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Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation


Leblond, Anne-Laure; Rechsteiner, Markus; Jones, Amy; Brajkovic, Saska; Dupouy, Diego; Soltermann, Alex (2019). Microfluidic-Based Immunohistochemistry Combined With Next-Generation Sequencing on Diagnostic Tissue Sections for Detection of Tumoral BRAF V600E Mutation. American Journal of Clinical Pathology, 152(1):59-73.

Abstract

OBJECTIVES

Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas.

METHODS

For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS.

RESULTS

After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification.

CONCLUSIONS

Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality.

Abstract

OBJECTIVES

Tailored diagnostics requires immunohistochemistry (IHC) and next generation sequencing (NGS). Here we combined on a single paraffin-embedded slide microfluidic-based IHC (micro-IHC) and NGS for BRAF V600E mutation detection in BRAFomas.

METHODS

For micro-IHC, we performed the primary antibody incubation step of conventional chromogenic IHC in a LabSat device (Lunaphore Technologies SA). Tumor areas immunoreactive for pan-cytokeratin, pan-melanoma, and BRAF V600E mutation-specific antibody were H-scored, microdissected, and analyzed by NGS.

RESULTS

After 2 minutes, pan-cytokeratin and BRAF micro-IHC increased exponentially (half-time values: 1.7 and 3.2 minutes). Pan-melanoma displayed a higher half-time value of 15 minutes. There was no significant difference in H-score and staining quality, respectively, between conventional and micro-IHC. BRAF V600E mutation was detected in all pan-cytokeratin and pan-melanoma stained samples without amplification but in only 40% of BRAF V600E stained samples with amplification.

CONCLUSIONS

Micro-IHC enables short antibody incubation times and subsequent NGS. Preprocessing is critical for preservation of DNA quality.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > University Hospital Zurich > Institute of Pathology and Molecular Pathology
Dewey Decimal Classification:610 Medicine & health
Language:English
Date:5 June 2019
Deposited On:25 Jul 2019 10:37
Last Modified:25 Sep 2019 00:37
Publisher:American Society for Clinical Pathology
ISSN:0002-9173
OA Status:Closed
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1093/ajcp/aqz028
PubMed ID:31065676

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