Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Filter by Categories
Case Report
Guest Editorial
Images in Clinical Practice
Invited Commentary
JSSTD Symposium
Letter to Editor
Letter to the Editor
Letter to the Editor - Study Letter
Media and News
Net Case
Net Consensus Statement
Net Educational Video for Residents
Net Image
Net Letter
Net Quiz in Dermatology
Original Article
Quiz in Dermatology
Resident’s Page
Review Article
Study Letter
Study Letter Case Series
Generic selectors
Exact matches only
Search in title
Search in content
Post Type Selectors
Filter by Categories
Case Report
Guest Editorial
Images in Clinical Practice
Invited Commentary
JSSTD Symposium
Letter to Editor
Letter to the Editor
Letter to the Editor - Study Letter
Media and News
Net Case
Net Consensus Statement
Net Educational Video for Residents
Net Image
Net Letter
Net Quiz in Dermatology
Original Article
Quiz in Dermatology
Resident’s Page
Review Article
Study Letter
Study Letter Case Series
View/Download PDF

Translate this page into:

Review Article
5 (
); 66-74

Immunofluorescence in dermatology: A brief review

Department of Pathology, Government Medical College, Manjeri, Malappuram, Kerala, India
Corresponding author: Swapna Balakrishnan, Department of Pathology, Government Medical College, Manjeri, Malappuram, Kerala, India.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-Share Alike 4.0 License, which allows others to remix, transform, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

How to cite this article: Balakrishnan S, Kalappurayil NB. Immunofluorescence in dermatology: A brief review. J Skin Sex Transm Dis 2023;5:66-74.


Immunofluorescence (IF) has been in use for the past five decades, both to investigate the pathophysiology of skin disorders and to help the dermatologists in the diagnosis of various bullous and connective tissue diseases. This review article, deals with different methods, applications, and recent advances in the IF methods used in dermatopathology. Here, we also discuss about the practical aspects of this technique such as handling of skin biopsy specimens and interpretation of direct and indirect IF findings.


Direct immunofluorescence
Indirect immunofluorescence
Bullous disease
Connective tissue disease


Immunofluorescence (IF) is a simple, reliable and reproducible technique in immunopathology. It detects the in situ and circulating immune deposits involved in the pathogenesis of various diseases, especially those affecting the renal system and skin. The beginning of direct IF (DIF) dates back to 1942, when Coons et al. showed the labeling of anti-pneumococcal antibodies with fluorescein in the pulmonary tissue.[1] IF was introduced in dermatology in the 1960s, when Beutner and Jordon revealed the tissue and circulating antibodies in pemphigus vulgaris (PV), pemphigus foliaceus (PF), and bullous pemphigoid (BP).[2-4] IF studies have now become an invaluable supplement to clinical and histopathological examination in a variety of dermatological diseases and are widely used both in research and clinical diagnostics.

In IF, the antibodies that are chemically conjugated with fluorescent dyes such as fluorescein isothiocyanate (FITC) or tetramethylrhodamine isothiocyanate bind directly or indirectly to the antigen of interest in cells or tissue sections. Then, it is visualized by fluorescence or confocal microscopy and quantified by a flow cytometer, an array scanner or an automated imaging instrument. In routine practice, the interpretation of IF study on tissue sections and serum samples is done by fluorescence microscopy.[5,6] There are mainly two types of IF analysis –DIF and indirect IF (IIF).


The most commonly used specimens for IF studies are: [5-7]

  1. Skin biopsy (1.3–6 mm punch biopsies of skin for DIF and 3 mm shave biopsy for IF antigen mapping, which is a type of IIF).

  2. Outer root sheath of anagen hair: When patient is not willing for a biopsy (useful in PV).

  3. Intact buccal mucosa: PV with isolated mucosal lesions (DIF).

  4. Serum: 3 ml of blood without anticoagulant (for IIF)

  5. Smears: Sputum, bronchoalveolar lavage, and tissue smears like Tzanck smear (DIF)


  1. To assist in the diagnosis and classification of various autoimmune blistering diseases (AIBDs) that may have a confusingly similar clinical profile.

  2. In evaluating the prognosis of AIBDs like PV: The intensity of DIF coincides fairly well with the clinical activity.[5]

  3. IIF can detect various circulating auto antibodies, which provides an indirect measure of the activity of the disease and helps to decide the most beneficial therapeutic option for the patient.

  4. IF antigen mapping (a type of IIF) marks for a particular protein, the presence or absence of which determines the disease classification in hereditary EB (epidermolysis bullosa).

  5. The clinical picture of some infections (infections due to herpes simplex virus, and Chlamydia) is often muddled and unclear in patients who are immunosuppressed. In such cases, DIF helps in confirming the diagnosis.[5]


  1. There is a necessity of fast reporting and documentation of findings using a digital camera as the fluorescence staining quenches rapidly on exposure to light, more so under the ultraviolet (UV) light of the fluorescence microscope.

  2. Three distinct forms of fluorescence (specific and nonspecific fluorescence and autofluorescence) should be considered while reading the IF assay especially with IIF.[5]

Specific fluorescence - Due to a reaction between the substrate and the protein labeled with fluorochrome (antigen-antibody reaction).[5]

Nonspecific fluorescence - Due to coloration of tissues by free fluorescent dye or fluorescent proteins or both.[5]

Autofluorescence - Due to the natural fluorescence of tissues (yellow, blue) when exposed to UV light.[5]


It is a single step procedure. DIF detects and localizes the immunoreactants deposited in vivo in the patient’s skin or mucosa. The primary antibody is labeled with a fluorescent dye. The results are analyzed with respect to the site, number, and the intensity of immune deposits. When multiple immune deposits are identified, the most intense deposit is noted. The sites of deposition (primary and other sites), are identified. Analysis of DIF staining of a biopsy specimen is called “immunohistologic study.”[5]


DIF is a useful diagnostic tool in many dermatoses [Table 1].[5,6]

Table 1: Significance of DIF microscopy in dermatology.
DIF is of diagnostic value
(A) Bullous diseases
Pemphigus (all forms)
Pemphigoid (all forms)
Herpes gestationis/gestational pemphigoid
Dermatitis herpetiformis
Linear IgA bullous dermatosis
Epidermolysis bullosa acquisita
(B) Connective tissue diseases
Discoid lupus erythematosus
Systemic lupus erythematosus
DIF is highly characteristic and of some diagnostic value
(A) Vascular diseases
Allergic vasculitis
Henoch–Schonlein purpura
Essential mixed cryoglobulinemia
Polyarteritis nodosa
(B) Other diseases
Porphyria cutanea tarda and other forms of porphyria
Lichen planus
DIF is not diagnostic but only suggestive of immunologic pathogenesis
(A) Connective tissue diseases
Mixed connective tissue diseases
Systemic sclerosis
(B) Inflammatory dermatosis
DIF negative bullous disorders
Subcorneal pustular dermatosis
Transient acantholytic dermatosis
Familial benign chronic pemphigus
Acropustulosis of infancy
Dermatopathies due to external energy (friction blistser, electric burns, etc.)

DIF: Direct immunofluorescence

It is important to collect DIF specimen from the appropriate site(s). The biopsy should be taken from a fresh blister/lesion, not older than 24–48 hours, for histopathology analysis in AIBDs. A biopsy of normal appearing uninvolved skin within 1 cm of a fresh blister is recommended for DIF in blistering conditions. In PV with isolated mucosal lesions, intact mucosal biopsy is preferred.[5]

Biopsy of a fresh lesion is recommended for DIF microscopy in connective tissue diseases and vasculitis. The appropriate sites of skin biopsy for DIF microscopy in various dermatological conditions are given in Table 2.[5-7]

Table 2: Ideal site of skin biopsy for DIF microscopy.
Diseases Types of specimen
Pemphigus (all forms)
Pemphigoid (all forms)
Epidermolysis bullosa acquisita (EBA)
IgA bullous dermatosis
Chronic bulllous disease of childhood
Peri-lesional skin within 1 cm of a bulla or intact mucosa (avoid erosions, ulcers, and large bullae)
Dermatitis herpetiformis Peri-lesional skin within 1 cm of a bulla or normal skin
Lupus erythematosus and other collagen vascular diseases Involved areas of skin (e.g., an erythematous or active border).
Involved, sun exposed skin, and uninvolved, non-sun exposed skin in systemic lupus erythematosus.
(Avoid ulcers and old lesions)
Vasculitis Erythematous or active border of a new lesion (<24 hours old), avoid ulcers and old lesions.
Porphyria cutanea tarda Involved skin (avoid ulcers and old lesions).

DIF: Direct immunofluorescence

DIF can also be performed on formalin-fixed paraffin-embedded tissue, which is called paraffin IF. This is done after antigen retrieval with proteases. This is less sensitive than DIF performed on a fresh tissue sample, with sensitivity varying from 0% to 79% in different diseases.[8,9]


Specimens for DIF should not be placed in formalin, as this alters the proteins and significantly diminishes the accuracy of results. The specimen should be transported to the laboratory as soon as possible. The biopsy specimens can be transported on a saline-soaked gauze, in saline solution or in special media (e.g., Michel’s or Zeus media). The specimens may also be transported frozen in liquid nitrogen (the specimen should not be allowed to thaw).[5]

Michel’s medium contains proteolytic enzyme inhibitors that prevent the autolysis of tissue structures and immunoreactants. The biopsies should be washed in distilled water or normal saline and placed in Michel’s medium to ensure the preservation of immunoreactants. Michel’s medium preserves immunoreactants for up to 6 months at ambient temperatures.[5,7,10]

Normal saline solution (0.9% NaCl) without addition of calcium or magnesium can also be used for transportation of biopsy specimens.[5,11] Tissue specimens can be kept in 5 ml saline solution for 24–48 hours at room temperature.[5,11] Each laboratory has its own protocols. From our experience, specimens in saline may yield superior sensitivity over other media if processed within 24–48 hours.


The prepared IF slides are examined by an experienced pathologist to identify the classes of immunoglobulin (Ig) or other immune deposits, the primary sites of immune deposition, and the patterns of deposition [Table 3]. It should be described under the following headings.[5-8]

Table 3: DIF findings in dermatoses.
Diseases Pattern and nature of immunoreactants
IgA Pemphigus
Epidermal cell surface deposits of IgG and C3
Epidermal cell surface deposits of IgA (upper epidermal layers in subcorneal pustular dermatoses type and throughout the epidermis or restricted to lower epidermis in intra-epidermal neutrophilic type)
Pemphigus erythematosus and paraneoplastic pemphigus Epidermal cell surface and DEJ, homogenous or non homogenous deposits of IgG and C3.
Bullous pemphigoid Linear homogenous deposits of IgG (epidermal side on salt –split skin ) and C3 at DEJ
Linear IgA dermatosis Linear homogenous deposits of IgA at DEJ
Pemphigoid gestationis Linear homogenous deposits of C3 at DEJ, rarely IgG
Epidermolysis bullosa acquisita Linear homogenous deposits of IgG (dermal side on salt – split skin) and C3 at DEJ
Mucous membrane pemphigoid Linear homogenous deposits of IgG and C3 at DEJ
Dermatitis herpetiformis Focal granular deposits of IgA at papillary dermis
Bullous eruption of lupus erythematosus Linear homogenous or non-homogeneous (lupus band), deposits of multiple immunoglobulins, C3 and fibrin at DEJ
Anti-p200 pemphigoid Linear deposition of IgG and C3 along the DEJ
Lichen planus Ragged or shaggy fibrinogen deposits at DEJ. Mostly IgM and rarely other deposits on colloid bodies in upper dermis
Lichen planus pemphigoides Linear IgG and C3 deposits along the BMZ
Porphyrias-(porphyria cutanea tarda, pseudo porphyria cutanea tarda, erythropoietic protoporphyria) Homogenous deposits of IgG, IgA and less frequently C3 at DEJ and within superficial blood vessels
Cutaneous small vessel vasculitis Most commonly C3 and fibrinogen deposits within the walls of post capillary venules in the superficial dermis
Henoch- Schonlein purpura Granular deposits of IgA on the vessel walls in the superficial dermis with or without other immune reactants
Psoriasis Bright continuous bands of granular positivity along the DEJ with IgG, IgM, C3, and fibrinogen
Mucocutaneous infections Cytoplasmic staining in HSV type 1-infected cells, nucleolar staining in HSV type 2-infected cells, elementary bodies in Chlamydia trachomatis infected samples and clusters of cysts in Pneumocystis jirovecii infected samples

DIF: Direct immunofluorescence, DEJ: dermoepidermal junction, CSVV: Cutaneous small vessel vasculitis, HSV: Herpes simplex virus, BMZ: Basement membrane zone

Type of immunoreactant

IgG, IgA, IgM, C3, fibrin, C5b-9.

Location of immune deposits

Staining patterns can be classed into five groups

  • Intercellular surface staining pattern [Figures 1 and 2].

  • Linear basement membrane zone (BMZ) pattern [Figure 3].

  • Granular BMZ pattern [Figures 4 and 5]: Granular papillary dermal staining of IgA is diagnostic of dermatitis herpetiformis as shown in Figure 4.

  • Shaggy BMZ pattern.

  • Vascular or other patterns [Figure 6].

Figure 1:
(a): A case of Pemphigus vulgaris, skin biopsy showing suprabasal bulla with basal layer of epithelial cells in “row of tomb stone” arrangement (H and E, 100×); (b): Direct immunofluorescence showing IgG reactive granular (+++) deposits at the intercellular spaces, in a fish net pattern in the epidermis (200×).
Figure 2:
A case of Pemphigus foliaceous (a): skin biopsy showing subcorneal bulla containing acantholytic cells (H and E, 100×); (b): subcorneal bulla containing acantholytic cells (H and E, 200×); (c): direct immunofluorescence showing IgG reactive granular (+++) deposits at the intercellular spaces (intense in the superficial part) in a fish net pattern in the epidermis (200×); (d): C3 reactive granular (++) deposits at the intercellular spaces (intense in the superficial part) in a fish net pattern in the epidermis (200×).
Figure 3:
(a): A case of bullous pemphigoid: Skin biopsy showing subepidermal bulla (H and E, 200×); (b): bulla with fibrinous material admixed with moderate amounts of neutrophils and occasional eosinophils (H and E, 400×); (c): direct immunofluorescence (DIF) showing IgG positive reactants - 3+ linear positivity along the basement membrane zone (400×); (d): DIF showing C3 positive reactants - 3+ linear positivity along the basement membrane zone (400×).
Figure 4:
(a): A case of dermatitis herpetiformis - skin biopsy showing sub-epidermal bulla with neutrophilic microabscess and superficial dermal infiltrate of neutrophils (H and E, 100×); (b): direct immunofluorescence showing IgA positive reactants - 3+ at the tip of dermal papilla (200×).
Figure 5:
(a): A case of discoid lupus erythematosus - epidermis showing hyperkeratosis, atrophy and vacuolization basal cells (H and E, 100×); (b): showing area with pigment incontinence in superficial dermis and lymphocytic infiltrate around the adnexa and vessels (H and E, 100×); (c): direct immunofluorescence (DIF) showing IgG positive reactive granular (+++) deposits at the dermoepidermal junction (400×); (d): DIF C3 positive reactive granular (+++) deposits at the dermoepidermal junction (400×).
Figure 6:
(a): A case of Henoch Schonlein purpura - skin biopsy showing leukocytoclastic vasculitis (H and E, 200×); (b): direct immunofluorescence showing IgA positive reactants in the superficial dermal vessels (400×).

Extent of staining

Focal or diffuse, homogenous, or non-homogenous.

Intensity of staining

Semi-quantitative grading of strength of fluorescence: + to ++++.

Pattern of immune complex deposits

Granular (coarse granules, speckles, threads, and fibrils) or linear.


Studying the serration pattern of BMZ deposits by DIF helps to distinguish between BP and EBA [Table 4]. It requires thinner frozen sections and a higher magnification (more than or equal to 600 fold higher magnification). Hence, it is very difficult to carry out serration pattern analysis in routine laboratory set up.[12,13]

Table 4: Serration pattern analysis on DIF microscopy.
Blistering diseases Serration pattern
• Epidermolysis bullosa acquisita
• Bullous Pemphigoid, anti-p200 pemphigoid, anti-laminin-332 mucous membrane pemphigoid
“u- serrated” pattern
“n-serrated” pattern

DIF: Direct immunofluorescence


Both false-negative and false-positive results can be observed in DIF microscopy.[5,6]

False-negative reactions mostly occur due to following reasons: [5]

  • Technical reasons such as formalin contamination of specimen, inappropriate transport medium, or a delay in shipping the sample to the laboratory.

  • The specimen may get dried up due to leakage of the fixative solution.

  • It is important to keep the biopsy sample in a moist environment because the immunoreactants in dried up biopsies will undergo degradation and may give a false negative result.

False-positive reactions mostly occur due to non-specific staining patterns. Cause for this include: [5-7]

  • Crushing and freezing artifacts can produce pemphigus-like pattern.[7]

  • Nonspecific granular BMZ staining is observed in conditions such as bullous mastocytosis (with IgM) and elastosis perforans serpiginosa (with IgG) due to intense inflammation along the BMZ or due to nonspecific binding of antibodies to altered elastic fibers.

  • Biopsy from the sun-exposed skin may show granular IgM staining along the BMZ resembling lupus band.[5]

  • Biopsy from the lower leg near the ankle, sometimes exhibits staining around the blood vessel wall, with fibrinogen. Hence, it is confused with vasculitis.[5] Hence, in suspected cases of vasculitis, it is ideal to take a biopsy (for DIF) from the lesion on the most proximal part of the lower limb.[5,14]

The processing of specimens for DIF microscopy can be carried out only in advanced laboratories having facilities such as cryostat for cutting frozen sections, deep freezers (−20°C/−80°C) to store these sections, and a fluorescence microscope to report the findings.[5,14]


IIF is a semi-quantitative method in which double immunolabeling is used. A secondary antibody labeled with fluorochrome is used to recognize a primary antibody. Here, a normal whole tissue (usually monkey esophagus) is used as the substrate. The patient’s serum is layered on the substrate and then fluoresceinated antibodies are applied. The method detects the circulating antibodies in the serum. IIF is 10– 15 times more sensitive than DIF. IIF titers may correlate with the disease severity. So in addition to diagnosis, IIF has prognostic value as well and is useful to monitor the response to therapy.[5]

The substrate is first incubated with a series of dilutions of patient’s serum in phosphate buffer solution for 30 minutes. Unlabeled primary antibodies from the patient serum are allowed to bind to the target molecule in pre-prepared tissue samples such as monkey esophagus or normal human skin. If circulating auto antibodies are present in the serum, it will bind to the respective antigens in the substrates and are detected by incubation with appropriate fluorescent (usually FITC) labeled, mouse anti-human IgG, and/or IgA. Screening for antinuclear antibody (ANA) should be done at dilutions of 1/40, as at lower dilutions, false-positive results can occur.[5]


Diagnosis of autoimmune bullous diseases

IIF is used to demonstrate the antibodies in the patient’s serum directed against specific antigens in the following diseases.[5]

  • PV

  • PF

  • Paraneoplastic pemphigus

  • Linear IgA bullous dermatosis

  • Chronic bullous disease of childhood

  • Dermatitis herpetiformis

  • BP

  • Pemphigoid gestationis

  • Mucous membrane pemphigoid

  • EBA

  • Bullous systemic lupus erythematosus (bullous SLE)

Diagnosis and assessment of disease activity in various autoimmune diseases

IIF is used to demonstrate ANAs in connective tissue diseases such as SLE, mixed connective tissue disease, and systemic sclerosis and to demonstrate antineutrophil cytoplasmic antibodies (ANCA) in ANCA associated small vessel vasculitis (AVV).[5,15-18]

The following precautions can yield an accurate result in IIF: [5]

  1. Adequate washing during each step of treatment with the antibody and fluorescent conjugate is very important to remove the unwanted immunodeposits.

  2. Appropriate storage of the tissue sections at adequate temperature (−70 °C to −80°C)

  3. Patient’s sera should be refrigerated until tests are performed. Repeated freezing and thawing should be avoided since this can lead to rapid loss of activity of the antibody.[5]

  4. It is important to compare the relative intensities between the positive and background staining in control slides while interpreting the IIF.[5]


ANA test is a standard screening assay for antibodies produced in autoimmune or ANA associated rheumatic disease and ANCA associated vasculitis.[15-17]

Hep-2 IIF is the method recommended by the American College of Rheumatology for ANA screening.[15,16] More than 100 autoantibodies to different nuclear and cytoplasmic antigens can be identified by this technique.[15-19] The nuclear patterns and the titer are the important aspects to be considered while interpreting IIF for ANA.

Patterns of ANA commonly reported by most laboratories are homogeneous, speckled, nucleolar, centromere, and proliferating cell nuclear antigen (PCNA).[15] The ANA patterns associated with specific diseases are mentioned below.


SLE, chronic autoimmune hepatitis, and juvenile idiopathic arthritis [Figure 7a].

Figure 7:
(a): Indirect immunofluorescence photomicrograph of Hep 2 cell lines showing homogenous pattern (400×); (b): Indirect immunoflourescence photomicrograph of Hep 2 cell lines showing nucleolar pattern of nuclear fluorescence (400×).


Patients with systemic sclerosis and those with clinical manifestations of other systemic autoimmune rheumatic diseases [Figure 7b].


In varying degrees in distinct systemic autoimmune rheumatic diseases, in particular, SLE, systemic sclerosis, mixed connective tissue disease, and undifferentiated connective tissue disease [Figure 8a].

Figure 8:
(a): Indirect immunofluorescence (IIF) photomicrograph of Hep 2 cell lines showing speckled pattern of fluorescence (400×); (b): IIF photomicrograph of Hep 2 cell lines showing centromere pattern of fluorescence (400×).


Limited cutaneous systemic sclerosis [Figure 8b].


Earlier, PCNA was considered highly specific for SLE, but this specificity is now debated. Clinically, it is associated with systemic sclerosis, autoimmune myopathy, rheumatoid arthritis, and hepatitis C virus infection [Figure 9].

Figure 9:
Indirect immunofluorescence photomicrograph of Hep 2 cell lines showing proliferating cell nuclear antigen pattern of nuclear fluorescence (400×).

Interpretation of ANCAs

ANCAs are important laboratory markers to support the diagnosis of ANCA-AAV, including granulomatosis with polyangiitis and microscopic polyangiitis. Conventionally, screening for ANCA by IIF is done and IIF positive samples are further evaluated for antibodies to proteinase 3 (PR3) or myeloperoxidase (MPO) by specific immunoassays.[17]

According to the IIF pattern on ethanol-fixed neutrophils, ANCAs are classified into[17]

  • C-ANCA (Poteinase 3 ANCA or PR3 ANCA): Granular cytoplasmic staining with accentuation between the nuclear lobes

  • P-ANCA (MPO-ANCA): Perinuclear and/or nuclear staining [Figure 10]

  • Atypical ANCA: Diffuse cytoplasmic and “perinuclear” staining. It is also termed as atypical P- ANCA or X-ANCA or snowdrift-ANCA.

Figure 10:
(a): A case of eosinophilic granulomatosis with polyangiitis, section of skin with eosinophilic granuloma and small vessel vasculitis in the dermis (H and E, 200×); (b): Indirect immunofluorescence photomicrograph of ethanol fixed neutrophil preparation showing anti neutrophil cytoplasmic antibody pattern of perinuclear fluorescence (400×).

The patterns correlate with varying degrees of specificity to specific neutrophil granule contents. ANCAs can be IgG (most frequent), IgM, or IgA.[17] In the revised 2017 international consensus on ANCA testing in small vessel vasculitis, the use of high-quality immunoassays with PR3-MPO-ANCA is recommended as the preferred method (without the categorical need for IIF) in suspected cases of ANCA-associated vasculitides.[17]


Antigen mapping or immunomapping

This modified IIF technique was described by Hintner et al. in 1981.[5,20] Patient’s own skin is used as the substrate. Immunofluorescence antigen mapping (IFM) helps to determine the exact site of cleavage in AIBDs and the abnormalities in the distribution of mutated structural proteins in hereditary EB. IFM has taken over transmission electron microscopy as the preferred method for preliminary diagnosis of EB.[20]

In IFM, frozen sections of shave biopsy of patient’s skin are stained with commercially available monoclonal antibodies (primary antibody) directed against different antigenic components of the BMZ/epidermis such as keratin 5/14, laminin 332, and type VII and 1V collagen.[20] A secondary antibody tagged with fluorochrome detects the immune complex. The staining pattern is then compared with that of normal healthy skin to confirm whether the antigen expression is normal, reduced, or absent.[5,20]

Complement fixation

Here, patient’s serum is layered on the substrate, and a source of complement is added. Then, fluoresceinated anti-complement antibodies are used to detect the presence of complement in the tissue. This type of IIF helps to detect small quantities of complement fixing antibodies.[5]

BIOCHIP mosaic slides (Euroimmun, Lubeck, Germany)

BIOCHIP mosaic slides are useful in patients with AIBDs to screen for autoantibodies. The commercially available, ready-to-use slides containing six different substrates (monkey esophagus, primate salt-split skin, recombinant BP180 NC16A, membrane-bound desmoglein (Dsg)1 ectodomain, Dsg3 ectodomain, and the C-terminal globular domain of BP230) in a miniature field are used. To these ready-to-use slides, serum from patients with suspected AIBD is added which is then examined under fluorescence microscope. This method does not require frozen sections of the substrate.[21]

Salt split technique (SST)

It is a procedure that reliably differentiates between various subtypes of immunobullous disorders that are characterized by separation at the dermoepidermal junction (DEJ). It was introduced by Gammon et al.[22] There are two types of SST - direct salt split skin test (D-SST) and indirect salt split skin test (I-SST).[5,23]

D-SST is performed on patient’s skin biopsy specimen. A fresh skin biopsy from clinically normal appearing “patient” skin can be used for D-SST or alternatively D-SST can be performed on the specimen that was used for routine DIF. Punch biopsy specimens are incubated in 5 ml of NaCl (1 mol/L) at 4°C for 24 hours. The epidermis is then teased from the dermis with the use of a fine forceps. The specimens are then processed in the same manner and treated with IgG and C3 conjugates as in DIF. D-SST aids to determine the site of in vivo deposition of bound auto antibodies within the DEJ (whether deposited on the blister roof or on the blister floor or on both sides of the artificial split).[5]

For I-SST, a sample of normal human skin is used as the substrate. After artificially inducing the junctional split, cryocut sections are prepared. Then IIF with patient’s serum is carried out. This is more sensitive than routine IIF and is very helpful when a lesional biopsy is not available.[5]


IF plays a significant role in the diagnosis of vesiculobullous disorders, connective tissue disorders and vasculitis. It should be interpreted and correlated with clinical features, other investigation results, and histopathology findings to arrive at the correct diagnosis.


The authors are grateful to Dr. KP. Aravindan, Professor and Former Head of the Department of Pathology and ICMR Emeritus Scientist, Government Medical College, Kozhikode and Medical Director of Microhealth Reference Laboratory, Kozhikode, Kerala for providing the histological diagnosis and immunofluorescene interpretation of the images described in the article. Also we are acknowledging Dr. Roshan Nasimudeen, Consultant Pathologist, Microhealth Reference Laboratory, Kozhikode, Kerala, for providing us some of the microphotographs used in this review.

Declaration of patient consent

Not required as patients identity is not disclosed or compromised.

Conflicts of interest

There are no conflicts of interest.

Financial support and sponsorship



  1. , , , . The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol. 1942;45:159-70.
    [Google Scholar]
  2. , . Demonstration of skin antibodies in sera of pemphigus vulgaris patients by indirect immunofluorescent staining. Proc Soc Exp Biol Med. 1964;117:505-10.
    [CrossRef] [PubMed] [Google Scholar]
  3. , , , , , , et al. Endemic pemphigus foliaceus (fogoselvagem) and pemphigus vulgaris: Immunoglobulin G heterogeneity detected by indirect immunofluorescence. Rev Hosp Clin Fac Med Sao Paulo. 2004;59:251-6.
    [CrossRef] [PubMed] [Google Scholar]
  4. , , , , , . Basement zone antibodies in bullous pemphigoid. JAMA. 1967;200:751-6.
    [CrossRef] [PubMed] [Google Scholar]
  5. , , . Immunofluorescence in dermatology. Indian J Dermatol Venerol Leprol. 2012;78:677-91.
    [CrossRef] [PubMed] [Google Scholar]
  6. . Direct immunofluorescence microscopy in the diagnosis of autoimmune bullous dermatoses. Clin Dermatol. 2001;19:607-13.
    [CrossRef] [Google Scholar]
  7. , , . Skin biopsy: Biopsy issues in specific diseases. J Am Acad Dermatol. 2016;74:1-16. quiz 17-8
    [CrossRef] [PubMed] [Google Scholar]
  8. , , . Direct immunofluorescence of skin using formalin fixed paraffin-embedded sections. J Clin Pathol. 1980;33:365-9.
    [CrossRef] [PubMed] [Google Scholar]
  9. , , , , . The value of direct immunofluorescence on proteinase-digested formalin-fixed paraffin-embedded skin biopsies. Am J Dermatopathol. 2018;40:111-7.
    [CrossRef] [PubMed] [Google Scholar]
  10. , , , , , . A retrospective analysis of tissue-fixed immunoreactants from skin biopsies maintained in Michel's medium. Dermatology. 1994;189(Suppl 1):131-2.
    [CrossRef] [PubMed] [Google Scholar]
  11. , , , , , . Enhanced diagnostic immunofluorescence using biopsies transported in saline. BMC Dermatol. 2004;4:10.
    [CrossRef] [PubMed] [Google Scholar]
  12. , , , . U-serrated immunodeposition pattern differentiates Type VII collagen targeting bullous diseases from other subepidermal bullous autoimmune diseases. Br J Dermatol. 2004;151:112-8.
    [CrossRef] [PubMed] [Google Scholar]
  13. . The many faces of epidermolysis bullosa acquisita after serration pattern analysis by direct immunofluorescence microscopy. Br J Dermatol. 2011;165:92-8.
    [CrossRef] [PubMed] [Google Scholar]
  14. , , , , . Direct immunofluorescence of skin biopsy: Perspective of an immunopathologist. Indian J Dermatol Venereol Leprol. 2010;76:150-8.
    [CrossRef] [PubMed] [Google Scholar]
  15. , , , , , , et al. Clinical relevance of HEp-2 indirect immunofluorescent patterns: The international consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78:879-89.
    [CrossRef] [PubMed] [Google Scholar]
  16. . Clinical relevance of HEp-2 indirect immunofluorescent patterns: The international consensus on ANA patterns (ICAP) perspective. Ann Rheum Dis. 2019;78:879-89.
    [CrossRef] [PubMed] [Google Scholar]
  17. , , , , , , et al. Revised 2017 international consensus on testing of ANCAs in granulomatosis with polyangiitis and microscopic polyangiitis. Nat Rev Rheumatol. 2017;13:683-92.
    [CrossRef] [PubMed] [Google Scholar]
  18. , , , . Interpretation of ANA indirect immunofluorescence test outside the darkroom using NOVA view compared to manual microscopy. J Immunol Res. 2014;2014:149316.
    [CrossRef] [PubMed] [Google Scholar]
  19. , . ANA screening: An old test with new recommendations. Ann Rheum Dis. 2010;69:1420-2.
    [CrossRef] [PubMed] [Google Scholar]
  20. , , , , , , et al. Immunofluorescence mapping of antigenic determinants within the dermal-epidermal junction in the mechanobullous diseases. J Invest Dermatol. 1981;76:113-8.
    [CrossRef] [PubMed] [Google Scholar]
  21. , , , , , , 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;7:49.
    [CrossRef] [PubMed] [Google Scholar]
  22. , , , , . Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol. 1984;82:139-44.
    [CrossRef] [PubMed] [Google Scholar]
  23. , , . Role of Immunofluorescence microscopy in dermatology. G Ital Dermatol Venereol. 2011;146:127-42.
    [Google Scholar]
Show Sections