Immunological Methods for the Diagnosis of Oral Mucosal Diseases

S. Sun; B. Zhong; W. Li; X. Jin; Y. Yao; J. Wang; J. Liu; H. Dan; Q. Chen; X. Zeng


The British Journal of Dermatology. 2019;181(1):23-36. 

In This Article

Immunological Methods

Direct Immunofluorescence

Direct immunofluorescence (DIF), which shows the immunoreaction of specific fluorescent antibodies against target antigens in tissues, can locate antigens. DIF is superior to clinical manifestation or histopathology in AIBD diagnosis,[3] and is also useful for OLP or DLE.

The preferred location of DIF biopsies for AIBDs in the oral mucosa is perilesional, normal-appearing mucosa, of 3–4 mm in diameter. However, for OLP or DLE, lesional biopsies are preferred.

For DIF, fresh frozen tissue is preferred compared with formalin-fixed paraffin-embedded (FFPE) tissue, but DIF on trypsin digestion of formalin-fixed sections can be an alternative method when fresh frozen tissue is unavailable.[4–6] In addition, if only FFPE tissues are available, C3d or C4d immunohistochemistry (IHC) has also been considered as a valuable diagnostic tool for bullous pemphigoid (BP) and MMP.[7–11] IHC for C4d or IgG4 may be diagnostically useful in pemphigus,[10,12] and C4d IHC can be helpful in the diagnosis of pemphigoid gestationis.[13] A reliable technique of IHC for IgA and IgG on FFPE tissue needs to be established to obtain higher sensitivity. Moreover, a case reported that DIF for C4d may be useful for BP in the case of negative results of DIF for IgG and C3.[14]

DIF samples stored in normal saline for transportation within 24 h may be optimal, because of reduction in IgG background fluorescence and enhancement of desired specific staining.[15] Michel's fixative, liquid nitrogen and saline are used for longer transportation periods of 24–48 h (liquid nitrogen for transportation presents a potential danger).[15,16] Cryocut sections (4–6 μm thick) should be read with a fluorescence microscope immediately. Sometimes, a second biopsy can rule out a false-negative result caused by biopsy location, confounding immune dysregulation, or tissue quality.[17] DIF fluorescence characteristics are shown in Table 1, and DIF diagnostic sensitivity in Table 3.[18–20]

Indirect Immunofluorescence

Indirect immunofluorescence (IIF) is a technique that involves incubating specimens successively in primary antibodies and fluorescein-conjugated secondary antibodies to detect circulating autoantibodies.

Monkey and guinea pig oesophagus and normal human skin are the most common substrates used in AIBDs, and optimal results can be obtained from a combination of monkey and guinea pig oesophagus rather than single substrate.[21] However, human epithelial type 2 cells (HEp-2) have been used to detect antinuclear antibody (ANA) in OLP or DLE. Substrate selection should also be determined according to diseases and experimental conditions.

Table 1 shows characteristics of IIF in these oral mucosal diseases. Detection of ANA contributes to evaluating the severity of OLP and DLE associated with the degree of erosions and the probability of developing systemic lupus erythematosus (SLE), respectively. Respective diagnostic sensitivities of IIF for PV, PNP, MMP and LAD are shown in Table 3.[18,22–31]

Salt-split Skin Test

In 1984, Gammon et al. first described IIF on salt-split skin (SSS) to differentiate AIBDs (samples were treated with 1·0 mol L−1 NaCl),[32] and DIF on SSS appeared later.[33] Artificial blisters were observed at the end of the experiment. Antibodies bound to either the epidermal and/or dermal sides of the SSS. An epidermal pattern reflects anti-lamina lucida and anti-hemidesmosome antibodies, and a dermal pattern shows anti-lamina densa and anti-sublamina densa antibodies (Table 2). In addition, SSS is a more sensitive substrate than intact mucosa for detecting anti-BMZ antibodies.[34]

The SSS test is quite useful for differential diagnosis of pemphigoid diseases. We mainly discuss characteristics of the SSS test in MMP, LAD and LPP (Table 1). The result of diagnostic sensitivity for LAD, which has been published, is listed in Table 3.[23]