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 Table of Contents  
Year : 2017  |  Volume : 7  |  Issue : 1  |  Page : 25-28

Immunofluorescence as a diagnostic tool

Department of Oral Pathology and Microbiology, Thaimoogambigai Dental College and Hospital, Chennai, Tamil Nadu, India

Date of Web Publication30-Jun-2017

Correspondence Address:
Aarthi Ramachandran
1857, 17th Street, I-Block, Thiruvalluvar Kudiyirupu, Anna Nagar, Chennai - 600 040, Tamil Nadu
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmd.ijmd_61_16

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Immunofluorescence (IF) is a molecular method for detection of antigen or antibody in a tissue section/serum sample. It is increasingly being used as an ancillary investigation that can add to the certainty of diagnosis, sometimes modify it and occasionally reveal a differential diagnosis. IF has also proved to be useful in assessing therapeutic response and predicting relapse. This review article discusses about the various techniques of IF and its diagnostic implication in mucocutaneous diseases.

Keywords: Fluorescent antibody technique; immunobullous; immunofluorescence

How to cite this article:
Ramachandran A, Radhika T, Jeddy N, Ananthalakshmi R. Immunofluorescence as a diagnostic tool. Indian J Multidiscip Dent 2017;7:25-8

How to cite this URL:
Ramachandran A, Radhika T, Jeddy N, Ananthalakshmi R. Immunofluorescence as a diagnostic tool. Indian J Multidiscip Dent [serial online] 2017 [cited 2022 Aug 12];7:25-8. Available from: https://www.ijmdent.com/text.asp?2017/7/1/25/209276

  Introduction Top

Immunofluorescence (IF) is a reliable histochemical staining technique used to demonstrate antibodies bound to antigens in tissues and circulating body fluids. It has gained a pivotal role as a diagnostic tool due to the pioneering works of Beutner and Jordon who introduced IF to dermatopathology in 1964, subsequent to its discovery by Coons et al. in 1941. IF is a valuable diagnostic tool used to differentiate lesions that are clinically and histologically similar like pemphigus, pemphigoid, lichen planus, lupus erythematosus, linear immunoglobulin A (IgA) disease, epidermolysis bullosa acquista.[1]

In IF, the antibody is chemically conjugated to a fluorochrome, these labelled antibodies bind to the specific antigen and allows detection through fluorescence technique. The diagnosis of various auto immune diseases can be established based on the presence of characteristic fluorescent patterns produced by several direct IF (DIF) reagents and the absence of which rules out these lesions, thereby strengthening the diagnosis of other oral mucosal diseases.[2] Fluorochrome is a dye that absorbs radiation, causing the molecule to attain an excited state which leads to electron redistribution and emission of radiation of a different wavelength. The most widely used fluorochrome is fluorescein isothiocyanate (FITC). In 1957, rhodamine was introduced as an alternative fluorescent label.[3]

There are three methods of labelling:[4]

  • DIF – The antibody specific to the antigen of interest is chemically conjugated to the fluorochrome
  • Indirect IF (IIF) – The primary antibody to the antigen is unlabelled, while a fluorochrome tagged second anti-Ig antibody (secondary antibody) is directed to the primary antibody
  • Complement binding IIF – The antigen-antibody complexes generate many molecules of complement 3, they are highlighted using labelled C3 antibodies. This is much more sensitive than indirect method.

  Direct Immunofluorescence Top

This single step method was introduced by Coons et al. The biopsy tissue should include an intact epithelium and basement membrane. Most vesiculobullous disorders present with erythema and ulceration, hence biopsy should be obtained from perilesional tissue. In collagenosis, biopsy should be from the site of active lesion (avoid recent lesions). In vasculitis, biopsy should be taken from site of recent lesion with up to 24 h of evolution.[3] The tissue should be immediately frozen or placed in transport medium-Michel's medium (ammonium sulphate, magnesium sulphate in a citrate buffer) for 24–48 h. Michel's medium maintains the Igs and few inflammatory proteins bound to tissue for 1–2 weeks.[1] Sections of 4–6 μ thickness are mounted and overlaid in a moist chamber with FITC conjugates with antilgG, antilgM, antilgA, antiC3 and antifibrin and each reagent is tested separately.[4] Positive and negative controls constitute an integral part of serologic method. The sections are then examined under a fluorescence microscope. Two main patterns of immunoreactants deposition is seen: as diffuse or focal deposition in the epidermal intercellular space and along the basement membrane zone as smooth-linear or granular-discontinuous deposits or a combination of both.[5]


The main advantage of this technique is a simple and short procedure time. This technique is very sensitive.


Expensive, less flexible and has lower signal. Labelling difficulties are encountered when commercial labelled conjugates are unavailable.[6]

  Indirect Immunofluorescence Top

This technique allows the evaluation of circulating autoantibodies as well as in autoimmune vesiculobullous dermatosis. Beutner and Jordon introduced this two stage procedure. IIF is ten times more sensitive than DIF, but is less applicable in the diagnosis of oral manifestation of mucocutaneous diseases.[5] The serum is incubated with the substrate (frozen tissue similar to human oral mucosa), this fixes theantibodies that are circulating to the substrate antigen. The following step is similar to DIF method-application of fluorescein labelled antihuman gammaglobulin to the substrate. Known positive-negative controls should be included in every step. The reading is done using epiluminescence microscopy.[3]


greater sensitivity than DIF and it has prognostic implications. Since more than one secondary antibody can adhere to the primary antibody there is amplification of signal. Secondary antibodies are commercially available in an array of colours and are relatively inexpensive.


difficulty in finding primary antibodies that are not obtained from the same species. There is also a potential for cross reactivity. Samples with endogenous Igs may exhibit a high background.[6],[7]

  Complement Binding Indirect Immunofluorescence Top

In this technique, the serum complement is inactivated and the serum antibody is fixed to the antigen in the substrate by incubation. The complement fixing primary antibody and antigen complex triggers the complement cascade. The section is then stained with FITC conjugate antihuman C3 antibody, and examined under fluorescence microscope. This technique is much more sensitive in comparison to routine IIF.[5]

  Variants of Indirect Immunofluorescence Top

Salt split technique

This technique is used to differentiate subepidermal blistering conditionsusing DIF findings. The biopsied tissue is split at the region of lamina lucida, either by suction blister creation or incubation in sodium chloride, trypsin and phosphour buffered saline. Antibodies bind to the floor and roof of the blister in bullous pemphigoid, while antibodies bind only to the floor in epidermolysis bullosa.[8]

Antigenic mapping method

It is a modified IIF technique that uses the patient's skin as the substrate. This method is performed using primary antibodies (polyclonal/monoclonal) directed against different antigens. The location of the antigens (roof/floor) in the mechanically induced blister determines the plane of cleavage.[9]

Double staining method

This method is used to demonstrate the co-distribution of two antigenic substances.

Sandwich technique

The section/smear is treated with unlabelled antigen which attaches itself to the antibody, the antigen is then subjected to fluorescent tagged antibody. The location of antibody is identified by the positive fluorescence seen in the original specimen. Sandwich technique is more sensitive than regular staining.[1]

  Application of Immunofluoresence Top

IF aids in diagnosis and provides additional prognostic information. Some applications of IF in diagnostic pathology are:[6]

  • Antigen analysis in frozen, fixed or fresh tissues; sub-cellular antigen localization in tissue culture; observation of parasitic or bacterial specimens
  • Detection and localization of specific chromosomal DNA sequences; and
  • Defining the spatial-temporal patterns of gene expression within cells/tissues.

The pathophysiology of blistering diseases show that autoantibodies target the cell to cell and cell to matrix adhesion molecules.

  Pemphigus Top

Positive IF is seen in 100% cases with active lesion with DIF, while only 80% positivity is seen in IIF. The diagnosis of pemphigus vulgaris is confirmed by DIF examination of tissue. IgG autoantibodies target desmoglein 3 (Dsg3), patients can also present antibodies against Dsg1, indicating worse prognosis of the disease. Active lesions show IgG4, in contrast to patients who are in remission, where IgG1 prevails. The pattern of fluorescence in pemphigus vulgaris is the deposition of IgG and C3 in the intercellular space of epidermal cells.[4] Williams in 1989 stated that DIF performed on perilesional tissue reveals a uniform “fishnet” or “chicken wire” pattern of binding.[10] The fluorescence is predominant in suprabasal area in pemphigus vulgaris and in the upper epidermal area in pemphigus foliaceus where the autoantibodies target Dsg1. In pemphigus erythematosus, a granular pattern is seen along the basement membrane zone in addition to network pattern in the intercellular space. This pattern is mimicked in paraneoplastic pemphigus, the pattern of fluorescence along the basement membrane zone is similar to that seen in bullous pemphigoid. It is difficult to distinguish between cases of paraneoplastic pemphigus and pemphigus erythematosus, especially when clinical or histologic information is not available. Frequently the intercellular space deposition in paraneoplastic pemphigus is diffuse or weak and nonspecific.[7],[11]

  Pemphigoid Top

Bullous pemphigoid autoantibodies bind to the basement membrane zone in stratified squamous epithelium in a linear pattern and can be demonstrated by direct and IIF. Deposition of C3 along the basement membrane is seen in 100% cases and IgG in 65%–95% in DIF.[12] The different patterns of deposition has been described as wavy, linear, granular and tubular. These variations in pattern result from variations in the angle at which cryosections are made, the site of biopsy and intensity of deposition.[7] Cicatricial pemphigoid is autoantibody induced and complement–mediated sequestration of leukocytes with resultant cytokine, thus causing detachment of basal cell from the basement membrane zone. The linear immune deposits along the basement membrane consist primarily of IgG and C3, although IgA and IgM may also be identified.[13] IIF is positive in only 5% of these patients, indicating a lack of circulating autoantibodies. Mucous membrane pemphigoid, also shows linear deposits along the basement membrane zone, it is rarely positive in IIF.[3] Weinberg et al. in 1999 reported that IIF studies are not reliable and may be negative or low in few cases.

  Lichen Planus Top

DIF can be used as an additional tool in the diagnosis of lichen planus. Immune deposits are evident within cytoid bodies in the superficial dermis, as well as along the basement membrane zone. Fibrinogen and IgM are the most frequently present immune deposits. Regezi and Sciubba in 1989 stated that DIF study demonstrated the presence of fibrinogen along the basement membrane zone in 90%–100% of cases. Richard et al. in 2002 and Lever et al. have mentioned that DIF of oral lichen planus interacts with anti-fibrinogen in the basement membrane zone with many irregular extensions into the superficial lamina propria. This pattern is characteristic for both lichen planus and systemic lupus erythematosus.[1],[14]

  Lupus Erythematosus Top

DIF may help in distinguishing among the various subsets of lupus erythematosus since the site of deposition, frequency of deposition and its morphology vary among the various subsets of lupus erythematosus. Antinuclear antibodies are directed against multiple nuclear antigens. IF patterns vary from homogeneous, fibrillar, linear to granulomatous, which can be focal or continuous. The combination of IgG and IgM along the basement membrane favours discoid lupus erythematosus. The presence of IgG, IgM, IgA and C3 along the basement membrane zone is suggestive of systemic lupus erythematosus.[3],[7]

  Limitations of Immunofluorescence Top

  • A significant problem with IF is phototoxicity or photobleaching, which is destruction of a flurophore due to the generation of reactive oxygen species. This can be overcome by – Decreasing the intensity and duration of excitation light, by adding singlet oxygen scavengerand by using low concentration of flurochrome [6]
  • Autofluorescence in mammalian cells due to flavin coenzyme and due to fixation with aldehydes, particularly glutaraldehyde, can be problematic in detection of IF [6]
  • The possibility that the emission signals overlap when measuring fluorescence of more than one color must be dealt with. It is necessary to electronically remove the overlapping signal by fluorescence compensation method.[6]

  Conclusion Top

IF is the visualization of antigens within cells using antibodies as fluorescent probes. It has proved to be a powerful tool in determining the cellular distribution of known antigens. Although histopathology still remains as the gold standard for diagnosis, IF proves to be a valuable adjunct, especially since circulating antibodies can be related to disease activity in few lesions. It is also useful in assessing the prognosis for certain conditions. The method has successfully combined high resolution and sensitivity in the visualization of antigens and will be a major diagnostic tool for many years to come.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Arvind Babu RS, Chandrasekar P, Chandra LP, Reddy S, Kumar K, Reddy R. Immunofluorescence and its application in dermatopathology with oral manifestations: Revisited. J Orofac Sci 2013;5:2-7.  Back to cited text no. 1
Culling CF. Handbook of Histological and Histochemical Techniques. 3rd ed. London: Butterworth and Co. Publishers Ltd.; 1974. p. 573-84.  Back to cited text no. 2
Aoki V, Sousa JX Jr., Fukumori LM, Périgo AM, Freitas EL, Oliveira ZN. Direct and indirect immunofluorescence. An Bras Dermatol 2010;85:490-500.  Back to cited text no. 3
Mohan KH, Pai S, Rao R, Sripathi H, Prabhu S. Techniques of immunofluorescence and their significance. Indian J Dermatol Venereol Leprol 2008;74:415-9.  Back to cited text no. 4
[PUBMED]  [Full text]  
Premalatha BR, Roopa SR, Vijaya M. Immunofluorescence in oral pathology: Part I-methodology. World J Dent 2011;2:326-31.  Back to cited text no. 5
Robinson JP, Jennifer Sturgis BS, Kumar GL. Immunofluorescence. Textbook of IHC Staining Methods. Vol. 5, Ch. 10. Science and Education Academic Publisher; 2009. p. 61-5.  Back to cited text no. 6
Priyanka S, Avinash K, Raghvendra B, Arati P. Immunofluorescence in oral mucosal diseases – A review. Oral Surg Oral Med Oral Radiol 2014;2:6-10.  Back to cited text no. 7
Satyapal S, Amladi S, Jerajani HR. Evaluation of salt split technique of immunofluorescence in bullous pemphigoid. Indian J Dermatol Venereol Leprol 2002;68:330-3.  Back to cited text no. 8
[PUBMED]  [Full text]  
Rao R, Mellerio J, Bhogal BS, Groves R. Immunofluorescence antigen mapping for hereditary epidermolysis bullosa. Indian J Dermatol Venereol Leprol 2012;78:692-7.  Back to cited text no. 9
[PUBMED]  [Full text]  
Anuradha CH, Malathi N, Anandan S, Magesh K. Current concepts of immunofluorescence in oral mucocutaneous diseases. J Oral Maxillofac Pathol 2011;15:261-6.  Back to cited text no. 10
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Rani Z, Hussain I. Immunofluorescence in immunobullous diseases. J Pak Assoc Dermatol 2003;13:76-88.  Back to cited text no. 11
Roopa SR, Premalatha BR, Vijaya M. Immunofluorescence in oral pathology: Part II-pathology and immunofluorescent patterns in subepidermal immunobullous disorders. World J Dent 2012;3:68-73.  Back to cited text no. 12
Bancroft JD, Gamble M, Carlton SJ. Theory and Practice of Histological Techniques. 5th ed. China: Elsevier; 2002. p. 579-80.  Back to cited text no. 13
Regezi J, Sciubba J. Oral Pathology: Clinical Pathologic Correlations. 2nd ed. Missouri: W.B. Saunders Company; 1989. p. 105-10.  Back to cited text no. 14


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Direct Immunoflu...
Indirect Immunof...
Complement Bindi...
Variants of Indi...
Application of I...
Lichen Planus
Lupus Erythematosus
Limitations of I...

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