Ultra-fast and automated immunohistofluorescent multistaining using a microfluidic tissue processor

• Published in: Scientific reports Vol. 9; no. 1; p. 4489
• Main Authors: Cappi, Giulia, Dupouy, Diego Gabriel, Comino, Marta Aurelia, Ciftlik, Ata Tuna
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 14.03.2019; Nature Publishing Group
• Subjects: 13/51; 13/56; 631/1647/350/877; 631/67/1347; 692/53/2421; 82/62; Automation; Automation, Laboratory; Biomarkers, Tumor - metabolism; Breast Neoplasms - metabolism; Female; Fluorescent Antibody Technique - instrumentation; Fluorescent Antibody Technique - methods; Humanities and Social Sciences; Humans; Immunohistochemistry; Keratins - metabolism; Microfluidics; Microfluidics - instrumentation; Microfluidics - methods; multidisciplinary; Peroxidase; Polymers; Proof of Concept Study; Receptor, ErbB-2 - metabolism; Receptors, Estrogen - metabolism; Receptors, Progesterone - metabolism; Science; Science (multidisciplinary)
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-41119-y

Multistaining of a tissue section targeting multiple markers allows to reveal complex interplays in a tumor environment. However, the resource-intensive and impractically long nature of iterative multiplexed immunostainings prohibits its practical implementation in daily routine, even when using work-flow automation systems. Here, we report a fully automated and ultra-fast multistaining using a microfluidic tissue processor (MTP) in as short as 20 minutes per marker, by immunofluorescent staining employing commercially available tyramide signal amplification polymer precipitation by horse-radish peroxidase (HRP) activation. The reported duration includes (i) 15 minutes for the entire fluidic exchange and reagent incubation necessary for the immunostaining and (ii) 5 minutes for the heat-induced removal of the applied antibodies. Using the automated MTP, we demonstrated a 4-plex automated multistaining with clinically relevant biomarkers within 84 minutes, showing perfect agreement with the state-of-the-art microwave treatment antibody removal. The presented HRP-based method is in principle extendable to multistaining by both tyramides accommodating higher number of fluorescent channels and multi-color chromogenic staining. We anticipate that our automated multi-staining with a turn-around time shorter than existing monoplex immunohistochemistry methods has the potential to enable multistaining in routine without disturbing the current laboratory workflow, opening perspectives for implementation of -omics approaches in tissue diagnostics.