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Published on: February 26, 2015
An ultrastructural comparison of dermo-epidermal separation techniques
E M Willsteed1, B S Bhogal, A Das
1Departsment of Immunofluorescence, St John's Hospital for Diseases of the Skin, London, England.
Insights
1 molar sodium chloride (NaCl) effectively separates skin layers at the lamina lucida for diagnostic studies. Thermolysin causes intra-epidermal separation, while suction blisters disrupt hemidesmosomes, making 1 M NaCl the most reliable method.
Area of Science:
- Dermatology
- Immunofluorescence
- Electron Microscopy
Background:
- Dermo-epidermal separation at the lamina lucida is crucial for immunoblotting and diagnosing autoimmune bullous diseases.
- Current methods include suction blister induction and 1 molar sodium chloride (NaCl) incubation.
- Thermolysin, a proteolytic enzyme, is a newer method for skin separation.
Purpose of the Study:
- To compare the electron microscopic appearance of skin separation using suction blisters, 1 M NaCl, and thermolysin.
- To evaluate the reliability and reproducibility of these methods for dermo-epidermal separation.
Main Methods:
- Electron microscopy was used to examine five specimens from each method: suction blister induction, 1 M NaCl incubation, and thermolysin treatment.
- Skin separation quality was assessed at the dermo-epidermal junction, specifically the lamina lucida.
Main Results:
- Both suction blister formation and 1 M NaCl treatment yielded clean separation through the lamina lucida in all specimens.
- Thermolysin treatment resulted in intra-epidermal separation in four cases and failed to achieve lamina lucida separation in three.
- Suction blister formation was associated with hemidesmosome disruption.
Conclusions:
- Incubation in 1 M NaCl is the most reproducible, convenient, and reliable method for laboratory dermo-epidermal separation.
- Thermolysin is less effective for achieving lamina lucida separation and can cause undesirable intra-epidermal cleavage.
- 1 M NaCl provides superior results for techniques requiring intact lamina lucida separation.
Abstract:
Dermo-epidermal separation through the lamina lucida is an essential technique for immunoblotting studies and for the diagnostic immunofluorescence of autoimmune bullous diseases. The most widely used methods of producing skin separation in the laboratory are suction blister induction and incubation in 1 molar sodium chloride. More recently the use of a proteolytic enzyme, thermolysin, has been described for this purpose. We examined the electron microscopic appearance of five suction blisters, five skin specimens separated by 1 M NaCl, and five treated with thermolysin. Both suction blister formation and treatment with 1 M NaCl resulted in a clean separation through the lamina lucida in all specimens examined. However specimens treated with thermolysin developed intra-epidermal separation in four cases without any lamina lucida separation in three. Suction blister formation was associated with hemidesmosome disruption. Incubation in 1 M NaCl remains the most reproducible, convenient, and reliable method of producing dermo-epidermal separation in the laboratory.
