Semi-automated approaches for interrogating spatial heterogeneity of tissue samples.

Tissues are spatially orchestrated ecosystems composed of heterogeneous cell populations and non-cellular elements. Tissue components' interactions shape the biological processes that govern homeostasis and disease, thus comprehensive insights into tissues' composition are crucial for understanding their biology. Recently, advancements in the spatial biology field enabled the in-depth analyses of tissue architecture at single-cell resolution, while preserving the structural context. The increasing number of biomarkers analyzed, together with whole tissue imaging, generate datasets approaching several hundreds of gigabytes in size, which are rich sources of valuable knowledge but require investments in infrastructure and resources for extracting quantitative information. The analysis of multiplex whole-tissue images requires extensive training and experience in data analysis. Here, we showcase how a set of open-source tools can allow semi-automated image data extraction to study the spatial composition of tissues with a focus on tumor microenvironment (TME). With the use of Lunaphore COMET platform, we interrogated lung cancer specimens where we examined the expression of 20 biomarkers. Subsequently, the tissue composition was interrogated using an in-house optimized nuclei detection algorithm followed by a newly developed image artifact exclusion approach. Thereafter, the data was processed using several publicly available tools, highlighting the compatibility of COMET-derived data with currently available image analysis frameworks. In summary, we showcased an innovative semi-automated workflow that highlights the ease of adoption of multiplex imaging to explore TME composition at single-cell resolution using a simple slide in, data out approach. Our workflow is easily transferrable to various cohorts of specimens to provide a toolset for spatial cellular dissection of the tissue composition.

Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Acoustofluidic large-scale mixing for enhanced microfluidic immunostaining for tissue diagnostics.

The usage of microfluidics for automated and fast immunoassays has gained a lot of interest in the last decades. This integration comes with certain challenges, like the reconciliation of laminar flow patterns of micro-scale systems with diffusion-limited mass transport. Several methods have been investigated to enhance microfluidic mixing in microsystems, including acoustic-based fluidic streaming. Here, we report both by numerical simulation and experiments on the beneficiary effect of acoustic agitation on the uniformity of immunostaining in large-size and thin microfluidic chambers. Moreover, we investigate by numerical simulation the impact of reducing the incubation times and the concentrations of the biochemical detection reagents on the obtained immunoassay signal. Finally, acoustofluidic mixing was successfully used to reduce by 80% the incubation time of the Her2 (human epidermal growth factor receptor 2) and CK (cytokeratins) biomarkers for the spatial immunostaining of breast cancer cell pellets, or reducing their concentration by 66% and achieving a higher signal-to-background ratio than comparable spatially resolved immunostaining with static incubation.

Efficient AC electrothermal flow (ACET) on-chip for enhanced immunoassays.

Biochemical reaction rates in microfluidic systems are known to be limited by the diffusional transport of reagents, leading often to lowered sensitivity and/or longer detection times in immunoassays. Several methods, including electrically powering electrodes to generate AC electrothermal flow (ACET) on-chip, have been adopted to enhance the mass transport of the reagents and improve microfluidic mixing. Here, we report a novel ACET electrode design concept for generating in-plane microfluidic mixing vortices that act over a large volume close to the reaction surface of interest. This is different from the traditional ACET parallel electrode design that provides rather local vertical mixing vortices directly above the electrodes. Both numerical simulation and experimental studies were performed to validate the new design. Moreover, numerical simulation was carried out to show the effects of experimental factors such as the reaction kinetics (association constant) and the reagent concentration on the ACET-enhanced surface-based assays. As a proof of concept, the new design for the ACET-enhanced immunoassays was used to improve the immunostaining signal of the HER2 (human epidermal growth factor receptor 2) cancer biomarker on breast cancer cells. Finally, the concept of scaling up the design has been validated by experiments (immunoassays on breast cancer cells for different ACET power and different assay times). In particular, we show that larger ACET in-plane designs can agitate and mix the fluid over large microfluidic volumes, which further enhances the immunoassay's output. We have achieved a 6-times enhancement in the assay signal with a 75% reduction in assay time.

Microfluidics-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections.

Because of the close interaction between tumors and the immune system, immunotherapies are nowadays considered as the most promising treatment against cancer. In order to define the diagnosis and the subsequent therapy, crucial information about the immune cells at the tumor site is needed. Indeed, different types or activation status of cells may be indicative for specific and personalized treatments. Here, we present a quantitative method to identify ten different immuno-markers in the same tumor cut section, thereby saving precious samples and enabling correlative analysis on several cell families and their activation status in a tumor microenvironment context. We designed and fabricated a microfluidic chip with optimal thermomechanical and optical properties for fast delivery of reagents on tissue slides and for fully automatic imaging by integration with an optical microscope. The multiplexing capability of the system is enabled by an optimized cyclic immunofluorescence protocol, with which we demonstrated quantitative sequential immunostaining of up to ten biomarkers on the same tissue section. Furthermore, we developed high-quality image-processing algorithms to map each cell in the entire tissue. As proof-of-concept analyses, we identified coexpression and colocalization patterns of biomarkers to classify the immune cells and their activation status. Thanks to the quantitativeness and the automation of both the experimental and analytical methods, we believe that this multiplexing approach will meet the increasing clinical need of personalized diagnostics and therapy in cancer pathology.

Microfluidic-based immunohistochemistry for breast cancer diagnosis: a comparative clinical study.

Breast cancer is a highly heterogeneous disease. The efficacy of tailored therapeutic strategies relies on the precise detection of diagnostic biomarkers by immunohistochemistry (IHC). Therefore, considering the increasing incidence of breast cancer cases, a concomitantly time-efficient and accurate diagnosis is clinically highly relevant. Microfluidics is a promising innovative technology in the field of tissue diagnostic, enabling for rapid, reliable, and automated immunostaining. We previously reported the microfluidic-based HER2 (human epidermal growth factor receptor 2) detection in breast carcinomas to greatly correlate with the HER2 gene amplification level. Here, we aimed to develop a panel of microfluidic-based IHC protocols for prognostic and therapeutic markers routinely assessed for breast cancer diagnosis, namely HER2, estrogen/progesterone receptor (ER/PR), and Ki67 proliferation factor. The microfluidic IHC protocol for each marker was optimized to reach high staining quality comparable to the standard procedure, while concomitantly shortening the staining time to 16 min-excluding deparaffinization and antigen retrieval step-with a turnaround time reduction up to 7 folds. Comparison of the diagnostic score on 50 formaldehyde-fixed paraffin-embedded breast tumor resections by microfluidic versus standard staining showed high concordance (overall agreement: HER2 94%, ER 95.9%, PR 93.6%, Ki67 93.7%) and strong correlation (ρ coefficient: ER 0.89, PR 0.88, Ki67 0.87; p < 0.0001) for all the analyzed markers. Importantly, HER2 genetic reflex test for all discordant cases confirmed the scores obtained by the microfluidic technique. Overall, the microfluidic-based IHC represents a clinically validated equivalent approach to the standard chromogenic staining for rapid, accurate, and automated breast cancer diagnosis.

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

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.

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

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.

Microfluidics-based immunofluorescence for fast staining of ALK in lung adenocarcinoma.

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.

Microfluidics for rapid cytokeratin immunohistochemical staining in frozen sections.

Frozen sections (FS) of tumor samples represent a cornerstone of pathological intraoperative consultation and have an important role in the microscopic analysis of specimens during surgery. So far, immunohistochemical (IHC) stainings on FS have been demonstrated for a few markers using manual methods. Microfluidic technologies have proven to bring substantial improvement in many fields of diagnostics, though only a few microfluidic devices have been designed to improve the performance of IHC assays. In this work, we show optimization of a complete pan-cytokeratin chromogenic immunostaining protocol on FS using a microfluidic tissue processor into a protocol taking <12 min. Our results showed specificity and low levels of background. The dimensions of the microfluidic prototype device are compatible with the space constraints of an intraoperative pathology laboratory. We therefore anticipate that the adoption of microfluidic technologies in the field of surgical pathology can significantly improve the way FSs influence surgical procedures.

Continuous quantification of HER2 expression by microfluidic precision immunofluorescence estimates HER2 gene amplification in breast cancer.

Chromogenic immunohistochemistry (IHC) is omnipresent in cancer diagnosis, but has also been criticized for its technical limit in quantifying the level of protein expression on tissue sections, thus potentially masking clinically relevant data. Shifting from qualitative to quantitative, immunofluorescence (IF) has recently gained attention, yet the question of how precisely IF can quantify antigen expression remains unanswered, regarding in particular its technical limitations and applicability to multiple markers. Here we introduce microfluidic precision IF, which accurately quantifies the target expression level in a continuous scale based on microfluidic IF staining of standard tissue sections and low-complexity automated image analysis. We show that the level of HER2 protein expression, as continuously quantified using microfluidic precision IF in 25 breast cancer cases, including several cases with equivocal IHC result, can predict the number of HER2 gene copies as assessed by fluorescence in situ hybridization (FISH). Finally, we demonstrate that the working principle of this technology is not restricted to HER2 but can be extended to other biomarkers. We anticipate that our method has the potential of providing automated, fast and high-quality quantitative in situ biomarker data using low-cost immunofluorescence assays, as increasingly required in the era of individually tailored cancer therapy.

Programmable parylene-C bonding layer fluorescence for storing information on microfluidic chips.

We demonstrate data storage on glass/silicon microfluidic devices fabricated using parylene-C as a bonding layer. In particular, we report intermediate parylene-C bonding layer fluorescence (iPBLF) and its use as an on-chip medium for data storage by dynamic programming of iPBLF intensity, using alternating exposure of parylene-C to UV and Green light. This technique allows data on the microfluidic chip to be read, written and erased by a common fluorescent microscope. Until now, no studies have focused on storing data like expiry date, protocol or operational parameters on a chip. However, this can be useful to overcome certain automation challenges in industrial applications for which communication of information is required, like needed during operation of remote microfluidic platforms. Finally, we also demonstrate the application of iPBLF for detecting channel dimensions and positions, and for marking on-chip zones of particular interest.