Recommended Stains and Ancillary Studies for Foot Lesion Biopsy
For a foot lesion biopsy, order routine hematoxylin-and-eosin (H&E) histology, periodic acid-Schiff (PAS) stain for fungal elements, and consider direct immunofluorescence (DIF) only if clinical features suggest a bullous dermatosis or autoimmune process.
Routine Histology: Hematoxylin and Eosin (H&E)
H&E staining is the essential foundation for all tissue diagnosis, providing broad visualization of nuclear detail, cytoplasmic features, and extracellular matrix that enables recognition of tissue types and morphologic changes 1, 2.
H&E sections should be cut at 2 mm thickness with at least two sections placed on each slide to optimize diagnostic yield 1.
Limitation of H&E alone: Fungal elements may be misinterpreted as artifacts when stained only with H&E, and hyphae are poorly visualized without special fungal stains 1.
Periodic Acid-Schiff (PAS) Stain for Fungal Elements
PAS staining should be performed routinely on all foot lesion biopsies, as fungal infections are frequently unsuspected clinically and may present with non-specific inflammatory patterns 3, 4.
In a study of 403 skin biopsies, routine PAS staining identified fungi in 10% of cases, but fungal infection had been suspected clinically in only 5 of those 34 positive cases 3.
Only 45% of PAS-positive tinea cases were diagnosed clinically, and H&E examination alone detected hyphal elements in only 45-68% of confirmed fungal infections 4.
PAS is sensitive, inexpensive, easy to perform, and reproducible for detecting fungal elements in skin biopsies 1.
Alternative fungal stains: Grocott methenamine silver (GMS) stain may be more sensitive than PAS (15% false negative rate for GMS versus 44% for PAS), though neither is statistically superior 5. Optical brighteners such as calcofluor white provide rapid visualization but require fluorescence microscopy 1.
Direct Immunofluorescence (DIF)
DIF should be requested selectively based on clinical presentation, not routinely for all foot lesions.
Indications for DIF:
Bullous dermatoses: If the foot lesion shows blistering, sloughing, erosions, or clinical features suggesting pemphigus, pemphigoid, or lichen planus, obtain additional biopsies specifically for DIF 1.
Autoimmune connective tissue diseases: If clinical history suggests lupus, dermatomyositis, or other autoimmune conditions with cutaneous manifestations, DIF may reveal immunoglobulin or complement deposition 1.
Vasculitis: If purpuric lesions or palpable purpura are present, DIF can detect vascular immunoglobulin or complement deposition 1.
Technical requirements for DIF:
DIF is best performed on unfixed, frozen sections transported fresh on saline-soaked gauze or in Michel's fixative 1.
Sections should be cut at 2-4 mm thickness in a cryostat 1.
Send DIF specimens to a pathology center of excellence with dermatopathology expertise to ensure proper interpretation 1.
Routine antigens examined include IgG, IgM, IgA, C3, C1q, C4, fibrin, and kappa/lambda light chains 1.
Common Pitfalls and How to Avoid Them
Do not rely on H&E alone to exclude fungal infection: Even experienced pathologists miss fungal elements on H&E in 32-55% of proven cases 4. Always order PAS staining when the clinical presentation is non-specific or inflammatory 3, 4.
Do not order DIF routinely for all foot lesions: DIF requires special handling (fresh or Michel's fixative) and is expensive 1. Reserve it for cases with clinical features suggesting bullous disease, vasculitis, or autoimmune conditions 1.
Ensure adequate tissue sampling: Submit sufficient tissue for both routine histology and special stains, as specimen loss during processing is common with small biopsies 1.
Provide detailed clinical information to the pathologist: Include duration of symptoms, distribution of lesions, systemic symptoms, immunosuppression status, recent travel, and suspected diagnoses to guide appropriate stain selection 1.
Serial sectioning increases diagnostic yield: Multiple sections from each biopsy sample improve detection of focal lesions, particularly for fungal infections 1, 3.