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The salt split separates at the lamina lucida with the following conditions staining on the dermal side: Laminin γ1 (p200 pemphigoid) Anti-laminin 332 (associated with mucuous membrane pemphigoid with higher risk of internal malignancy) Collagen VII (EBA, Bullous Lupus) When examining the linear fluorescence on salt split skin at higher magnification, the serration pattern of the undulating basement membrane zone can help distinguish between these deeper conditions It separates conditions based on whether the antigens at/above the lamina densa or below Recap: Most helpful when salt split shows dermal-side binding (floor) and need to differentiate the conditions further Memory aid: 'u' for ‘under’ the lamina densa BLOOD TESTS Blood tests can be performed for the serologic detection of antibodies The conventional method performed is: Initially do indirect immunoflourescence (IIF) microscopy as a screening test This can be followed by a more antigen-specific serologic assay (eg ELISA) based on the clinical suspicion and IIF screening results INDIRECT IMMUNOFLOURESCENCE Detectes circulating antibodies in serum Very sensitive - good for screening Not as specific for determining causative antibody as ELISA IIF is a technique used to detect circulating antibodies in a patient’s serum blood test: Antibodies/complement (eg IgG, IgM IgA, C3 ) from the patient’s serum (primary antibodies) bind to the target molecules (the substrate) in pre-prepared tissue samples (eg monkey oesophagus) Then antibodies labelled with dye bind to the above antibodies The pattern of staining then seen using a fluorescent microscope Choice of substrate tissue to use may depend on clinical suspicion of the suspected disease For instance, classic prepared tissues which are better for certain diseases are: Sub-epidermal blistering diseases - Normal human skin Pemphigus vulgaris - monkey oesophagus - monkey oesophagus has high expression of Dsg 3 Pemphigus foilaceous - guinea pig oesophagus - good for dsg 1 (think of a guinea pig running in foliage) Paraneoplastic pemphigus - rat bladder - plakins (envoplakin, periplakin, desmoplakin) are highly expressed in bladder ELISA To achieve a more precise diagnosis, clinicians can identify antibodies directed against specific target antigens using ELISA (Enzyme-Linked Immunosorbent Assay) , Immunoblotting , or monospecific Indirect Immunofluorescence (IIF)