Tissue Multiplex Analyte Detection in Anatomic Pathology - Pathways to Clinical Implementation.

Background: Multiplex tissue analysis has revolutionized our understanding of the tumor microenvironment (TME) with implications for biomarker development and diagnostic testing. Multiplex labeling is used for specific clinical situations, but there remain barriers to expanded use in anatomic pathology practice. Methods: We review immunohistochemistry (IHC) and related assays used to localize molecules in tissues, with reference to United States regulatory and practice landscapes. We review multiplex methods and strategies used in clinical diagnosis and in research, particularly in immuno-oncology. Within the framework of assay design and testing phases, we examine the suitability of multiplex immunofluorescence (mIF) for clinical diagnostic workflows, considering its advantages and challenges to implementation. Results: Multiplex labeling is poised to radically transform pathologic diagnosis because it can answer questions about tissue-level biology and single-cell phenotypes that cannot be addressed with traditional IHC biomarker panels. Widespread implementation will require improved detection chemistry, illustrated by InSituPlex technology (Ultivue, Inc., Cambridge, MA) that allows coregistration of hematoxylin and eosin (H&E) and mIF images, greater standardization and interoperability of workflow and data pipelines to facilitate consistent interpretation by pathologists, and integration of multichannel images into digital pathology whole slide imaging (WSI) systems, including interpretation aided by artificial intelligence (AI). Adoption will also be facilitated by evidence that justifies incorporation into clinical practice, an ability to navigate regulatory pathways, and adequate health care budgets and reimbursement. We expand the brightfield WSI system "pixel pathway" concept to multiplex workflows, suggesting that adoption might be accelerated by data standardization centered on cell phenotypes defined by coexpression of multiple molecules. Conclusion: Multiplex labeling has the potential to complement next generation sequencing in cancer diagnosis by allowing pathologists to visualize and understand every cell in a tissue biopsy slide. Until mIF reagents, digital pathology systems including fluorescence scanners, and data pipelines are standardized, we propose that diagnostic labs will play a crucial role in driving adoption of multiplex tissue diagnostics by using retrospective data from tissue collections as a foundation for laboratory-developed test (LDT) implementation and use in prospective trials as companion diagnostics (CDx).

The InSituPlex® Staining Method for Multiplexed Immunofluorescence Cell Phenotyping and Spatial Profiling of Tumor FFPE Samples.

Multiplexed immunohistochemistry (mIHC) enables the detection, quantification, and localization of many markers within cell or tissue samples, leading to a better understanding of the architecture of a disease at the cellular level. Current mIHC techniques involve long staining and assay times, require dedicated and/or captive instrumentation, and entail tedious assay optimization, hindering their establishment as routine methods. Here, we demonstrate the use of the InSituPlex® method for spatial profiling of immuno-oncology targets in FFPE tumor tissue with the UltiMapper™ I/O PD-L1 multiplex assay. The panel consists of five protein markers to profile immune infiltration and PD-L1 expression and includes CD8, CD68, PD-L1, pan CK, and SOX10 markers. The assay shows benefits of high and low expression of markers, coexpression and colocalization of proteins in single cells, and completion of staining and image acquisition in 5.5 h. Through the combination of multiplexed characterization of protein expression in whole tissue sections, fast staining workflow, and compatibility with existing instrumentation, the InSituPlex method provides a robust modality for deep phenotyping of the tumor and its microenvironment.

Protein Counting in Single Cancer Cells.

The cell is the basic unit of biology and protein expression drives cellular function. Tracking protein expression in single cells enables the study of cellular pathways and behavior but requires methodologies sensitive enough to detect low numbers of protein molecules with a wide dynamic range to distinguish unique cells and quantify population distributions. This study presents an ultrasensitive and automated approach for quantifying phenotypic responses with single cell resolution using single molecule array (SiMoA) technology. We demonstrate how prostate specific antigen (PSA) expression varies over several orders of magnitude between single prostate cancer cells and how PSA expression shifts with genetic drift. Single cell SiMoA introduces a straightforward process that is capable of detecting both high and low protein expression levels. This technique could be useful for understanding fundamental biology and may eventually enable both earlier disease detection and targeted therapy.

Measuring atomic emission from beacons for long-distance chemical signaling.

In an effort to exploit chemistry for information science, we have constructed a system to send a message powered by a combustion reaction. Our system uses the thermal excitation of alkali metals to transmit an encoded signal over long distances. A message is transmitted by burning a methanol-soaked cotton string embedded with combinations of high, low, or zero levels of potassium, rubidium, and/or cesium ions. By measuring the intensities at the characteristic emission wavelengths of each metal in the near-infrared, 19 unique signals can be distinguished. We have built a custom telescope to detect these signals from 1 km away for nearly 10 min. The signal is isotropic, is self-powered, and has a low background. A potential application of this platform is for search and rescue signaling where another layer of information can be transmitted, in addition to the location of the beacon. This work, which seeks to encode and transmit information using chemistry instead of electronics, is part of the new field of "infochemistry".

Assessing the stochastic intermittency of single quantum dot luminescence for robust quantification of biomolecules.

Single molecule detection schemes promise that one has the ability to reach the ultimate limit of detection: one molecule. In this paper, we use the stochastic luminescence of single semiconductor nanocrystals (quantum dots, QDs) to detect and localize particles as digital counts. These digital counts can be correlated to the concentration of analytes in solution. Here, we use total internal reflection fluorescence (TIRF) microscopy to probe individual QDs immobilized on a functionalized substrate. QDs have found their niche in the bioanalytical community due to their remarkable brightness and stability. Despite their numerous outstanding photophysical properties, QDs at the single particle level display a pronounced intermittent luminescence, posing a challenge for the detection of individual particles. In this paper, we demonstrate a reliable method for detecting QDs that takes advantage of these signal fluctuations by comparing the variations in the QD's fluorescence signals against variations of the background signal. The quantitative methodology developed here results in signal-to-background ratios up to 90:1, which is at least 8-times higher than the ratios obtained using methodologies relying solely on signal integration. This enhanced signal-to-background ratio facilitates a robust thresholding process and results in femtomolar limits of detection.

Dynamic microbead arrays for biosensing applications.

In this paper we present the development of an optical tweezers platform capable of creating on-demand dynamic microbead arrays for the multiplexed detection of biomolecules. We demonstrate the use of time-shared optical tweezers to dynamically assemble arrays of sensing microspheres, while simultaneously recording fluorescence signals in real time. The detection system is able to achieve multiplexing by using quantum dot nanocrystals as both signaling probes and encoding labels on the surface of the trapped microbeads. The encoding can be further extended by using a range of bead sizes. Finally, the platform is used to detect and identify three genes expressed by pathogenic strains of Escherichia coli O157:H7. The in situ actuation enabled by the optical tweezers, combined with multiplexed fluorescence detection offers a new tool, readily adaptable to biosensing applications in microfluidic devices, and could potentially enable the development of on-demand diagnostics platforms.

InfoBiology by printed arrays of microorganism colonies for timed and on-demand release of messages.

This paper presents a proof-of-principle method, called InfoBiology, to write and encode data using arrays of genetically engineered strains of Escherichia coli with fluorescent proteins (FPs) as phenotypic markers. In InfoBiology, we encode, send, and release information using living organisms as carriers of data. Genetically engineered systems offer exquisite control of both genotype and phenotype. Living systems also offer the possibility for timed release of information as phenotypic features can take hours or days to develop. We use growth media and chemically induced gene expression as cipher keys or "biociphers" to develop encoded messages. The messages, called Steganography by Printed Arrays of Microbes (SPAM), consist of a matrix of spots generated by seven strains of E. coli, with each strain expressing a different FP. The coding scheme for these arrays relies on strings of paired, septenary digits, where each pair represents an alphanumeric character. In addition, the photophysical properties of the FPs offer another method for ciphering messages. Unique combinations of excited and emitted wavelengths generate distinct fluorescent patterns from the Steganography by Printed Arrays of Microbes (SPAM). This paper shows a new form of steganography based on information from engineered living systems. The combination of bio- and "photociphers" along with controlled timed-release exemplify the capabilities of InfoBiology, which could enable biometrics, communication through compromised channels, easy-to-read barcoding of biological products, or provide a deterrent to counterfeiting.

Preparation and characterization of silver substrates coated with antimony-doped SnO2 thin films for surface plasmon resonance studies.

This paper reports on the preparation of silver/antimony-doped tin oxide (Ag/SnO(2):Sb) hybrid interfaces using magnetron sputtering and their characterization. The influence of the Sn target composition (doping with 2 or 5% Sb) on the electrochemical and electrical characteristics of the hybrid interface was investigated using X-ray photoelectron spectroscopy (XPS), sheet resistance measurements, cyclic voltammetry, scanning tunneling microscopy (STM) and surface plasmon resonance (SPR). The best interface in terms of electrical conductivity and SPR signal is a hybrid interface with a 8.5 +/- 0.3 nm thick SnO(2):Sb layer obtained from a Sn target with 2% Sb deposited on 38 nm thick silver film. Different strategies to link functional groups onto the Ag/SnO(2):Sb interface are also presented.

Selective detection of hexachromium ions by localized surface plasmon resonance measurements using gold nanoparticles/chitosan composite interfaces.

Selective removal of hexavalent chromium ions from aqueous solutions using a chitosan/gold nanoparticles composite film was demonstrated. Localized surface plasmon resonance (LSPR) was used to measure the interface stability and detect the incorporation of chromium ions over time. The effects of pH, ethylenediaminetetraacetic acid (EDTA), and various foreign ions such as trivalent chromium, sodium, calcium, phosphate, sulfate and chloride on the adsorption of hexavalent chromium were investigated.

Electrochemical impedance spectroscopy and surface plasmon resonance studies of DNA hybridization on gold/SiOx interfaces.

The use of Au/SiO(x) interfaces for the investigation of DNA hybridization using electrochemical impedance spectroscopy (EIS) and surface plasmon resonance (SPR) simultaneously is demonstrated. Standard glass chemistry was used to link single-stranded DNA (ss-DNA) on aldehyde-terminated Au/SiO(x) interfaces. The layer thickness and amount of grafted oligonucleotides (ODNs) were calculated from SPR on the basis of a multilayer system of glass/Ti/Au/SiO(x)/grafted molecule. Capacitance and resistance values of the modified interface before and after hybridization were calculated from EIS data using an equivalent circuit and allowed the affinity rate constant, K(A) = 4.07 x 10(5) M(-1), to be determined. The EIS results were comparable to those obtained by SPR hybridization kinetics recorded in parallel.

Gold island films on indium tin oxide for localized surface plasmon sensing.

Mechanically, chemically and optically stable gold island films were prepared on indium tin oxide (ITO) substrates by direct thermal evaporation of thin gold films (2-6 nm) without the need for pre- or post-coating. The effect of mild thermal annealing (150 °C, 12 h) or short high temperature annealing (500 °C, 1 min) on the morphology of the gold nanostructures was investigated. ITO covered with 2 nm gold nanoislands and annealed at 500 °C for 1 min was investigated for its ability to detect the adsorption of biotinylated bovine serum albumin using local surface plasmon resonance (LSPR), and its subsequent molecular recognition of avidin.

Localized electropolymerization on oxidized boron-doped diamond electrodes modified with pyrrolyl units.

This paper describes the functionalization of oxidized boron-doped diamond (BDD) electrodes with N-(3-trimethoxysilylpropyl)pyrrole (TMPP) and the influence of this layer on the electrochemical transfer kinetics as well as on the possibility of forming strongly adhesive polypyrrole films on the BDD interface through electropolymerization. Furthermore, localized polymer formation was achieved on the TMPP-modified BDD interface using the direct mode of a scanning electrochemical microscope (SECM) as well as an electrochemical scanning near-field optical microscope (E-SNOM). Depending on the method used polypyrrole dots with diameters in the range of 1-250 microm are electrogenerated.