|Year : 2013 | Volume
| Issue : 1 | Page : 2-8
Immunofluorescence and its application in dermatopathology with oral manifestations: Revisited
RS Arvind Babu1, P Chandrasekar2, K Lalith Prakash Chandra2, G Sridhar Reddy2, K Krian Kumar2, BV Ramana Reddy2
1 Oral and Maxillofacial Pathologist, Lecturer in Dentistry, Faculty of Medical Sciences - Undergraduate Dental Programme, The University of the West Indies, Mona Campus, Kingston, Jamaica
2 SIBAR Institute of Dental Sciences, Takkellapadu, Guntur, Andhra Pradesh, India
|Date of Web Publication||20-Jun-2013|
R S Arvind Babu
Oral and Maxillofacial Pathologist, Lecturer in Dentistry, Faculty of Medical Sciences - Undergraduate Dental Programme, The University of the West Indies, Mona Campus, Kingston
Source of Support: None, Conflict of Interest: None
The use of fluorescence probes the field of cell and molecular biology. Immunofluorescence is a molecular method for detection of antigen or antibody in a tissue section/serum sample. Although histopathology remains gold standard for most of auto immune, immune mediated and vesiculo - bullous diseases. Immunofluorescence is an ancillary investigation, which are used to support clinical diagnosis more evidently. This method provides additional diagnostic, prognostic information and remains more specific diagnostic aid for vesiculo - bullous diseases of skin. This review article discusses about immunofluorescence techniques and its application in dermatopathology with oral manifestations.
Keywords: Autoimmunity, dermatological diseases, fluorescent antibody technique, immunofluorescence, vesiculo - bullous diseases
|How to cite this article:|
Arvind Babu R S, Chandrasekar P, Chandra K L, Reddy G S, Kumar K K, Ramana Reddy B V. Immunofluorescence and its application in dermatopathology with oral manifestations: Revisited. J Orofac Sci 2013;5:2-8
|How to cite this URL:|
Arvind Babu R S, Chandrasekar P, Chandra K L, Reddy G S, Kumar K K, Ramana Reddy B V. Immunofluorescence and its application in dermatopathology with oral manifestations: Revisited. J Orofac Sci [serial online] 2013 [cited 2017 Mar 25];5:2-8. Available from: http://www.jofs.in/text.asp?2013/5/1/2/113680
| Introduction|| |
Immunofluorescence has been used for over 70 years to localize antigenically distinct molecules in tissue section in microscopy. This is achieved by the use of specific antibody which, when prepared appropriately, is used to detect subtle differences at the molecular level. The credit of work by Coons, Creech and Jones in 1941, immunofluorescent techniques have been extensively developed and applied widely in recent days.  The word "Immunofluorescence" means "A method of determining the location of antigen or antibody in a tissue section or smear by the pattern of fluorescence resulting when the specimen is exposed to the specific antibody or antigen labeled with a fluorochrome".  The combination of antibody with its specific antigen does not lead to visible change and therefore a readily identifiable label must be irreversibly bound to the antibody so that its localization can be recognized. In immunofluorescence this label takes the form of "Fluorochrome", for example fluorescein or rhodamine, which has the property of absorbing radiation in the form of ultraviolet or visible light. This absorbed radiation causes the molecule to attain an excited state leading to electron redistribution and the emission of radiation of a different wave length. Thus, the emitted light is longer wave length and within the visible spectrum. 
This leads to important practical considerations in selecting suitable light sources and filters to obtain optimum results. The method introduced by Coons is termed as the Direct Technique, where the antibody was conjugated directly with the flurochrome (B anthracence) and used to detect the antigen in the slide preparation when examined under ultraviolet light microscopy.  Later in 1954, Weller andcoons introduced the sandwich or indirect technique was a major advance to the field of immunofluorescent technique, here it involves the interaction between the antigen and serum containing antibody. 
This review article discusses about the various procedures and techniques employed in immunofluorescent and its diagnostic application in dermatopathology with oral manifestations.
| Direct Immunofluorescent Technique|| |
Use of Direct immunofluorescent technique (DIF) for diagnosis in Oral and Maxillofacial Pathology began about 30 years ago. In this technique tissue is related directly with a fluorescein conjugated antibody. The principal application of DIF is in the identification of antibodies and other inflammatory proteins in tissue sections. DIF can be valuable tool for the diagnosis of groups of diseases, that are often difficult to separate clinically and those cases that are histologically similar like pemphigus, pemphigoid, lupus erythematosus, lichen planus, erythema multiforme, linear IgA disease, epidermolysis bullosa acquista.  The presence of characteristic fluorescent patterns produced by several DIF reagents can definitely establish the diagnosis of various auto immune diseases. The absence of these fluorescent patterns rules out these conditions, thereby strengthening the diagnosis of other oral mucosal diseases. 
Tissue preparation for DIF
Tissue sampled for testing should not be ulcerated because the presence of epithelium and an intact basement membrane zone is essential for definitive DIF examination. When considering a vesiculobullous disorder, where predominantly erythematous and ulcerated mucosal lesions occur, a biopsy specimen should be obtained a few millimeters away from an ulcer so that the specimen's epithelium and connective tissue will be intact. A biopsy specimen of lesions with a hyperkeratotic component (Lichen Planus, Lupus Erythematosus.) should include lesional tissue along with its adjacent erythematous mucosa. Biopsy specimen to be examined by DIF can be immediately frozen or placed in a transport media (Michel's Medium) under room temperature. Specimens must not be placed in conventional fixative such as formalin because this will produce cross - linking of tissue proteins including antigens, reducing their detectability by DIF. To freeze a specimen, it should first be placed with epithelial side down on a small piece of aluminum foil which is then enclosed in a suitable cryovial, immersed in liquid nitrogen and stored at 70°C until crysectioning. Studies have shown that the Michel's medium is able to effectively maintain tissue bound immunoglobulins and other inflammatory proteins for 1-2 weeks before DIF examination. Because there is no tissue fixation produced by Michel's medium, progressive cytolysis of epithelial cells begins after few days. However human skin basement membrane zone components, Tonofilaments and basal epithelial cells are well preserved in this medium even upto 4 weeks.
Fixative solutions for immunofluorescent techniques
It contains a very high concentration of ammonium sulphate, which precipitates antibodies and other inflammatory proteins within the tissue. It also contains an enzyme inhibitor in citrate buffer - magnesium sulphate.  Ideally fixation time for immunofluorescence by Michel's medium is 24-48 hrs. This is followed by washing of tissue in buffer, 3 times over 10 minutes and freeze at -70°C. The composition of Michel's medium is given in [Table 1].
Normal saline solution (0.9% Nacl) without addition of calcium or magnesium can be used. Tissue specimens can be kept in 5 ml saline solution for 24 h or 48 h at room temperature. 
Steps in direct immunofluorescence
- The procedures are performed on thin (<6 μm) cryostat sections of fresh unfixed material.
- Sections are washed with 0.1 M phosphate buffered saline with three changes over a period of 30 min.
- Drain off excess PBS and wipe around section with cellulose tissue. Cover section with diluted conjugated and allow to react for at least 30 min at room temperature.
- Drain off conjugate and wash with 3 changes of PBS over a period of 30 min. Although ideally the PBS should be gently agitated, this is not essential and too much agitation can easily remove sections from the slides.
- Excess PBS is drained off and the area around the section carefully dried with cellulose tissue.
The sections are mounted using a PBS/Glycerol/1,4 - Diazabicyclooctane DABCO solution. It is important not to maneuver the cover slip since this will distort unfixed tissue.
- The edges of the cover slip are then sealed using nail varnish. A more permanent sealant using polyvinyl alcohol has also been used with success.
- Read the preparations on a fluorescent microscope as soon as possible or store at 4°C. Avoid direct sunlight on the slides at all times. Provided bacterial/fungal contamination is avoided and drying out is prevented, it is possible to re-evaluate slides after more than 1 year's storage at 4°C, even where weakly fluorescent reactions are involved.
Indirect immunofluorescence (IIF) is a test in which a patient's serum is examined for the presence of antibodies to a defined antigen. It is common to use a multiblock technique, in which a single serum sample can be tested simultaneously against a number of tissue antigens, this approach gives extra relevant information.  The selection of suitable tissues to build up the multiblock is often dictated by local interest and expertise, a suitable selection is rat liver, stomach, kidney and human or monkey thyroid. Sections should be about 5 μm in thickness. This allows the detection of one or more of the following antibody reactions in a single dilution of patient serum. 
This is the 2 stage procedure for in vitro demonstration of circulating antibodies in the patient's serum. The serum is added to substrate (the frozen section of tissue that is similar to human oral mucosa as monkey oesophagus) and incubated resulting in fixation of circulating antibodies to the antigen in the substrate. The next step consists of the application of fluorescein labeled antihuman gammaglobulin to the substrate. 
Steps in indirect immunofluorescence
- Prepare patient serum dilution in PBS. In most routine tests a 1:10 dilution is used, conveniently 50 μl + 450 μl PBS.
- Expose sections to serum dilution for atleast 30 min, at room temperature. This is adequate for most reactions, the use of a longer time will not adversely affect results, although drying - out must be prevented. Some workers prefer a longer incubation step at a lower temperature e.g., 4°C overnight, when weak reactions are expected, e.g., Islet cell antibodies.
- Wash over 30 min, in PBS with 3 changes.
- Remove excess PBS with cellulose tissue.
- Cover sections with appropriately diluted antihuman Ig/FITC conjugate. Routinely a broad spectrum polyvalent conjugate which has been shown to detect all the major classes of immunoglobulins is used. After 30 min drain off excess conjugated and wash over 30 min with three changes of PBS.
- Mount cover slip as outlined under direct technique.
Procedure for formaldehyde fixed tissue and frozen sections
Conventionally frozen sections are used in immunofluorescent technique. Whereas, formalin fixatives are not employed due to fixation of proteins. However, enzyme digest is done to promote proteolytic treatment and there by exposing the proteins.  Steps in conventional frozen section and formalin fixed paraffin embedded tissue discussed in [Table 2].
|Table 2: Steps in frozen sections and formalin-fixed paraffin embedded tissue for immunofluorescence|
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Variants in immunofluorescence techniques
The smear or section is first treated with unlabeled antigen which will attach itself to antibody. After an appropriate time this is washed off and replace by a fluorescent tagged antibody. The tagged antibody will react (or bind) with the antigen which has attached itself in the first stage to the antibody in the original specimen, thus making a sandwich. The presence and location of positive fluorescence now indicates antibody sites in the original specimen. Sandwich staining is more sensitive than simple staining. 
Multiple layer technique
This is an extension of the sandwich technique. This technique for antigen (or antibody) can be made more sensitive by successive layering of antibodies; for example, if mouse antibody (globulin) is to be detected, the specimen is treated with unlabeled rabbit - anti-mouse gamma globulin. This will combine with any mouse globulin that may be present. Uncombined antisera are washed off. Then tissue is treated with unlabeled goat - anti - rabbit sera which will combine with the rabbit globulin used in step 1. 
Salt split technique: (Variant of IIF)
This technique is used to distinguish between sub-epidermal blistering conditions with similar direct immunofluorescence findings. In this procedure, normal human skin is incubated in 1M NaCl (sodium chloride) for 48-72 h to split it at the level of the lamina lucida. Bullous pemphigoid antibodies bind to the roof and floor of the blister while epidermolysis bullosa aquisita antibodies bind solely to the dermal (floor) side of the split skin. ,
Antigens and antibodies bind to one another to generate many molecules of complement 3 (C3). This amplification principle is used in complement IIF. It is a 3 step technique; normal tissue substrate is overlaid with plasma, serum/other tissue fluid which has been heated to 56°C for 30 min to destroy the complement-fixing activity of antigen-antibody complexes. Specimens are then washed. Tissue sections are incubated with a source of complement such as fresh human serum. Complement-fixing IgG/IgM antibodies that have bound to the antigen in the first step can now activate complement, giving rise to numerous C3 molecules that are bound at the antigen-antibody binding site in the tissue. Specimens are then washed again. Sections are then incubated with fluorescein-labeled antihuman C3 antibodies. These bind to C3 generated in the second step. After washing the C3-antibody conjugates can be examined under a fluorescence microscope. ,
Applications of immunofluorescence
Immunofluorescent technique possess practical considerations in wide variety of pathology [Table 3]. This ancillary investigatory study is used to support diagnosis and to provide additional diagnostic and prognostic information in autoimmune diseases, vesiculobullous diseases, transplantation and glomerular disease. 
The diagnosis of Pemphigus vulgaris should be confirmed by direct immunofluorescence examination of fresh perilesional tissue or tissue submitted in Michel's solution. With this procedure, antibodies (usually IgG or IgM) and Complements (usually C3) can be demonstrated in the intercellular spaces between the epithelial cells, in almost all patients with this disease.  Essentially, all patients with active pemphigus vulgaris have a positive direct immunofluorescence test for IgG on the cell surface of epithelial cells in perilesional mucosa, including even those who are in the very early stage of the disease and show only a few lesions.  Indirect immunofluorescence is also typically positive in 80-90% of cases, demonstrating the presence of circulating autoantibodies in the patient's serum. It is critical that perilesional tissue be obtained for both light microscopy and direct immunofluorescence to maximize the probability of a diagnostic sample. If ulcerated mucosa is submitted for testing, the results are often inconclusive because of either a lack of an intact interface between the epithelium and connective tissue or a great deal of non-specific inflammation. William's in, 1999, stated that direct immunofluorescence performed on perilesional tissue reveals a uniform "fish net" pattern. IgG deposition was seen in majority of cases. IgM and IgA occur in approximately 50% of cases. IgG4 subclass may also be of pathogenic significance. Indirect immunofluorescence also shows a similar appearance.  Direct immunofluorescence study of pemphigus revealed deposition of IgG in 86% and C3 positivity in 14% of cases within the intercellular spaces of epithelium resulting in a reticular pattern. 
Cicatricial Pemphigoid is auto-antibody induced and complement-mediated sequestration of leukocytes with resultant cytokine, thus causing detachment of basal cell from the basement membrane zone (BMZ). It is characterized by the presence of IgG subclass, particularly IgG4. The two major antigens associated with Cicatricial Pemphigoid are Bullous Pemphigoid Antigen 2 (BPAG 2) and Epiligrin (Laminin - 5). Association of HLA DQB1 * 0301 is noted with the cases of ocular Pemphigoid.  Direct immunofluorescence studies of perilesional mucosa show a continuous linear band of immunoreactants at the basement membrane zone. The immune deposits consist primarily of IgG and C3, although IgA and IgM may also be identified.  One study has suggested that, when IgG and IgA deposits are found in the same patient, the disease may be more severe.  Immunoreactants such as IgG, IgG4 and IgA may play a role in the pathogenesis of the sub-epithelial vesicle formation by weakening the attachment of the basement membrane through a variety of mechanism, including complement activation with recruitment of inflammatory cells, particularly neutrophills.  Indirect immunofluorescence is positive in only 5% of these patients, indicating a relatively consistent lack of circulating autoantibodies. 
Bullous Pemphigoid autoantibodies bind to basement membrane zone of stratified squamous epithelium in a linear pattern. This linear pattern can be demonstrated by direct and indirect immunofluorescence. The other patterns are tubular, cytoplasmic and membranous. The pattern of basement membrane zone staining depends on its ultra-structural morphology in each tissue.  Bullous Pemphigoid is characterized by the presence of Immunoglobulin G (IgG); autoantibodies specific for the hemidesmosomal Bullous Pemphigoid antigens BP 230 (BPAg1) and BP 180 (BPAg2) directed against components of basement membrane.  Direct immunofluorescence studies show a continuous linear band of immunoreactants, usually IgG and C3, localized to the basement membrane zone in 90% to 100% of affected patients. These antibodies may bind to proteins associated with hemisdesmosomes, structures that bind the basal cell layer of the epithelium to the basement membrane and the underlying connective tissue.  These proteins have been designated as bullous pemphigoid antigens (BP 180 and BP 230) and immunoelectron microscopy has demonstrated the localization of BP 180 to the upper portion of the lamina lucida of the basement membrane. Indirect immunofluorescence studies show the tissue bound autoantibodies, 40%-70% of patients also have circulating autoantibodies in the serum. Indirect immunofluorescence pattern that is identical to that of the direct immunofluorescence. Unlike Pemphigus vulgaris, the antibody titers seen in bullous pemphigoid do not appear to correlate with disease activity. Serum levels of autoantibodies to Bullous Pemphigoid 180 (BP 180) correlates with disease activity. Assaying reactivity to Bullous Pemphigoid 180 (BP 180) should be helpful guide for the therapeutic management of patients with this disease, thus it can be considered as a prognostic marker.  Studies with direct immunofluorescence using perilesional mucosa showed a linear continuous band at the basement membrane zone usually with IgG and C3. ,
Direct immunofluorescent studies of lichen planus shown that nearly all specimens from oral lesions of this disease react with anti-fibrinogen and exhibit an intensely positive fluorescence that outlines the basement membrane zone with numerous irregular extensions into the superficial lamina propria. This particular pattern is characteristic of both lichen planus and lupus erythematosus. This is not present in pemphigoid or erythema multiforme.  It can be concluded that the pattern of fibrinogen deposition, in the absence of fluorescence by other reagents, is sufficiently unique to be used as a diagnostic criterion for oral mucosal lichen planus. Richard et al., in 2002, in his review on advanced diagnostic method stated that lichen planusshows a characteristic pattern of fibrinogen deposition along the basement zone and extending irregularly in to the superficial lamina propria described as shaggy or fibrillar pattern.  A study on direct immunofluorescence of Lichen Planus demonstrates a ragged band of fibrinogen in the basement membrane in 100% of cases.  Lever et al., in 2005, mentioned that direct immunofluorescence of oral lichen planus react with anti-fibrinogen at basement membrane zone with numerous irregular extensions into the superficial lamina propria. This particular pattern is characteristic for both systemic lupus erythematosus and lichen planus.  One study stated that direct immunofluorescence revealed deposition of fibrinogen in 100% of cases, with the characteristic pattern of fibrinogen outlining the basement membrane zone and extending irregularly into the superficial lamina propria. 
Systemic lupus erythematosus
Hahn BH in 1998, in his review stated that cause of Systemic Lupus Erythematosus remains uncertain, but the existence of limitless number of antibodies in these patients against self - constituents indicates that the fundamental defect is a failure of the mechanism that maintain self-tolerance. Antibodies have been identified against an array of nuclear, cytoplasmic and cell surface antigens of blood cells. Antinuclear antibodies (ANA) are directed against several nuclear antigens and can be grouped into 4 categories: Antibodies to DNA, Antibodies to histones, Antibodies to non-histone proteins bound to RNA and antibodies to nucleolar antigens.  Direct immunofluorescence testing of lesional tissue shows deposition of 1 or more immunoreactants (usually IgM, IgG, or C3.) in a shaggy or granular band at the basement membrane zone. In addition, DIF testing of clinically normal skin of SLE patients often shows a similar deposition of IgG, IgM or Complement components. This finding is known as a positive lupus band test.  The appearance of the immunoglobulins deposited in this located in discoid lupus generally is the "particulate" (or 'speckled') pattern.  A study stated that Direct immunofluorescence in Discoid Lupus Erythematosus revealed granular deposits of IgG, C3 and faint deposition of IgM along the Basement Membrane Zone in 25% of cases. She suggested that the Direct immunofluorescence can be used as an additional diagnostic tool. 
Stevens Johnson syndrome
Immunofluorescence studies on Stevens Johnson syndrome cases showed negative results. It can be concluded that immunofluorescent technique can be employed in these cases to rule out other vesiculo bullous lesions of skin. 
Chronic ulcerative stomatitis
This immune mediated disorder affects oral mucosa. These patients develop autoantibodies against a 70KD nuclear protein that is similar to p63 and may play a role in epithelial growth and differentiation. Direct immunofluorescence studies, suggested autoantibodies (usually IgG) that are directed against the nuclei of stratified squamous epithelial cells in basal and parabasal regions of the epithelium are detected. Indirect immunofluorescence studies are also positive for these stratified epithelium - specific antinuclear antibodies (ANA). ,
| Conclusion|| |
Immunofluorescence is a helpful diagnostic aid for autoimmune - vesiculo bullous disease of skin. It is advanced laboratory method that detects immune deposits. Although histopathology is considered as gold standard in diagnostic protocol, but may be inconclusive. This molecular technique helps in exact diagnosis and also prognostic tool, since circulating antibodies can be correlated with disease activity in few lesions.
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[Table 1], [Table 2], [Table 3]