in distinguishing alternative or overlapping diagnoses. ^, Delayed diagnosis frequently leads to a corresponding delay in the initiation of systemic immunosuppressive therapy (IMT), increasing the risk of ocular sequelae and vision loss. However, in patients with characteristic progressive cicatrization and exclusion of common mimickers based on history and examination, the diagnosis can be made clinically, and treatment should be instituted in a timely manner without pending biopsy confirmation. This perspective will focus on the diagnostic challenges of oMMP, particularly the role and limitations of direct immunofluorescence (DIF) in conjunctival biopsy.

Narrowing down the differentials in a case of cicatrizing conjunctivitis hinges on meticulous history taking. This includes ruling out congenital causes such as ectodermal dysplasia or epidermolysis bullosa and acquired causes such as postsurgery, postradiation, and ocular burns. History of trachoma, atopy, radiation, allogeneic bone marrow transplant, and antiglaucoma medication use suggests cicatrization secondary to these causes. Other rare causes include paraneoplastic pemphigoid and cicatrization in the setting of autoimmune diseases such as Sjogren disease. Once history excludes these possible causes, the clinician is primarily left with differentiating between Stevens-Johnson syndrome (SJS) and MMP. The acute presentations of these 2 clinical entities are often distinctly different. If the clinician has evaluated the patient during this phase or has access to reliable prior documentation, the diagnosis is usually straightforward. However, this is not always possible, as patients may present years after the initial episode or without adequate medical records. In such situations, the scoring system proposed by Shanbhag and associates may assist in differentiating between the two. This system incorporates key historical features such as hospitalization, cutaneous desquamation, and nail shedding, along with clinical findings such as distichiasis, which collectively favor SJS over MMP. A score >5 has been suggested to be indicative of SJS.

The diagnosis of MMP is established through a combination of clinical assessment, DIF microscopy, histopathology, and serologic testing. The fundamental clinical prerequisite is the predominant involvement of mucosal surfaces, compared with limited or absent skin disease. Once clinical suspicion is raised, the cornerstone investigation is DIF of a perilesional biopsy, which typically demonstrates linear deposition of IgG, IgA, and/or C3 along the subepithelial basement membrane zone. ^, A perilesional punch biopsy is recommended for patients with skin lesions, whereas in isolated mucosal disease, the biopsy should be taken from perilesional or normal-appearing mucosa (eg, buccal mucosa, inner lip). Because the sensitivity of DIF ranges from approximately 60% to 90% and may initially be negative, current guidelines recommend repeating the biopsy at least once, and even multiple times, if clinical suspicion remains high. ^, Histopathologic examination of a lesional specimen with routine hematoxylin-eosin staining supports the diagnosis by demonstrating subepithelial blister formation and helps differentiate MMP from intraepithelial blistering disorders such as pemphigus vulgaris. Serologic evaluation complements tissue diagnosis and includes indirect immunofluorescence microscopy, particularly using salt-split human skin. This substrate is prepared by inducing a split at the dermal-epidermal junction, thereby separating the epidermal and dermal components and exposing target antigens at the basement membrane zone. This improves assay sensitivity for detecting circulating autoantibodies in MMP and related subepidermal blistering disorders and is generally considered more sensitive than alternative epithelial substrates, where antigen accessibility may be more limited. In addition, the pattern of antibody binding on salt-split skin helps localize target antigens to either the blister roof (epidermal side; eg, BP180, BP230, and α6β4 integrin) or the blister floor (dermal side; eg, laminin 332 and type VII collagen), thereby aiding in further disease classification. ^,

Conjunctival biopsy with DIF has been widely used as a diagnostic tool for oMMP. In practice, however, clinical series demonstrate that the sensitivity of DIF is limited in ocular-predominant disease, with false-negative results occurring in a substantial proportion of patients who otherwise meet the clinical diagnostic criteria. ^,, Across studies using conjunctival specimens, reported DIF positivity ranges from approximately 40% to nearly 80%, reflecting heterogeneity in patient populations, disease distribution, and biopsy practice. ^,,,, Negative DIF results occur across a spectrum of disease severity, limiting the reliability of DIF as a discriminator of the disease. ^,

Several disease- and sampling-related factors contribute to reduced or inconsistent DIF sensitivity. First, DIF results may vary over time, and a single biopsy may not reflect the immunopathologic status across the disease course. Second, immunoreactant deposition may be focal, such that single-site conjunctival sampling can miss diagnostically informative areas. Consistent with this, bilateral conjunctival biopsy has been associated with higher positivity than unilateral sampling. Third, patients with isolated ocular involvement seem more likely to have negative biopsy results than those with additional extraocular disease, and conjunctival biopsies may yield negative DIF results more frequently than biopsies taken from other sites. ^,

Importantly, patients with either negative or positive biopsy results have been reported to have similar outcomes, and isolated ocular disease may still progress despite negative immunopathology result. This clinicopathologic discordance challenges test-centered diagnostic paradigms and underscores the need for judicious correlation. Although DIF remains an important diagnostic tool for oMMP, false-positive linear staining may occasionally occur in healthy conjunctival tissue; therefore, DIF results should always be interpreted in conjunction with clinical findings and histopathologic correlation. ^,

A conjunctival biopsy is performed under topical or local anesthesia. Obtaining biopsies from both eyes simultaneously is recommended to increase the rate of positivity. A conjunctival strip measuring approximately 4 to 5 mm in length and 2 to 3 mm in width is harvested from the inferior bulbar conjunctiva in a clinically uninvolved area adjacent to the area of conjunctival scarring. Alternatively, biopsy of the oral or inner lip mucosa can be considered. The excised tissue is divided into 2 equal portions for histopathologic examination and DIF analysis. One portion is fixed in 10% formalin for routine histopathology, whereas the second portion is first transported in Michel’s medium and later embedded in optimal cutting temperature compound, laid flat within the medium, and transferred immediately for snap freezing. Furthermore, ∼10 cryosections of 3 to 4 µm in thickness are prepared on poly- l -lysine–coated slides, wrapped in aluminum foil, and stored at −20°C until further processing. Sections are stained with rapid periodic acid–Schiff stain and processed for immunofluorescence using antibodies against IgG, IgA, IgM, C3, and C4, along with positive and negative controls. Antibody dilutions are standardized using known controls, with IgG, IgA, IgM, and C4 typically diluted at 1:10 and C3 at 1:5.

For DIF staining, slides are thawed at room temperature for 10 minutes, fixed in 100% cold acetone for 10 minutes, and washed 3 times with 1 × phosphate-buffered saline (PBS). Fluorescein-conjugated antibodies diluted in PBS are then applied to the tissue sections, whereas negative controls are processed identically without antibody application. The slides are incubated in a humidified chamber at 37°C for 45 to 60 minutes, washed with PBS, counterstained with 4′,6-diamidino-2-phenylindole–containing mounting medium, and coverslipped before examination under a fluorescence microscope. Interpretation of DIF requires confirmation of an intact basement membrane on periodic acid–Schiff staining, where it appears as a bright pink linear structure beneath the conjunctival epithelium. DIF positivity is identified by bright green linear fluorescence along the basement membrane, indicating deposition of IgG, IgA, IgM, C3, or C4. Positivity for any 1 immunoreactant is considered DIF positive.

Considering the limitations of conjunctival biopsy with DIF, optimization of biopsy site selection, specimen adequacy, and ancillary testing strategies is essential to improve diagnostic yield. A biopsy should be obtained from conjunctiva or oral mucosa that is clinically uninvolved or minimally involved, avoiding densely scarred or keratinized areas, as end-stage cicatricial tissue may yield limited diagnostic information and may compromise conjunctival reserve. ^, Most technique descriptions favor bulbar over tarsal conjunctiva, particularly in regions that remain mobile and structurally preserved. Superior bulbar conjunctiva away from the limbus is commonly selected to balance diagnostic yield with limbal preservation, whereas inferior bulbar conjunctiva may serve as an alternative when needed. These preferences reflect established surgical practices aimed at preserving ocular surface function while maintaining diagnostic utility.

Specimen adequacy and orientation also influence interpretation. Very small or fragmented specimens may preclude reliable assessment of linear immunoreactant deposition along the epithelial basement membrane. ^, Conversely, unnecessarily large biopsies may increase tissue manipulation and conjunctival morbidity without clear benefit. A single, well-oriented specimen of adequate size is therefore preferred, particularly in eyes with limited conjunctival reserve. Prompt and appropriate tissue handling, including immediate placement into suitable transport media and avoidance of fixation delays, is key to preserving immunoreactant integrity and optimizing DIF yield. ^,

Initiation of immunomodulatory therapy before performing a mucosal biopsy can reduce tissue immunoreactivity and lower the sensitivity of DIF testing. Biopsies performed after prolonged clinical remission, as observed in autoimmune blistering disorders, may yield negative results despite ongoing or relapsing disease, likely reflecting suppression of immunologic activity. ^,, These findings highlight that prior or ongoing immunosuppressive treatment can influence immunopathologic outcomes and should be carefully considered when interpreting negative biopsy results. When initial DIF results are negative despite high clinical suspicion, additional strategies may be considered. Immunoperoxidase staining on paraffin-embedded tissue may provide complementary information when fresh tissue is unavailable or results are equivocal. A repeat biopsy or biopsy from alternative mucosal sites, such as the oral mucosa, may increase diagnostic yield in selected patients with multisite disease involvement. ^,,

Although conjunctival biopsy with DIF is often regarded as a minor diagnostic procedure, it carries potential risks in patients with oMMP, where conjunctival inflammation, fragility, and impaired healing are common. Reported adverse effects relate primarily to disease exacerbation and conjunctival scarring rather than to acute surgical complications. ^, Surgical trauma has been proposed as a trigger for increased inflammatory activity, particularly when the disease is active or insufficiently controlled at the time of biopsy. These observations underscore the importance of careful timing, perioperative disease control, and adjunctive anti-inflammatory management. Longer-term concerns center on conjunctival scarring and loss of conjunctival reserve. Biopsy-associated progression of cicatrization, including symblepharon formation and fornix shortening, has been reported, although causality is difficult to establish in a progressive fibrotic disease. The risk seems higher with repeated conjunctival interventions, and multiple biopsies from the same eye have been cautioned against because of cumulative scarring. ^, Conversely, large clinical series suggest that conjunctival biopsy can be performed safely when appropriate technique and site selection are used. In a cohort of >300 bulbar conjunctival biopsies, no significant biopsy-related complications were reported, supporting the relative safety of the procedure when the fornices are avoided. ^, When clinically indicated, performing a biopsy under conditions of controlled inflammation and with appropriate perioperative anti-inflammatory therapy may reduce biopsy-related flares.

Establishing a diagnosis of oMMP does not mandate a positive conjunctival biopsy result. The diagnosis can be made clinically in the presence of progressive conjunctival cicatrization after exclusion of alternative causes such as SJS, chemical injury, topical medication–induced toxicity (eg, antiglaucoma medications), or systemic agents such as gliptins. This raises the question of whether DIF of conjunctival biopsy specimens is redundant. A recent survey of practice patterns for oMMP diagnosis demonstrated substantial heterogeneity among ophthalmologists. Although most respondents reported performing conjunctival biopsy with DIF, more than one quarter did not perform biopsies, citing concerns about exacerbating inflammation, limited impact on management, or lack of access to appropriate laboratory testing. Among those who performed biopsies, practices varied with respect to unilateral vs bilateral sampling, lesional vs perilesional site selection, and the use of routine histopathology or immunoperoxidase staining. European ophthalmologists were more likely than their North American counterparts to perform bilateral and perilesional biopsies and to recommend extraocular biopsies. Routine serologic testing for circulating autoantibodies was not commonly performed overall but was used more frequently by less experienced clinicians. Most respondents managed oMMP outside of a multidisciplinary setting. Overall, the findings highlighted discrepancies between real-world practice and published guideline recommendations, suggesting that variability is influenced by geographic location, experience level, and resource availability.

Although not obligatory for diagnosis, conjunctival biopsy retains clinical utility in selected scenarios. Its value is greatest in patients with very early disease or atypical presentations, where clinical findings may be inconclusive. ^, In cases of suspected drug-induced cicatrizing conjunctivitis, a positive DIF result can help secure the diagnosis of oMMP. ^, This distinction is critical, as it supports the initiation of systemic IMT while permitting the continuation of necessary topical antiglaucoma medications, rather than prompting premature surgical intervention solely to eliminate exposure to the suspected offending agent. Furthermore, histopathologic confirmation may facilitate patient counseling, particularly in individuals with early-stage disease confined to the ocular surface and preserved visual acuity. Such patients may be reluctant to accept systemic IMT with potentially significant adverse effects when visual function remains good. A positive biopsy result can help justify the need for systemic treatment and underscore the inadequacy of topical therapy alone in preventing disease progression. In addition, when clinically indicated, biopsy with adjunctive histopathology can facilitate the identification of alternative or concurrent diagnoses, such as sarcoidosis or ocular malignancy. ^,

Clinical assessment and conjunctival biopsy represent complementary components of oMMP diagnosis. From a management perspective, the need for systemic IMT is often determined by clinical behavior rather than histopathologic confirmation alone. A major limitation of a biopsy-driven diagnostic approach is the potential delay in initiating IMT. Such delays are often logistical. Patients may defer or decline a biopsy for personal reasons. In resource-limited settings, access to laboratories capable of performing and interpreting DIF may be restricted. Furthermore, initiation of IMT is frequently contingent upon medical consultation, adding another step to the treatment pathway. Importantly, a negative biopsy result may discourage treatment initiation despite strong clinical suspicion, thereby risking ongoing disease progression.

Conversely, clinician-initiated IMT based solely on clinical assessment carries a risk of overdiagnosis in cases that are not MMP. This risk can be mitigated when management is undertaken by clinicians with specific expertise in oMMP or through timely referral to specialized centers. For clinicians developing experience in managing this condition, a structured approach is advisable. The diagnostic algorithm presented in Figure 1 is proposed by the authors as a pragmatic clinical framework to guide the assessment and management of oMMP. It is based on expert clinical experience and available literature. Patients presenting with cicatrizing conjunctivitis should undergo comprehensive history-taking to exclude alternative etiologies such as SJS, chemical injury, graft-versus-host disease, atopic disease, and drug-induced pemphigoid. In general, clinical features that favor oMMP over other differential diagnoses include conjunctival cicatrization that is disproportionate to, or exceeds, eyelid margin disease and corneal involvement, medial canthal cicatrization, and keratinization. In cases with high clinical suspicion, biopsy and IMT may be initiated concurrently. In early or subtle disease, a stepwise approach with a conjunctival biopsy followed by IMT is reasonable. In advanced disease with extensive fibrosis, immediate initiation of IMT while deferring biopsy may be justified because its yield is often low due to the scarred conjunctiva.

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