**5. Diagnosis**

#### **5.1. Histopathology**

In PV, biopsy of lesional skin demonstrates intraepidermal splitting that occurs suprabasally forming an intraepidermal blister [34]. A single layer of basal keratinocytes remains attached

**Figure 6.** Medium power view of a skin biopsy from a patient with pemphigus vulgaris showing acantholysis of the suprabasal keratinocytes leaving a single layer of basal keratinocytes attached to the basement membrane (tomb‐ stone pattern), which forms the floor of an intraepidermal blister. The roof of the blister comprises relatively intact superficial epidermal layers with the stratum corneum. A sparse perivascular lymphocytic and eosinophilic inflamma‐ tory infiltrate is found within the upper dermis.

to the basement membrane (tombstone pattern), which forms the floor of the blister. The roof of the blister comprises relatively intact superficial epidermal layers with the stratum corneum showing a basketweave pattern. Hematoxylin and eosin staining demonstrates suprabasalar acantholysis and infiltration with predominantly neutrophils and eosinophils. A sparse perivascular lymphocytic and eosinophilic inflammatory infiltrate is found in the upper dermis [35] (Figure 6). In contrast, intraepidermal splitting is found subcorneally in PF with the initial formation of vacuoles within the intercellular spaces of the granular and/or upper spinous layers of the epidermis. [34] The vacuoles become larger and eventually lead to subcorneal blister formation within the upper epidermis (Figure 7). Hematoxylin and eosin staining show variable amounts of acantholytic keratinocytes, neutrophils, and fibrin within the blisters (Figure 8). Chronic PF lesions may show evidence of papillomatosis, acanthosis, hyperkeratosis, parakeratosis, and follicular plugging. The papillary dermis contains an inflammatory infiltrate composed of small numbers of neutrophils, eosinophils, and lympho‐ cytes.

#### **5.2. Direct immunofluorescence**

**Figure 5.** Pemphigus foliaceus with large scaly and crusted erosions over the trunk giving a 'corn flakes' appearance

PF causes significant morbidity as the lesions are painful and patients are prone to secondary infection, dehydration and metabolic disturbances with fatalities reported for those cases

Clinically, the differential diagnosis for mucosal lesions of PV (before the development of cutaneous disease) includes herpes simplex virus, lichen planus, mucous membrane (cicatri‐ cial) pemphigoid: erythema multiforme, and paraneoplastic pemphigus. The differential diagnosis for cutaneous involvement include other autoimmune blistering skin conditions, such as pemphigus foliaceus, pemphigus vegetans, IgA pemphigus, paraneoplastic pemphi‐ gus, bullous pemphigoid, linear IgA disease, erythema multiforme, Grover's disease, and

The differential diagnosis of endemic and nonendemic PF includes bullous impetigo, IgA pemphigus, pemphigus herpetiformis, drug eruptions, subcorneal pustular dermatosis, and systemic lupus erythematosus. If lesions are localized to the face or scalp with abundant scaling and yellow crusting, seborrheic dermatitis needs to be considered. The differential diagnosis of exfoliative erythroderma as the manifestation of PF includes papulosquamous diseases such as psoriasis, pityriasis rubra pilaris, and Drug Reaction with Eosinophilia and Systemic

In PV, biopsy of lesional skin demonstrates intraepidermal splitting that occurs suprabasally forming an intraepidermal blister [34]. A single layer of basal keratinocytes remains attached

involving exfoliative erythroderma.

**4.3. Differential diagnosis**

112 Skin Biopsy - Diagnosis and Treatment

Hailey-Hailey disease.

Symptoms.

**5. Diagnosis**

**5.1. Histopathology**

Biopsy for direct immunofluorescence should be taken from perilesional mucosa and/orskin [36]. Biopsy of lesional tissue is not useful as immunoreactants are rapidly degraded by inflammatory activity [37]. To prevent the destruction of immunoreactants, specimens must be snap frozen and stored at temperatures below -70°C or placed in special transport media (such as Michel's medium) [36]. In direct immunofluorescence, specimens are incubated with fluorescein isothiocyanate-labelled antibodies against immunoglobulins, complement or fibrinogen, and examined with a fluorescent microscope [36]. In PV, direct immunofluores‐

**Figure 7.** Medium power view of a skin biopsy from a patient with pemphigus foliaceus showing vacuoles within the intercellular spaces of the stratum granulosum and the upper stratum spinosum of the epidermis. The papillary dermis contains an inflammatory infiltrate comprising neutrophils, eosinophils, and lymphocytes (hematoxylin and eosin staining).

**Figure 9.** Direct immunofluorescence of a skin biopsy from a patient with pemphigus vulgaris revealing deposition of

Pemphigus Vulgaris and Pemphigus Foliaceus

http://dx.doi.org/10.5772/56423

115

The target of pemphigus is desmoglein. Desmoglein-1 and -3 are restricted to stratified squamous epithelium, whereas desmoglein-2 is expressed in all desmosome possessing tissues [8]. In indirect immunofluorescence, patient serum is incubated with epithelial substrates containing the target antigen. In pemphigus, the most frequently used substrates are monkey esophagus, which is more sensitive for the detection of PV autoantibodies, and guinea pig esophagus and rabbit lip mucosa, which are more sensitive for the diagnosis of PF [39]. In PV, indirect immunofluorescence produces a characteristic chicken-wire pattern predominantly on the lowermost epithelial layers. The result is invariably positive in active disease. PF autoantibodies produce a chicken wire pattern in the superficial epithelial layers (Figure 10). The positivity rate is approximately 79-90% [38]. False positives can result from patient serum containing antibodies to cell surface antigens[39]; antibodies can be found in patients with staphylococcal scalded skin, penicillin adverse drug reactions, toxic epidermolysis necrosis, and burns. Importantly, patients with blood group O who have antibodies to blood groups A and B may give low false positives on indirect immunofluorescence testing, which is avoided by preabsorbing their sera with these blood group antigens. Indirect immunofluorescence

Indirect immunofluorescence is not reactive with all pemphigus sera and does not differentiate between desmoglein-3 and desmoglein-1[41]. In response, ELISAs have been developed using the recombinant ectodomains of desmoglein-1 and -3 [42]. In commercially available assays,

IgG throughout the epidermis resulting in a chicken wire appearance.

titers can be used as a marker of disease activity in patients[38],[40].

**6. Enzyme-Linked Immunosorbent Assay (ELISA)**

**5.3. Indirect immunofluorescence**

**Figure 8.** High power view of a skin biopsy from a patient with pemphigus foliaceus showing clefting with acantholy‐ sis and spongiosis (hematoxylin and eosin staining).

cence shows intercellular immunoglobulin G (IgG) throughout the epidermis as a result of deposition of both anti-desmoglein-1 and -3 antibodies (Figure 9). Approximately 50% of biopsies may display complement 3 (C3) [36]. In contrast, PF is characterized by both inter‐ cellular IgG and C3 predominantly in the upper half of the epidermis due to the increased density of desmoglein-1 and subsequent antibody deposition in the superficial epidermis. The diagnostic sensitivity of direct immunofluorescence in pemphigus disease is 80-95% [38].

**Figure 9.** Direct immunofluorescence of a skin biopsy from a patient with pemphigus vulgaris revealing deposition of IgG throughout the epidermis resulting in a chicken wire appearance.

#### **5.3. Indirect immunofluorescence**

cence shows intercellular immunoglobulin G (IgG) throughout the epidermis as a result of deposition of both anti-desmoglein-1 and -3 antibodies (Figure 9). Approximately 50% of biopsies may display complement 3 (C3) [36]. In contrast, PF is characterized by both inter‐ cellular IgG and C3 predominantly in the upper half of the epidermis due to the increased density of desmoglein-1 and subsequent antibody deposition in the superficial epidermis. The diagnostic sensitivity of direct immunofluorescence in pemphigus disease is 80-95% [38].

**Figure 8.** High power view of a skin biopsy from a patient with pemphigus foliaceus showing clefting with acantholy‐

sis and spongiosis (hematoxylin and eosin staining).

**Figure 7.** Medium power view of a skin biopsy from a patient with pemphigus foliaceus showing vacuoles within the intercellular spaces of the stratum granulosum and the upper stratum spinosum of the epidermis. The papillary dermis contains an inflammatory infiltrate comprising neutrophils, eosinophils, and lymphocytes (hematoxylin and eosin

staining).

114 Skin Biopsy - Diagnosis and Treatment

The target of pemphigus is desmoglein. Desmoglein-1 and -3 are restricted to stratified squamous epithelium, whereas desmoglein-2 is expressed in all desmosome possessing tissues [8]. In indirect immunofluorescence, patient serum is incubated with epithelial substrates containing the target antigen. In pemphigus, the most frequently used substrates are monkey esophagus, which is more sensitive for the detection of PV autoantibodies, and guinea pig esophagus and rabbit lip mucosa, which are more sensitive for the diagnosis of PF [39]. In PV, indirect immunofluorescence produces a characteristic chicken-wire pattern predominantly on the lowermost epithelial layers. The result is invariably positive in active disease. PF autoantibodies produce a chicken wire pattern in the superficial epithelial layers (Figure 10). The positivity rate is approximately 79-90% [38]. False positives can result from patient serum containing antibodies to cell surface antigens[39]; antibodies can be found in patients with staphylococcal scalded skin, penicillin adverse drug reactions, toxic epidermolysis necrosis, and burns. Importantly, patients with blood group O who have antibodies to blood groups A and B may give low false positives on indirect immunofluorescence testing, which is avoided by preabsorbing their sera with these blood group antigens. Indirect immunofluorescence titers can be used as a marker of disease activity in patients[38],[40].
