title: A novel, automated technology for multiplex biomarker imaging and application to breast cancer.
abstract: OBJECTIVE
  Multiplexed immunofluorescence is a powerful tool for validating multigene assays and understanding the complex interplay of proteins implicated in breast cancer within a morphological context. We describe a novel technology for imaging an extended panel of biomarkers on a single, formalin-fixed paraffin-embedded breast sample and evaluating biomarker interaction at a single-cell level, and demonstrate proof-of-concept on a small set of breast tumours, including those which co-express hormone receptors with Her2/neu and Ki-67.
  
  
  RESULTS
  Using a microfluidic flow cell, reagent exchange was automated and consisted of serial rounds of staining with dye-conjugated antibodies, imaging and chemical deactivation. A two-step antigen retrieval process was developed to satisfy all epitopes simultaneously, and key parameters were optimized. The imaging sequence was applied to seven breast tumours, and compared with conventional immunohistochemistry. Single-cell correlation analysis was performed with automated image processing.
  
  
  CONCLUSIONS
  We have described a novel platform for evaluating biomarker co-localization. Expression in multiplexed images is consistent with conventional immunohistochemistry. Automation reduces inconsistencies in staining and positional shifts, while the fluorescent dye cycling approach dramatically expands the number of biomarkers which can be visualized and quantified on a single tissue section.
authors: Sunnybrook Research Institute, Toronto, ON, Canada.
authors: Gina M
authors: GM
authors: Clarke
authors: Judit T
authors: JT
authors: Zubovits
authors: Kashan A
authors: KA
authors: Shaikh
authors: Dan
authors: D
authors: Wang
authors: Sean R
authors: SR
authors: Dinn
authors: Alex D
authors: AD
authors: Corwin
authors: Alberto
authors: A
authors: Santamaria-Pang
authors: Qing
authors: Q
authors: Li
authors: Sharon
authors: S
authors: Nofech-Mozes
authors: Kela
authors: K
authors: Liu
authors: Zhengyu
authors: Z
authors: Pang
authors: Robert J
authors: RJ
authors: Filkins
authors: Martin J
authors: MJ
authors: Yaffe
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D001940:Breast / Q000378:metabolism* / Q000473:pathology; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*
chemical_list: D014408:Biomarkers, Tumor
keywords: biomarker multiplex imaging; breast cancer; computer-assisted detection; immunofluorescence; microfluidics