Best Practices for Technical Reproducibility Assessment of Multiplex Immunofluorescence

Multiplex immunofluorescence (mIF) tyramide signal amplification is a new and useful tool for the study of cancer that combines the staining of multiple markers in a single slide. Several technical requirements are important to performing high-quality staining and analysis and to obtaining high internal and external reproducibility of the results. This review manuscript aimed to describe the mIF panel workflow and discuss the challenges and solutions for ensuring that mIF panels have the highest reproducibility possible. Although this platform has shown high flexibility in cancer studies, it presents several challenges in pre-analytic, analytic, and post-analytic evaluation, as well as with external comparisons. Adequate antibody selection, antibody optimization and validation, panel design, staining optimization and validation, analysis strategies, and correct data generation are important for reproducibility and to minimize or identify possible issues during the mIF staining process that sometimes are not completely under our control, such as the tissue fixation process, storage, and cutting procedures.

On the problematic placement of the fossil arthropod Devonopilio hutchinsoni in Opiliones (Arachnida)

Harvestmen (Arachnida: Opiliones) were among the earliest terrestrial arthropods but their unmineralized exoskeletons are scarce and often fragmentary as fossils (Palencia et al. 2019). Consequently, the discovery and interpretations of fossil harvestmen from the early Palaeozoic can have disproportionate effects on the understanding of evolution in Opiliones. Recently, Devonopilio hutchinsoni Tihelka, Tian & Cai, 2020, was described as a new fossil harvestman from the well-known Rhynie chert deposits of Scotland, an important source of information on freshwater and terrestrial ecosystems of the early Devonian (Tihelka et al. 2020). This species would be one of the earliest records of harvestmen. The description was based on a single slide showing fragments of arthropod cuticle, which Tihelka et al. interpreted as a harvestmen penis and unspecified associated body parts. As specialists on harvestman morphology and systematics, we were intrigued by these conclusions. However, based on the material presented by Tihelka et al. we find no compelling evidence supporting the proposal that the specimen is a harvestman.

Locations optimization of multiple cable-drivers for an anti-wind disturbance system in large antenna

Since the structural vibration deformation of a large antenna under wind disturbance leads to the pointing deterioration, an adaptive anti-wind disturbance system was presented by Liang et al. (Int. J. Antennas Propag., Article ID 2015341, 2019). To further improve the vibration suppression performance, this paper presents a locations-optimization algorithm of multiple cable-drivers for the anti-wind disturbance system. First, according to the spatial geometric relationship among the antenna structure, single slide track, four cables and drivers, the feasible domain of the drivers is determined. Next, the separate optimization method, and the joint optimization method for the locations of the four drivers are proposed, respectively. Finally, the simulation implementation of a 7.3 m antenna under various wind conditions is used to compare the joint optimization method and the separate optimization method.

Procedural Requirements and Recommendations for Multiplex Immunofluorescence Tyramide Signal Amplification Assays to Support Translational Oncology Studies

In the development of a multiplex immunofluorescence (IF) platform and the optimization and validation of new multiplex IF panels using a tyramide signal amplification system, several technical requirements are important for high-quality staining, analysis, and results. The aim of this review is to discuss the basic requirements for performing multiplex IF tyramide signal amplification (TSA) in formalin-fixed, paraffin-embedded cancer tissues to support translational oncology research. Our laboratory has stained approximately 4000 formalin-fixed, paraffin-embedded tumor samples using the multiplex IF TSA system for immune profiling of several labeled biomarkers in a single slide to elucidate cancer biology at a protein level and identify therapeutic targets and biomarkers. By analyzing several proteins in thousands of cells on a single slide, this technique provides a systems-level view of various processes in various tumor tissues. Although this technology shows high flexibility in cancer studies, it presents several challenges when applied to study different histology cancers. Our experience shows that adequate antibody validation, staining optimization, analysis strategies, and data generation are important steps for generating quality results. Tissue management, fixation procedures, storage, and cutting can also affect the results of the assay and must be standardized. Overall, this method is reliable for supporting translational research given a precise, step-by-step approach.

Microarray Embedding/Sectioning for Parallel Analysis of 3D Cell Spheroids

Three-dimensional cell spheroid models can be used to predict the effect of drugs and therapeutics and to model tissue development and regeneration. The utility of these models is enhanced by high throughput 3D spheroid culture technologies allowing researchers to efficiently culture numerous spheroids under varied experimental conditions. Detailed analysis of high throughput spheroid culture is much less efficient and generally limited to narrow outputs, such as metabolic viability. We describe a microarray approach that makes traditional histological embedding/sectioning/staining feasible for large 3D cell spheroid sample sets. Detailed methodology to apply this technology is provided. Analysis of the technique validates the potential for efficient histological analysis of up to 96 spheroids in parallel. By integrating high throughput 3D spheroid culture technologies with advanced immunohistochemical techniques, this approach will allow researchers to efficiently probe expression of multiple biomarkers with spatial localization within 3D structures. Quantitative comparison of staining will have improved inter- and intra-experimental reproducibility as multiple samples are collectively processed, stained, and imaged on a single slide.

Multiplex fluorescence in situ hybridisation to detect anaplastic lymphoma kinase and ROS proto-oncogene 1 receptor tyrosine kinase rearrangements in lung cancer cytological samples

AimsSeveral predictive biomarkers of response to specific inhibitors have become mandatory for the therapeutic choice in non-small-cell lung cancer (NSCLC). In most lung cancer patients, the biological materials available to morphological and molecular diagnosis are exclusively cytological samples and minimum tumour wastage is necessary. Multiplex fluorescence in situ hybridisation (mFISH) to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide could be useful in clinical practice to save cytological samples for further molecular analysis. In this study, we aim to validate diagnostic performance of multiplex ALK/ROS1 fluorescence in situ hybridisation (FISH) approach in lung adenocarcinoma cytological series compared with classic single break apart probes.MethodsWe collected a series of 61 lung adenocarcinoma cytological specimens enriched in tumours harbouring ALK-rearrangement and ROS1-rearrangement. ALK and ROS1 status were previously assessed by classic FISH test using single break apart probes and immunohistochemistry. Study population was composed of 6 ALK-positive, 2 ROS1-positive and 53 ALK/ROS1-wild type. All specimens were analysed by multiplex FISH assay using FlexISH ALK/ROS1 DistinguISH Probe Zytovision.ResultsThe dual ALK/ROS1 FISH probe test results were fully concordant with the results of previous single ALK and ROS1 FISH tests on two different slides. 6 ALK-positive and 2 ROS1-positive were confirmed through multiplex FISH test, without false-positive and false-negative results. Multiplex ALK/ROS1 FISH test results agreed with immunohistochemistry assay staining results.ConclusionMultiplex ALK/ROS1 FISH probe test is a useful tool to detect simultaneously ALK-rearrangement and ROS1-rearrangement on a single slide in cytological specimens with a small amount of biomaterial.