Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.
- Brajkovic S Lunaphore Technologies SA, EPFL Innovation Park-Building A, CH-1015, Lausanne, Switzerland. saska.brajkovic@lunaphore.com.
- Pelz B Microsystems Laboratory 2, Swiss Federal Institute of Technology, CH-1015, Lausanne, Switzerland.
- Procopio MG Institute of Pathology and Molecular Pathology, University Hospital Zurich, CH-8091, Zurich, Switzerland.
- Leblond AL Institute of Pathology and Molecular Pathology, University Hospital Zurich, CH-8091, Zurich, Switzerland.
- Repond G Lunaphore Technologies SA, EPFL Innovation Park-Building A, CH-1015, Lausanne, Switzerland.
- Schaub-Clerigué A Lunaphore Technologies SA, EPFL Innovation Park-Building A, CH-1015, Lausanne, Switzerland.
- Dupouy DG Lunaphore Technologies SA, EPFL Innovation Park-Building A, CH-1015, Lausanne, Switzerland.
- Soltermann A Institute of Pathology and Molecular Pathology, University Hospital Zurich, CH-8091, Zurich, Switzerland.
- 2018-10-18
Published in:
- Diagnostic pathology. - 2018
Anaplastic lymphoma kinase
Immunofluorescence
Lung adenocarcinoma
Microfluidic tissue processor
Adenocarcinoma of Lung
Anaplastic Lymphoma Kinase
Carcinoma, Non-Small-Cell Lung
Gene Rearrangement
Humans
In Situ Hybridization, Fluorescence
Lung Neoplasms
Receptor Protein-Tyrosine Kinases
Sensitivity and Specificity
English
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.
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.
- Language
-
- English
- Open access status
- gold
- Identifiers
-
- DOI 10.1186/s13000-018-0757-1
- PMID 30326973
- Persistent URL
- https://folia.unifr.ch/global/documents/3674
Statistics
Document views: 25
File downloads:
- fulltext.pdf: 0