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Direct immunofluorescence (DIF) microscopy of a skin biopsy specimen is the reference standard for the diagnosis of pemphigoid diseases (PDs). Serration pattern analysis enables the differentiation of epidermolysis bullosa acquisita (EBA) from other PDs using DIF microscopy alone. However, practice gaps need to be addressed in order to implement this technique in the routine diagnostic procedure.We sought to determine and optimize the technical requirements for serration pattern analysis of DIF microscopy and determine interrater conformity of serration pattern analysis.We compared serration pattern analysis of routine DIF microscopy from laboratories in Groningen, The Netherlands and Lübeck, Germany with 4 blinded observers. Skin biopsy specimens from 20 patients with EBA and other PDs were exchanged and analyzed. Various factors were evaluated, including section thickness, transport medium, and biopsy specimen processing.The interrater conformity of our 4 observers was 95.7%. Recognition of serration patterns was comparable in samples transported in saline and in Michel's medium and with section thicknesses of 4, 6, and 8 μm.Limitations include our small sample size and the availability of 20 samples that were compared retrospectively.DIF serration pattern analysis is not restricted by variation in laboratory procedures, transport medium, or experience of observers. This learnable technique can be implemented as a routine diagnostic method as an extension of DIF microscopy for subtyping PD.