title: Young investigator challenge: Validation and optimization of immunohistochemistry protocols for use on cellient cell block specimens.
abstract: BACKGROUND
  The objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system.
  
  
  METHODS
  Thirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin-fixed, paraffin-embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions.
  
  
  RESULTS
  Thirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC-31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber-EP4, D2-40, and paired box gene 8 [PAX8]) were not successfully validated.
  
  
  CONCLUSIONS
  Nearly one-half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol-fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs.
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Sauter
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Karen L
authors.initials: KL
authors.lastname: Grogg
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Vrana
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Mark E
authors.initials: ME
authors.lastname: Law
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Halvorson
authors.affiliation: Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Henry
mesh_terms: D000906:Antibodies / Q000276:immunology*; D003581:Cytodiagnosis / Q000379:methods*; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D009369:Neoplasms / Q000473:pathology; D012108:Research Personnel; D012680:Sensitivity and Specificity; D013048:Specimen Handling; D016610:Tissue Embedding; D016707:Tissue Fixation / Q000379:methods
chemical_list: D000906:Antibodies
keywords: Cellient; antibodies; cell block; exfoliative cytology; immunohistochemistry; validation