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LITHUANIAN UNIVERSITY OF HEALTH SCIENCE
Department of Skin and Venereal Diseases
Serological Immunoassays for Diagnosis of Autoimmune Bullous Diseases
Aviram Albagly
Faculty of Medicine VI
Group No. 35
Supervisor: assoc. prof. Vesta Kucinskiene, MD, PhD
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TABLE OF CONTENT
1. Title Page……….1 2. Summary of Thesis………3-4 3. Acknowledgment……….5 4. Conflict of Interest………...5 5. Abbreviation………6 6. Introduction………7-8 7. Aim and Objectives……….….9 8. Literature Review……….10-20 9. Materials and Methodology………..21-22 10. Results and their Discussion……….23-24 11. Conclusion………...253
SUMMARY OF THESIS
Serological Immunoassays for Diagnosis of Autoimmune Bullous Diseases Aviram Albagly
Aim: To review the accuracy of serological immunoassays when diagnosing autoimmune bullous
diseases (AIBD).
Objectives:
1. To identify the accuracy of immune-enzyme methods for the diagnosis of AIBD.
2.
To assess the accuracy of immunofluorescence method for diagnosis of AIBD.3. To analyze the new serological methods for the diagnosis of AIBD.
Methods:
The main literature search was conducted using MEDLINE PubMed online research engine, including studies that were published in the last 10 years in the English languish for serological immunoassays, as well as studies for new methods (BIOCHIP), for the diagnosis of autoimmune bullous diseases.
Results:
For ELISA method for BP, sensitivity values for anti-BP180 autoantibodies were in a range of 69.1%-97.9%, with specificity of 90.3%-96%. For anti-BP230 sensitivity values were 48%-72.3%, with a specificity between 85.5%-100%.
For ELISA method for PV, sensitivity values were 84.8% and 90.2%, and specificity values were 96.7% and none.
IIF method for BP showed sensitivity values for ME substrates of 76.5% and 73.2%, with a specificity of 100% and 96.5%. For SSS substrate, sensitivity values were 100% and 73.3%, with a specificity of 100% and 97.1%.
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IIF method for PV using ME substrates showed sensitivity values of 87.4% and 84.8%, with a specificity of 93.5% and 91.8%.
BIOCHIP method for diagnosis of BP showed sensitivity values for anti-BP180 autoantibodies of 76.5%, 83.3% and 55.3%, with specificity of 100%, 100% and 97.7%. Sensitivity values for anti-BP230 autoantibodies were 45.1%, 39% and 65.8%, with specificity of 100%, 100% and 86.5%.
BIOCHIP method for diagnosis of PV yield sensitivity for anti-DSG3 autoantibodies of 60.9% and 97.62%, with a specificity of none and 100%, while for anti-DSG1 autoantibodies sensitivity results were 13% and 19%, with a specificity of none and 100%.
Conclusions:
1. ELISA method for BP gives satisfactory results for detection of anti-BP180 autoantibodies, but the combination of anti-BP180 and anti-BP230 autoantibodies will give better possibility to diagnose the disease.
2. In case of diagnosis of PV using ELISA method, it has been suggested that ELISA system possess a very high sensitivity and specificity for diagnosis, comparing to other techniques such as direct and indirect IF.
3. IIF for diagnosis of BP will give better results of sensitivity and specificity if used substrate SSS more than substrates ME.
4. IIF for diagnosis of PV had high sensitivity and specificity values by using ME substrates. 5. For BIOCHIP methods, the difference between the BP autoantibodies sensitivity and
specificity is very high in favor of anti-BP180, with very poor results for anti-BP230. The same goes for PV autoantibodies, with even larger differences in sensitivity and specificity in favor of anti-DSG3 autoantibodies.
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AKNOWLEDGMENT
I would like to thank my supervisor for her guidance and advices on this review. Her
knowledge and experience within the field of Dermatology helped me a great deal on completing this literature review.
CONFLICT OF INTEREST
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ABBREVIATIONS
AIBD- Autoimmune Bullous Disease.
BP- Bullous Pemphigoid.
DIF- Direct Immunofluorescence.
DSG- Desmoglein.
ELISA- Enzyme-Linked Immunosorbent Assay.
FITC- Fluorescein Isothiocyanate.
HEK- Human Embryonic Kidney.
HRPO- Horseradish Peroxidase.
IF- Immunofluorescence.
IgG- Immunoglobulin G.
IIF- Indirect Immunofluorescence.
MBL- Medical and Biological Laboratories CO.
ME- Monkey Esophagus.
PBS- Phosphate-Buffered Solution.
PV- Pemphigus Vulgaris.
SSS- Human Salt-Split Skin.
TMB- Tetramethylbenzidine.
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INTRODUCTION
Autoimmune bullous disorders (AIBD) is a group of rare disorders with clinical manifestation of blisters and erosions on the skin and/or mucous membrane [1].
AIBD occurs when patient's autoantibodies bind to structural proteins located in the skin and mucous membrane [2]. These proteins function as part of the desmosome (mainly desmogleins, desmocollins, plakins) and also part of hemidesmosomes (for example, BP180, BP230, plectin, type VII collagen) [3].
Autoimmune process interrupt the intracellular connections as well as the anchoring mechanism inside the epidermis and the junction between dermal and epidermal, causing the skin layer to separate with the development of blisters with or without erosions [4].
AIBD are categorized into 2 groups of disorders: intra-epidermal, the most common subtype
Pemphigus Vulgaris (PV) with IgG antibodies against desmoglein, and sub-epidermal, most common type Bullous Pemphigoid (BP) with IgG antibodies against hemidesmosome.
There is a wide range of PV incidence in Europe, which vary from 0.5 cases/million per year in Germany to 8 cases/million per year in Greece. Eastern European countries have a higher incidence than northern European countries. In Asia the incidence is much greater and varies from 1.6
cases/million per year in Saudi-Arabia up to 16.1 cases/million per year in Israel. Also, there are differences in incidence based on ethnic group, mainly high incidence in Ashkenazy Jews as well as people of Mediterranean origin [1].
BP typically affects the elderly, mostly between the ages 60-70 years old, and the incidence of BP varies between 2.4 to 21.7 cases/million per year, up to recent studies that suggest an even higher incidence of 42.8 causes/million per year [5].
Diagnosis of AIBD, including BP and PV, is a multi-step process that is primarily based on clinical symptoms, histopathology, serology such as DIF, IIF and ELISA, and less commonly immunoblotting techniques [3].
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The diagnostic gold standard for AIBD is histology HE stain and direct immunofluorescence (IF) microscopy for the detection of autoantibodies in the skin. Each one of the subtypes of AIBD has its own autoantibodies.
In PV, the location of the intracellular binding of IgG and C3 is found in the epidermis, and the autoantibodies are mainly anti-Desmoglein 3 and anti-Desmoglein 1, While in BP, the staining of IgG and C3 is linear, located at the dermal-epidermal junction, with common antibodies against BP180 and BP230 [6].
Another screening tool is for circulating autoantibodies such as indirect IF (IIF) microscopy or ELISA system.
For IIF in PV, the most sensitive and widely used substrate is either guinea pig or monkey esophagus, while in BP case the substrate is composed of human salt split skin [6].
ELISA system is leaning on recombinant target antigens, which are greatly available, and its application in serological diagnosis of AIBD had increased. There are several commercial ELISA
and Euroimmun, that are used for molecular and biological laboratories CO.)
system, such as MBL (
the detection of autoantibodies specific for PV and BP [2].
In recent years, new serological immunoassays were produced for diagnosis of AIBD, based on mosaic-based Indirect IF technique called the BIOCHIP. It has a unique design that allows differential diagnosis of all the AIBD subgroups, via combination of screening of several autoantibodies with target antigen-specific substrate, all dense into a single miniature incubation field [7].
The thesis aim is to analyze the serological methods of immunoassays for diagnosis of AIBD, by identifying immune-enzyme methods and assessing immunofluorescence methods for the diagnosis of AIBD, and analysing the new serological methods for the diagnosis of AIBD.
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AIM AND OBJECTIVES
Aim: To review the accuracy of serological immunoassays when diagnosing autoimmune bullous
diseases (AIBD).
Objectives:
1. To identify the accuracy of immune-enzyme methods for the diagnosis of AIBD.
2.
To assess the accuracy of immunofluorescence method for diagnosis of AIBD.10
LITREATURE REVIEW
Enzyme-Linked Immunosorbent Assay (ELISA):
The predominant antigens PV exhibit called desmoglein 3 (DSG-3) and desmoglein 1 (DSG-1) [2]. The types and level of antigens in PV may vary between several phenotypes of the disease [8]. In PV involving mucous membrane lesion alone, the autoantibodies react to DSG-3, where is in PV involving mucosal and skin lesions as well, the 2 types of autoantibodies, DSG-3 and DSG-1, will be present [9].
BP is activated by serum autoantibodies that target the basement membrane zone. This includes the intracellular 230 kD ingredient of hemidesmosome, known as BP230, and also a transmembrane protein 180 kD that link the hemidesmosome of basal keratinocyte with the lamina densa, and known as BP180 [10].
Enzyme-Linked Immunosorbent Assays (ELISA) is a diagnostic tool that brings a quantitative method for measuring specific levels of autoantibodies, as well as monitoring disease activity [11]. Other methods of serological diagnosis, including Western immunoblotting and immunoprecipitation, are considered as time-consuming, and are not practical for routine screening involving multiple serum samples, as compared to ELISA [12].
The principle behind ELISA technique is the formation of antigen-antibody complex by adding patient serum that contains antibodies to the antigen-coated ELISA plate. Then adding enzyme (HRPO) that conjugate anti-antibody to form an antigen-antibody-anti antibody complex. By adding a substrate (TMB) to the enzyme, the substrate will be converted to a detectable colored product, usually a yellow color that is related to the amount of circulating autoantibodies [13].
For PV, ELISA made it possible for production of recombinant DSG-1 and DGS-3, and by so,
detecting pemphigus autoantibodies [2]. For BP, ELISA executes recombinant BP180 and BP230, and are commercially available via several companies, MBL located in Japan and Euroimmun located in Germany, with a relatively high percentage of diagnostic sensitivity for BP180 [8].
ELISA technique had many studies over the years, regarding the usefulness of the result, as reflected in sensitivity and specificity criteria.
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BP:
According to different studies around the world, ELISA method is more used to detect anti-BP180 autoantibodies, due to higher sensitivity than anti-BP230.
In a prospective study that was held in 2015 at the Razi Dermatology Hospital in Teheran, Iran, 50 newly diagnosed BP patients were assessed using commercial ELISA kit Euroimmun, between the years 2011 and 2013, along with a control group of 50 people. The study suggests that anti-BP180 has a better sensitivity value than anti-BP230, with 88% sensitivity for anti-BP180 and 48% sensitivity for anti-BP30, while the specificity for both was 96% [14].
However, the measuring of BP230 alone gives lower accuracy for diagnosis of BP. However, ELISA method involving both BP180 and BP230 autoantibodies giver better results [15].
In a 2012 study from the Department of Dermatology, Yonsei University College of Medicine, Seoul, Korea, there were measured serum IgG levels of anti-BP180 and anti-BP230 autoantibodies by using ELISA method in patients with BP. The study was performed on a sample of 47 patients with BP, and 15 healthy volunteers, as well as 16 patients with epidermolysis bullosa aquisita, who served as a control group, between the years 2008 to 2010. The results showed 97.9% sensitivity and 90.3% specificity for anti-BP180, while anti-BP230 autoantibodies showed sensitivity of 72.3% and
specificity of 100%. The combination of BP180 and BP230 gave better results, with 100% sensitivity and 90.3% specificity [16].
In another research that was held at the outpatient department of Dermatology, Mahidol University in Bangkok, Thailand, 2020, a total of 131 patients were included. 68 of them were BP patients, while 63 served as a control group. The study was conducted between 2014 and 2016, and the patients were diagnosed using commercial ELISA kits of Euroimmun. The result shows that sensitivity and
specificity of anti-BP180 were 69.1% and 90.5%, respectfully, while the sensitivity and specificity and anti-BP230 were 55.9% and 85.5%, respectfully. However, using the two antibodies combined will give an about 7% rise in sensitivity, result in 76.5%, compared to anti-BP 180 alone [17].
Consequently, in a situation of clinically suspected BP case, when anti-BP180 testing is negative, it is highly recommended to assess anti-BP230 autoantibodies as well [18].
12 Fig. 1. Summary of sensitivity for autoantibodies anti-BP180 and anti-BP230 for ELISA diagnosis
for BP
Fig. 2. Summary of specificity for autoantibodies anti-BP180 and anti-BP230 for ELISA diagnosis for BP
PV:
In case of PV, it has been suggested that ELISA system has very high sensitivity and specificity for diagnosis, comparing to other techniques such as direct and indirect IF.
In a single-center retrospective study, held in 2016, 33 patients that were diagnosed as pemphigus patients from the Department of Oral Medicine, Peking university school and hospital of stomatology were included in the study, along with a control group of 61 people, between the years 2010 and 2014. Anti DSG-1 and anti DSG-3 were detected using the MBL, a commercially available ELISA test. The results showed the ELISA holds a sensitivity for PV autoantibodies of 84.8% and a specificity of 96.7% [19]. 88% 97.90% 69.10% 48% 72.30% 55.90% 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
Razi Hospital, Iran, 2015
Yonsei University, 2012
Mahidol University, Thailand 2020
anti-BP180 sensitivity anti-BP230 sensitivity
96% 90.30% 90.50% 96% 100% 85.50% 75% 80% 85% 90% 95% 100%
Razi Hospital, Iran, 2015
Yonsei University, 2012
Mahidol University, Thailand 2020
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Department of Dermatology, Kasturba Medical College, Manipal University, Another study from the
consist of 61 patients enlisted, 50 of them are PV patients and 11 are
,
Karnataka, India, held in 2017
pemphigus foliaceous patients. A serum sample of anti-DSG1 and anti-DSG3 autoantibodies was collected, and the results showed a sensitivity of 90.2%. The study did not include a control group, and therefore specificity was not possible to assess [20].
Fig. 3. Sensitivity and specificity for anti-DSG1 and anti-DSG3 for ELISA diagnosis for PV
Indirect Immunofluorescence (IIF):
Indirect immunofluorescence (IIF) is considered an essential method for quick and reliable diagnosis of AIBD. It works by a way of using the patient's serum along with a substrate in order to better visualize the circulating autoantibodies that are present in the blood of PV or BP patients [21].
For the IIF process, a sample from patient blood is collected. Usually, about 3 ml of blood is drawn without anticoagulants, and the clotted blood is separated from the serum. The serum should be stored in freezing until the test is executed. Overall, one or two freeze-thaw cycles are considered tolerable, because addition cycles can damage the process, when five or more will completely lose the activity. Then, the substrates is incubated with series of dilutions of the patient's serum with PBS (phosphate-buffered solutions), usually started with a 1:10 ratio, for 30 minutes at room temperature, with washing them after. Any autoantibodies that are present in the serum will bind to the respective antigens in the substrates [22]. The next step in the process is incubation for 30 minutes with a second autoantibody that direct against the immunoglobulin's classes that have bound in the first step, which are conjugated to a fluorescent dye such as FITC. For the result, the slides are examined under a fluorescence
microscope [23]. 84.80% 90.20% 96.70% 75.00% 80.00% 85.00% 90.00% 95.00% 100.00%
Peking University, China, 2016 Manipal University, India, 2017 sensitivity specificity
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A suitable substrate is essential in order to detect and better visualized the circulating antibodies. For PV and BP the most used substrates are esophagus and salt-split human skin.
As for the esophagus, the mainly used acceptable substrate is esophagus from either monkey or guinea pig. It presents high sensitivity for PV autoantibodies (mainly anti-DSG3, anti-DSG1 is in a lower grade) and satisfactory sensitivity for BP. The PV-specific autoantibodies will develop a honeycomb-like fluorescence of the intercellular substance along the Stratum Spinosum. The anti-BM zone
autoantibodies, mainly anti-BP180 and anti-BP230 which are associated with BP, will appear as a fine linear staining between the Stratum Basale and the connective tissue surrounding it [2].
Salt-split skin (SSS) is considered as the IIF substrate of choice for the detection of anti-BM zone autoantibodies for screening of BP, with high sensitivity rates. Salt-split skin is produced when partial dermal-epidermal splitting is induced by incubating it with a solution of 1 M NaCl solution [2]. This solution separates the skin between the epidermis and the dermis along the Lamina Lucida. Direct SSS is performed on the patient skin, while indirect SSS is performed on another NHS (normal human skin) using the patient's serum [23]. Anti-BP180 and anti-BP230 will stain the epidermal side of the artificial split, known as the "blister roof" [2].
Fig. 4. Different tissue substrates of IIF staining for diagnosis of BP or PV.
A. represent staining on monkey esophagus susbtrate for PV autoantibodies. B. represent staining on monkey esophagus susbtrate for BP autoantibodies. C. represent staining on salt-split human skin susbtrate for BP autoantibodies.
Saschenbrecker S, Karl I, Komorowski L, Probst C, Dähnrich C, Fechner K, et al. Serological diagnosis of autoimmune bullous skin diseases. Front Immunol. 2019;10(AUG):1–18.
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BP:
As mentioned above, in most cases, the substrate of choice for diagnosis of BP using IIF method will be salt-split human skin (SSS), with satisfactory results, although monkey esophagus (ME) can be used as well.
In a study published in 2019, from the Department of Dermatology and Immunodermatology, Medical University of Warsaw, Poland, 51 patients with confirmed BP were recruited between the years 2013-2018, along with 39 individuals, 35 are pemphigus patients and 4 who are healthy, served as a control group. The subjects tested using salt split human skin (SSS) and monkey esophagus (ME) substrate for IIF method. The results showed that as for ME, sensitivity was 76.5% with 100% specificity, while in SSS substrate, the sensitivity raised to 100%, also with 100% specificity [24].
In another retrospective analysis for diagnosis of BP using IIF method, held in the Department of Dermatology and Allergology at the Ludwig Maximilian University, Munich, Germany, 2013, 313 patients with BP were recruited between the years 2008-2012, along with 488 individuals served as a control group. The subjects were tested for BP using different substrates for IIF, including SSS and ME. The results showed that for ME substrate was 73.2%, with 96.5% specificity, while sensitivity for SSS was 73.3% with 97.1% specificity [25].
Fig. 5. Sensitivity for differential substrates for IIF diagnosis of BP
PV:
As mentioned above, the substrates of choice for diagnosis of PV by IIF method is esophagus substrates, mainly monkey esophagus, to detect anti-DSG3 autoantibodies.
76.50% 73.20% 100% 73.30% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00%
Warsaw, Poland, 2019 Munich, Germany, 2013 ME SSS
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A retrospective cohort study was held at Rambam health care campus, Haifa, Israel, collect 770 serum samples that were tested for the presence of PV autoantibodies using IIF method. The study published in the year 2018 and include the serum sample from patients between the years 2000-2017. Out of the 770 samples, 174 patients diagnosed with PV, 29 diagnosed with PF, and 567 patients did not present a confirmed diagnosis for either of the pemphigus diseases. The result showed a sensitivity of 87.4% for PV anti-DSG3 autoantibodies using monkey esophagus, and a specificity of 93.5% [26].
Another study from the Department of Oral Medicine, Peking university school and hospital of stomatology, China, 2016, includes 33 pemphigus patients along with a control group of 61
individuals, between the years 2010 and 2014. The IIF technique was conducted via a standard method using monkey esophagus as a substrate as well. The result showed a sensitivity of 84.8%, along with specificity of 91.8% [19].
Fig. 6. Sensitivity and specificity for anti-DSG3 autoantibodies in diagnosis of PV using ME substrate for IIF
BIOCHIP:
In recent years, a new method for serological diagnosis of AIBD was developed called BIOCHIP. It is a mosaic-based 7 IIF, with a design that makes it possible for differentiation between diagnoses of several different AIBD subtypes, all from one single serum sample. It allows a quick and precise diagnosis of PV or BP, which especially useful in cases that requires aggressive, immediate treatment [27]. 87.40% 84.80% 93.5% 91.80% 75.00% 80.00% 85.00% 90.00% 95.00% 100.00%
Haifa, Israel, 2018 Peking, China, 2016 sensitivity specificity
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The BIOCHIP is essentially a multiplex IIF mosaic that combines screening along with targeting antigen-specific substrates, all in a single miniature incubation field. On a standard-sized slide lies down 10 incubation fields, each contains 6 different BIOCHIPs, represent staining after incubation with BP and PV serum. Anti-DSG1, anti-DSG3, and anti-BP230 are expressed in human HEK293 (Human Embryonic Kidney) cells. Anti-BP180 is directly coated on the BIOCHIP. The other two are monkey esophagus and primate salt-split skin substrates [28].
Fig. 7. BIOCHIP mosaic for diagnosis of AIBD.
A. a standard size slide, with 10 incubation fields, each contains 6 different BIOCHIP. B. representative staining after incubation with BP.
C. representative staining after incubation with PV.
Van Beek N, Rentzsch K, Probst C, Komorowski L, Kasperkiewicz M, Fechner K, et al. Serological diagnosis of autoimmune bullous skin diseases: Prospective comparison of the BIOCHIP mosaic-based indirect immunofluorescence technique with the conventional multi-step single test strategy. Orphanet J Rare Dis. 2012;7(1):1–10.
This unique structure makes it possible for screening 10 patients for autoantibodies for PV and BP at the same time, providing a more efficient and cost-saving way [7]. In this way, the BIOCHIP is a faster, cheaper, easy to use technique, which allows easy differentiation among the different autoantibodies specificities. Due to that, the BIOCHIP would be able to allow initial screening for identification of patients with AIBD, when indecisive results can then be confirmed by more traditional methods, such as ELISA [29].
BP:
As in other serological methods for diagnosis of BP, the BIOCHIP also contain different results when it comes to the 2 most used autoantibodies in BP, anti-BP180 and anti-BP230.
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In a study from 2019, held at the Department of Dermatology and Immunodermatology, Medical University of Warsaw, Warsaw, Poland, 51 patients with confirmed BP were enlisted, along with 39 participants, 35 PV patients and 4 healthy individuals, which served as a control group. The
participants were recruited in the years 2013-2018. A sample from the participant's serum was taken, and levels of anti-BP180 and anti-BP230 were measured using BIOCHIP. The results showed that anti-BP180 holds a sensitivity of 76.5%, while anti-BP230 holds a sensitivity of 45.1% only. Specificity in both groups was 100% [30].
Another small study from the University of Padua, Unit of Dermatology, Italy, held in 2012, had a small group of 18 patients with BP, along with 3 healthy individuals and 2 PV patients which served as a control group. The study found that sensitivity for anti-BP180 was 83.3% compared to 39%
sensitivity for anti-BP230. Specificity was 100% in both groups [29].
Although it has been proved that the detection of BP180 in most cases is more precise than anti-BP230, it has been suggested that validation of either the autoantibodies can give better results. In a study that was held at the Department of Dermatology, St George Hospital, Sydney, Australia, 2019, 38 patients with BP were enlisted, along with 63 disease control and 39 healthy individuals served as a control group. The study found sensitivity anti-BP180 was 55.3% and specificity was 97.7%, while the sensitivity for anti-BP230 was 65.8% with a specificity of 86.5%. however, the values of sensitivity and specificity for either anti-BP180 or anti-BP230 were 86.8% and 70.7%, respectfully [27].
Fig. 8. Summary of sensitivity for anti-BP180 and anti-BP230 for BIOCHIP diagnosis of BP 76.50% 83.30% 55.30% 45.10% 39.00% 65.80% 0.00% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00%
Warsaw University, Poland, 2019
Padua University, Italy, 2012 St George Hospital, Sydney, Australia, 2019 anti-BP180 sensitivity anti-BP230 sensitivity
19 Fig. 9. Summary of specificity for anti-BP180 and anti-BP230 for BIOCHIP diagnosis of BP
PV:
In the serological diagnosis of PV, it has been suggested that the use of autoantibodies anti-DSG3 is much more sensitive than the use of anti-DSG1.
In a study published in 2019, held at the Department of Dermatology, St George Hospital, Sydney, Australia, sera from 23 patients with PV was used, along with 63 diseased control patients and 39 healthy participants that served as a control group. The results showed that BIOCHIP for either DSG1 or DSG3 substrates had a sensitivity of 65.2% and specificity of 69.6%. However, anti-DSG3 alone had a sensitivity of 60.9%, while anti-DSG1 held a sensitivity of only 13% [27].
Another study that was held in 2013 at the Department of Medicine, Dermatology Unit, University of Padua, Italy, consist of 42 patients with PV, along with 10 normal participants that served as a control group. The study showed sensitivity of 97.62% in detection anti-DSG3, while detection of anti-DSG1 yield only 19% sensitivity. Specificity was 100% in both groups [31].
The BIOCHIP technique was researched as a diagnostic tool not only for diagnosing PV, but also to differentiate between the different subtypes of PV, mucosal PV (m-PV), and mucocutaneous PV (mc-PV).
A study that was held in the Department of Dermatology and Immunodermatology, Medical
University of Warsaw, Poland, 2019, recruited participants between the years 2013-2017 to evaluate the usefulness of BIOCHIP for diagnosing PV autoantibodies, anti-DSG3 and anti-DSG1. The study involved 35 patients, 21 of them with PV, and out of the PV patients, 13 were m-PV patients and 8
100.00% 100.00% 97.70% 100.00% 100.00% 86.50% 80.00% 82.00% 84.00% 86.00% 88.00% 90.00% 92.00% 94.00% 96.00% 98.00% 100.00%
Warsaw University, Poland, 2019
Padua University, Italy, 2012 St George Hospital, Sydney, Australia, 2019 anti-BP180 specificity anti-BP230 specificity
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were mc-PV patients. Along with them, 48 participants serve as a control group. The result showed anti-DSG3 sensitivity to be 100%, while sensitivity for anti-DSG1 was 42.8%. Specificity in both groups was 100% as well.
Overall, the study showed sensitivity of 81% in detecting PV, with 76.9% sensitivity for m-PV and 87.5% sensitivity for mc-PV. Thus, The BIOCHIP technique is a useful method not only for diagnosis of PV, but also for differentiation between the types of PV [24].
Fig. 10. Summary of sensitivity for anti-DSG3 and anti-DSG1 for BIOCHIP diagnosis of PV 60.90% 97.62% 100% 13.00% 19% 42.80% 10.00% 20.00% 30.00% 40.00% 50.00% 60.00% 70.00% 80.00% 90.00% 100.00%
St George Hospital, Sydney, Australia, 2019
Padua University, Italy, 2013 Warsaw University, Poland, 2019
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MATERIALS AND METHODOLOGY:
The Medline (PubMed) online research engines along with Up-To-Date database engine and Wiley online library were searched for studies of any serological diagnosis of autoimmune bullous diseases, mostly of type's Pemphigus Vulgaris and Bullous Pemphigoid. Other variants of AIBD such as Paraneoplastic Pemphigus, Pemphigoid Gestational, or Mucous Membrane Pemphigoid can be present in some of the articles, but were excluded from the review.
The research protocol included the following steps: identification and establishment of databases online engines to be searched, determining search strategy, searching for references, reading the abstract as a first stage, reading the full text as a second stage, extract the data from the relevant articles, evaluation of the data inside the articles, and summarizing the findings as the final stage.
Keywords:
The process was started with the establishment and searching of keywords such as "autoimmune bullous diseases", "Bullous Pemphigoid", "Pemphigus Vulgaris", "serological immunoassay's", "direct/indirect immunofluorescence", "ELISA", "BIOCHIP", and the combination of the mentioned keywords.
Searching for references:
The searching for articles started in 2020 and included articles from the last 10 years, that are only in the English language, and articles where patients were diagnosed with AIBD, both PV and/or BP, on the routine multistep diagnostic algorithm including serology. Articles that were based on clinical studies and clinical trials were used for results and discussion.
First and Second-stage search:
During the first stage of searching, abstracts from the above-mentioned articles were read. Abstracts that were selected for the second stage screening were required to meet the following
criteria: 1)PV and/or BP. 2) diagnosis of adult patients with AIBD. 3) Methods for diagnosis of AIBD in the forms of serological, immunofluorescence, ELISA, or BIOCHIP.
Moreover, the studies cited in the articles were also reviewed in order to find more published researches that are suitable for this review.
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Data extraction:
Full texts of the relevant articles mentioned above were read and the data regarding the subject was taken under consideration using the flowing variables: name of the disease in which the patients were affected, number of participants attending the trial, serological method in which the diagnosis of the disease was done, the outcome as reflected by the efficiency of the serological diagnostic tool to identify the disease, and conclusion.
Evaluation and summarization of the data:
Studies for evaluation of serological immunoassays for the diagnosis of AIBD were measured as the primary outcome of sensitivity and specificity for each diagnostic tool reviewed in this thesis. This was summarized in order to determine the accuracy of the diagnostic methods to identify the autoantibodies of AIBDs.
Fig. 11. Research protocol used for this review total articles extracted from: PubMed Up-To-Date Wiley total articles chosen based on key words (52): BP/PV serological immunoassay's IIF ELISA BIOCHIP full-text articles that were excluded
(21): 1. articles on patients diagnosed with other diseases
than BP or PV. 2. articles not in the english language. 3. articles older than 10 years. potenitially relevant full-text articles based on inclusion criteria's (31)
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RESULTS AND THEIR DISCUSSION:
This review of published papers has yielded 31 studies that investigate the different serological techniques for the diagnosis of AIBD, both BP and PV, by detecting their corresponded
autoantibodies.
The different serological techniques for the diagnosis of BP and PV were ELISA, IIF method using the substrate monkey esophagus for PV and monkey esophagus and salt-split human skin for BP, and the relatively new BIOCHIP technique. Each technique had 2 or 3 studies that, by using samples from BP and PV patients as well control group in most of the studies, evaluating the efficacy of the different techniques, as measured by sensitivity and specificity.
Looking at the results of the studies for the different techniques of serological diagnosis of AIBD, there are different values of sensitivity and specificity for each technique, as well as different results for each autoantibodies for BP or PV, or in case of IIF, different substrates.
For ELISA diagnosis of BP, different studies suggest that sensitivity for anti-BP180 autoantibodies is of greater value than sensitivity for anti-BP230, with values of 88%, 97.9%, 69.1% comparing to 48%, 72.3%, and 55.9% sensitivity for anti-BP230 autoantibodies. However, the combination of the two autoantibodies gives better results for sensitivity and specificity, with 100% and 76.5% sensitivity of the combined autoantibodies, and 90.3% specificity [(14), (16), (17)].
thus, in a situation of clinically suspected BP case, when anti-BP180 testing is negative, it is highly recommended to assess anti-BP230 autoantibodies as well [18].
For ELISA diagnosis for PV, evaluating the autoantibodies anti-DSG3 and anti-DSG1 will yield relatively high sensitivity values, 84.8% and 90.2%, compared to more traditional serological methods such as IIF, when only one article give specificity of 96.7%, due to a lack of control group in one of the articles [(19), (20)].
In IIF method, it has been suggested that a suitable substrate for either BP or PV is essential for the detection and better visualization of the circulating autoantibodies.
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For BP, sensitivity for ME was 76.5% and 73.2%, with a specificity of 100% and 96.5%, while for SSS, sensitivity values rise to 100% and 73.3%, with a specificity of 100% and 97.1%. thus, the use of SSS as a substrate gives better results for evaluating BP autoantibodies [(24), (25)].
For PV, the usefulness of IIF method is measured mainly by ME substrates, with a sensitivity of 87.4% and 84.8%, and specificity of 93.5% and 91.8% [(26), (19)].
Overall, IIF method is considered an essential method for quick and reliable serological diagnosis for AIBD [21].
The BIOCHIP method has a unique structure, comprised of 10 incubation fields, each containing 6 different BIOCHIPs for autoantibodies for AIBD. such structure provide a more efficient and cost-saving way for diagnosis of AIBD [7]. The unique design also makes it possible for easy
differentiation between the different autoantibodies specificities, and due to the fact that it is a relatively quick method, it is especially useful in cases that requires aggressive, immediate treatment [27].
BIOCHIP method for diagnosis of BP relies mainly on autoantibodies anti-BP180, with sensitivity values of 76.5%, 83.3%, and 55.3%, and specificity of 100%, 100%, and 97.7%. This are much higher values than the autoantibodies anti-BP230, which yield 45.1%, 39%, and 65.8% sensitivity, along with 100%, 100% and 86.5% specificity [(30), (29), (27)].
Diagnosis of PV using BIOCHIP method also relies on one autoantibody, anti-DSG3, more than the other, anti-DSG1, and is more distinct, with anti-DSG3 sensitivity values of 69.9% and 97.62%, comparing to poor results of 13% and 19% for anti-DSG1 autoantibodies [(27), (31)].
Limitations for this review manifest mainly on the different serum samples for this review, regarding each technique. Some of the articles had not so large difference between each study, but others had a very large difference between the serum samples of each study for each method, manifest by over than a 100 individuals differences in some of the studies.
Also, due to fact that IIF and ELISA methods are relatively classic methods, not so many studies from the last 10 years were available. This creates only 2 or 3 studies for each method to extract data from.
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CONCLUSIONS:
1. ELISA method for BP gives satisfactory results for detection of anti-BP180 autoantibodies, but the combination of anti-BP180 and anti-BP230 autoantibodies will give better possibility to diagnose the disease.
2. In case of diagnosis of PV using ELISA method, it has been suggested that ELISA system possess a very high sensitivity and specificity for diagnosis, comparing to other techniques such as direct and indirect IF.
3. IIF for diagnosis of BP will give better results of sensitivity and specificity if used substrate SSS more than substrates ME.
4. IIF for diagnosis of PV had high sensitivity and specificity values by using ME substrates. 5. For BIOCHIP methods, the difference between the BP autoantibodies sensitivity and
specificity is very high in favor of anti-BP180, with very poor results for anti-BP230. The same goes for PV autoantibodies, with even larger differences in sensitivity and specificity in favor of anti-DSG3 autoantibodies.
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