**8. Diagnosis**

Early and accurate diagnosis is important. This is dependent on the co-detection of clinical presentation and tissue-bound and/or circulating autoantibodies.

The diagnosis of pemphigus vulgaris begins with a thorough history and physical examination. Since the presence of mucosal lesions is more common in PV than in other pemphigus, it should detect the presence of mucosal involvement. Mucosal involvement may not always be in the areas to be seen during the examination. Therefore, clinicians should question the presence of ocular symptoms, hoarseness, dysphagia and dyspareunia to assess the involvement of all mucosal surfaces [3]. Drug use should be questioned.

After a thorough history and physical examination, two separate biopsies should be obtained from the patient for both histopathological examination and DIF.

A 4 mm punch biopsy should be taken from the edge of the early lesion or erosion for hematoxylin and eosin (H&E) staining and routine histopathological examination. For direct immunofluorescence (DIF), an additional perilesional skin biopsy should be taken from normal-appearing skin 4 mm from a vesicle or erosion. Lesions of skin biopsies for DIF are more likely to be associated with false-negative results as a result of the elimination of immunoreactions involved in the inflammatory process of the underlying pemphigus disease. DIF biopsies should not be placed in formalin. Michel medium or Zeus medium should be used. Fresh specimens also may be sent to the laboratory, provided they are kept moist with saline and processing within 24 hours.

The characteristic histopathological finding in PV is acantholysis due to loss of intercellular adhesion without necrosis in keratinocytes and thus the formation of intraepidermal bullae. Although acantholysis is often located just above the basal layer (suprabasal), intraepidermal localization at the level of the stratum spinosum has also been reported to a lesser extent. There are epidermal cell clumps and

acantholytic cells in the bulla cavity. Although basal keratinocytes lose their connection with the adjacent cell, they do not lose their bond with the basal membrane through hemidesmosomes, leading to the appearance of a 'tombstone pattern' [19]. Sparse inflammatory infiltrates in the dermis with eosinophils.

The gold standard in the diagnosis of PV is direct immunofluorescence (DIF) microscopy, which can detect tissue-bound autoantibodies [4]. In DIF, there is the accumulation of IgG and C3 in the intercellular space throughout the mid-lower or entire epidermis.

IIF and ELISA are serological tests that detect circulating autoantibodies that bind epithelial cell surface antigens. These tests are used to further support the diagnosis of pemphigus in patients with a positive DIF result. More than 80% of pemphigus vulgaris patients have circulating antibodies detectable by IIF. The substrate used affects the test sensitivity [25]. The monkey esophagus is the preferred substrate for the diagnosis of pemphigus vulgaris. Intercellular 'honeycomb' or 'coil wire' IgG accumulation is observed in PV [26]. The enzyme-linked immunosorbent assay (ELISA) test can be used for IgG antibodies to Dsg 1 and Dsg 3. ELISA is more sensitive and specific than IIF in the diagnosis of pemphigus vulgaris [25]. The ELISA test can also be used to monitor disease course and response to therapy in PV. In a study in which Dsg 1 and Dsg 3 autoantibody levels were followed in patients under treatment, it was found that Dsg 1 level more clearly showed the course of the disease. Dsg 3 antibody levels remained elevated during remission in some patients with mucosal pemphigus vulgaris [27]. Antibodies can be detected in patients who do not yet have clinical signs of pemphigus and can be found in patients with staphylococcal scalded skin, penicillin adverse drug reactions, toxic epidermolysis necrosis and burns [1].

Additional serological tests that may be used to diagnose pemphigus vulgaris include immunoblotting and immunoprecipitation. However, these tests are more difficult to perform than IIF and ELISA. Therefore, they are rarely used in the clinical setting.

In addition, cytological examination (Tzanck smear) stained with hematoxylin and eosin is useful for rapid demonstration of acantholytic keratinocytes of the spinous layer (abundant eosinophilic cytoplasm and rounded central nucleus) [20].
