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Analysis of The Immunoserological Profile of Autoimmune Skin Diseases

(Reference – 2013.03.003)

Notice of Assessment

April 2014

http://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Avril_2014/Analyse_profil_immunoserologique_maladies_cutanees_auto-immune.pdfhttp://www.inesss.qc.ca/fileadmin/doc/INESSS/Analyse_biomedicale/Avril_2014/Analyse_profil_immunoserologique_maladies_cutanees_auto-immune.pdf

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1 GENERAL INFORMATION

1.1 Requestor: CHUM

1.2 Application for Review Submitted to MSSS: April 11, 2013

1.3 Application Received by INESSS: November 1, 2013

1.4 Notice Issued: February 28, 2014

Note:

This notice is based on the scientific and commercial information submitted by the requestor and on a complementary review of the literature according to the data available at the time that this test was assessed by INESSS.

2 TECHNOLOGY, COMPANY, AND LICENCE(S)

2.1 Name of the Technology

Analysis of the immunoserological profile of autoimmune skin diseases using indirect immunofluorescence on specific substrates and ELISA (enzyme-linked immunosorbent assay) microplates.

2.2 Brief Description of the Technology, and Clinical and Technical Specifications

The immunoserological analysis of autoimmune skin diseases consists of detecting serum autoantibodies that target protein components of the skin. Two methods are included in the requestor's application:

Indirect Immunofluorescence on a Mosaic of Six Substrates (BIOCHIP Mosaics™ by EUROIMMUN)11: monkey esophagus, salt-split human skin, cells expressing desmoglein 1 and 3, BP23012 (C-terminal globular domain), and BP180 (Figure 1). Each substrate is in a microarray13 available for simultaneous analysis.

The technique consists of bringing into contact the serum dilution (1:10) and the substrates. The serum autoantibodies attach themselves to the antigenic determinant of the target protein. The antigen-antibody complex is detected by an (IgG) antibody tagged with a fluorochrome, fluorescein isothiocyanate, and examined using a conventional fluorescence microscope.

The anti-intercellular substance antibodies against desmoglein 1 and desmoglein 3 react with the surface antigens of keratinocytes. The analysis of the esophagus tissue section displays a granular fluorescence. As it is difficult to differentiate between desmoglein 1 and desmoglein 3, specific transfected cells are used to establish an accurate diagnosis. When the autoantibodies against BP180 or BP230 are present, a fluorescence is observed on the epidermis of the skin sample separated at the dermal-epidermal junction by a saline solution. The epidermal basal membrane of the esophagus is visible as a fine linear colouring between the stratum basale and the connective tissue. These

11 EUROIMMUN. Autoantibodies against antigens of the skin [website]. Available at: http://www.euroimmun.de/index.php?id=aak_gegen_antikoerper_der_haut&L=1 (viewed November 21, 2013). 12 BP: bullous pemphigoid. 13 A microarray is an analytical and diagnostic tool measuring approximately 1 cm2. It consists of a glass or silicon support on which proteins are fixed. (Universcience glossary [website]. Available at: http://www.universcience.fr/fr/lexique/definition/c/1248117915087/-/p/1239022830869/).

http://www.euroimmun.de/index.php?id=aak_gegen_antikoerper_der_haut&L=1

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antibodies can be differentiated by means of BP180-NC16A-4X coated BIOCHIPs and cells specifically transfected with BP230 (C-terminal globular domain) [EUROIMMUN, 2014].

Figure 1: BIOCHIP mosaic

Source: Zarian et al., 2012. Figure reproduced with permission of the author.

ELISA Microplate Method for the specific detection of autoantibodies: various ELISA kits using 5 recombinant antigens are available for a quantitative analysis of the target proteins, with the exception of envoplakin, for which the analysis is semiquantitative (characterization of the autoantibodies or identification of the target epitopes). These antigens are extracellular domain of desmoglein 1 or desmoglein 3, envoplakin, tetramer of the NC16A domain of BP180 (BP180-NC16A-4X), and the C-terminal segment of BP230 (BP230-CF).

The ELISA method comprises a microplate with 8 individual reagent wells. The detection of monospecific antibodies is carried out in several steps: 1) serum dilution 1:100; 2) incubation with the recombinant antigen on solid support; and 3) incubation with a conjugate (monoclonal anti-human IgG antibody) coupled with peroxidase. If the samples are positive, the specific IgG, IgA, and IgM antibodies will attach to the corresponding antigenic site. The quantitative results, expressed in U/mL, are obtained by spectrophotometric measurement of the intensity of the colorimetric reaction [EUROIMMUN, 2014]. Other kits from the Japanese company Medical & Biological Laboratories (MBL) are available.

2.3 Company or Developer

EUROIMMUN (Medizinische Labordiagnostika AG) (Germany).

2.4 Licence(s): Not applicable.

2.5 Patent, If Any: Not applicable.

2.6 Approval Status (Health Canada, FDA)

The ELISA (IgG) kits from EUROIMMUN have been approved by Health Canada: anti-desmoglein 1 (No. 82882); anti-desmoglein 3 (No. 82881); anti-envoplakin (No. 91661); anti-BP180-NC16A-4X (No. 74292); anti-BP230-CF (No. 91721).

IIFT: Dermatology Mosaic 7 (EUROIMMUN) was approved by Health Canada in July 2013 (No. 91721).

The FDA has approved the ELISA (IgG) semi-quantitative test kits for anti-BP180 and anti-BP230, MBL International Corporation (k07196114); the qualitative or semi-quantitative kits for anti-BP180-4X ELISA (IgG), EUROIMMUN US Inc. (k08361515); and the semi-quantitative and qualitative kits for the

14 Food and Drug Administration (FDA). 510(k) Substantial equivalence determination decision summary. 510(k) Number: k071961. Available at: http://www.accessdata.fda.gov/cdrh_docs/reviews/K071961.pdf. 15 510(k) Number: k083615. Available at: http://www.accessdata.fda.gov/cdrh_docs/reviews/K083615.pdf.

http://www.accessdata.fda.gov/cdrh_docs/reviews/K071961.pdfhttp://www.accessdata.fda.gov/cdrh_docs/reviews/K083615.pdf

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measurement of anti-desmoglein 1 and anti-desmoglein 3 (IgG), EUROIMMUN US Inc. (k09196916). No approval was found for the Dermatology Mosaic 7 (EUROIMMUN) kit.

2.7 Weighted Value: 148

3 CLINICAL INDICATIONS, PRACTICE SETTINGS, AND TESTING PROCEDURES

3.1 Targeted Patient Group

Patients with autoimmune bullous diseases who are being treated in an oncodermatology service.

3.2 Targeted Disease(s)

Acquired autoimmune bullous diseases are a heterogeneous group of rare disorders of the skin and external mucous membranes characterized by the production of autoantibodies against the surface antigens of keratinocytes and the components of the desmosomes17 (anti-intercellular substance) and other antigens that are not part of the junctional complexes [Grando, 2012]. These autoantibodies are responsible for the loss of intercellular structure by acantholysis (or loss of adhesion between skin cells18) and the formation of intraepidermal bullae (pemphigus). The autoantibodies may be directed against the dermal-epidermal junction (anti-basement membrane), causing a change in one of the components (hemidesmosomes, anchoring filaments) and the formation of subepidermal bullae (bullous pemphigoid). Different antigents (proteins) are associated with various clinical forms [HAS, 2011b; Humbel, 2003]:

pemphigus foliaceus: desmoglein 1;

pemphigus vulgaris: desmogleins 1 and 3;

paraneoplastic pemphigus: plakins (desmoplakins 1 and 2, envoplakin, and periplakin), desmogleins 1 and 3, BPAG119, plectin (HD1);

bullous pemphigoid: BPAG1 and BPAG 2;

cicatricial pemphigoid: BPAG1 and BPAG2, LABD20 Antigen 1, integrin, laminin 5, type VII collagen;

Linear IgA bullous dermatosis: BPAG1 or BPAG2, LABD, type VII collagen;

IgA pemphigus: desmocollins 1 and 2;

Pemphigoid gestationis or herpes gestationis: BPAG2, BP180-NC16A;

Acquired epidermolysis bullosa: type XVII collagen. Bullous pemphigoid is the most common clinical form,21 with an incidence of 43 cases per million inhabitants per year in the United Kingdom and 7 to 13 cases per million inhabitants per year in other parts of Europe [Venning et al., 2012]. It affects individuals aged 70 and over and manifests in its classical form as pruritus, erythema, and the formation of bullae, with no mucosal involvement or Nikolsky’s sign.22 Atypical forms are accompanied by mucosal involvement, particularly of the buccal mucosa [HAS, 2011a]. These forms are associated with significant morbidity (e.g., severe itching and

16 510(k) Number: k091969. Available at: http://www.accessdata.fda.gov/cdrh_docs/reviews/K091969.pdf. 17 Desmosome: protein ensuring intercellular adhesion and interkeratinocyte junctions. 18 Two main isoforms are targeted: desmoglein 1, expressed primarily in the superficial epithelium, and, to a lesser extent, in the oral mucosa; desmoglein 3 expressed primarily in the epithelium of the oral musoca and the suprabasal epidermal layer. 19 BPAG1: bullous pemphigoid Antigen 1. 20 LABD: linear IgA bullous dermatosis. 21 Bernard P. Bullous pemphigoid [website]. Available at: http://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=703 (viewed January 24, 2014). 22 Nikolsky’s sign is a skin finding in which the top layers of the skin slip away from the lower healthy layers when rubbed. It indicates intraepidermal cleavage (acantholysis) during pemphigus. (Collège National des Enseignants de Dermatologie. Item 116: Dermatoses bulleuses auto-immunes. Available at: http://umvf.univ-nantes.fr/dermatologie/enseignement/dermato_45/site/html/cours.pdf).

http://www.accessdata.fda.gov/cdrh_docs/reviews/K091969.pdf

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impetiginization23), which affects quality of life. The mortality rates at 1 year, as reported in four studies reviewed by Schmidt et al. [2012], range between 19% and 29%.

Gestational pemphigoid is a rare form that occurs in the second or third trimester of pregnancy and manifests as severe pruritus, urticaria, and blisters that first appear on the abdomen [Ingen-Housz-Oro et al., 2011].

Pemphigus is an even rarer disease. According to the literature, its incidence ranges from 1 to 16 new cases per million inhabitants per year [Joly and Sin, 2011]. The form known as pemphigus vulgaris presents as delayed mucosal (especially buccal erosions) and cutaneous (bullous eruptions) involvement, with a positive Nikolsky’s sign when involvement is severe. The sequelae, mainly due to cutaneous, ophthalmologic, and buccal involvement, and the side effects of immunosuppressive therapy, are irreversible. When there is no mucosal involvement, it is referred to as superficial pemphigus or pemphigus foliaceus [HAS, 2011b].

Paraneoplastic pemphigus is the most severe form, associated with other neoplastic, especially lymphoproliferative, diseases [Humbel, 2003]. It is extremely rare, and its prevalence is unknown.24

3.3 Number of Patients Targeted

The requestor estimates the provincial volume for the next three years to be approximately 100 patients per year.

3.4 Medical Specialties and Other Professions Involved

Oncodermatology and hematology-immunology.

3.5 Testing Procedure

Blood tests are performed in a test centre using venipuncture.

4 TECHNOLOGY BACKGROUND

4.1 Nature of the Diagnostic Technology: Single test.

4.2 Brief Description of the Current Technological Context

Two tests are currently available to confirm the diagnosis of pemphigus or bullous pemphigoid: the histological examination of a skin sample from an intact blister, and direct immunofluorescence of a skin biopsy. The histological examination is used to specify the epidermal location of the bullae [Joly et al., 2011; Humbel, 2003]. Direct immunofluorescence of peribullous skin shows linear deposits of IgG and complement C3 along the dermal-epidermal junction with a linear fluorescence pattern (bullous pemphigoid) or on the surface of keratinocytes with a honeycomb fluorescence pattern of the epithelium (pemphigus) [Ingen-Housz-Oro et al., 2011; Joly et al., 2011].

Indirect immunofluorescence has long served as a reference method to detect anti-skin autoantibodies in serum, using monkey esophagus or human salt-split skin as a substrate [Tampoia et al., 2012a]. This technique allows the detection of anti-basement membrane zone (anti-BMZ) antibodies in the malpighian epithelium with a linear fluorescence (bullous pemphigoid) and of anti-intercellular substance antibodies (anti-ICS) in the malpighian epithelium with a honeycomb-patterned fluorescence of the epithelium (pemphigus) [Humbel, 2003]. According to certain authors, these procedures are difficult to standardize and depend on the skills of the individuals using them [Charneux et al., 2011].

23 Microbial secondary infection resembling impetigo on affected skin that was not initially infected, such as eczema. 24 Dubertret L. Paraneoplastic pemphigus [website]. Available at: http://www.orpha.net/consor/cgi-bin/OC_Exp.php?lng=EN&Expert=63455 (viewed January 24, 2014).

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4.3 Brief Description of the Advantages Cited for the New Technology

BIOCHIP Mosaics offers a full antigen spectrum in a single analysis and can be used to differentiate between pemphigus vulgaris and pemphigus foliaceus.

ELISA is a simple technology that yields results in one day, which allows a large number of samples to be tested in a relatively short time [Lee et al., 2012]. It provides quantitative results and does not require experienced personnel [Atzori et al., 2008].

Based on the information provided in the application submitted by the requestor, BIOCHIP Mosaics and ELISA technology provide a specific approach that allows an accurate diagnosis to be established in 90% of patients.

4.4 Cost of Technology and Options: Not assessed.

5 EVIDENCE

5.1 Clinical Relevance

5.1.1 Other Tests Replaced

This test could replace the detection of cutaneous anti-basement membrane antibodies (pemphigoid) (code 20700) and anti-intercellular substance antibodies (pemphigus) (code 20715) by indirect immunofluorescence.

5.1.2 Diagnostic or Prognostic Value

Analysis of the immunoserological profile confirms the clinical diagnosis of autoimmune skin diseases by identifying the antigen targeted by the autoantibodies. Analysis also distinguishes various clinical forms, particularly pemphigus vulgaris or pemphigus foliaceus and atypical forms of bullous pemphigoid.

In the case of pemphigus, a correlation was reported between the immunological profile of the autoantibodies and the clinical phenotype. The presence of anti-desmoglein 3 is specific to mucosal involvement, and the presence of anti-desmoglein 1 and 3 is specific to mucocutaneous involvement [Joly et Sin, 2011]. Moreover, the higher the levels of autoantibodies, the more severe the clinical condition [HAS, 2011b; Joly and Sin, 2011].

In the early stages or in atypical forms, bullous pemphigoid may be mistaken for various types of dermatoses and localized drug reactions. Clinical and histopathological criteria, and particularly the development of immunological techniques involving ELISA, allow a serological diagnosis to be made for most patients [Schmidt et al., 2012].

Correlation Between Antidesmoglein 1 and 3 Titres (Using ELISA) and the Severity of Pemphigus

The degree to which the disease has spread is clinically assessed according to severity, the number and extent of mucocutaneous lesions, the number of erosions (mainly oral), the number of new blisters since diagnosis, and the intensity of the pruritus.

Pemphigus Vulgaris In the case of pemphigus vulgaris, most of the studies show a significant correlation between levels of anti-desmoglein 1 and 3 and the extension or activity of the disease when the diagnosis is established (Table 1).

Belloni-Fortina et al. [2009] did not observe a correlation between anti-desmoglein 1 and 3 titres and mucocutaneous spread as they monitored the progression of the disease. By contrast, Herrero-González et al. [2010] reported a significant correlation between levels of anti-desmoglein 3 and an

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unfavourable disease prognosis (several refractory episodes, chronic disease) (p < 0.001), but they did not observe any association with levels of anti-desmoglein 1 antibodies (p value not available).

Another study [Abasq et al., 2009] reported a reduction in levels of anti-desmoglein 1 (from 113.8 U/mL to 22.3 U/mL; p < 0.001) in patients with pemphigus vulgaris who had mucocutaneous lesions and showed complete remission of their skin lesions. Levels of anti-desmoglein 3 decreased from 148.4 U/mL to 99.4 U/mL per day (p < 0.001) in patients who showed complete remission of their mucosal lesions. The decrease in levels of anti-desmoglein 3 was not as significant as that of anti-desmoglein 1 in patients with mucocutaneous pemphigus or pemphigus foliaceus (cutaneous). For 11 patients with pemphigus foliaceus or pemphigus vulgaris who had recurrent mucocutaneous lesions, anti-desmoglein 1 levels were significantly higher (p = 0.03) during periods of relapse than during periods of remission. However, no significant difference was noted in levels of anti-desmoglein 3 between periods of complete remission and periods of relapse (p = 0.13) of pemphigus vulgaris mucosal lesions.

Table 1: Relationship between anti-desmogleins 1 and 3 titres by ELISA and the severity of pemphigus vulgaris

STUDY ASSESSMENT OF THE SEVERITY OR EXTENT

OF THE DISEASE

AUTOANTIBODIES TEST P VALUE

TYPE RESULT

Herrero-González et al., 2010

Extent of mucocutaneous lesions

Anti-Dsg1 Anti-Dsg3

Kruskal-Wallis

n/a < 0.001 < 0.001

Schmidt et al., 2010 Number of mucocutaneous lesions (PDAI

25)

Anti-Dsg1 Anti-Dsg3

Spearman NR

< 0.001 < 0.001

Belloni-Fortina et al., 2009

Number of mucosal lesions

Anti-Dsg1 Anti-Dsg3

Spearman

NR

0.001 0.006

Extent of cutaneous lesions

Anti-Dsg1 Anti-Dsg3

0.102 0.009

Daneshpazhooh et al., 2007

Severity of cutaneous involvement

Anti-Dsg1 Anti-Dsg3

Spearman 0.746 0.382

< 0.001 < 0.001

Number of oral erosions

Anti-Dsg1 Anti-Dsg3

–0.245 0.295

< 0.036 < 0.011

Sharma et al., 2006 Cutaneous surface affected by vesicles, bullae, or erosions

Anti-Dsg1 Anti-Dsg3

Pearson 0.429 0.217

0.026 0.278

Number of oral erosions

Anti-Dsg3 ANOVA test

n/a 0.039

Abbreviations: Dsg = desmoglein; n/a = not applicable; NR = not reported.

Avgerinou et al. [2013] reported the mean difference in anti-desmoglein 1 and 3 titres between the beginning of treatment and 12 months after treatment in 40 patients with pemphigus vulgaris. They observed a significant decrease in anti-desmoglein 1 (from 140.82 to 31.56; p = 0.003) and anti-desmoglein 3 (from 178.08 to 126.43; p = 0.009) titres in 25 patients who showed clinical improvement. In the 7 patients who showed clinical deterioration, anti-desmoglein 1 titres did not change significantly, whereas anti-desmoglein 3 increased from 176.70 to 245.92; p = 0.028. No changes were observed in the 8 patients in stable clinical condition. The authors conclude that anti-

25 PDAI (pemphigus disease area index): score 3: > 3 lesions, score 2: 2-3 lesions; score 1: 1 lesion; score 0: no lesions.

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desmoglein 1 and 3 can be useful for the clinical follow-up of pemphigus vulgaris and to direct therapeutic treatment.

Pemphigus Foliaceus

Two studies showed divergent results (Table 2). Although a correlation was established between levels of anti-desmoglein 1 and the extent of cutaneous involvement for a small number of patients (n = 5) in one study [Schmidt et al., 2010], no correlation was observed by Herrero-González et al. [2010], who noted that this might be a result of the small sample size (n = 7).

Table 2: Relationship between anti-desmoglein1 titres by ELISA and the severity of pemphigus foliaceus

STUDY ASSSESSMENT OF THE SEVERITY OR EXTENT OF THE

DISEASE

AUTOANTIBODIES

TEST COEFFICIENT

P VALUE

Herrero-González et al., 2010

Extent of cutaneous lesions

Anti-Dsg1 Kruskal-Wallis

n/a 0.091

Schmidt et al., 2010 Number of cutaneous lesions

Anti-Dsg1 Spearman

NR < 0.001

Abbreviations: Dsg = desmoglein; n/a = not applicable; NR = not reported.

Abasq et al. [2009] reported a significant decrease in the level of anti-desmoglein 1, from 123.8 U/mL to 76.8 U/mL (p = 0.03), in patients with pemphigus foliaceus who had a complete remission of their skin lesions, after initial treatment and after a 90-day follow-up.

Correlation Between Anti-BP180 and Anti-BP230 Titres by ELISA and the Severity of Bullous Pemphigoid

As anti-BP180 is strongly associated with bullous pemphigoid activity and severity (Table 9), and is recommended for monitoring of the progression of the disease.

An anti-BP180 titre of ≥ 27 UI/mL is a good indicator of clinical recurrence during the year following cessation of treatment, with a positive predictive value of 90.9% and a negative predictive value of 51.2% [Bernard et al., 2009].

A study reported significantly lower values of anti-BP180 during follow-up of patients without recurrences (p = 0.03) [Le Saché-de Peufeilhoux et al., 2012]. The significance of anti-BP230 has not been demonstrated in typical cases of bullous pemphigoid [Le Saché-de Peufeilhoux et al., 2012].

Correlation Between BIOCHIP Mosaic-based Indirect Immunofluorescence and Anti-desmoglein 1 and 3 Titres by ELISA

For 9 patients (3 cases of bullous pemphigoid, 3 cases of pemphigus foliaceus, and 3 cases of pemphigus vulgaris), Van Beek et al. [2012] reported a correlation between anti-BP180 and anti-desmoglein 1 and 3 titres—measured using the two methods (BIOCHIP Mosaic-based indirect immunofluorescence and ELISA)—and the activity of the disease26 during follow-up. The authors concluded that ELISA is the most appropriate test to monitor autoantibody titres.

26 Score 4: > 10 lesions; score 3: 4 to 10 lesions; score 2: 1 to 3 lesions; score 1: clinical remission with immunosuppressive therapy; score 0: clinical remission without immunosuppressive therapy.

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Table 3: Relationship between anti-BP180 and anti-BP230 titres by ELISA and the severity of bullous pemphigoid

STUDY ASSESSMENT OF THE SEVERITY OF THE DISEASE

AUTOANTIBODIES

TEST COEFFICIENT

P VALUE OF P

Lee et al., 2012 Disease activity score* Anti-BP180 Anti-BP230

Spearman 0.45

0.002 0.002 0.99

Severity of the disease based on score (mild, moderate, severe)

Anti-BP180 Anti-BP230 Wilcoxon

rank-sum test

n/a 0.006 0.54

Intensity of pruritus Anti-BP180 Anti-BP230

0.04 0.56

Patsatsi et al., 2012 BPDAI27

BPDAI (intensity of

pruritus)

ABSIS28

Anti-BP180

Pearson Spearman Pearson

0.557 0.530 0.570

< 0.0001 0.001

< 0.0001

BPDAI BPDAI (intensity of

pruritus)

ABSIS

Anti-BP230 Spearman

0.206 0.192 0.245

0.208 0.242

0

Charneux et al., 2011

Number of new vesicles Anti-BP180 Anti-BP230

Spearman 0.37 -0.06

< 0.001 0.51

Roussel et al., 2011 Number of new blisters each day

Anti-BP180 Anti-BP230

Spearman 0.44 0.16

< 0.001 0.03

Tampoia et al., 2009 Number of new blisters over two consecutive days

Anti-BP180 Not available

0.616 0.0042

Feng et al., 2008 Number of blisters, erythema, erosion, % of cutaneous involvement

Anti-BP180

Spearman 0.609 0.006

Abbreviations: BP = bullous pemphigoid; n/a = not applicable.

*Score based on the extent of the disease and the intensity of treatment.

5.1.3 Therapeutic Value

Recommendations for the diagnosis and treatment of bullous pemphigoid, published in France by Bernard et al. [2011], include confirming with ELISA that anti-PB180 titre levels are not higher than 27 UI before cessation of treatment. Recommendations for the diagnosis and treatment of pemphigus, published in France by Joly et al. [2011], indicate that, prior to cessation of treatment, the absence or presence of circulating antibody levels lower than 1/20 should be confirmed by IIF, and high anti-desmoglein 1 titre levels should also be ruled out by ELISA. These antibodies are predictive of cutaneous recurrence [Joly et al., 2011].

27 BP Disease Area Index Score (from 0 to 120): number and size of bullous lesions, number and size of nonbullous erythematous lesions, number and size of mucosal lesions. BPDAI score (intensity of pruritus) (from 0 to 30): assessment of pruritus severity during the last 24 hours (from 0 to 10), in preceding weeks (from 0 to 10), in previous month (from 0 to 10). 28 Autoimmune Bullous Skin Disorder Intensity Score (from 0 to 206): extent and quality of cutaneous lesions.

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5.2 Clinical Validity

COMPONENT PRESENCE ABSENCE NOT APPLICABLE

Sensitivity X

Specificity X

Positive predictive value (PPV) X

Negative predictive value (NPV) X

Likelihood ratio (LR) X

ROC curve X

Accuracy

5.2.1 Clinical Performance of BIOCHIP Mosaic-based Indirect Immunofluorescence in the Diagnosis of Bullous Pemphigoid and Pemphigus Vulgaris and Foliaceus

Bullous Pemphigoid

Four recent studies29 were identified (Tables 4 and 5). BIOCHIP Mosaic allows the detection of the NC16A domain of the BP180 protein, the main target of the antibodies found in the serum of patients with bullous pemphigoid, with a sensitivity of 83.3% to 100% and a specificity of 96.5% to 100%. BIOCHIP Mosaic has a sensitivity of 38% to 54.8% and a specificity of 98% to 100% for the detection of the C-terminal globular domain of BP230.

Pemphigus Vulgaris

In the case of pemphigus vulgaris, the antibodies are directed against desmoglein 1 and 3. Therefore, anti-desmoglein 1 and 3 are useful in distinguishing cases of pemphigus vulgaris from cases of pemphigus foliaceus. Results from BIOCHIP mosaic-based indirect immunofluorescence provide confirmation, since in pemphigus vulgaris anti-desmoglein 3 is detected in 98.5% to 100% of cases, with a specificity of 99.6% to 100%, while anti-desmoglein 1 has a sensitivity of 33.3% to 52.3%, with a specificity of 98.5% to 100%.

Pemphigus Foliaceus

In pemphigus foliaceus, desmoglein 1 is the only antibody target. It is present in 90% of cases with a specificity of 100%.

According to certain authors [Tampoia et al., 2012b; Van Beek et al., 2012], BIOCHIP Mosaic shows a high sensitivity and specificity comparable to those published by other studies using conventional ELISA methods for bullous pemphigoid, pemphigus vulgaris, and pemphigus foliaceus. They conclude that BIOCHIP Mosaic-based indirect immunofluorescence performs well in the diagnosis of bullous autoimmune skin disease. The method is simple to execute and allows the results for several substrates to be obtained in a single incubation. It is also more cost-effective than ELISA, although no cost analyses have been published. However, it is important to note that, although the results are similar to those obtained with ELISA, they show low sensitivity in certain cases and are of a qualitative nature. Quantitative results with ELISA are still necessary, particularly for uncertain cases.

29 The authors of two of these studies have a stake in EUROIMMUN [Van Beek et al., 2012; Damoiseaux et al., 2012].

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Table 4: Clinical performance of BIOCHIP mosaic (EUROIMMUN) with indirect immunofluorescence for the detection of anti-BP180 and anti-BP230 in the diagnosis of bullous pemphigoid

STUDY PATIENTS (N)

CONTROL GROUP (N)

AUTOANTIBODIES SE % (n/N)

SP %

LR

+ -

Damoiseaux et al., 2012

60 AISD (22)* Other ISD (35)†

Anti-BP180 NC16A Anti-BP230gC in HEK293 cells

88 (53/60) 38 (23/60)

96.5 98

25.2 22.5

0.12 0.63

Zarian et al., 2012 18 HV (3) pemphigus vulgaris (2)

Anti-BP180-NC16A Anti-BP230-gC

83.3 (15/18) 39 (7/18)

100 100

NA NA

Van Beek et al., 2012 42 HV (100) Other NISD (97)‡

Anti-BP180-NC16A Anti-BP230-gC

100 (42/42) 54.8 (23/42)

98.2 100

NA NA

Tampoia et al., 2012b

40 HV (40) Other SD (54)§

Anti-BP180-NC16A Anti-BP230

║

90 40

100 100

NA NA

Abbreviations: AISD = autoimmune skin diseases; BP = bullous pemphigoid; HV = healthy volunteers; ISD = Inflammatory skin diseases; LR = likelihood ratio; N = total number of patients; n = number of patients tested; NA = not available; NISD = noninflammatory skin diseases; SD = skin diseases; Se = Sensitivity; Sp = Specificity. *AISD: pemphigus vulgaris and pemphigus foliaceus (8); subacute or chronic cutaneous lupus erythematosus (5); vascularity (4); psoriasis (2); IgA dermatosis (1); dermatitis herpetiformis (2). †ISD (35): eczema (11); prurigo (6); erythema (3); various other skin manifestations (15). ‡ NISD: linear IgA dermatosis; noninflammatory skin diseases including vascular leg ulcers, basal cell carcinoma, and squamous cell carcinoma. § SD: psoriasis (38), discoid lupus erythematosus or lichen planus (16). ║ The entire molecule and C-terminal domain of anti-BP230.

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Table 5: Clinical performance of BIOCHIP mosaic (EUROIMMUN) for the detection of anti-desmoglein 1 and 3 for the diagnosis of pemphigus vulgaris and foliaceus

STUDY PATIENTS

(N)

CONTROL GROUP (N)

AUTOANTIBODIES

SE % (n/N)

SP %

Pemphigus vulgaris

Van Beek et al., 2012 65 HV (100) Other NISD (97)

*

Anti-Dsg1 Anti-Dsg3

52.3 (33/65) 98.5 (64/65)

100 99.6

Tampoia et al., 2012b

36 HV (40) Other SD (54)†

Anti-Dsg1 Anti-Dsg3

33.3 100

98.5 100

Pemphigus foliaceus

Van Beek et al., 2012 50 HV (100) Other NISD (97)‡

Anti-Dsg1 Anti-Dsg3

90 (45/50) n/a

100 99.6

Abbreviations: Dsg = desmoglein; HV = healthy volunteers; N = total number of patients; n = number of patients with the autoantibodies; n/a = not applicable; NISD = noninflammatory skin diseases; SD = skin diseases; Se = sensitivity; Sp = Specificity.

* NISD: linear IgA dermatosis; noninflammatory skin diseases including vascular leg ulcers, basal cell carcinoma, and squamous cell carcinoma.

† SD: psoriasis (38), discoid lupus erythematosus or lichen planus (16).

‡ NISD: linear IgA dermatosis; noninflammatory skin diseases including vascular leg ulcers and carcinoma.

5.2.2 Clinical Performance of Anti-BP180 and BP230, Anti-desmoglein 1 and 3 by Conventional Indirect Immunofluorescence and ELISA

The results of studies of the diagnostic performance of ELISA commercial kits are shown in Table 6 for cases of bullous pemphigoid and Table 7 for cases of pemphigus. Most of the studies used the conventional method, indirect immunofluorescence with a substrate (monkey esophagus or human salt-split skin). This method does not differentiate between different autoantibodies, and the substrate used may not react with all of the sera of patients with bullous pemphigoid or pemphigus. Quantitative analysis of the autoantibodies with the various ELISA kits completes the diagnostic process.

Bullous Pemphigoid

The meta-analysis of the results of 17 studies published between 1994 and 2011 [Tampoia et al., 2012a] on bullous pemphigoid show that ELISA kits using BP180 antigens for the detection of anti-BP180 have a sensitivity of 87% (between 85% and 89%) and a specificity of 98% (between 98% and 99%). The area under the curve is 0.988. The authors conclude that this method is highly sensitive and specific for the detection of autoantibodies in the case of bullous pemphigoid.

The results of nine other studies reviewed report a better clinical performance for the detection of anti-BP180, with a sensitivity and specificity of 80% to 100% and 90% to 100%, respectively, compared with the results of indirect immunofluorescence on a substrate, which has a sensitivity and specificity of 50% to 100% and 63% to 100%, respectively. Among these studies, that of Damoiseaux et al. [2012] emphasizes that the specificity of the anti-BP180 ELISA kit (EUROIMMUN) could be increased from 93.0% to 94.7%, without compromising its sensitivity, for threshold values ranging from 20 RU/mL to 25 RU/mL, according to an analysis of the ROC curve.

Tampoia et al. [2012b] compared ELISA kits from two different companies (EUROIMMUN and MBL) and reported statistically significantly different mean concentrations of anti-BP180 and anti-BP230 between the groups of patients who were ill and the control groups (p < 0.0001 and p < 0.04, respectively). The area under the curve for the ELISA (EUROIMMUN) kits versus ELISA (MBL) kits is 0.964 (0.907 to 0.990) versus 0.945 (0.882 to 0.981) (p = 0.610) for anti-BP180 and 0.748 (0.617 to 0.853) versus 0.889 (0.779 to 0.956) (p = 0.058) for anti-BP230.

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For anti-BP230, the sensitivity is 33.3% to 72.3%, with a specificity of 82.5% to 100%. Damoiseaux et al. [2012] point out that, according to an analysis of the ROC curve, an increase in the threshold values of 20 RU/mL to 25 RU/mL for anti-BP230 ELISA (EUROIMMUN) would yield a sensitivity of 56.7% and a specificity of 87.7%. They also report that all the specific tests for BP180 antigen have a positive likelihood ratio of > 10, which makes the results clinically significant. However, the significance is limited, since the negative likelihood ratios are between 0.1 and 0.2.

Pemphigus Vulgaris

The meta-analysis of 13 studies [Tampoia et al., 2012b] of pemphigus vulgaris and the detection of anti-desmoglein 3 with ELISA showed a sensitivity of 97% and a specificity of 98%. The area under the ROC curve is 0.995. The authors concluded that ELISA tests using recombinant desmoglein 3 antigens are highly sensitive and specific for the detection of autoantibodies in the diagnosis of pemphigus vulgaris.

For five other studies, the ELISA kits for the detection of anti-desmoglein 3 had a sensitivity of 83% to 100% and a specificity of 95% to 98.5%. Indirect immunofluorescence on a substrate had a sensitivity of 68.5% to 100% and a specificity of 95.5% (only one study) for the detection of anti-intercellular substance antibodies typical of pemphigus. Anti-desmoglein 1 by ELISA showed a sensitivity of 25% to 77.8% and a specificity of 95.5% to 100%. The performance of immunofluorescence on a substrate was also low, between 42% and 81%, but the specificity was 100% (only one study).

Pemphigus Foliaceus

Two studies showed an anti-desmoglein 1 sensitivity of 75% to 91% for the diagnosis of pemphigus foliaceus and a specificity of 100% (only one study). The sensitivity of indirect immunofluorescence on a substrate was between 42% and 81%, and the specificity was 100% (only one study). The same studies report very low sensitivities, between 12% and 37.5%, for anti-desmoglein 3, and a specificity of 100% (only one study) [Cozzani et al., 2013; Cunha et al., 2006]. Cunha et al. [2006] noted that, although previous studies have associated the presence of anti-desmoglein 3 with pemphigus vulgaris, there are no tests offering 100% precision for the differential diagnosis of the two forms of pemphigus.

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Table 6: Clinical performance of IIF and ELISA in the diagnosis of bullous pemphigoid

STUDY GROUP RESULT BASED ON METHOD

Experimental N

Control (N) IIF Se % (n/N)

Sp %

ELISA (Threshold)

Se % (n/N)

Sp. %

Meta-analysis

Tampoia et al., 2012a

*

1,178 HV (2,058) or other NISD

‡ (1,113)

NR Anti-BP180 (5 U/mL to 20 U/mL or OD between 0.207

and 0.443)

87 98

Primary studies

Damoiseaux et al., 2012

60 AISD (22)§

other ISD (35)║

Anti-BMZ

(ESO) Anti-BMZ (HS NaCl)

95 (57/60) 95 (57/60)

84.2 77.2

Anti-BP180-NC16A (20 RU/mL) ¶

Anti-BP230 (20 RU/mL) ¶

86.7 (52/60) 60 (NA)

93 82.5

Anti-BP180-NC16A (9 U/mL)**

Anti- BP230 (9 U/mL)

**

80 (48/60) 58.3 (NA)

98.2 93

Le Saché-de Peufeilhoux et al.,

2012

74 NR Anti-BMZ (ESO)

Anti-BMZ (HS NaCl)

93 (69/74) 95 (70/74)

Anti-BP180-NC16A (9 AU) Anti-BP230 (9 AU)

92 (68/74) 58 (43/74)

Lee et al., 2012 47 HV (15) AEB (16)

Anti-BMZ (HS NaCl)

100 (47/47)

NA

Anti-BP180-NC16A (9 U/mL) Anti-BP230 N-terminus (9

U/mL)

97.9 (46/47) 72.3 (34/47)

90.3 100

Patsatsi et al., 2012 39 NR NR Anti-BP180-NC16A (9 U/mL) Anti-BP230 (9 U/mL)

100 (39/39) 66.7 (26/39)

NA

Tampoia et al., 2012b 40 HV (40) Other SD (54)

‡‡

Anti-BMZ (ESO)

50

100

Anti-BP180†

Anti-BP230†

85 33.3

100†

100†

Yang et al., 2012 62 HV (32); PV (19); PF (11)

Anti-BMZ (HS NaCl)

83.9 (52/62)

100 Anti-BP180-NC16A (> 9 U/mL) Anti-BP230 N-terminal and C-

terminal (> 9 U/mL)

87.1 (54/62) 56.5 (35/62)

100 100

Zarian et al., 2012 18 HV (3) PV (2)

NR Anti-BP180-NC16A (9 U/mL) 100 (18/18) 100

Charneux et al., 2011 138 0†† Anti-BMZ (ESO)

Anti-BMZ (HS NaCl)

68 (90/132)

62 (70/113)

Anti-BP180-NC16A (9 U/mL) Anti-BP230 (9 U/mL)

86 (119/138)

59 (81/138)

14

STUDY GROUP RESULT BASED ON METHOD

Roussel et al., 2011 190 Pemphigus (40) Cicatricial

pemphigoid (22); EBA (16)

Anti-BMZ (HS)

81 (153/190)

63

Anti-BP180 (9 U/mL) Anti-BP230 (9 U/mL)

79 (150/190)

61 (115/190)

90 96

Abbreviations: AEB = acquired epidermolysis bullosa; AISD = autoimmune skin diseases; AU = arbitrary unit; BMZ = basal membrane zone; BP = bullous pemphigoid; ESO = monkey esophagus; HS = salt-split human skin; HV = healthy volunteers; IIF = conventional indirect immunofluorescence; ISD = inflammatory skin diseases; N = total number of patients; n = number of patients with the autoantibodies; NA = not available; NISD = noninflammatory skin diseases; NR = not reported; OD = optical density; RU = relative unit.

* Of 17 studies: ELISA (MBL) for 11 studies and ELISA (in-house) for 6 studies. The total number of patients was calculated using Table 1 from the study, due to the discrepancies in the data provided in the text and in Table 1. ‡ Diseases such as pemphigus, epidermolysis bullosa. § AISD: pemphigus vulgaris and pemphigus foliaceus (8); subacute or chronic cutaneous lupus erythematosus (5); vascularity (4); psoriasis (2); IgA dermatosis (1); dermatitis herpetiformis (2). ║ ISD (35): eczema (11); prurigo (6); erythema (3); various other skin manifestations (15). ¶ ELISA (Euroimmun). **ELISA (MBL). †† The authors state that the specificity of these tests on control samples was not assessed, since it had already been analyzed in other studies. ‡‡ SD: psoriasis (38), discoid lupus erythematosus or lichen planus (16). † The authors calculated the sensitivity values using a specificity set at 98.8% [Tampoia et al., 2012b].

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Table 7: Results of the clinical performance of the ELISA method for the diagnosis of pemphigus vulgaris and pemphigus foliaceus

STUDY GROUP RESULT BASED ON METHOD

EXPERIMENTAL N

CONTROL (N) IIF SE % (n/N)

SP %

ELISA (THRESHOLD)

SE % (n/N)

SP %

Pemphigus vulgaris

Meta-analysis

Tampoia et al., 2012a*

654

HV (926) or other NISD‡ (1354)

NR Anti-Dsg 3 (7 to 20 U/mL) 97 98

Primary study

Avgerinou et al., 2013

54 NR Anti-ICS 68.5 Anti-Dsg 1 (20 U/mL) Anti-Dsg 3 (14 U/mL)

77.8 87

Cozzani et al., 2013

12 NR Anti-ICS (ESO)

100 (12/12) Anti-Dsg 1 (20 U/mL) Anti-Dsg 3 (20 U/mL)

25 (3/12) 83 (10/12)

Tampoia et al., 2012b

36 HV (40) Other SD (54) §

Anti-ICS (ESO)

83.3 95.5 Anti-Dsg 1 Anti-Dsg 3

44.4 100

95.5║ 98.5║

Belloni-Fortina et al.,

2009

20 HV (20) NR Anti-Dsg 1 (20 U/mL) Anti-Dsg 3 (20 U/mL)

60 (12/20) 90 (18/20)

100 95

Daneshpazhooh et al., 2007

73

NR NR Anti-Dsg 1 (>20 U/mL) Anti-Dsg 3 (>20 U/mL)

76.7 (56/73) 94.5 (69/73)

Pemphigus foliaceus

Cozzani et al., 2013

8 NR Anti-ICS (ESO)

42 (5/12)

Anti-Dsg 1 (20 U/mL) Anti-Dsg 3 (20 U/mL)

75 (6/8) 37.5 (3/8)

Cuhna et al., 2006

32 HV (15) Anti-ICS (HS NaCl)

81 (26/32) 100 Anti-Dsg 1 (20 U/mL) Anti-Dsg 3 (20 U/mL)

91 (29/32) 12 (4/32)

100 100

Abbreviations: Dsg = desmoglein; ESO = monkey esophagus; HS = human skin; HV = healthy volunteers; ICS = intercellular substance; IIF = conventional indirect immunofluorescence; N = total number of patients; n = number of patients with the autoantibodies; NISD = noninflammatory skin diseases; NR = not reported; SD = skin diseases; Se = sensitivity; Sp = specificity. * Of the 13 studies: ELISA (MBL) (9 studies), ELISA (EUROIMMUN) (1 study), and ELISA (in-house method) (3 studies). The total number of patients was calculated using Table 1 from the study, due to discrepancies between the data provided in the text and in Table 1. ‡ Diseases such as pemphigus, epidermolysis bullosa. § SD: psoriasis (38), discoid lupus erythematosus or lichen planus (16). ║ The authors calculated the sensitivity values using a specificity set at 98.8% [Tampoia et al., 2012b].

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Mucocutaneous Recurrence

Abasq et al. [2009] evaluated the sensitivity and specificity of anti-desmoglein 1 and 3 for the diagnosis of a mucocutaneous recurrence of pemphigus vulgaris and pemphigus foliaceus under treatment. Based on the threshold values proposed by the manufacturer (> 20 U/mL for anti-desmoglein 1 and > 14 U/mL for anti-desmoglein 3), the sensitivity and specificity of anti-desmoglein 1 were 86% and 78%, with positive and negative predictive values of 79% and 85%, respectively. Anti-desmoglein 3 had a sensitivity of 100%, with a low specificity (23%) for the diagnosis of mucosal recurrence. Analysis of the ROC curve determined that to obtain an optimal sensitivity and specificity (80% and 84%, respectively), the threshold value of anti-desmoglein 3 must be 130 U/mL. The authors concluded that anti-desmoglein 1 has a good predictive value for cutaneous recurrence, while anti-desmoglein 3 does not predict mucosal recurrence adequately enough and should not be relied upon exclusively to make a therapeutic decision when there are no clinical symptoms.

5.2.3 Diagnosis of Paraneoplastic Pemphigus by ELISA Detection of Anti-envoplakin and Anti-periplakin Antibodies

The two studies presented in Table 8 focus on paraneoplastic pemphigus. ELISA tests using recombinant envoplakin and periplakin proteins are highly sensitive and specific for the diagnosis of paraneoplastic pemphigus. However, the number of available patients is very low, and a larger sample size is required before the diagnostic value of the two ELISA kits can be promoted [Huang et al., 2009].

Table 8: Results of the clinical performance of the ELISA method for the diagnosis of paraneoplastic pemphigus

STUDY GROUP RESULT BASED ON METHOD

Experimental N

Control (N)

IIF Se % (n/N)

Sp %

ELISA (Threshold)

Se %

(n/N)

Sp %

Huang et al., 2009

16 PV (20) PF (12) BP (20) CT

*(2)

HV (20)

Rat bladder

envoplakin (4.3) periplakin (6.4)

100 100

100 100

Probst et al., 2009

31 PV (30) BP (50)

HV (140)

Rat ESO or

bladder

NR NR envoplakin1–481 (NA)

80.6 98.8

envoplakin1626–2033 (NA)

80.6 97.5

periplakin1–324 (NA) 74.2 96.3

Abbreviations: BP = bullous pemphigoid; ESO = esophagus; HV = healthy volunteers; IIF = conventional indirect immunofluorescence; N = total number of patients; n = number of patients with the autoantibodies; NR = not reported; PF = pemphigus foliaceus; PV = pemphigus vulgaris; Se = sensitivity; Sp = specificity. *Castleman’s tumours without symptoms of paraneoplastic pemphigus.

In Probst et al. [2009], the ELISA test based on an N-terminal envoplakin fragment shows a high diagnostic accuracy to detect circulating anti-envoplakin antibodies in paraneoplastic pemphigus. The sensitivity of envoplakin by the ELISA method (envoplakin1–481 and envoplakin1626–2033) is 80.6% and the specificity is 97.5% to 98.8%. For periplakin1–324, the sensitivity is 74.2% and the specificity is 96.3%. The diagnostic accuracy of envoplakin is 93.7% and that of periplakin1–324 is 92.8% (p < 0.0001).

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5.3 Analytical (or Technical) Validity

COMPONENT PRESENCE ABSENCE NOT APPLICABLE

Repeatability

Reproducibility x

Analytical sensitivity

Analytical specificity x

Matrix effect x

Concordance x

Correlation between test and comparator x

Other, depending on type of test

No studies on the technical validity of BIOCHIP Mosaic-based indirect immunofluorescence or ELISA were identified. The data on the analytical validity of the ELISA kits are drawn from the manufacturer’s product monograph [EUROIMMUN, 2014] and the FDA’s notices of decision.

Reproducibility

The reproducibility data from the ELISA kits (EUROIMMUN) are presented in Table 9.

Table 9: Intra- and inter-assay coefficients of variation observed at different threshold values for the detection of various autoantibodies (ELISA, EUROIMMUN)

AUTOANTIBODIES INTRA-ASSAY INTER-ASSAY

Mean threshold value

(RU/mL)

% CV * Mean threshold

value (RU/mL)

% CV†

Anti-BP 180 27 61

119 177

3.0 1.2 1.6 2.1

26 61

119 174

4.8 3.1 4.2 2.6

Anti-BP230

24 61 97

114

5.0 4.6 3.4 3.0

23 60

101 111

6.8 3.5 6.1 3.4

Anti-Dsg3 42 63

155

4.4 2.6 5.9

44 69

163

6.1 5.3 3.3

Anti-Dsg1 27 73

111

4.0 3.1 3.3

46 70

114

3.7 6.1 6.0

Anti-envoplakin 2.2‡

2.9‡

3.4‡

2.9 1.6 2.4

2.2‡ 3.0‡ 3.4‡

3.9 2.8 3.9

Abbreviations: BP = bullous pemphigoid; CV = coefficient of variation; Dsg = desmoglein; mL = millilitre; RU = relative unit. *The intra-assay variation was determined by 20 serum measurements. †The inter-assay variation was determined by 4 measurements from 6 different series of tests. ‡ Mean value of the ratio.

18

Accuracy

The assessment of the ELISA kits from the company MBL (Medical & Biological Laboratories) showed intra- and inter-assay coefficients of variation of < 5% for anti-BP180 and < 10% for anti-BP230 (Table 10).

Table 10: Intra- and inter-assay coefficients of variation observed at different threshold values for the detection of anti-BP180 and 230 (ELISA MESACUP)

AUTOANTIBODIES INTRA-ASSAY INTER-ASSAY

Mean threshold value

(RU/mL)

% CV*

Mean threshold value

(RU/mL)

% CV†

Anti-BP180 Lot A Lot B Lot A Lot B Lot A Lot B Lot A Lot B

5.4 5.3 5.8 1.3 8.1 7.7 3.4 4.1

9.4 10.0 2.9 1.6 11.9 12.1 4.1 4.7

17.8 18.0 3.1 3.2 23.0 23.2 2.4 2.4

50.7 52.2 1.1 1.6 46.1 46.7 1.5 1.5

116.6 117.0 0.6 1.3 89.5 90.3 1.7 1.7

201.5 191.8 1.2 2.2

Lot A Lot B Lot A Lot B Lot A Lot B Lot A Lot B

Anti-BP230 5.9 5.2 2.2 4.5 8.2 8.7 5.0 6.9

11.3 10.6 2.1 3.2 17.2 17.5 1.6 2.1

16.7 17.0 3.4 2.6 19.1 22.4 9.7 9.1

20.6 23.5 1.9 3.9 71.3 74.2 7.8 6.7

66.0 70.3 3.2 1.5 141.3 130.0 4.9 4.2

139.6 130.9 1.1 3.4 Abbreviations: BP = bullous pemphigoid; CV = coefficient of variation; RU = relative unit. *The intra-assay variability was tested on 3 samples, 8 times for 3 distinct tests using two different lots. † The inter-assay variability was tested on 3 samples in 8 repetitive assays for 5 consecutive days.

Linearity

Analysis of the ELISA (EUROIMMUN) kits’ reproducibility of expected results for a series of 4 dilutions of 6 serum samples yielded a correlation coefficient R2 > 0.95. Autoantibody titres are linear for threshold values of at least 10 RU/mL to 199 RU/mL for anti-BP180, 17 RU/mL to 190 RU/mL for anti-BP230, 9 RU/mL to 197 RU/mL for anti-desmoglein 1, and 14 RU/mL to 195 RU/mL for anti-desmoglein 3.

Detection Limit

For the various ELISA (EUROIMMUN) kits, the lower detection limit is 0.6 RU/mL (anti-BP180), 1.0 RU/mL (anti-BP230), 0.5 RU/mL (anti-desmoglein 1), 0.3 RU/mL (anti-desmoglein 3), and 0.07 (ratio) (envoplakin).

Analytical Specificity

Interference

Anti-BP180, anti-BP230, anti-desmoglein 1 and 3, and envoplakin ELISA (EUROIMMUN) kits

There was no interference with hemolytic, lipemic, and icteric samples for concentrations of up to 10 mg/mL of hemoglobin, 20 mg/mL of triglycerides, and 0.4 mg/mL of bilirubin.

19

ELISA (MESACUP, MBL) kits for anti-BP180 and anti-BP230

No significant effect on assay results was noted by the addition of interfering substances (bilirubin C: 39.0 mg/dL, bilirubin F: 37.2 mg/dL, hemoglobin: 440.0 mg/dL, chyle: 2,350 units) in three patient samples (negative, weakly positive, and strongly positive).

Cross Reactivity

ELISA (EUROIMMUN) kits: anti-BP180, anti-BP230, anti-desmoglein 1 and 3

No cross reactivity was noted with other autoantibodies in the serum samples of patients with:

scleroderma (n = 18) for BP180;

scleroderma (n = 12) and pemphigus vulgaris (n = 15) for BP230;

bullous pemphigoid (n = 20) and linear IgA dermatosis (n = 20) for desmoglein 1 and desmoglein 3;

bullous pemphigoid (n = 30) and pemphigus vulgaris (n = 20) for envoplakin.

ELISA (MESACUP, MBL) kits for anti-BP180 and anti-BP230

There was no cross reactivity with the serum samples of patients with Epstein-Barr virus, Treponema pallidum, systemic lupus erythematosus, rheumatoid arthritis, Sjögren syndrome, and other diseases, with the exception of pemphigus (positive reaction of 7%) and acquired epidermolysis bullosa (33.3%) for PB180, and of pemphigus (positive reaction of 3.9%) for BP230.

Matrix effect

Anti-BP180 ELISA (EUROIMMUN) kit

The effect of the sample type, serum versus plasma, with various anticoagulants (EDTA30, heparin, citrate) was tested with a Passing-Bablok regression analysis. The 95% confidence interval for the slope contains the value 1.0, and the 95% confidence interval of the y-intercept contains the value 0, indicating an equivalence between the serum and plasma concentrations of the corresponding templates.

ELISA (EUROIMMUN) kit

The purpose of the analysis was to demonstrate that analyte recovery from plasma on anticoagulants such as lithium heparin, citrate, and EDTA was equivalent to analyte recovery from serum samples. Samples drawn from the same patient were collected in the four test tubes and analyzed. The regression analysis confirms the hypothesis and yields coefficients R2 of 0.994 (lithium heparin), 0.992 (citrate), and 0.988 (EDTA) for anti-desmoglein 1 and coefficients R2 of 0.994 (lithium heparin), 0.991 (citrate), and 0.983 (EDTA) for anti-desmoglein 3.

Correlation of Conventional Indirect Immunofluorescence and ELISA Test Results

Bullous Pemphigoid

Le Saché-de Peufeilhoux et al. [2012] reported a significant correlation between conventional indirect immunofluorescence and anti-BP230 ELISA results in 74 patients (Table 11).

Table 11: Results of the correlation between conventional indirect immunofluorescence and the ELISA method for bullous pemphigoid

METHOD CORRELATION COEFFICIENT (SPEARMAN R) P VALUE

IIF Anti-BP180 ELISA –0.12 0.32

IIF Anti-BP230 ELISA 0.75 0.0001

Abbreviation: IIF = conventional indirect immunofluorescence.

30 EDTA: Ethylenediaminetetraacetic acid.

20

Pemphigus Vulgaris

Herrero-González et al. [2010] reported a significant correlation between anti-desmoglein 3 and conventional indirect immunofluorescence in 40 patients (Table 12).

Table 12: Results of the correlation between conventional indirect immunofluorescence and the ELISA method for pemphigus vulgaris

METHOD CORRELATION COEFFICIENT (SPEARMAN RHO) P VALEUR

IIF Anti-Dsg3 ELISA 0.736 < 0.001

IIF Anti-Dsg1 ELISA 0.082 0.650

Abbreviation: IIF = conventional indirect immunofluorescence.

Concordance

BIOCHIP Mosaic-based Indirect Immunofluorescence Versus Conventional Indirect Immunofluorescence and ELISA

The study by Van Beek et al. [2012] (n = 454) is the only study to have compared indirect immunofluorescence on six substrates (BIOCHIP Mosaic) with conventional indirect immunofluorescence (on monkey esophagus or salt-split human skin substrate) completed by the ELISA method with different substrate kits (anti-desmoglein 1 and 3, anti-BP180 and 230, and envoplakin).

A high concordance (93.6%) in the results of the two methods was observed. There was concordance for some positive sera (kappa = 0.97) and 322 negative sera without autoantibodies (kappa = 0.88). However, there was no reaction with BIOCHIP Mosaic for 21 patients (4.6%), whereas the conventional method identified cases of autoimmune bullous diseases. Every divergent result was analyzed.

BIOCHIP Mosaic-based Indirect Immunofluorescence and ELISA

High concordance was reported between the results of the BIOCHIP Mosaic and ELISA for:

bullous pemphigoid:

the detection of anti-BP180: between 79.5% and 93.2% [Damoiseaux et al., 2012] and 90% [Tampoia et al., 2012b];

the detection of anti-BP230: 87.2% [Damoiseaux et al., 2012].

pemphigus vulgaris [Tampoia et al., 2012b] : the detection of anti-desmoglein 3: 100% (kappa ranging from 0.901 to 1.0).

5.4 Recommendations from Other Organizations

In France, reference centres for the diagnosis and monitoring of pemphigus and bullous pemphigoid recommend serological testing with indirect immunofluorescence on a substrate and the detection of anti-PB180-NC16A [Bernard et al., 2011] or anti-desmoglein 1 and 3 [Joly et al., 2011] with ELISA. In the case of pemphigus, ELISA is one of the recommended options [Joly et al., 2011].

The detection of BP180-NC16A antibodies by ELISA is useful as a complement to direct immunofluorescence when addressing an atypical urticarial eruption during pregnancy (gestational pemphigoid). The diagnostic value of ELISA alone remains to be determined [Ingen-Housz-Oro et al., 2011].

In the United Kingdom, the clinical guidelines for the management of bullous pemphigoid state that, although the ELISA method is not widely available, it is useful in selected cases (no further details on these cases) and for research purposes. Direct and indirect immunofluorescence on a substrate (salt-split human skin) remains the reference method for the diagnosis [Venning et al., 2012].

21

The European Autoimmunity Standardisation Initiative (EASI) also published practice guidelines that presented indirect immunofluorescence on monkey esophagus and ELISA kits as the methods to diagnose pemphigus and bullous pemphigoid [Shoenfeld and Meroni, 2012].

6 ANTICIPATED OUTCOMES OF INTRODUCING THE TEST

6.1 Impact on Material and Human Resources: Not assessed.

6.2 Economic Consequences of Introducing Test Into Quebec’s Health Care and Social Services System: Not assessed.

6.3 Main Organizational, Ethical, and Other (Social, Legal, Political) Issues: Not assessed.

7 IN BRIEF

7.1 Clinical Relevance

BIOCHIP Mosaic-based indirect immunofluorescence and ELISA kits allow the detection of bullous pemphigoid and the differential diagnosis of pemphigus vulgaris and pemphigus foliaceus. ELISA kits can be reserved for uncertain and atypical cases of bullous pemphigoid and are suitable to monitor autoantibody titres during the progression of the disease and patient follow-up. Although anti-BP230 testing should not be done systematically, it may be used for the minority of patients who test negative for anti-BP180 or atypical forms with the ELISA method. The utility of anti-desmoglein 1 in the monitoring of pemphigus foliaceus has not been demonstrated.

7.2 Clinical Validity

The BIOCHIP Mosaic shows a sensitivity and specificity comparable to those of conventional indirect immunofluorescence (on a substrate) and various ELISA kits. However, the sensitivity of these two methods is low to moderate in certain cases.

7.3 Analytical Validity

The ELISA kits assessed showed good analytical validity. Limited data indicate a high concordance (93.6%) between BIOCHIP Mosaic-based indirect immunofluorescence and conventional indirect immunofluorescence (on a substrate) with various ELISA kits for the diagnosis of bullous pemphigoid, pemphigus vulgaris, and pemphigus foliaceus.

7.4 Recommendations from Other Organizations

Current practice guidelines do not address the use of the BIOCHIP Mosaic method, although they recommend ELISA kits as an option or as a complementary method to conventional indirect immunofluorescence.

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8 INESSS NOTICE IN BRIEF

Analysis of the Immunoserological Profile of Autoimmune Skin Diseases

Status of the Diagnostic Technology

Established: ELISA for 2 of the 6 substrates

Innovative: BIOCHIP

Experimental (for research purposes only)

Replacement for technology: , which becomes obsolete

INESSS Recommendation

Include test in the Index

Reject request as submitted

Reassess test

Additional Recommendation

Draw connection with listing of drugs, if companion test

Produce an optimal use manual

Identify indicators, when monitoring is required

NOTE

It would have been preferable to:

present each test separately, by marker and disease, to establish its clinical relevance;

present an algorithm for the indications for the test;

define the weighted value for each test more clearly;

define the threshold values;

justify the added clinical value of BIOCHIP compared with the available conventional immunofluorescence test.

23

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