nad a22 4500 551468335 20181215081820.0 cr unu---uuuuu 181215e20181016xx s 000 0 eng 10.5167/uzh-159447 doi 10.1186/s13000-018-0757-1 doi (ZORA)oai:www.zora.uzh.ch:159447 610 ddc 30326973 pmid Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma [Elektronische Daten] [Saška Brajkovic, Benjamin Pelz, Maria-Giuseppina Procopio, Anne-Laure Leblond, Grégoire Repond, Ariane Schaub-Clerigué, Diego G Dupouy, Alex Soltermann] openAccess eu-repo BACKGROUND Anaplastic lymphoma kinase (ALK) is a key oncogenic driver in lung adenocarcinoma patients and its fusion proteins are routinely assessed. The microfluidic tissue processor (MTP) device is based on a chip-confined low-volume technology allowing for rapid immunohistochemistry/immunofluorescence (IHC/IF) stainings of formalin-fixed paraffin-embedded (FFPE) or frozen tissue samples. METHODS A novel ALK IF protocol was developed for the MTP device using the primary mouse anti-human ALK antibody clone 5A4. FFPE tumor whole sections from 14 resected lung adenocarcinoma patients documented to be ALK positive (ALK+) by automated chromogenic IHC and/or FISH were used. MTP-derived IF immunoreactivity was measured by computerized analysis of digitalized images on individual frames of tumor epithelia and surrounding stroma, using an ImageJ plug-in. RESULTS The 5A4 antibody yielded saturated immunoreactivity at an incubation time of 4 min on a titration curve ranging from 2 to 32 min. Total staining time on the MTP device was 18 min including secondary IgG Alexa Fluor 647. MTP-based ALK IF confirmed all 12 cases; with epithelial signal above stromal staining based on computerized pixel-based measurement. MTP-IF (mean intensity levels 458 to 1301) and chromogenic IHC (H-score 120 to 300) showed an equal range of variation of 2.8 and 2.5 folds, respectively, and a trend for direct correlation (p-value 0.051). CONCLUSION The newly developed protocol for immunofluorescent detection of ALK protein with the MTP device confirms chromogenic IHC results on FFPE lung adenocarcinoma specimens. MTP-based IF is fast and reliable. We foresee this study to be a first step opening the road for further realization of microfluidic-based assays for rapid simultaneous detection of targetable oncogenic and immune-system related markers in their topographical context to investigate tumour heterogeneity and micro-environmental interactions. Institute of Pathology and Molecular Pathology zora Brajkovic Saška joint author Pelz Benjamin joint author Procopio Maria-Giuseppina joint author Leblond Anne-Laure joint author Repond Grégoire joint author Schaub-Clerigué Ariane joint author Dupouy Diego G. joint author Soltermann Alex joint author Diagnostic Pathology 13:79 https://www.zora.uzh.ch/id/eprint/159447/1/Brajkovic_diagnostic_pathology_2018.pdf text/html WWW-Backlink auf das Repository 1 Journal Article PeerReviewed zora ZORA 856 40 https://www.zora.uzh.ch/id/eprint/159447/1/Brajkovic_diagnostic_pathology_2018.pdf text/html WWW-Backlink auf das Repository ZORA 700 1- Brajkovic Saška joint author ZORA 700 1- Pelz Benjamin joint author ZORA 700 1- Procopio Maria-Giuseppina joint author ZORA 700 1- Leblond Anne-Laure joint author ZORA 700 1- Repond Grégoire joint author ZORA 700 1- Schaub-Clerigué Ariane joint author ZORA 700 1- Dupouy Diego G. joint author ZORA 700 1- Soltermann Alex joint author ZORA 773 0- Diagnostic Pathology 13:79 BK010053 XK010053 XK010000 ZORA ZORA ZORA Journal Article openAccess