Immunofluorescence in dermatology: A brief review

INTRODUCTION

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).

SPECIMENS FOR IF

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)

INDICATIONS FOR IF IN DERMATOLOGY

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]

TECHNICAL CONSIDERATIONS

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]

DIF

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]

INDICATIONS FOR DIF

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

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]

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]

TRANSPORT AND STORAGE OF BIOPSY SPECIMEN

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.

HOW TO REPORT A FLUORESCENT STAINED SKIN BIOPSY SPECIMEN

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]

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].

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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×).

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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×).

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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×).

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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×).

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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×).

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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×).

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SERRATION PATTERN ANALYSIS

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]

PITFALLS OF DIF

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

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]

INDICATIONS FOR IIF

INTERPRETATION OF ANA TEST

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.

Homogeneous

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

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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×).

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Speckled

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].

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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×).

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PCNA

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].

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Figure 9:

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

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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.

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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×).

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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]

OTHER FORMS OF IIF

CONCLUSION

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.