A 52-year-old woman presents with recurrent painful erosions on the buccal mucosa and palate for 3 weeks, followed by flaccid blisters on the trunk and flexural areas. Clinical examination reveals positive Nikolsky sign. Which investigation is most appropriate to confirm the diagnosis of pemphigus vulgaris?

## Investigation of Choice in Pemphigus Vulgaris **Key Point:** Direct immunofluorescence (DIF) of perilesional skin is the gold standard and most specific investigation for confirming pemphigus vulgaris. ### Why DIF is the Investigation of Choice DIF detects **in situ IgG and C3 antibodies** deposited on the surface of keratinocytes in a characteristic **intercellular (chicken-wire) pattern**. This pattern is pathognomonic for pemphigus and distinguishes it from other bullous disorders. **High-Yield:** The perilesional skin (clinically normal-appearing skin adjacent to the blister) is preferred over the blister itself because: - Antibodies are concentrated in viable epidermis - The blister roof may have sparse or absent antibody deposits - Sensitivity is highest in perilesional tissue ### Diagnostic Investigations in Pemphigus Vulgaris | Investigation | Finding | Sensitivity | Specificity | Role | | --- | --- | --- | --- | --- | | **DIF (perilesional)** | IgG + C3 in intercellular pattern | 95–100% | Nearly 100% | **Gold standard** | | H&E histopathology | Acantholysis, suprabasal clefting | 90–95% | Not specific (also in pemphigoid, herpetiform) | Supportive | | IIF (serum) | Circulating IgG anti-Dsg3 ± anti-Dsg1 | 80–90% | Specific but lower sensitivity | Confirms autoimmunity, prognostic | | Tzanck smear | Acantholytic cells | Low sensitivity | Non-specific (also HSV, VZV) | Rapid screening only | **Clinical Pearl:** DIF is positive even on clinically normal-appearing skin in pemphigus, making it invaluable for diagnosis when clinical features are atypical or early. ### Diagnostic Algorithm ```mermaid flowchart TD A[Clinical suspicion: Pemphigus]:::outcome --> B{Perform DIF on perilesional skin}:::decision B -->|Intercellular IgG + C3| C[Pemphigus confirmed]:::action C --> D[Perform H&E histology for subtype]:::action D --> E[Perform IIF serum for Dsg3/Dsg1 profile]:::action E --> F[Classify: Mucosal vs Mucocutaneous]:::outcome B -->|Negative DIF| G[Consider other diagnosis]:::urgent G --> H[Pemphigoid, Linear IgA, Dermatitis herpetiformis]:::outcome ``` **Mnemonic:** **DIF = Direct In situ Fluorescence** — directly on tissue, not serum. ### Why Other Investigations Are Secondary - **H&E histopathology** shows acantholysis and suprabasal clefting but is NOT specific (similar findings in pemphigoid, herpetiform dermatitis) - **IIF (serum)** is confirmatory and prognostically useful (Dsg3/Dsg1 antibody profile) but has lower sensitivity than DIF - **Tzanck smear** is rapid but non-specific; acantholytic cells are seen in HSV, VZV, and other acantholytic disorders