title: Reduced plakoglobin immunoreactivity in arrhythmogenic cardiomyopathy: methodological considerations.
abstract: BACKGROUND
  Arrhythmogenic cardiomyopathy (AC) primarily is considered to be a desmosomal disease with a predominant right ventricular phenotype. Reduced signal intensity for junctional plakoglobin (JUP) at the intercalated disks has been proposed as a marker that contributes to diagnosis of the disease. In this technical study, we investigated how methodology-related differences caused by tissue preservation and antibody dilutions affect an appropriate diagnosis.
  
  
  METHODS
  Autopsy and biopsy material was available from a total of 7 control and 25 AC patients that fulfilled the diagnostic Task Force Criteria as proposed in 2010. Immunohistochemical analysis was performed on cryosections and formalin-fixed material using antibodies against JUP and N-Cadherin.
  
  
  RESULTS
  Immunohistochemistry (1:1000 antibody dilution) on formalin-fixed material showed a reduced signal for JUP in 7/10 AC patients in a bidirectional, double-blinded exchange experiment in which 77% of individuals were correctly classified. Unmasking this disturbed JUP pattern was highly dependent on tissue preservation and antibody dilution since on cryosections the disturbed pattern in patients could only be unmasked at a very strong antibody dilution of 1:100000.
  
  
  CONCLUSIONS
  Reduced immunoreactive signal of JUP at the intercalated disks can be observed in a majority of AC patients. These changes can comparably be detected on both cryo- (74%) and formalin-fixed material (70%) but demand a different, highly defined, and uniformly used approach.
authors.affiliation: Department of Medical Physiology, University Medical Center Utrecht, 3584CM Utrecht, The Netherlands; Interuniversity Cardiology Institute, The Netherlands.
authors.forename: Maartje
authors.initials: M
authors.lastname: Noorman
authors.forename: Sara
authors.initials: S
authors.lastname: Hakim
authors.forename: Angeliki
authors.initials: A
authors.lastname: Asimaki
authors.forename: Arnold
authors.initials: A
authors.lastname: Vreeker
authors.forename: Harold V M
authors.initials: HV
authors.lastname: van Rijen
authors.forename: Marcel A G
authors.initials: MA
authors.lastname: van der Heyden
authors.forename: Nicolaas
authors.initials: N
authors.lastname: de Jonge
authors.forename: Roel A
authors.initials: RA
authors.lastname: de Weger
authors.forename: Richard N W
authors.initials: RN
authors.lastname: Hauer
authors.forename: Jeffrey E
authors.initials: JE
authors.lastname: Saffitz
authors.forename: Toon A B
authors.initials: TA
authors.lastname: van Veen
mesh_terms: D000906:Antibodies; D019571:Arrhythmogenic Right Ventricular Dysplasia / Q000175:diagnosis* / Q000378:metabolism*; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D051180:Desmoplakins / Q000378:metabolism*; D003896:Desmosomes / Q000378:metabolism; D004311:Double-Blind Method; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D014021:Tissue Preservation; D051185:gamma Catenin
chemical_list: D000906:Antibodies; D015415:Biomarkers; D051180:Desmoplakins; C495292:JUP protein, human; D051185:gamma Catenin
keywords: Arrhythmogenic cardiomyopathy; Biopsy; Immunohistochemistry; Plakoglobin