Direct immunofluorescence demystified: Essential insights and recent advances for dermatologists

Oral cavity as a site of biopsy

Oral biopsies for DIF are technically difficult to perform due to poor accessibility, the need for minimal tissue handling, and the innate fragility of the mucosa. A recent study has shown that a routine buccal punch biopsy of the uninvolved mucosa is as sensitive as a perilesional biopsy (within 1 cm of lesion) in the diagnosis of oral pemphigus vulgaris and multi-site mucous membrane pemphigoid.¹⁴

The intact epithelium is easier to obtain from the buccal mucosa as compared to the thin and friable gingiva. Gingival biopsies often result in a falsely negative DIF due to chronic inflammation around the gingiva. Additionally, they may cause a permanent periodontal defect leading to plaque accumulation.¹⁴

In pure gingival mucous membrane pemphigoid, a biopsy from the reflected alveolar mucosa adjacent to the area of gingival inflammation is preferred. Obtaining gingival specimens with an intact epithelium is critical for histological and DIF evaluation. In addition to the standard perilesional 3-mm punch biopsy, a “stab-and-roll” technique and a “peeling” technique have also been used to obtain a mucosal biopsy.¹¹

Other substrates for DIF

• 1.

  Hair

  The outer root sheath of the hair follicle is structurally analogous to the epidermis. The diameter of scalp terminal hair is almost twice that of vellus hair at other body sites. Thus, the total volume of desmosomal structures is greater per unit area of the scalp than at any other epidermal site. Pemphigus antigens are found throughout the outer root sheath of the hair follicle and in the dermal bulb matrix.

  Wilson et al. first described the intercellular deposition of immunoreactants in the outer root sheath of the terminal hair follicle in longitudinal sections of follicular-oriented scalp biopsies.¹⁵ Subsequently, Tanasilovic et al. showed the utility of DIF of plucked hair in the diagnosis of pemphigus and observed that the sensitivity and specificity of DIF of plucked hair are comparable with that of anti-desmoglein 1 and 3 antibodies as detected by ELISA.¹⁶

  Five or more anagen hairs, plucked with forceps, are collected. The DIF of both fresh and frozen hair samples (stored for two weeks at –20°C) showed positive results in all patients with active PV and PF. Mounting entire hairs, rather than sections is easier. The sensitivity of DIF of plucked hair is 80–100% and positive results have been documented in cases with only mucosal involvement. ^17,18

  When monitoring immunological remission in pemphigus, plucked hair provides a simpler, more specific, sensitive, and less invasive alternative to skin biopsy regardless of scalp involvement, thereby avoiding repeated skin biopsies.^17,18 However, inadequate samples or lack of expertise in processing hair specimens may result in negative outcomes.

• 2.

  Formalin fixed paraffin-embedded (FFPE) tissue

  If only formalin-fixed paraffin-embedded (FFPE) tissue is available (e.g., immunobullous or connective tissue disease is not initially suspected, or if frozen tissue is unavailable) DIF can be performed after digestion with proteinase or protease K. However, DIF performed on FFPE samples may show decreased staining and sensitivity and the presence of artefactual background staining of IgG in the epidermis could potentially result in a misdiagnosis of pemphigus.

  This technique shows best results in diagnosing bullous pemphigoid and pemphigoid gestationis (with a sensitivity of 79%).¹⁹ The diagnosis of IgA vasculitis and dermatitis herpetiformis can pose challenges due to the difficulty in demonstrating IgA deposits.

• 3.

  Tzanck smears

  DIF may also be performed on Tzanck smears particularly in settings where skin biopsy facilities are not available or lesions are located on mucosal sites that are not easily accessible for biopsy. It provides a straightforward, quick, and non-invasive option compared to skin or mucosal biopsy, with sensitivity ranging from 40% to 87.8%.^20,21 The antigenicity in the air-dried smears kept at room temperature is retained for up to 10 days. Positive smears show bright green fluorescence at the margins of the acantholytic cells or in the intercellular spaces if clumps of acantholytic cells are present. However, non-specific background staining in smears prepared from inflammatory lesions is an important limitation.

Transportation

• 1.

  Michel’s medium (MM) Michel’s medium is a pH-neutral buffered solution containing ammonium sulfate, N-ethylmaleimide, potassium citrate buffer, magnesium sulfate, and distilled water. It is commonly utilised to transport specimens for DIF testing. Unused media should be stored in a refrigerator (2–8°C). If there is ammonium sulfate precipitation, crystals should be dissolved at room temperature before use. The expiration date of the media ranges from 12 to 18 months. Specimens preserved in Michel’s solution can be stored at room temperature for at least 2 weeks and potentially for 1 to 6 months without compromising signal integrity.¹¹

• 2.

  Saline:

  Phosphate buffered saline (PBS) or normal saline is routinely used to transport biopsy samples that can be transported to the laboratory within a few hours after taking the biopsy. Normal saline has shown superior results in DIF than in MM.²²

  Advantages of saline as a transport medium include its inexpensive nature and ready availability in all clinics. It significantly reduces background fluorescence in the dermis, particularly with IgG, and may enhance the cutting properties of skin biopsies. Also, saline could potentially induce an artificial split at the dermo-epidermal junction (DEJ) facilitating the detection of immunoreactants at the basement membrane zone (BMZ) and subepidermal blood vessel walls.

  Disadvantages include the need to be transported to the laboratory within 24 hours, as immunoreactants undergo rapid degradation in saline after 48 hours especially in tropical and hot climates. Additionally, it is not a suitable medium for antigen mapping in genetic disorders because it may induce an artificial split at the DEJ.^4,11

• 3.

  Honey:

  Honey has recently been shown to serve as an effective transport medium for DIF specimens in resource-poor settings. It is believed to preserve cells by preventing autolysis and putrefaction. The optimum preservation time for samples is approximately two weeks; beyond this period, there is a deterioration of immunoreactants in the biopsy sample.²³

Tissue processing and staining

Processing and staining of skin biopsy specimens for DIF has been detailed in Figure 1.

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Figure 1:

Processing and staining skin biopsy for DIF.

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Sensitivity of DIF and causes of false negative results

In general, DIF is highly sensitive for autoimmune bullous diseases (AIBDs) and cutaneous vasculitis (such as IgA vasculitis) (>90%). DIF is highly sensitive but has limited specificity in the pemphigus group of diseases, while it is very sensitive and specific (90–98%) in the pemphigoid group of disorders.¹¹ However, the sensitivity of DIF is low (27–41%) for the diagnosis of paraneoplastic pemphigus.

The lupus band test has variable sensitivity and specificity in cutaneous lupus erythematosus. It is more sensitive (80–93%) in SLE as compared to DLE (40–60%), however, it has high specificity (80–90%) for acute cutaneous LE.^27,28 The sensitivity and specificity of DIF is almost similar for non-lesional sun protected as well as sun exposed areas in SLE.²⁸

False negative results in DIF can result from:

Preanalytical Factors

• 1.

  Contamination with formalin

• 2.

  Delay in transporting biopsies, especially when sent in saline

• 3.

  Use of inadequate transport medium

• 4.

  Delay in obtaining biopsies (eg., DIF results are likely to be negative in IgA vasculitis if the sample is obtained more than 7 days after the onset of symptoms ^29,30

• 5.

  Low antibody titres or prior treatment

• 6.

  Absence of epidermis or epithelium (common in biopsies taken from the buccal mucosa in autoimmune blistering diseases (AIBDs)

• 7.

  Lesional specimens in AIBDs where immunoreactants have already been consumed

The advantages of DIF include:

• 1.

  Enhanced intensity of illumination

• 2.

  Clearer and brighter image resolution

• 3.

  Easy to perform

• 4.

  Flexibility to utilise different antibodies for fluorescence

• 5.

  Signal amplification capabilities

• 6.

  Specific targeting ability

• 7.

  Diagnostic and analytical capacity (when clinical features or histopathology are inconclusive, a diagnosis can be solely based on DIF findings. The demonstration of immune complexes in skin biopsies at various locations such as intraepidermal, dermoepidermal junction (DEJ), and dermal blood vessels aids in diagnosis)³¹

• 8.

  Discrimination between immune-mediated bullous lesions and others

• 9.

  Definitive diagnosis in certain diseases, such as the fish-net pattern of pemphigus and IgA positivity in linear IgA dermatosis¹¹

• 10.

  Facilitates monitoring of response to therapy and can predict relapse in patients in remission (in pemphigus, early positivity in DIF and increased complement deposition indicate relapse in patients)

• 11.

  Initial use of DIF on hair for patients in clinical remission to assess immunological status. If positive, DIF on skin may be avoided initially^16,17,32

Disadvantages of DIF:

• 1.

  Expensive

• 2.

  Photobleaching: Fading occurs under intense illumination

• 3.

  Formation of protein cross-links in fixed tissue samples

• 4.

  Loss of tissue morphology and relevant antigens

• 5.

  Requires strict processing and optimal antibody usage determination

• 6.

  Immunoreactivity restoration may be necessary if not stored in darkness ³³