
==== Front
BMC Oral Health
BMC Oral Health
BMC Oral Health
1472-6831
BioMed Central London

39285275
4853
10.1186/s12903-024-04853-y
Systematic Review
Oral biopsy in mucous membrane pemphigoid and pemphigus vulgaris with gingival expression: the optimal site. A systematic review and meta-analysis
Dridi Sophie-Myriam 123
Lutz Claire Manon 456
Gaultier Frédérick 47
Bellakhdar Fadel 7
Jungo Sébastien 589
http://orcid.org/0000-0002-0396-3836
Ejeil Anne Laure anne-laure.ejeil@u-paris.fr

4510
1 Faculty of Odontology, Department of Periodontology, Côte d’Azur, Nice, France
2 grid.410528.a 0000 0001 2322 4179 Institute of Oral Medicine Riquier, CHU Nice, France
3 Oral Microbiology, Immunotherapy and Health, Nice, EA 7354 France
4 Faculty of Odontology, Department of Oral Surgery, Paris Cité, France
5 https://ror.org/0146pps37 grid.411777.3 0000 0004 1765 1563 Department of Odontology, Bretonneau Hospital AP-HP, 2 rue Carpeaux, Paris, 75018 France
6 https://ror.org/008e1sp22 Human Immunology Pathophysiology and Immunotherapy, UMRS 976, Paris, France
7 grid.412116.1 0000 0004 1799 3934 Department of Odontology, Henri Mondor Hospital AP-HP, Créteil, France
8 Faculty of Odontology, Department of Periodontology, Paris Cité, France
9 Centre de Recherche Epidémiologique et Biostatistiques, UMR 1153, Paris, France
10 https://ror.org/05f82e368 grid.508487.6 0000 0004 7885 7602 Université Paris Cité, Laboratory URP2496 Orofacial Pathologies, Imaging and Biotherapies, Paris, France
16 9 2024
16 9 2024
2024
24 10938 4 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Purpose

In order to diagnose mucous membrane pemphigoid (MMP) and pemphigus vulgaris (PV) with gingival expression, clinical data must be compared with immunohistochemical data obtained using direct immunofluorescence (DIF). It is therefore essential to carry out a good quality mucosal biopsy for this vital additional test. To date, no study has been able to effectively guide clinicians in their choice of oral site for biopsy to guarantee the efficient contribution of DIF to diagnosis. We propose a systematic review of the literature and a meta-analysis to clarify this issue.

Materials and Methods

Electronic databases and bibliographies of articles were searched in April 2023. The primary outcome was the rate of DIF + contribution to diagnosis according to the location of the oral site biopsied.

Results

16 studies were included. Gingival biopsies showed a rate of DIF + 100% [97%-100%] p = 0.998 I2 = 0.0% with no heterogeneity for PV, and 90.2% [66.5%-100%] p < 0.001 I2 = 89.6% with high heterogeneity for MMP. For the other oral sites, this rate was 95.7% [87.4%- 100%] p = 0.011 I2 = 73.0% with moderate heterogeneity for PV, and 87.4% [70.1%- 98.7%] p < 0.001 I2 = 92.6% with high heterogeneity for MMP. In addition, meta-regression confirmed the significant association between the appearance of the biopsied mucosa and the rate of DIF + in MMP (p < 0.001), with no influence on residual heterogeneity.

Conclusion

The nature of the oral mucosa biopsied does not influence the rate of DIF + to diagnosis. The choice of biopsy site should only take into account the characteristics of the clinical picture and the benefit/risk balance of the surgical protocol.

The sample must be taken in healthy aeras as close as possible of active lesions: on the gingiva if the MMP and PV are strictly gingival, on the alveolar mucosa if the whole gingiva is altered and on any healthy mucosa if a large number of oral sites are affected.

Clinical trials

CRD42023392345.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12903-024-04853-y.

Keywords

Oral biopsy
Desquamative gingivitis
Mucous membrane pemphigoid
Pemphigus vulgaris
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcIntroduction

Numerous mucocutaneous diseases affect the oral mucosa, including autoimmune bullous diseases (AIBD), a rare and heterogeneous group of chronic diseases that represent a diagnostic challenge due to their variable clinical presentations and potentially severe prognosis [1]. These diseases are characterized by the formation of intra- or sub-epidermal/epithelial bullae caused by autoantibodies or complement compounds directed against chorioepithelial or cellularjunctions. Some of these diseases, such as subepithelial cicatricial pemphigoid (MMP) and intraepithelial pemphigus vulgaris (PV) [2], may present inaugural oral involvement. In these clinical forms, they often lead to an isolated or predominant erosive gingivitis (commonly known as desquamative gingivitis) [3]. Thus, it can be clinically indistinguishable from other oral pathologies with gingival expression such as erosive gingival lichen planus plasma cell gingivitis [4, 5] and plaque-induced gingivopathies [6, 7].

Early diagnosis of AIBD is essential to prevent disease progression and potential complications. Indeed, delays in diagnosis vary from a few months to a few years in the case of oral AIBD [7, 8]. While clinical examination is crucial, definitive diagnosis relies on histopathological and immunohistochemical analysis, with direct immunofluorescence (DIF) which is considered the gold standard [9, 10]. In addition, the diagnostic value of DIF is greater than that of serological tests which, for this reason, are indicated as second-line tests to confirm the subtype of AIBD [11]. However, the quality of the biopsy directly impacts the sensitivity of DIF, making the selection of both the biopsy site and the surgical technique critical for accurate diagnosis, which requires that an informative tissue sample is obtained. Several authors have attempted to address these issues through clinical studies, including Zeng et al. (2021) [12]. However, currently there is no clear consensus within the literature regarding the optimal oral mucosal biopsy site for maximizing DIF sensitivity in diagnosing AIBD, particularly in cases of severe erosive gingivitis.

This lack of guidance often leads to diagnostic uncertainty and delays in appropriate treatment.

This systematic review of the literature and meta-analysis addresses this clinical challenge.

Its primary objective was to determine the optimal oral mucosal biopsy site to achieve the highest diagnostic yield of positive direct immunofluorescence staining (DIF +) in cases of MMP or PV with erosive gingivitis (This analysis compared the diagnostic accuracy of biopsies taken from the attached gingiva, papillary gingiva versus various mucosal sites including alveolar mucosa, jugal mucosa, and labial mucosa).

Its secondary objectives were to:Assess whether the rate of DIF + (percentage of direct immunofluorescence-positive staining compared to the total sample) is influenced by the inflammation status and mucosal involvement;

Assess the impact of different surgical techniques on DIF results.

This comprehensive review synthesizes existing evidence and provides clinicians faced with a clear and practical guide for patients with suspected oral AIBD, particularly those with gingival involvement. This guidance will ultimately improve diagnostic accuracy, minimize patient discomfort and facilitate timely and effective treatment.

Material and methods

Recording the protocol

The literature review was prepared in accordance with the PRISMA recommendations, "Preferred Reporting Items of Systematic Reviews and Meta-Analysis", updated in 2021 [13]. The protocol was registered in the international PROSPERO database (Prospective Register of Systematic Review) under number CRD42023392345.

Research question and eligibility criteria

The main research question was: What is the optimal oral biopsy site to obtain a DIF + (immunostaining clearly visible) that contributes to the diagnosis of MMP and PV with gingival expression?

The selected studies eligible for reviewing were based on the PICOS criteria as follows:(P) Population: adults aged at least 18 years old with MMP or PV with gingival expression, whatever their general state of health, age and gender;

(I) Procedure: oral biopsy for diagnostic purposes using DIF, whatever the surgical protocol;

(C) Control: no comparison or comparison based on oral biopsy sites;

(O) Primary endpoint: rate of direct immunofluorescence-positive (DIF +) labelling.

(S) Type of study: retrospective, cross-sectional, or longitudinal study.

Search strategy and equations

This systematic review was based on three electronic bibliographic databases: PubMed (Medline), Embase (Experta Medical Database by Elsevier) and Scopus.

The search equations were as follows:PubMed (Medline): ((biopsy technique) OR (biopsy site) OR (fluorescent antibody technique [MeSH Terms]) OR (immunofluorescence)) AND ((pemphigus) OR (pemphigus [MeSH Terms]) OR (pemphigus vulgaris) OR (desquamative gingivitis) OR (mucous membrane pemphigoid) OR (blistering disease) OR (bullous pemphigoid) OR (mucous membrane pemphigoid, benign [MeSH Terms])) AND ((oral mucosa) OR (mouth mucosa [MeSH Terms]) OR (gingiva [MeSH Terms]).

Embase: ( 'biopsy technique'/exp OR 'biopsy technique'/br OR 'fluorescent antibody technique'/exp OR 'fluorescent antibody technique'/br OR 'immunofluorescence'/exp OR 'immunofluorescence'/br) AND ('pemphigus'/exp OR 'pemphigus'/br OR 'desquamative gingivitis'/exp OR 'desquamative gingivitis'/br OR 'bullous skin disease'/exp OR 'bullous skin disease'/br OR 'bullous pemphigoid'/exp OR 'bullous pemphigoid'/br OR 'mucous membrane pemphigoid'/exp OR 'mucous membrane pemphigoid'/br) AND ('mouth mucosa'/exp OR 'mouth mucosa'/br OR 'gingiva'/exp OR 'gingiva'/br OR 'oral mucosa'/br).

Scopus: TITLE-ABS-KEY (biopsy) AND TITLE-ABS-KEY ((pemphigus) OR (pemphigoid) OR (desquamative AND gingivitis) OR (vesiculo AND erosive AND diseases)) AND TITLE-ABS-KEY technique).

In addition, two additional manual searches were carried out, one using the bibliographies of articles eligible for full reading, and the other using several specialist odontology or dermatology journals indexed in PubMed (J Clin Periodontol, J Periodontol, J Periodontal Res, J Dent Res, J Dentistry, J Dent Sci, Oral Surg, J Oral Pathol, JAMA Dermatol, BJD).

The search for articles was completed on 30 April 2023.

Selection of articles and data extraction

The two principal investigators (SMD, ALE) assessed the eligibility of the articles independently according to a standardised protocol. In the event of disagreement that could not be resolved through discussion, a 3rd reviewer (CML) was called on to make a final decision on the selection of contentious studies. The selection protocol had to comply with the following stages: after eliminating duplicates between databases using bibliographic reference management software (Zotero 5.0.96.3), articles were selected on the basis of their title, then their abstract and finally their full reading. Single-centre or multi-centre study, written in English or French, with no limit on the date of publication, except case reports, systematic reviews and meta-analyses, authors' opinions, expert opinions, questionnaires, and editorials were selected.

Data collection and synthesis were carried out independently and in a standardised manner by the two principal reviewers on the basis of articles that met the inclusion criteria.

Risk of bias and level of scientific evidence

The search for bias was carried out independently by two investigators (CML, SJ), using the Newcastle Ottawa Scale as recommended in the Cochrane handbook 5.1 for observational studies [14]. The accurate checklist was used according to the study design. The following threshold values were used to determine the risk of bias: low risk: 7 to 9 points, Moderate risk: 4 to 6 points, High risk: 0 to 3 points. The total score is the sum of the stars attributed to each section: Selection – 4 stars (representativeness; exposure assessment-cohort/cross-sectional; control selection – case–control), Comparability – 2 stars (adjustment for main/additional confounder), Outcome/Exposure-3 stars (adequacy of outcome and exposure). The level of scientific evidence was assessed using the GRADE system [15]. Observational studies are initially classified as low evidence. Then they could be re-upgrade according to the presence of a large effect size or a dose effect gradient. Conversely, they can be downgraded due to a high risk of bias, of a publication bias, inconsistency or heterogeneity across the results, or an imprecision due to a small sample size or a large confidence interval.

Quantitative analysis

The meta-analysis of the results of the contribution rates (% of positive DIF) was carried out using random-effects models. Given the non-normal distribution of the proportions and in order to stabilise the variances, a Freeman and Tukey double ArcSine transformation [16] was performed. The results were obtained using the Miller’s inverse transformation (1978) [17]. The studies were weighted according to the inverse of their variance. The heterogeneity of the results was assessed using the I2 statistic. It was considered non-existent below a value of 25%, low between 25 and 50%, moderate between 50 and 75% and high above 75%. From the outset, we planned to perform analyses stratified according to pathology (PV vs MMP), oral sampling site (gingiva vs other oral sites), sampling technique (slide vs punch), and mucosal condition (peri-lesional vs healthy). Publication bias was estimated using funnel plots of effect sizes and Egger tests. For stratified analyses, we planned to perform meta-regressions in the event of moderate heterogeneity and non-overlap of confidence intervals. All the analyses were performed using R software (4.2.2) and the Metafor package [18]. Analyses were considered significant when the p was < 0.05 or when the 95% IC did not overlap. Inter-rater reliability was calculated using kappa (κ) statistics.

Results

Our initial search strategy across tree database yielded an initial list of 4025 results. After removing. 339 duplicates and screening title, 134 articles remained. Abstract review led to the exclusion of 37 leaving 97 articles eligible for full reading. An addition, manual search identified a further 21 studies of which one was retained. Finally, 16 articles were eligible [8, 19–32]. Agreement for the final list was considered excellent (k > 0.90); only two articles [25, 32] required discussion between the principal investigators and the 3rd investigator to reach a conclusion. The discussion focused on the complex presentation or dispersion of the clinical data provided by the authors.

Details of the study selection process are shown in Fig. 1. The list of articles not included and the reasons for their non-inclusion is provided in supplementary data 1.Fig. 1 Flow chart of the research process according to the PRISMA 2021 recommendations [13]

General characteristics of the studies

Type of study

The studies were published between 1981 and 2022, including 5 from 2018 onwards. Twelve studies were retrospective [20–33], two were prospective [24, 29] and two were diagnostic [8, 19]. In addition, apart from 2 studies which were bicentric [32, 33], all the others were monocentrics. The characteristics of all the studies included are described in Table 1. Table 1 General characteristics of the studies included

Authors
references	Type of study
country	Inclusion and non-inclusion criteria	All subjects included (pathology)	Intervention	
Gaultier et al

[33]

	A 8-½ -year retrospective

Bicentric

France

	Inclusion

Patients with chronic erosive gingivitis in the active phase, whether isolated or predominant in the case of multisite disease, whatever their age or state of health, who have undergone a biopsy

Non-inclusion

Patients referred who had already had one or more biopsies performed in a department outside the two hospitals involved in the study

Patients on corticosteroid treatment

	n = 100

(MMP, PV, OLP, other pathologies)

	Biopsy for DIF: in the papillary gingiva

Site selection criteria: least inflamed papilla, at a distance from erosion

Procedure: excision of a vestibular papilla, using a n°15 or 15C scalpel blade, haemostasis control without sutures, Michel's medium for transport. Short-term follow-up (10–21 days post-operatively) and after 3 months or more

Type and number of investigators: variable, specialists in periodontology or oral surgery, interns or students in the specialty

Laboratory: specialised laboratories in both hospitals (slides analysed by the same pathologist for each hospital)

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunolabelling of IgG, IgA and/or C3 in the basement membrane zone; it is considered negative (-) if there is no labelling

DIF interpretation in the case of PV: the test is considered positive ( +) if an IgM, IgG, IgA or C3 immunolabelling is present in the intercellular space (honeycomb pattern); it is considered negative (-) if the labelling is absent

Additional tests: standard histology

	
Carey et al

[32]

	Retrospective, number of years ND

Bicentric

United Kingdom

	Inclusion

Patients referred for clinical suspicion of MBAI with DIF + 

Non-inclusion

ND

	n = 328

(MMP, PV)

	Biopsy for DIF: in the jugal, alveolar mucosa or gum

Site selection criteria: peri-injury (radius < 1 cm from a lesion) or healthy jugal mucosa

Procedure: use of a 4 mm diameter scalpel punch, Michel's medium for transport

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP and PV: ND

Additional tests: standard histology, IFI, ELISA (anti Dsg1 and 3, anti BP180 and BP 230)

	
Endo et al

[31]

	A 13-year retrospective

Monocentric

Japan

	Inclusion

Clinical diagnosis of PV with desquamative gingivitis (± other oral sites)

Non-inclusion

ND

	n = 10

(PV)

	Biopsy for DIF: sampling site ND

Site selection criteria: in a healthy area

Procedure: ND

Laboratory: ND

DIF interpretation in case of PV: by certified dermatologists, IgG deposits in intercellular areas

Additional examinations: standard histology, IFI peri-lesional, cytological study to look for Tzanck cells

	
Kamaguchi et al [30]	Transversal

Monocentric

Japan

	Inclusion

Patients with suspected MMP and need for an DIF that can be performed in the healthy jugal mucosa

Non-inclusion

ND

	n = 7

(MMP)

	Biopsy for DIF: in the jugal mucosa

Site selection criteria: healthy mucosa

Procedure: use of a 4 mm diameter scalpel punch, then freezing, ND transport medium

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is linear immunolabelling of IgG and/or C3 in the basement membrane zone; it is considered negative (-) if there is no labelling

Additional tests: standard histology, IFI, ELISA (anti BP180), Immunoblot

	
Rameshkumar et al

[29]

	Retrospective,

Monocentric

India

	Inclusion

Patients with ulcerative or erosive vesiculobullous disease requiring histological examination and DIF

Non-inclusion

ND

	n = 12

(MMP, PV, OLP, other pathologies)

	Biopsy for DIF: in the gum and jugal mucosa

Site selection criteria: peri-injury

Procedure: ND

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunostaining, IgG and/or IgC3 in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive ( +) in the presence of an IgG and/or C3 immunolabelling in the intercellular region

Additional tests: standard histology

	
Endo et al

[28]

	A 11-year retrospective

Monocentric

Japan

	Inclusion

painful desquamative gingivitis (± other intra- or extra-oral sites)

Non-inclusion

ND

	n = 27

(MMP, PV, OPL)

	Biopsy for DIF: in the attached gingiva

Site selection criteria: in a healthy area

Procedure: stab and roll technique, followed by freezing

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: by certified dermatologists, the test is considered positive ( +) in the presence of an IgG and/or C3, IgA immunostaining along the basal lamina

DIF interpretation in the case of PV: by certified dermatologists, the test is considered positive ( +) in the presence of an IgG immunostaining in the intercellular region

Additional tests: standard histology

	
Sano et al

[27]

	A 5-year retrospective

Monocentric

Argentina

	Inclusion

Patients with bullous disease (PV, MMP or Bullous Pemphigoid) or oral lichen planus, of any age and sex, who have undergone a histopathological study and an DIF

Non-inclusion

Patients without MBAI or OLP

Biopsy site not specified for DIF

Concomitant diagnoses: carcinoma, candidiasis, leukoplakia

	n = 125

(MBAI, OPL)

	Biopsy for DIF: in the gum, jugal, labial, lingual and palatal mucosa and the floor of the mouth

Site selection criteria: peri-injury (radius < 1 cm from a lesion) or distant

Procedure: use of a scalpel punch or scalpel blade, placement of the biopsy in saline solution, stored at 3–5 °C before being transported immediately to the laboratory. A specific biopsy or 1 half-biopsy (biopsy divided in two) for DIF

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunostaining for IgM, IgG, IgA and/or IgC3 in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive ( +) in the presence of an IgM, IgG, IgA or C3 immunostaining in the intercellular space (honeycomb pattern)

Additional tests: standard histology

	
Yih et al

[26]

	A 10-year retrospective

Monocentric

USA

	Inclusion

Patients with desquamative gingivitis

Non-inclusion

ND

	n = 72

(MMP, PV, OPL, linear IgA)

	Biopsy for DIF: in the gum

Site selection criteria: peri-lesional for vesiculobullous or erosive areas and striated area if lichenoid appearance

Procedure: ND, frozen at -20 °C (IgG, IgA and C3 tests)

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is an immunostaining for IgG and/or C3 and/or fibrinogen, linear in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive if an IgG and C3c immunolabelling are present in the intercellular space

Additional tests: standard histology

	
Helander et al

[25]

	A 5-year retrospective

Monocentric

United Kingdom

	Inclusion

Patients who have had an DIF

Non-inclusion

ND

	n = 342

(MBAI, OPL, other pathologies)

	Biopsy for DIF: in the gum, jugal mucosa or other oral mucosa

Site selection criteria: peri-injury

Procedure: ND, freezing in liquid nitrogen

Type and number of investigators: ND

Laboratory: specialised within the hospital (DTI performed blind to clinical diagnosis)

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunostaining for IgG and/or IgC3 and/or IgA in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive ( +) in the presence of a strong IgG and/or C3 immunolabelling in the intercellular region

Additional tests: standard histology

	
Venning et al

[24]

	Transversal

Monocentric

United Kingdom

	Inclusion

Patients referred either to dermatology (if skin bullae) or oral surgery (if bullae or ulcers in the oral mucosa, including the gums)

Non-inclusion

ND

	n = 51

(PB, MMP)

	Biopsy for DIF: in the lower labial mucosa

Site selection criteria: in healthy mucosa

Procedure: 3 mm scalpel punch, frozen in liquid nitrogen

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunostaining for IgG, IgA and/or C3 in the epidermal or epithelial basement membrane zone (skin, lip or conjunctiva biopsy

Additional tests: eye biopsy

	
Rogers and Van Hale

[23]

	A 11 ½ -year retrospective

Monocentric

USA

	Inclusion

Patients with ulcerative, erosive or bullous diseases of the oral mucosa who have undergone DIF

Non-inclusion

	n = 451

(MMP, PV, OPL, lupus,

other pathologies)

	Biopsy for DIF: in the oral mucosa ( location ND)

Site selection criteria: peri-injury

Procedure: freezing in liquid nitrogen

Type and number of investigators: ND

Laboratory: specialised within the hospital

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear immunostaining for IgG and/or C3 and/or IgA in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive ( +) in the presence of an IgG and/or C3 immunolabelling in the intercellular region

Additional tests: ND

	
Daniels and Quadra-White

[22]

	Retrospective, number of years: ND

Monocentric

USA

	Inclusion

Patients with chronic or recurrent ulcerative or erosive disease of the oral mucosa, diagnosed clinically and histopathologically, without knowledge of DIF results

Non-inclusion

Trauma, mouth ulcers, herpes, leukoplakia or ulcerations or erosions without an established diagnosis

	n = 130

(MMP, PV, OPL, other pathologies)

	Biopsy for DIF: in the gum or other mucous membranes ( location ND)

Site selection criteria: peri-injury

Procedure: care to preserve attached epithelium, freezing in liquid nitrogen

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is a linear IgG and/or C3 immunostaining in the area of the basement membrane with an intensity greater than that of the background; it is considered negative (-) if there is no staining with an intensity greater than that of the background and equivocal if the immunostaining is slightly greater than that of the background

DIF interpretation in PV: the test is considered positive ( +) if there is an intercellular IgG immunostaining with the 3 types of intensity as in pemphigoid

Additional tests: standard histology

	
Niesengard et al

[21]

	Retrospective number of years: ND

Monocentric

USA

	Inclusion

Clinical diagnosis of desquamative gingivitis

Non-inclusion

ND

	n = 100

(MMP, PV, OPL, psoriasis and other pathologies)

	Biopsy for DIF: in the gum or other oral mucosa (ND)

Site selection criteria: peri-injury or healthy mucosa

Procedure: transport in Michel's medium, or freezing at -20°

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is an IgG and/or linear C3 immunostaining in the basement membrane zone

DIF interpretation in the case of PV: the test is considered positive if an IgG immunolabelling is present in the intercellular region

Additional tests: standard histology, IFI

	
Laskaris and Angelopoulos

[20]

	Transversal

Monocentric

Greece

	Inclusion

Patients with MMP

Non-inclusion

ND

	n = 104

(33 MMP, other non-bullous pathologies)

	Biopsy for DIF: in the oral mucosa ( location ND)

Site selection criteria: ND

Procedure: biopsy taken with a slide divided in two (1 piece for DIF, 1 piece for histology) peri-lesionally, frozen at -30 °C

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of MMP: the test is considered positive ( +) if there is linear IgG and/or C3 immunostaining in the basement membrane zone

Additional tests: standard histology, IFI

	
Laskaris

[19]

	A 7-year retrospective

Monocentric

Greece

	Inclusion

Patients with PV with isolated oral involvement only

Non-inclusion

ND

	n = 129

(PV, other non-bullous pathologies)

	Biopsy for DIF: in the oral mucosa ( location ND)

Site selection criteria: peri-injury

Procedure: biopsy taken from the slide divided in two (1 piece for DIF, 1 piece for histology), frozen at -30 °C

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of PV: the test is considered positive if an IgG immunolabelling is present in the intercellular region

Additional tests: standard histology, IFI

	
Laskaris et al

[8]

	A 3 to 6- year retrospective

Monocentric

Greece

	Inclusion

Patients with severe desquamative gingivitis

Non-inclusion

ND

	n = 54

(MMP, PV, OPL, other non-bullous pathologies)

	Biopsy for DIF: in the gum

Site selection criteria: peri-injury

Procedure: biopsy taken with a slide divided in two (1 piece for DIF, 1 piece for histology) freezing at -30 °C

Type and number of investigators: ND

Laboratory: ND

DIF interpretation in the case of PV: the test is considered positive if an IgG immunolabelling is present in the intercellular region

Additional tests: standard histology, IFI

	
n number, ELISA Enzyme-linked immunosorbent assay, DIF Direct immunofluorescence, IFI Indirect immunofluorescence, ND Not determined, Ig Immunoglobulin, AIBD Autoimmune bullous disease, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris, OLP Oral lichen planus

Geographical location of populations

Four studies were carried out in the United States [21–23, 26], 3 in Greece [8, 19, 20], 3 in the United Kingdom [24, 25, 32], 3 in Japan [28, 30], 1 in France [33], 1 in India [29] and 1 in Argentina [27].

Characteristics of subjects

It was not possible to accurately determine the total number of MMP and PV patients because some studies counted these patients with those diagnosed with others oral pathologies such as oral lichen planus. Similarly, the distribution of women and men, as well as the average age of the subjects, could not be accurately assessed because in 5 studies these parameters were not mentioned [8, 23, 25, 30, 32]. Nevertheless, all the subjects were adults.

Number of biopsies contributing to the diagnosis of AIBD

The cohorts of the 8 PV patients in the study by Endo et al. (2014) [28] and the cohort of 15 MMP patients in the study by Laskaris et al. (1981) [8] were excluded as they were repeated by the authors in other studies.

Finally, all the studies together yielded a total of 859 oral biopsies contributing to the diagnosis of PV and MMP: 255 DIF + for PV and 604 DIF + for MMP.

Type of oral mucosa biopsied

The gingival and jugal mucosa were the most frequently sampled, followed by the labial, alveolar, lingual, palatal and floor of the mouth mucosa.

Risk of bias and level of evidence

Risk of bias are summarized in Table 2. Inter-examiner agreement in assessing risk of bias was judged to be excellent (k ˃ 0.90). All studies presented a moderate risk of bias except one, Laskaris et al. (1981) [8]. Level of evidence from main and secondary outcomes are presented in Tables 3, 4, 5 and 6 Certainty ranged from very low (for 2 main outcomes and 2 secondary outcomes) to moderate (for 2 main outcomes and 1 secondary outcome), with the remaining outcomes being classified as low. All outcomes were upgraded due to a high or very high effect size. Downgrades due to heterogeneity, a small number of studies, indirectness, wide confidence intervals, or publication bias are specified in the tables for each outcome. Table 2 Risk of bias estimation was done according to the Newcastle Ottawa Scale with the following values: Low risk: 7 to 9 points, Moderate risk: 4 to 6 points, High risk: 0 to 3 points. For each section, maximum stars are attributed the following way: Selection – 4 stars (representativeness; exposure assessment – cohort/cross-sectional; control selection – case–control), Comparability – 2 stars (adjustment for main/ additional confounder), Outcome/ Exposure – 3 stars (adequacy of outcome and exposure)

Authors
References	Selection	Comparability	Exposure	NOS score	Risk of bias	
Gaultier et al. [33]	**	*	**	5	Moderate	
Carey et al. [32]	**	*	**	5	Moderate	
Endo et al. [31]	**	*	**	5	Moderate	
Kamaguchi et al

[30]

	*	*	**	4	Moderate	
Rameshkumar et al. [29]	***	*	**	6	Moderate	
Endo et al. [28]	**	*	**	5	Moderate	
Sano et al. [27]	**	**	**	6	Moderate	
Yih et al. [26]	**	**	**	6	Moderate	
Helander et al. [25]	*	*	**	4	Moderate	
Venning et al. [24]	***	*	*	5	Moderate	
Rogers et van Hale [23]	**	*	**	5	Moderate	
Daniels et Quadra-White [22]	*	**	**	5	Moderate	
Niesengard et al

[21]

	*	*	**	4	Moderate	
Laskaris et Angelopoulos [20]	*	*	**	4	Moderate	
Laskaris [19]	**	*	*	4	Moderate	
Laskaris et al. [8]	*	*	*	3	High	

Table 3 Evaluation of the certainty of evidence for the primary outcomes according to GRADE guidelines (I)

Certainty assessment	№ of patients	Effect	Certainty	Importance	
№ of studies	Study design	Risk of bias	Inconsistency	Indirectness	Imprecision	Other considerations	[intervention]	[comparaison]	Absolute
(95% CI)	Relative
(95% CI)	
DIF + (assessed with: % of DIF +)	
16	non-randomised studies	not serious	seriousa	seriousb	not serious	very strong association

all plausible residual confounding would reduce the demonstrated effect

	859/975 (88.1%)	-	% 91.5

(83.2 to 97.6)

	-	⨁⨁⨁◯

Moderate

	4-IMPORTANT	
DIF + in PV (assessed with: (% of DIF +))	
12	non-randomised studies	not serious	not serious	seriousb	not serious	very strong association	255/266 (95.9%)	-	% 95.6

(96.0 to 100.0)

	-	⨁⨁⨁◯

Moderate

	6-IMPORTANT	
DIF + in MMP (assessed with: (% of DIF +))	
12	non-randomised studies	not serious	seriousa	not serious	seriousc	strong association

all plausible residual confounding would reduce the demonstrated effect

	606/711 (85.2%)	-	% 88.5

(75.8 to 97.5)

	-	⨁⨁◯◯

Low

	6-IMPORTANT	
Moderate certainty: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different

Low certainty: further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate

Explanations

CI Confidence interval, DIF + Positive direct immunofluorescence, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris

aHigh heterogeneity, p < 0.001, I2 > 75%

bMost studies published before year 2000

cWide 95%CI

Table 4 Evaluation of the certainty of evidence for the primary outcomes according to GRADE guidelines (II)

Certainty assessment	№ of patients	Effect	Certainty	Importance	
№ of studies	Study design	Risk of bias	Inconsistency	Indirectness	Imprecision	Other considerations	[intervention]	[comparaison]	Relative
(95% CI)	Absolute
(95% CI)	
DIF + in gingival biopsies in PV (assessed with: (% of DIF +))	
7	non-randomised studies	not serious	not serious	seriousb	seriousd	very strong association	43/43 (100.0%)	-	% 100

(97 to 100)

	-	⨁⨁◯◯

Low

	9-CRITICAL	
DIF + in gingival biopsies in MMP (assessed with: (% of DIF +))	
5	non-randomised studies	not serious	seriousa	not serious	very seriousc,d	very strong association

all plausible residual confounding would reduce the demonstrated effect

	165/199 (82.9%)	-	% 90.3

(73.1 to 99.7)

	-	⨁⨁◯◯

Low

	9-CRITICAL	
DIF + in other oral sites biopsies in PV (assessed with: (% of DIF +))	
5	non-randomised studies	not serious	seriouse	seriousb	seriousd	very strong association

all plausible residual confounding would reduce the demonstrated effect

	212/223 (95.1%)	-	% 95.7

(87.5 to 100.0)

	-	⨁⨁◯◯

Low

	9-CRITICAL	
DIF + in other oral sites biopsies in MMP (assessed with: (% of DIF +))	
8	non-randomised studies	not serious	seriousa	not serious	very seriousc,d	strong association

all plausible residual confounding would reduce the demonstrated effect

	441/512 (86.1%)	-	% 87.4

(70.1 to 98.7)

	-	⨁◯◯◯

Very low

	9-CRITICAL	
Low certainty: further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate

Very low certainty: we are very uncertain about the estimate

Explanations

CI Confidence interval, DIF + Positive direct immunofluorescence, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris

aHigh heterogeneity, p < 0.001, I2 > 75%

bMost studies published before year 2000

cWide 95%CI

dModerate number of studies (5–10)

eModerate heterogeneity, p < 0.05, I2 > 50%

Table 5 Evaluation of the certainty of evidence for the secondary outcomes according to GRADE guidelines (I)

Certainty assessment	№ of patients	Effect	Certainty	Importance	
№ of studies	Study design	Risk of bias	Inconsistency	Indirectness	Imprecision	Other considerations	[intervention]	[comparaison]	Absolute
(95% CI)	Relative (95% CI)	
DIF + of perilesional biopsies in PV (assessed with: (% of DIF +))	
7	non-randomised studies	not serious	not serious	seriousb	seriousd	very strong association	183/194 (94.3%)	-	% 97.8

(91.6 to 100.0)

	-	⨁⨁◯◯

Low

	9-CRITICAL	
DIF + of perilesional biopsies in MMP (assessed with: (% of DIF +))	
5	non-randomised studies	not serious	seriousa	seriousb	seriousd	publication bias strongly suspected

very strong association

all plausible residual confounding would reduce the demonstrated effectf

	410/469 (87.4%)	-	% 95.4

(86.6 to 99.9)

	-	⨁◯◯◯

Very low

	9-CRITICAL	
DIF + of normal-appearing mucosa biopsies in PV (assessed with: (% of DIF +))	
3	non-randomised studies	not serious	not serious	not serious	very seriouse	very strong association

all plausible residual confounding would reduce the demonstrated effect

	58/58 (100.0%)	-	% 100.0

(98.2 to 100.0)

	-	⨁⨁⨁◯

Moderate

	9-CRITICAL	
DIF + of normal-appearing mucosa biopsies in MMP (assessed with: (% of DIF +))	
5	non-randomised studies	not serious	seriousa	not serious	very seriousc,d	strong association

all plausible residual confounding would reduce the demonstrated effect

	181/219 (82.6%)	-	% 87.9

(66.8 to 99.8)

	-	⨁◯◯◯

Very low

	9-CRITICAL	
Moderate certainty: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different

Low certainty: further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate

Very low certainty: we are very uncertain about the estimate

Explanations

CI Confidence interval, DIF + Positive direct immunofluorescence, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris

aHigh heterogeneity, p < 0.001, I2 > 75%

bMost studies published before year 2000

cWide 95%CI

dModerate number of studies (5–10)

eSmall number of studies (< 5)

fPublication bias identified with funnel plot and Egger test (p < 0.001)

Table 6 Evaluation of the certainty of evidence for the secondary outcomes according to GRADE guidelines (II)

Certainty assessment	№ of patients	Effect	Certainty	Importance	
№ of studies	Study design	Risk of bias	Inconsistency	Indirectness	Imprecision	Other considerations	[intervention]	[comparaison]	Absolute
(95% CI)	Relative (95% CI)	
DIF + in scalpel biopsies in PV (assessed with: (% of DIF +))	
2	non-randomised studies	not serious	not serious	not serious	extremely seriousb,c	very strong association

all plausible residual confounding would reduce the demonstrated effect

	8/8 (100.0%)	-	% 100.0

(78.8 to 100.0)

	-	⨁⨁◯◯

Low

	5-IMPORTANT	
DIF + in scalpel biopsies in MMP (assessed with: (% of DIF +))	
4	non-randomised studies	not serious	not serious	not serious	very seriousc	very strong association	97/102 (95.1%)	-	% 97.1

(89.1 to 100.0)

	-	⨁⨁◯◯

Low

	5-IMORTANT	
DIF + in punch biopsies in PV (assessed with: (% of DIF +))	
1	non-randomised studies	not serious	not serious	not serious	very seriousc	very strong association	84/85 (98.8%)	-	% 98.8

(95.0 to 100.0)

	-	⨁⨁◯◯

Low

	5-IMPORTANT	
DIF + in punch biopsies in MMP (assessed with: (% of DIF +))	
3	non-randomised studies	not serious	seriousa	not serious	extremely seriousb,c	strong association	293/325 (90.2%)	-	% 83.8

(44.0 to 100.0)

	-	⨁◯◯◯

Very low

	5-IMPORTANT	
Low certainty: further research is very likely to have an important impact on our confidence in the estimate of effect and is likely to change the estimate

Very low certainty: we are very uncertain about the estimate

Explanations

CI Confidence interval, DIF + Positive direct immunofluorescence, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris

aHigh heterogeneity, p < 0.001, I2 > 75%

bWide 95%CI

cSmall number of studies (< 5)

Meta-analysis: main results

The main results of the meta-analysis models are summarised in Table 7 and illustrated in the forest plots (Fig. 2 a-g) and funnel plots (Supplementary Data 2). Except when specified, no publication bias was identified by funnel plots or Egger’s tests. Table 7 Results of studies included. The mean age is that determined at the time of diagnosis. All biopsies were taken for diagnostic purposes

Authors	Biopsy results	DIF results	Diagnostic performance of dif by sampling sites
Sensitivity %
Other clinical data	
Gaultier et al

[33]

	Papillary gingiva biopsies (max and md)

n = 148

Papillary gingiva for DIF

n = 53 for DIF (27 for MMP, 6 for PV, 20 for other diagnoses)

	-MMP

n = 27 patients, 13 men (48%), 14 women (52%)

Average age = 64.8 y

DIF + 

3 biopsies/5 for the first hospital (60%)

20 biopsies/22 for the second hospital (90.9%)

23 biopsies/27 for both hospitals (85.1%)

-PV

n = 6 patients, 1 male, 5 women

Average age = 46 y

DIF + 

3 biopsies/3 for the first hospital

3 biopsies/3 for the second hospital

6 biopsies/6 for both hospitals

	-MMP

Papillary gingiva

sensitivity for the 2 hospitals = 86

Sensitivity is higher for the second hospital

-PV

Papillary gingiva

sensitivity for both hospitals = 100

Sensitivity is comparable for both hospitals

No post-operative complications at 3 months in 29 MMP + PV patients (gingival bleeding > 24 h and ecchymosis, no pain). Moderate transient bleeding after sampling in 4 patients (two patients on anti-haemorrhagic medication, two non-compliant patients). No postoperative analgesic medication for the 33 MMP + PV patients

Restitution ad integrum of the biopsied papilla at 21 days for the 33 MMP and PV patients

	
Carey et al

[32]

	Oral mucosa biopsy (all sites)

n = 381 (304 for MMP, 77 for PV)

Peri-lesional jugal mucosa for DIF

n = 186 (143 for MMP, 43 for PV)

Normal jugal mucosa for DIF

n = 186 (144 for MMP, 42 for PV)

Alveolar mucosa for DIF

n = 17 (only in cases of MMP with isolated gingival involvement)

	-MMP with gingival involvement only

n = 133 patients, gender and mean age = ND

DIF + 

63 peri-lesional biopsies /69

63 biopsies in normal-appearing mucosa/75

17 alveolar mucosa biopsies/17

-MMP with multisite involvement, including gingiva

n = 118 patients, gender and mean age = ND

DIF + 

71 peri-injury biopsies/74

66 healthy mucosa biopsies/69

-PV

n = 77 patients, gender and age = ND

DIF + 

42 peri-lesional biopsies/43

42 biopsies in healthy mucosa/42

	-MMP with gingival involvement only

Peri-lesional jugal mucosa

sensitivity = 91

Normal-appearing healthy mucosa of the cheek

sensitivity = 84

Alveolar mucosa

sensitivity = 100

-MMP with multisite involvement, including the gum

Peri-lesional jugal mucosa

sensitivity = 96

Normal-appearing healthy mucosa of the cheek

sensitivity = 96

Histology and IFI contributed less to the diagnosis (69% and 64% respectively). Elisa tests also contribute less (from 21 to 10% depending on the Ac)

-PV

Peri-lesional jugal mucosa

sensitivity = 98

Normal-appearing healthy mucosa of the cheek

sensitivity = 100

The contribution of histology and IFI to diagnosis is slightly lower (93% and 92% respectively). Elisa tests, on the other hand, contributed significantly less

	
Endo et al

[31]

	Gum biopsy for DIF

n = 10

	-PV

n = 10 patients, 1 male, 9 women

Average age = 46.3 (24–73) y

DIF + 

10 biopsies /10

	-PV

Gum

sensitivity = 100%

	
Kamaguchi et al

[30]

	Biopsy of the oral mucosa

n = 11 (7 for DIF)

Healthy jugal mucosa for DIF

n = 7

	-MMP

n = 7 patients, gender = ND

Average age = 70 y

DIF + 

7 biopsies/7

	-MMP

Healthy jugal mucosa

sensitivity = 100

Histology accounts for 14% of the diagnosis, while IFI and immunoblot make no contribution

	
Rameshkumar et al

[29]

	Oral mucosa biopsy (all sites)

n = 4 (2 for MMP, 2 for PV)

Gum biopsy for DIF

n = 2 for PV

Jugal mucosa for DIF

n = 2 in the case of MMP

	-MMP

n = 2 patients, 2 men

Average age = 46 y

DIF + 

1 biopsy/2

-PV

n = 2, 1 woman, 1 man

Average age = 52 y

DIF + 

2 biopsy/2

	-MMP

Jugal mucosa

sensitivity = 50

-PV

Gum

sensitivity = 10

The DIF was used to correct the histological and clinical diagnoses for 2 PV and 1 MMP out of a total of 4 AIBDs

	
Endo et al

[28]

	Gum biopsy for DIF

n = 25

	-MMP

n = 13 patients, 4 men, 9 women

Average age = 65 y

DIF + 

13 biopsies/13

-PV:

N = 8 patients, 8 women

Average age = 39 y

DIF + 

8 biopsies/8

	-MMP

Gum

Sensitivity = 100

-PV

Gum

Sensitivity = 100

	
Sano et al

[27]

	Oral mucosa biopsy (all sites)

n = 129 for DFI (disease distribution = ND)

Gingival mucosa for DIF

n = 17 (AIBD and others)

Jugal mucosa for DIF

n = 70 (AIBD and others)

Lingual mucosa for DIF

n = 21 (AIBD and others)

Labial mucosa for DIF

n = 13 (AIBD and others)

Other oral sites for DFI

n = 8 (AIBD and others)

	-All types of illness (AIBD and others)

n = 125 patients

93 women (74.4%)

32 men (25.6%)

Average age = ND (from 11 to 95 y)

-DIF + , compatible with the clinical picture of an MMP

14/21 biopsies (all sites combined)

-DIF + , compatible with the clinical picture of PV

9/9 biopsies (all sites combined)

	-MMP

sensitivity = 67 (all biopsy sites combined)

-PV

sensitivity = 100 (all biopsy sites combined)

Comparable rate of diagnostic contribution regardless of biopsy site (difference NS)

Comparable rate of diagnostic contribution for peri- and distant biopsies (difference NS)

Higher rate of contribution to the diagnosis if the biopsy is not split in two compared to the ½ biopsy (72.70% versus 40%)

	
Yih et al

[26]

	Biopsy of the oral mucosa

n = 72 for DIF (including 30 LP, 29 MMP, 2 linear IgA, 2 PV, 1 paraneoplastic pemphigus, 7 bacterial pathologies)

Gum biopsy for DIF

n = 2 for PV

n = 29 in case of MMP

	-MMP

n = 29 patients, 5 men, 24 women

Average age = 59 y

DIF + 

29 biopsies/29

-PV

n = 2 patients, gender and mean age = ND

DIF + 

2 biopsies /2

	-MMP

Gum

sensitivity = 100

-PV

Gum

sensitivity = 100

	
Helander et al

[25]

	Biopsy of the oral mucosa

n = 264 for DIF (including 70 for all types of pemphigoid, 18 for PV, 176 for OLP and lichenoid lesions)

Gum biopsy for DIF

n = 39 for all types of pemphigoid, n = ND for PV

Jugal mucosa for DIF

n = 18 for all types of pemphigoid, n = ND for PV

Other oral sites for DIF

n = 13 for all types of pemphigoid

	-Pemphigoid

n = 81 patients, including 69 MMP with oral involvement, 10 MMP exclusively oral, 2 bullous pemphigoid, gender = ND, mean age = ND

DIF + 

49 biopsies/70 (for all types of pemphigoid)

-PV

n = 21 patients, gender and mean age = ND

DIF + 

16 biopsies /21

	-Pemphigoid

Gum

sensitivity = 77

Peri-lesional jugal mucosa

sensitivity = 61

Other oral mucosa

sensitivity = 62

For gingival biopsy sites, the sensitivity of DIF is better than that of standard histology (74%) but poorer than that of clinical impression (85%)

For jugal biopsy sites, the sensitivity of DIF is better than that of standard histology (50%) but worse than that of clinical impression (72%)

For other oral mucosal sites, the sensitivity of DIFI is better than that of standard histology (54%) but worse than that of clinical impression (77%)

-PV

Peri-lesional jugal and gingival mucosa (ND distribution)

sensitivity = 89

For all biopsy sites, the sensitivity of DIF is better than that of standard histology (66%) and clinical impression (50%)

	
Venning et al

[24]

	Biopsy of the oral mucosa

n = 49 for DIF (35 for Bullous Pemphigoid, 14 for MMP)

Labial mucosa for DIF

n = 14 biopsies for MMP

	-MMP

n = 14 patients, 4 men, 10 women

Average age = 59 (45–78) y

DIF + 

6 biopsies/14

	-MMP

Labial mucosa

3 patients with several negative biopsies before diagnosis

Sensitivity = 43

	
Rogers and Van Hale

[23]

	Biopsy of the oral mucosa for DIF:

n = 734 (of which 50 for PV, 167 for MMP)

	-MMP

n = 167 patients, gender and mean age: NA

DIF + 

131 biopsies/167

-PV

n = 50 patients, gender and mean age = ND

DIF + 

46 biopsies /50

	-MMP

sensitivity = 78

DIF is more sensitive than histology and IIF

-PV

sensitivity = 92

The sensitivity of DIF is greater than that of IIF (83%) and similar to that of histology (90%)

	
Daniels and Quadra-White

[22]

	Oral mucosa biopsies (all sites)

n = ND

Gum biopsy for DIF

n = 22 in case of MMP

n = 2 for PV

Other sites for DIF (location ND)

n = 11 in case of MMP

n = 8 in case of PV

	-MMP

n = 33 patients, 7 men (21%), 26 women (79%)

Average age = 64.9 y

DIF + 

33 biopsies/33

-PV

n = 10 patients, 3 men (30%), 7 women (70%)

Average age = ND

DIF + 

10 biopsies /10

	-MMP

Gum and other mucous membranes

sensitivity = 100

The sensitivity of DIF is excellent whatever the sampling site, provided that the epithelium remains attached

Histology contributes less to the diagnosis of MMP (74%)

-PV

Gum and other mucous membranes

sensitivity = 100

The sensitivity of the DIF is excellent whatever the sampling site

Histology also makes an excellent contribution to the diagnosis of PV (100%)

	
Niesengard et al

[21]

	Biopsy of the oral mucosa

n = 170

Breakdown by pathology = ND

Gum biopsy for DIF

n = 2 for PV

n = 59 for MMP

	-MMP

n = 35 patients, 9 men, 26 women

Average age = 42 y (men) and 49 y (women)

DIF + 

In healthy mucosa: 16/27

In lesional/peri-lesional mucosa: 19/32

-PV

n = 3 patients, 3 women

Average age = 43

DIF + 

3 biopsies /3

	-MMP

Healthy gums

sensitivity = 59

Peri-lesional gingiva

Sensitivity: 59

The biopsies of the two biopsies taken from the patients give a sensitivity of 100%

-PV

Gum

sensitivity = 100

	
Laskaris and Angelopoulos

[20]

	Biopsies of the oral mucosa

n = 104 (33 in the case of MMP)

Oral mucosa for DIF

n = 33 in case of MMP

	-MMP

n = 33 patients, 18 women (55%), 15 men (45%)

Average age = 65 y

DIF + 

32 biopsies /33

	-MMP

sensitivity = 97

Histology made a smaller contribution to diagnosis (76%), while IFI made a much smaller contribution (36%)

	
Laskaris

[19]

	Biopsies of the oral mucosa

n = 129 (58 in case of PV)

Oral mucosa for DIF

n = 58 for PV

	-PV

N = 58 patients, 35 women (60%), 23 men (40%)

Average age = 55 y

DIF + 

57 biopsies /58

	-PV

sensitivity = 98%

The contribution of histology to diagnosis is lower (93%), while that of IFI varies from 48 to 86% depending on the type of substrate

	
Laskaris et al

[8]

	Oral mucosa biopsies (all sites)

n = ND

Gum biopsy for DIF

n = 10 for PV

	-PV

n = 10, gender and average age = ND

DIF + 

10 biopsies /10

	-PV

Gum

sensitivity = 100

The contribution to the diagnosis of IFI is also excellent (100%)

	
DIF Direct immunofluorescence, IFI Indirect immunofluorescence, MMP Mucous membrane pemphigoid, PV Pemphigus vulgaris, ND Not determined, S Significant, NS Not significant

Fig. 2 Forest plots of biopsy contribution rates for DIF. a overall contribution rate, MMP + PV. b overall contribution rate, MMP only. c overall contribution rate, PV only. d contributory rate for gingival biopsies, PV only. e contributory rate for gingival biopsies, MMP only. f: contribution rate for oral sites other than gingival, PV only. g contribution rate for oral sites other than gingival, MMP only. h contribution rate for peri-lesional biopsies, PV only. i contributory rate of biopsies in healthy mucosa, PV only. j contribution rate for peri-lesional biopsies, MMP only. k contributory rate of biopsies in healthy mucosa, MMP only. l contributory rate for slide biopsies, PV only. m contributory rate for slide biopsies, MMP only. n contributory rate for biopsies performed with the scalpel punch, PV only. o contributory rate for biopsies performed with the scalpel punch, MMP only

Overall rate of DIF + 

The overall rate of DIF + was 91.5% [83.2%- 97.6%] Q = 106.35, df = 1, p < 0.00; I2 = 89.5% (Fig. 2a). Certainty is moderate (Table 3).

For the diagnosis of MMP, the rate of DIF + was 88.5% [75.8%- 97.5%] Q = 90.42, df = 11, p < 0.001; I2 = 91.6% with a high degree of heterogeneity (Fig. 2b). This rate fluctuated between 53 and 100%, with a value of over 60% in 6 studies [8, 22, 25, 27, 31–33]. Certainty is low (Table 3). This rate was even higher for the diagnosis of PV: 99.6% [96.0%-100%], Q = 13.64, df = 11, p = 0.253; I2 = 35.9% (Fig. 2c) with little heterogeneity, varying from 86 to 100% for all the studies. Certainty is moderate (Table 3).

Given the potential difference in contribution according to diagnosis, although not significant, all the analyses were stratified according to diagnosis.

These results are indicated irrespective of the precise location of the oral mucosa biopsied. They could be analysed globally because the DIF evaluation criteria were homogeneous from one study to another. In addition, in order to limit bias in the calculation of the DIF + rate, only the first biopsies were taken into account in all the results when several biopsies had to be repeated by the authors.

Rate of DIF + according to the nature of the oral mucosa biopsied

Gingival biopsies achieved a rate of 100% [97%-100%], Q = 0.51, df = 6, p = 0.998, I2 = 0.0% with no heterogeneity for PV (Fig. 2d), and 90.3%[ 73.1%- 99.7%], Q = 37.72, df = 4, p < 0.001; I2 = 88.6% with high heterogeneity for MMP (Fig. 2e). Certainty is low (Table 4).

For oral sites other than the gingiva, the rate was 95.7% [87.4%-100%], Q = 12.98, df = 4, p = 0.011, I2 = 73.0% with moderate heterogeneity for PV (Fig. 2f), and 87.4% [70.1%-98.7%], Q = 52.74, df = 7, p < 0.001; I2 = 92.6% with high heterogeneity for MMP (Fig. 2g). Certainty is low in PV and very low in MMP (Table 4).

Meta-analysis: secondary results

Rate of DIF + according to inflammatory aspect of the sample

To assess whether the DIF + rate varied with the mucosal involvement, we categorized biopsies as lesional, perilesional or healthy-appear.

The heterogeneity of results was greater for PV if the sample was taken from peri-lesional mucosa 97.8%[ 91.6%- 100%], Q = 10.47, df = 6, p = 0.106; I2 = 48.3% (Fig. 2h) rather than from healthy mucosa 100% [ 98.2%- 100%]Q = 0.44, df = 2, p = 0.803; I2 = 0.0% (Fig. 2 i). Certainty is low in perilesional and moderate in healthy mucosa (Table 6). Conversely, a better contribution was found for MMP if the samples were peri-lesional 95.4%[ 86.6%- 99.9%], Q = 36.86, df = 4, p < 0.001; I2 = 87.5% (Fig. 2j) compared with samples taken from apparently healthy mucosa 87.9%[ 66.8%- 99.8%], Q = 18.47, df = 4, p < 0.001; I2 = 85.5% (Fig. 2k), in both cases with high heterogeneity. Meta-regression confirmed the significant relationship between mucosal appearance and contribution to MMP (p < 0.001 for each modality), with no influence on residual heterogeneity. However, we noted a potential publication bias for peri-lesional MMP (p < 0.037). Certainty is very low for both outcomes (Table 5). 

Rate of DIF+ according to surgical technique

When the biopsies were taken with a slide, the rate of DIF+ was 100% with no heterogeneity for PV ([78.8%-100%], Q = 0.09, df = 1, p = 0.759, I² = 0.0%) (Fig. 2 l) and 97.1% with moderate heterogeneity for MMP ([89.1%-100%], Q = 6.40, df = 3, p = 0.094; I² = 53.5%) (Fig. 2 m). In contrast, punch samples gave less satisfactory results for PV (Fig. 2 n) and MMP (Fig. 2 o). Certainty is low for all outcomes and very low for the use of punch in MMP (Table 6).

Discussion

This systematic review aimed to provide clarity on the choice of the optimal biopsy site for obtain DIF + testing in oral AIBD, specifically MMP and PV. Of the 118 articles retrieved for full reading, 16 were selected for qualitative and quantitative analysis. However, most of the following discussed findings presented a moderately low level of scientific evidence, which was often lower in MMP than in PV. Consequently, they generate hypotheses that require cautious interpretation.

Contribution of the DIF to the establishment of a diagnosis

For many authors, this rate of DIF + is equivalent to the sensitivity of the test [25, 27]. In the case of oral AIBD, it is essential to take into account the rate of DIF + , as there can be no false positives. When autoantibody labelling exists their presence in situ is real and confirms that the patient does indeed suffer from AIBD. However, it is difficult to differentiate a true negative from a false negative due to poor choice of site (if the sample is taken too far from the site where the autoantibodies are located, where inflammation has destroyed them) and/or an inappropriate surgical technique if the epithelium has completely detached from the chorion at the time of sampling. For this reason, we analysed only the DIF + rates for each study included.

We can therefore conclude that all oral sites, in particular the gingiva, alveolar and jugal mucosa, can provide tissue specimens compatible with the performance of DIF.

However, the choice of biopsy site and the surgical protocol are parameters to be taken into account.

Biopsy site selection: balancing sensitivity and practicality

Peri-lesional or apparently healthy mucosal biopsy: a balance of yield and integrity

The review supports the widely held view that biopsies should never be taken from areas of active erosive, pseudomembrane-covered or intense inflammation. Such sites compromise tissue integrity and increase the risk of immunoglobulin and complement degradation, potentially leading to false-negative results [27, 34, 35].

Peri-lesional biopsy, targeting tissue 2–3 mm from the erosion edge within a 1 cm radius, or much further away, from an area of apparently healthy mucosa emerges as a balanced approach. This strategy increases the likelihood of capturing the immune reactants while preserving tissue quality. But harvesting from apparently healthy mucosa provided that the self-reactive elements are widely and effectively disseminated. If certainty appears low or very low according to the mucosa aspect, it is moderate in healthy mucosa in PV.

Gingival biopsy: advantages and limitations

In case of of strictly gingival AIBD, peri-lesional biopsies of the vestibular papilla (Fig. 3) attached gingiva (Fig. 4) offer high diagnostic yield. These sites directly target the affected tissue and demonstrate good clinical tolerance with rapid healing [33]. However, extensive gingival involvement, severe periodontitis, or significant plaque accumulation make gingival biopsies less suitable. The increased fragility of the mucosa in these situations elevates the risk of tissue damage during the procedure [26]. Thus, the certainty of evidence for those considerations is low to very low.Fig. 3 MMP with gingival expression: biopsy of papillary gingiva. A clinical situation, B schematic illustration, C histological section (HE coloration)

Fig. 4 MMP with gingival expression: biopsy of the attached gingiva. A clinical situation, B schematic illustration, C histological section (HE coloration)

Alternative sites: alveolar and jugal or labial mucosa

When gingiva is completely damaged, the alveolar mucosa (Fig. 5) and intact labial mucosa (Fig. 6) opposite the lesions, present viable alternatives. These sites maintain a high probability of detecting autoantibodies [32]. although, patients should be informed about the potential for mucosal flange formation in the medium to long term [33].Fig. 5 MMP with gingival expression: biopsy in healthy alveolar mucosa. A clinical situation, B schematic illustration

Fig. 6 MMP with gingival expression: biopsy of healthy labial mucosa

Furthermore, in cases of multi-site mucosal involvement, the widespread distribution of autoantibodies allows for biopsy of the apparently healthy jugal mucosa. This approach demonstrates comparable DIF positivity rates to peri-lesional biopsies [32] with a simplified surgical procedure. The level of evidence appears to be equivalent regardless of the site from which the biopsy stems.

Our results differ slightly from those of Zeng et al. (2021) [12]. For MMP, their quantitative analysis showed that there was no significant difference in the DIF positivity rate between biopsies taken from apparently healthy mucosa and those taken from the edges of an erosion (odds ratio 1.91, 95% IC 0.91–4.01, I2 = 0%).On the other hand, for PV, the peri-lesional mucosa would represent the optimal biopsy site for DIF. This discrepancy in results may be explained by the fact that their study focused on DIF + rates solely as a function of the inflammatory state of the biopsied mucosa. In addition, their analysis included overlapping cohorts and took into account all biopsies performed, even if they were repeated, which may introduce bias.

However, their meta-analysis found no significant difference in DIF sensitivity between peri-injury sites examined in the jugal mucosa or in the gingival mucosa (89.3%; 95% IC, 70.6%-97.2% vs 100%; 95% IC, 80.0%-100%; p = 0.22), which is consistent with our findings.

Surgical technique: minimizing tissue damage

Beyond site selection, meticulous surgical technique is paramount in ensuring a high-quality tissue specimen with preserved epithelium. An inadequate procedure can undermine the entire diagnostic process, even with optimal site selection.

Key Considerations for Minimizing Tissue Damage:Dedicated Biopsy. A separate biopsy, 5–10 mm in diameter depending on the mucosal site, should be dedicated solely for DIF analysis [27].

Atraumatic Anesthesia. Local anesthesia with vasoconstriction, administered at a distance from the biopsy site, minimizes tissue distortion and prevents the formation of a reaction bubble [33].

Respecting Anatomical Boundaries. When harvesting from the attached gingiva, preserving the marginal gingiva is crucial to avoid irreversible gingival recession (supplementary data 3). The papillary biopsy technique is reproducible and simple to perform, as the protocol does not require the use of suction or sutures for hemostasis [33].

Sharp Dissection. Mucosal borders should be incised to ensure clean and precise tissue removal. This study shows that biopsies obtained using a scalpel blade tend to be more contributory than those obtained using a punch scalpel.

Gentle Handling. Tissue should be grasped delicately at the edges using a tissue grasper without a pick to prevent damaging the epithelium.

Distant Blood Aspiration. Blood should be aspirated away from the biopsy area using gentle suction to avoid disrupting the delicate epithelial layer and potentially washing away immune reactants.

In addition to the importance of carrying out all these steps correctly, it is important to stress that several biopsies may sometimes be indicated. As pointed out by Shimanovich et al. (2017) [36], a negative initial DIF result should not definitively rule out AIBD in the presence of suggestive clinical signs. Sano et al. (2008) [27] supports this practice. In all their patients included, while the number of DIF + was comparable after 1 or 2 biopsies (72.70 and 61.10% respectively, a non-significant difference), its value rose to 100% after 4 biopsies. Gaultier et al. (2021) [33] also corroborated this phenomenon by showing, for their 4 MMP patients with a negative initial DIF, that a second biopsy enabled them to obtain a DIF + .

Limitations of the review

Several studies had to be excluded because of our eligibility criteria. On the other hand, most studies included in this review presented a low level of scientific evidence. This limitation stems from the rarity of oral AIBD and the inherent challenges in conducting large-scale, high-powered studies. Consequently, the findings presented generate only hypotheses. The lack of robust evidence also prevented the calculation of more informative statistical measures like likelihood ratios or predictive values.

Additionally, a potential publication bias favoring peri-lesional biopsy for MMP (p < 0.001) raises questions. This bias could lead to an overestimation of the effectiveness of peri-lesional biopsy for MMP diagnosis. However, the only identified publication bias concerns a data extraction on a secondary outcome. While it is, of course, necessary to temper the validity of this study, it does not indicate a general publication bias related to this article.

The ideal solution to overcome these limitations – conducting randomized controlled trials – is challenging in this context for ethical considerations.

Conclusion

This systematic review suggests that the location of oral mucosa biopsy (gingival, jugal, or alveolar) does not significantly impact the DIF + positivity rate in diagnosing oral AIBD. This finding supports clinicians in prioritizing biopsy site selection based on clinical presentation to obtain a high-quality tissue specimen and the benefit/risk balance of the surgical protocol. However, further controlled studies are needed to confirm these findings.

Supplementary Information

Supplementary Material 1.

Supplementary Material 2.

Supplementary Material 3.

Abbreviations

AIBD Autoimmune bullous diseases

PV Pemphigus vulgaris

MMP Mucous membrane pemphigoid

DIF +  Positive direct immunofluorescence

DIF- Negative direct immunofluorescence

IFI Indirect immunofluorescence

Acknowledgements

Not applicable.

Compliance with ethical standards

Conflicts of Interest: Pr. S.M Dridi declares that she has no conflict of interest. Dr. CM LUTZ declares that she has no conflict of interest. Dr. S Jungo declares that he has no conflict of interest. Dr. F Bellakhdar declares that he has no conflict of interest. Dr. F Gaultier declares that he has no conflict of interest. Dr. A.L Ejeil declares that she has no conflict of interest.

Authors’ contributions

Concept and design: SMD, ALE; Acquisition, analysis, and interpretation of data: SMD, CML, SJ, ALE; Writing—original draft preparation, SMD, ALE; Writing—review and editing,: SMD, CML, SJ, FB, FG, ALE; Statistical analyzes: SJ; All authors have read and approved the final manuscript.

Funding

Not applicable.

Availability of data and materials

All data analysed during this study are included in supplementary data 1 and in Tables 1 and 2.

The others datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Sophie-Myriam Dridi and Claire Manon Lutz contributed equally to this work and are co-first authors.

Sébastien Jungo and Anne-Laure Ejeil contributed equally to this work and are co-last authors.
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