1

Diagnostic relevance of direct immunofluorescence in ocular mucous membrane pemphigoid

Short Title: DIF in ocular MMP

Authors: Tarun Mehra1,5*, Emmanuella Guenova1,2, Frieder Dechent3, Florian Würth4, Manfred Zierhut4, Martin Röcken1, Martin Schaller1, Cristoph Deuter4

Affiliations: 1) Department of Dermatology, Eberhard-Karls-University, Liebermeisterstr. 25, D-72076 Tübingen, Germany.

2) Department of Dermatology, University Hospital of Zürich, Gloriastr. 31, 8091 Zürich, Switzerland

3) Department of Psychiatry, University of Basel, Wilhelm Klein-Strasse 27, 4012 Basel, Switzerland

4) Centre for Ophtalmology, Eberhard- Karls-University, Liebermeisterstr. 25, D-72076 Tübingen, Germany.

5) Medical Directorate, University Hospital of Zürich, Rämistrasse 100, 8091 Zürich, Switzerland*) Corresponding Author

Word Count Article: 2576 Word Count Abstract: 146 Tables: 3 Figures: 1

Funding: This work was partially supported by the Deutsche Forschungsgemeinschaft (GU1271/2-1)

Conflict of interests: None declared

Corresponding Author: Tarun Mehra, MDMedical Directorate, Department of Medical Controlling, University Hospital of ZürichRämistrasse 100, 8091 Zürich, Switzerlandtarun.mehra@usz.ch Tel.: +41 44 255 95 29Fax: +41 44 255 93 66

Key Words: ocular pemphigoid, bullous pemphigoid, direct immunofluorescence,

inflammatory disorders

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ABSTRACT

Background and objectives: The objective was to determine the diagnostic value of

direct immunofluorescence (DIF) in ocular mucous membrane pemphigoid (ocular

MMP), taking into account immunofluorescence patterns and biopsy sites.

Patients and methods: DIF results and medical records of 54 patients with a

suspected diagnosis of ocular MMP were reviewed.

Results: There was an overall prevalence of ocular MMP in 70.4 % of cases. Linear

deposition of IgA, IgG, or C3 showed a high positive predictive value (84–100 %).

Sensitivity and negative predictive value of IgG, IgM, IgG, and C3 in DIF were higher

in cutaneous samples than in conjunctival biopsies, thus yielding a higher diagnostic

accuracy. The sensitivity of DIF in ocular MMP seems to be lower than in bullous

pemphigoid.

Conclusions: The diagnostic value of DIF in the workup of ocular MMP was

confirmed. However, biopsies taken from non-conjunctival, cutaneous tissue appear

to yield more accurate results.

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INTRODUCTION

Ocular mucous membrane pemphigoid (ocular MMP), is a sub-entity of blistering

autoimmune disorders predominantly affecting mucous membranes [1]. Mucous

membrane pemphigoids are a rare group of diseases affecting roughly one to two in

a million per year [2-4]. There is an apex of cases diagnosed in the seventh decade

[5]. In about 60 % of patients with mucous membrane pemphigoid, the conjunctiva is

affected [6].

Clinically, patients usually first present with unilateral, recurrent conjunctivitis later

affecting the second eye, followed by subconjunctival fibrosis, often of the medial

canthus and subsequent shortening of fornices, symblepharon, ankyloblepharon,

entropion, trichiasis, eventually leading to cicatrisation and loss of vision [7, 8].

Direct immunofluorescence (DIF) is an important part of the diagnostic workup of

blistering autoimmune disorders [2]. Linear disposition of IgG and C3 is published as

being the characteristic finding in ocular MMP [9, 10]. On the other hand, linear

deposition of IgA is highly suggestive for linear IgA disease [11] but may also occur

in MMP [7]. Mucosal lichen planus presents with cytoid bodies and a characteristic

thick band of fibrinogen [12]. Epidermolysis bullosa acquisita (EBA) can be

distinguished from ocular MMP through DIF in salt-split-skin, However, the existence

of two different clinical entities for ocular involvement is be arguable, given a certain

overlap and the extent of clinical differences [13, 14]. Anti-laminin 332 blistering

disease, or anti-epiligrin disease as it was formerly known, was thought to be a

separate clinical entity but is now classified as a subgroup of MMP [15]. Taken

together, DIF is essential for the differential diagnosis of blistering diseases that may

have mucosal involvement (e.g. ocular MMP, bullous pemphigoid (BP), EBA).

Despite being the gold standard in the diagnosis of ocular MMP, the diagnostic

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accuracy of DIF in ocular MMP is still controversial. Furthermore, although the

literature recommends biopsies to be taken from a perilesional [16] localization in

cutaneous autoimmune blistering diseases, data on the comparison of DIF results

from samples of different localizations is not available. Due to the different

characteristics of conjunctival tissue compared to skin or even other mucosa in DIF,

differences can be expected [17].

We therefore retrospectively analysed the immunofluorescence findings of specimen

with suspected diagnosis of ocular MMP as to calculate the sensitivity and specificity

of DIF diagnostic criteria in ocular MMP, discerning between biopsies taken from

lesional conjunctiva, perilesional conjunctiva or from cutaneous biopsies. To our

knowledge, our subset of 54 cases is one of the largest analysed so far.

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MATERIAL AND METHODS

Institutional review board approval of the study was obtained prior to beginning the

retrospective analysis.

We retrospectively reviewed the direct immunofluorescence results of the ocular

biopsies of 54 patients which had been sent to the Department of Dermatology by the

Centre for Ophthalmology, both at the University Hospital of Tübingen, between

August 2001 and May 2011 with a suspected diagnosis of ocular MMP.

Biopsies measuring 1mm × 3 mm had been obtained from bulbar lesional (inflamed,

n=41 , non-lesional (not-inflamed, n=32) conjunctiva and/or non-lesional cutaneous

(n=44) biopsies of a total of 54 patients with a suspected ocular pemphigoid

presenting in the Department of Ophthalmology, University Hospital of Tübingen.

Specimen had been placed into 0.9 % NaCl and sent to the Department of

Dermatology for analysis. Samples were frozen at -20°C and subsequently cut with a

thickness of 5-7 µm. Antibodies for IgA, IgG, IgM, C3 and fibrinogen were obtained

from Instrumentation Laboratory and used undiluted for IgA, IgG and IgM and 1:20

for C3 and fibrinogen. Stainings with the antibodies were established using

epidermis specimen of patients without blistering autoimmune disorders as negative

controls and epidermis specimen of cases with previous positive stainings as positive

controls. Routine staining of samples was done in batches assuring at least one

positive and one negative staining per antibody per batch as positive and negative

controls. If no negative or no positive staining for one antibody per batch was found,

the samples were stained again and if necessary, the staining protocol re-

established.

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Specifically, the presence or absence as well as the pattern of IgG, IgA, IgM, C3 and

fibrinogen recorded in the immunofluorescence test were reviewed from the DIF

analysis results. All immunofluorescence results had been interpreted at time of

diagnosis by an experienced dermatologist at the Department of Dermatology,

University Hospital of Tübingen. The definite diagnosis had been made clinically by

an ophthalmologist at the same hospital according to published diagnostic criteria [2],

taking all available diagnostic results into account.

Subsequently, the medical records of all 54 patients were searched for age, sex,

duration of symptoms, extra-ocular involvement of other mucous membranes and

cutaneous affection. The sensitivity, specificity, positive predictive value (PPV) and

the negative predictive value (NPV) were calculated for each subset.

Data was processed with Microsoft Excel.

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RESULTS

Study population

Of the 54 patients analysed, 23 were female. The median age at biopsy was 72

years, ranging from 37-90 years. In this group, 38 had a definitive clinical diagnosis

of ocular MMP and 16 ultimately had a different diagnosis (Table 1). Therefore, the

prevalence of ocular MMP in our patient sample was 70.4%. Of the 16 other patients,

5 had a sympblepharon, 3 had chronic keratoconjunctivitis. Further diagnoses are

reported in Table 1. Importantly, of the 54 cases included, 35.2% had a typical DIF

allowing the diagnosis of ocular MMP (n=19). Of those with a definitive clinical

diagnosis ocular MMP (n=38), less than half (44.7%, 17 cases) had a positive DIF

diagnosis. On the contrary, of the cases without a definitive clinical diagnosis of

ocular MMP (n=16), 12.5% had a DIF findings which were typical for ocular MMP (2

cases).

The median duration of symptoms until biopsy was longer in the ocular MMP cohort

than for non-ocular MMP cases (2.3 years vs. 1.8 years). In total, 4 patients from the

ocular MMP-group had extra-ocular, mucosal manifestations of blistering

autoimmune disease (7.9% of ocular MMP cases) and none of the cases without

ocular MMP. Cutaneous findings of blistering autoimmune disease were present in

44.7% of ocular MMP cases (n=17) and in 12.5% of non- ocular MMP cases (n = 2).

DIF findings

Of the 54 cases in our study, 50 had a complete immunofluorescence panel and

were considered for the evaluation of immunofluorescence findings. Four cases of

the ocular MMP-group had an incomplete panel and were missing at least one of the

tests for IgA, IgG, IgM or C3 and therefore could not be considered for the evaluation

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of predictive values. Of the 50 cases included, 34 had a definitive clinical diagnosis of

ocular MMP, 16 were non- ocular MMP cases. When analysing the ocular MMP

cases, 47.1% showed a linear basal membrane zone (BMZ) IgG band (n=16), 23.5%

a linear BMZ IgA band (n=8), and 14.7% a linear BMZ C3 band (n=5) (Figure 1).

Nineteen had a linear fibrinogen band along the BMZ (55.9%). Three cases (8.8%)

were positive for a pattern of distribution other than a linear BMZ band for IgM (two of

which showed cytoid bodies and one a lupus band) and three for C3 (one linear

granular pattern, one lupus band and one finding with cytoid bodies). Two cases

(5.9%) were positive for a pattern of distribution other than a linear BMZ band for IgA

(cytoid bodies and interepethelial distribution respectively), two cases for IgG (both of

an interepithelial pattern) and fibrinogen (one of which had a lichen planus-type

deposition as well as cytoid bodies, in the other case, only cytoid bodies were found).

In contrast, 18.8% of the non- ocular MMP cases displayed a linear BMZ IgG band

(n=3), 6.3% a linear BMZ IgA band (n=1) and 75% showed a linear fibrinogen BMZ

band (n=12). One case was positive for a pattern of distribution other than a linear

BMZ band for IgG and one for fibrinogen. Of the 50 cases with a complete

immunofluorescence panel included for statistical analysis, 72.0% had a positive

finding for a fibrinogen BMZ band of any type (n=36). All cases with a linear BMZ

band in the C3 staining (n=5) also showed a linear BMZ band in the IgG staining. The

linear BMZ C3 band had a specificity and a PPV of 100%, but a low sensitivity of

14.7% (Table 2). A linear IgA BMZ IgA band had a sensitivity of 25.5% and a

specificity of 93.8% as well as a PPV of 88.9%. As a linear BMZ band for IgM could

not be found in any non- ocular MMP-cases, we refrained from calculating the

sensitivity or specificity. Nonetheless, as three cases had a positive staining different

from a linear BMZ-band in the ocular MMP-group, a malfunctioning test throughout

the period considered was considered unlikely. Linear BMZ IgG distribution had a

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sensitivity of 47.1%, a specificity of 81.3% and a PPV of 84.2%. The negative

predictive values for all diagnostic parameters ranged between 16.7% (fibrinogen,

band of any type) and 41.9% for linear BMZ IgG.

Biopsy localization

We then reviewed the medical records of all 54 cases for DIF results to determine the

difference of DIF results of biopsies taken from different localizations. The sources of

data for this part of the study were expanded from the dermatological DIF reports to

include all sources of medical documents. Findings from 41 DIF-results of lesional

conjunctiva biopsies, 32 non-lesional conjunctiva biopsies and 44 cutaneous biopsies

where reviewed. There were no major differences in the findings for IgA, IgM, C3 or

fibrinogen between lesional and non-lesional conjunctival biopsies (Table 3). The

sensitivity of IgG testing in lesional conjunctiva (21.9%) was half as high as in the

non-lesional conjunctival biopsy (40.9%). For all immunological stainings excluding

fibrinogen, where results from cutaneous samples were not obtainable, the sensitivity

and NPV were higher in the cutaneous samples obtained compared to the

conjunctival samples. The largest differences in sensitivity were found in IgG staining

in cutaneous biopsies in comparison to IgG staining in lesional biopsies (sensitivity of

47.8% vs. 21.9%) as well as in C3 staining in cutaneous biopsies compared to C3

staining in biopsies taken from lesional and perilesional conjunctiva alike (sensitivity

of 17.4% vs. 6.5% and 4.8% respectively). The differences in NPV ranged from 6.8%

to 19.6%.

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DISCUSSION

Generally, it is considered that for mucosal pemphigoid, linear deposition of IgG

and/or of C3 along the BMZ are the most common findings and are sufficient for

diagnosis [12, 18, 19], with occasional findings of an IgA band along the BMZ [1, 19,

20]. Linear, “shaggy” dispositions of fibrinogen along the BMZ are discussed as

suggestive of ocular lichen planus [21, 22]. In line with previous data we found that

apart from fibrinogen, linear IgG along the BMZ is the most common finding in

patients with ocular MMP. A linear BMZ band of IgA was the second most frequent

finding and all positive results for linear C3 bands along the BMZ were conjointly

found with linear depositions of IgG at the BMZ. In two cases of patients with ocular

MMP, the deposition of IgG followed an interepithelial pattern, which is usually

considered to characterize pemphigus vulgaris. However, the diagnosis of ocular

MMP was made by the Department of Ophthalmology on the basis of clinical

appearances as well as other laboratory analysis not considered in this study.

Nonetheless, we found lower sensitivities for DIF than those reported in the literature

[12, 20]. Helander et al. published a sensitivity of 68% and a specificity of 98% for

DIF findings in mucous membrane pemphigoid [23]. However, when judging the

diagnostic value of DIF, the literature often either considers all localizations taken

together [12] or provides measures of statistical validity for mucous membrane

pemphigoid based on samples from oral mucosa alone [23]. Conjunctiva samples

react differently in immunofluorescence testing. For example, Mehta et al. found

linear deposition of fibrinogen along the BMZ in all cases, irrespective of conjunctival

inflammation [17]. The unspecific fibrinogen findings are in line with our results, as

we found t a fibrinogen band of any morphology had a specificity of 18.8% and a

negative predictive value of 16.7%. Our results are therefore in line with the accepted

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scientific school of thought that a fibrinogen band in conjunctival biopsies should is to

rather be considered a normal finding without no or little diagnostic relevance.

The publications which specifically analyse immunofluorescence results in ocular

MMP patients show findings which are more consistent with our results. Bernauer et

al. found linear depositions of immunoglobulins or C3 in 50% of ocular MMP cases.

The reported lower sensitivity rate of linear BMZ-patterns in DIF in ocular MMP

patients in comparison to MMP patients in general support our findings where 56% of

cases were positive [24]. Nonetheless, Bernauer et al. found the most frequent

pattern to be IgA (91%) followed by C3 (54%). The ratio of IgG deposition along the

BMZ was found to be similar to ours (45% vs. 47%). Chan et al. also found a higher

rate for IgA deposits and lower rates for IgG and C3 in ocular MMP cases in

comparison to samples from cases with cutaneous BP only [25]. Frith et al. reported

linear BMZ deposits (IgG, IgA. IgM or C3) in 46% of ocular MMP cases (n=14) and

in 73% of cases with cutaneous BP (n=18) [26]. In samples taken from patients with

ocular MMP (n=13), linear IgG at the BMZ was present in 38%, C3 in 23% and IgA in

8%. IgM did not stain positively. Thus, our results confirm the lower rate of positive

immunofluorescence findings as compared to those reported for cutaneous BP

published in the literature [24, 25].

Serological confirmatory testing for antibodies against MMP target antigens, such as

anti-BP180, anti-laminin 332, anti-BP230, anti-α6-integrin or anti-β4-integrin, with

ELISA, immunoblot or immunoprecipitation assays, is recommended [27]. Indirect

immunofluorescence can be useful in determining circulating autoantibodies for

which diagnostic kits are so far not commercially available, or which so far have not

been characterized.

Indeed, it is known that for skin biopsies from BP patients, the sampling locations

(lesional vs. non-lesional) have an impact on the positivity of DIF testing [12]. In this

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report we investigated for the first time the impact of the biopsy location of lesional

and non-lesional conjunctiva samples and non-conjunctiva samples of 54 patients

with and without ocular MMP. Our results indicate a higher sensitivity and specificity

of biopsies obtained from non-lesional conjunctiva and are even higher still for

cutaneous samples. Our findings indicate that the biopsy site strongly influences the

sensitivity of DIF. Prospective controlled trials are needed to identify the optimal

biopsy site for DIF in ocular MMP.

Our results indicate that the biopsy site strongly influences the sensitivity of DIF, as

results obtained from cutaneous samples show markedly higher sensitivities and

negative predictive values. Furthermore, a linear deposition of IgA, IgG or C3 along

the BMZ in conjunction with the appropriate clinical presentation has a PPV high

enough to diagnose ocular MMP, although an absence of the former does not rule

out ocular MMP. DIF for IgA, IgG or C3 can be considered specific. Nonetheless,

sensitivity in DIF for ocular pemphigoid patterns is not as high as in other forms of

MMP or BP. Thus in the presence of clinical findings typical for ocular MMP and

negative DIF-finding, further testing such as indirect immunofluorescence or repeated

biopsies are warranted.

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ACKNOWLEDGEMENTS

We would like to thank Wolfram Hötzenecker and Rudolf Moos for their help in

critically revising the manuscript, Birgit Fehrenbacher and Eva-Müller-Hermelink for

their help in identifying cases of our cohort and the Deutsche

Forschungsgemeinschaft (grant number GU1271/2-1) for partially funding this work.

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FIGURE LEGENDS

Fig. 1 Venn diagram of DIF results for patients with suspected ocular mucosal

membrane pemphigoid (ocular MMP) (n=50).

Results are shown for patients with a clinically confirmed diagnosis of ocular MMP (A) in comparison

to patients with a definite diagnosis different to ocular MMP (B).

The definite diagnosis of ocular cicatricial pemphigoid was made after the biopsy for DIF had been

taken, clinically, by an ophthalmologist considering all available diagnostic test results. Definite

diagnoses for patients without ocular cicatricial pemphigoid included sympblepharon,

keratoconjunctivitis, blepharitis marginalis cataracta provecta, chronic blepharoconjunctivitis,

pemphigus vulgaris, sicca syndrome without keratopathy and corneal hypervascularization.

Positivity and negativity of DIF results refer to a linear pattern of deposits along the basal membrane

zone only. Solely cases with a full DIF panel were included. F: fibrinogen

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Table 1

Patient characteristics

Total(n=54)

Ocular MMP(n=38)

Other*(n=16)

Age, median (years) 72 72 67.5

Age, range (years) 37-90 44-90 37-86

Sex (female) 42.6% 42.1% 43.8%

DIF diagnosis, ocular cicatrical pemphigoid

positive

19 (35.2%) 17 (44.7%) 2 (12.5%)

Duration of symptoms (years, median)**

2.2 2.3 1.8

Extraocular involvement, mucosal

3 (5.6%) 3 (7.9%) 0 (0.0%)

Extraocular involvement, cutaneous

19 (35.2%) 17 (44.7%) 2 (12.5%)

*including sympblepharon, keratoconjunctivitis, blepharitis marginalis cataracta provecta, chronic blepharoconjunctivitis, pemphigus vulgaris, sicca syndrome without keratopathy and corneal hypervascularization.

**in one case of ocular MMP, the duration of symptoms could not be determined

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Table 2

Statistical testing (n=50), immunofluorescence findings

Sensitivity Specificity PPV NPV

IgA positive,linear BMZ band 25.5% 93.8% 88.9% 36.6%

IgG positive,linear BMZ band 47.1% 81.3% 84.2% 41.9%

IgM positive,linear BMZ band N/A N/A N/A 32.0%

C3 positive,linear BMZ band 14.7% 100.0% 100.0% 35.6%

Fibrinogen positive,linear BMZ band 55.9% 25.0% 61.3% 21.1%

Fibrinogen positive,any band 58.8% 18.8% 62.5% 16.7%

N/A: Not available; PPV: positive predictive value; NPV: negative predictive value

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Table 3

Immunofluorescence findings per localization

samplesconjunctiva,

lesional (n=41)conjunctiva,

non-lesional (n=32)Cutaneous

(n=44)negative positive negative positive negative positive

IgA

not ococular MMP* 15 0 12 0 15 1

ococular MMP 27 5 19 3 18 4

Sensitivity 15.6% 13.6% 18.2%

Specificity 100.0% 100.0% 93.8%

PPV 100.0% 100.0% 80.0%

NPV 35.7% 38.7% 45.5%

IgG

not ococular MMP 12 2 10 2 13 3

ococular MMP 25 7 13 9 12 11

Sensitivity 21.9% 40.9% 47.8%

Specificity 85.7% 83.3% 81.3%

PPV 77.8% 81.8% 78.6%

NPV 32.4% 43.5% 52.0%

IgM

not ococular MMP 14 0 12 0 16 0

ococular MMP 29 1 22 0 20 1

sensitivity 3.3% 0.0% 4.8%

specificity 100.0% 100.0% 100.0%

PPV 100.0% N/A 100.0%

NPV 32.6% 35.3% 44.4%

C3

not ococular MMP 14 0 12 0 16 0

ococular MMP 29 2 20 1 19 4

sensitivity 6.5% 4.8% 17.4%

specificity 100.0% 100.0% 100.0%

PPV 100.0% 100.0% 100.0%

NPV 32.6% 37.5% 45.7%

Fibrinogen

not ococular MMP 5 9 4 8 N/A N/A

ococular MMP 13 19 10 12 N/A N/A

sensitivity 59.4% 54.5% N/A

specificity 35.7% 33.3% N/A

PPV 67.9% 60.0% N/A

NPV 27.8% 28.6% N/A

*including sympblepharon, keratoconjunctivitis, blepharitis marginalis cataracta provecta, chronic blepharoconjunctivitis, pemphigus vulgaris, sicca syndrome without keratopathy and corneal hypervascularization.N/A: Not available. PPV: positive predictive value. NPV: negative predictive value.