An ultrastructural comparison of dermo‐epidermal separation techniques

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.

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol

“…In contrast to the findings of Walzer et al [29], our results indicate that a 1-2 -h incubation with ther molysin at 4°C does not provide a reliable dermoepidermal separation. This is supported by WilJsteed et al [30] who observed intraepidermal separation in four of five cases after thermolysin incubation for 1 h at 4°C. Even in cubation for 2 h at 37°C, as used by Germain et al [10], does not provide a reliable separation in psoriatic skin.…”

“…junction, providing an epidermal sheet with a com plete basal layer 129|. Subsequent incubation with 204 trypsin provides single-cell suspensions suitable for flow cytometric analysis [10], Various incubation times and temperatures have been proposed by different authors ( 10,25,29,30], So far, thermolysin-trypsin separation has not been validated in hyperproliferative skin.…”

Mucosal keratinocyte isolation: a short comparative study on thermolysin and dispase

“…Suprabasal split of the epidermis using thermolysin in keratinizing skin has been reported previously. 21 No histological analysis has been reported on the use of thermolysin for nonkeratinizing mucosa.…”

“…The keratinocytes located in the basal layer are the cells with the highest proliferative capacities in vitro; therefore these are the cells that need to be isolated to establish successful 2,18,20 Widely used cell isolation techniques include explantation and enzymatic dissociation. Enzymatic protocols often involve a two-step procedure, using either dispase or thermolysin 4,5,7,14,21 to separate the epidermis from the dermis. Keratinocytes are typically cultured on a feeder layer of lethally irradiated fibroblasts.…”

Linear IgA Disease: A Heterogeneous Disease

“…This pattern has previously been described by us with suction blisters and prolonged incuba tion with salt-split skin [2,30], These results suggest that there are at least 2 antigens. The change from the epidermal to the combined pattern with different methods of splitting may indicate 2 antigens or 2 epitopes, as there are differ ences in ultrastructure after salt and suction splitting [31].…”