title: Immunohistochemistry of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded skeletal muscle tissue: a promising tool for the diagnostic evaluation of common muscular dystrophies.
abstract: BACKGROUND
  The analysis of fresh frozen muscle specimens is standard following routine muscle biopsy, but this service is not widely available in countries with limited medical facilities, such as Thailand. Nevertheless, immunohistochemistry (IHC) analysis is essential for the diagnosis of patients with a strong clinical suspicion of muscular dystrophy, in the absence of mutations detected by molecular genetics. As the successful labelling of sarcolemmal membrane-associated proteins in formalin-fixed and paraffin-embedded (FFPE) muscle sections using IHC staining has rarely been described, this study aimed to develop a reproducible IHC method for such an analysis.
  
  
  METHODS
  Thirteen cases were studied from the files of the Department of Pathology, Mahidol University. Diagnoses included three Duchenne muscular dystrophy (DMD), one Becker muscular dystrophy (BMD), one dysferlinopathy, and several not-specified muscular dystrophies. IHC was performed on FFPE sections at different thicknesses (3 μm, 5 μm, and 8 μm) using the heat-mediated antigen retrieval method with citrate/EDTA buffer, followed by an overnight incubation with primary antibodies at room temperature. Antibodies against spectrin, dystrophin (rod domain, C-terminus, and N-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan were used. Frozen sections were tested in parallel for comparative analysis.
  
  
  RESULTS
  Antibodies labelling spectrin, dystrophin (rod domain and C-terminus), dysferlin, sarcoglycans (α, β, and γ), and β-dystroglycan clearly exhibited sarcolemmal staining in FFPE sections. However, staining of FFPE sections using the antibody directed against the N-terminus of dystrophin was unsuccessful. The absence of labeling for dystrophins and dysferlin in FFPE sections was documented in all three DMD patients and the dysferlinopathy patient. The BMD diagnosis could not be made using IHC in FFPE sections alone because of a lack of staining for the dystrophin N-terminus, indicating a limitation of this method.
  
  
  CONCLUSIONS
  We developed a reliable and reproducible IHC technique using FFPE muscle. This could become a valuable tool for the diagnosis of some muscular dystrophies, dystrophinopathies, sarcoglycanopathies (LGMD2D, LGMD2E, and LGMD2C), and dysferlinopathy, especially in situations where the analysis of fresh frozen muscle samples is not routinely available.
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.|Department of Anatomy, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Chinnawut
authors.initials: C
authors.lastname: Suriyonplengsaeng
authors.affiliation: Division of Neurology, Department of Medicine, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Charungthai
authors.initials: C
authors.lastname: Dejthevaporn
authors.affiliation: Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Chaiyos
authors.initials: C
authors.lastname: Khongkhatithum
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Suda
authors.initials: S
authors.lastname: Sanpapant
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Nattha
authors.initials: N
authors.lastname: Tubthong
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Koset
authors.initials: K
authors.lastname: Pinpradap
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand.
authors.forename: Nippa
authors.initials: N
authors.lastname: Srinark
authors.affiliation: Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand. jariya.wai@mahidol.ac.th.
authors.forename: Jariya
authors.initials: J
authors.lastname: Waisayarat
mesh_terms: D000293:Adolescent; D000328:Adult; D002648:Child; D002675:Child, Preschool; D000073939:Dysferlin; D049030:Dystroglycans / Q000378:metabolism*; D016189:Dystrophin / Q000378:metabolism*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry; D007223:Infant; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008875:Middle Aged; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000473:pathology; D009136:Muscular Dystrophies / Q000175:diagnosis* / Q000378:metabolism; D049288:Muscular Dystrophies, Limb-Girdle / Q000175:diagnosis* / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000175:diagnosis / Q000378:metabolism; D016612:Paraffin Embedding; D015203:Reproducibility of Results; D058088:Sarcoglycanopathies / Q000175:diagnosis / Q000378:metabolism; D049031:Sarcoglycans / Q000378:metabolism*
chemical_list: C483081:DAG1 protein, human; C114839:DYSF protein, human; D000073939:Dysferlin; D016189:Dystrophin; D008565:Membrane Proteins; D009124:Muscle Proteins; D049031:Sarcoglycans; D049030:Dystroglycans
keywords: Dysferlin; Dystroglycan; Dystrophin; Dystrophinopathy; Formalin-fixed and paraffin-embedded; Immunohistochemistry; Muscle biopsy; Muscular dystrophy; Sarcoglycan; Snap frozen section