High Sample Throughput Research Articles

Quantitation using HRMS: A new tool for rapid, specific and sensitive determination of catecholamines and deconjugated methanephrines metanephrines in urine

Urinary catecholamines and their methylated metabolites are biochemical indicators of pheochromocytoma, paraganglioma and neuroblastoma. A rapid and precise analytical method based on solid-phase extraction (SPE) and liquid chromatography separation coupled to high-resolution mass spectrometry (LC-HRMS) was developed and validated to measure urinary catecholamines (epinephrine (E), norepinephrine (NorE), dopamine (D)) and total methylated metabolites (normetanephrine (NorMN), metanephrine(MN) and 3-methoxytyramine (3-MT)) in a clinical setting. Results of 51 urine specimens measured using this LC-HRMS method were compared with a liquid chromatography assay with electrochemical detection (LC-EC). Urine samples (200 μL) were spiked with an internal standard solution followed by SPE purification. In the case of total methylated metabolites, urine was hydrolyzed before SPE purification. Separation was achieved on an Acclaim Mixed Mode WCX column, with an 8.5 min runtime. All compounds were detected in electrospray positive ionization mode with a parallel reaction monitoring acquisition and quantified with a linear regression (r2 > 0.998) between 2 and 200 µg/L (10.9–1090; 11.8–1182 nmol/L) for E and NorE respectively and between 10 and 1000 µg/L for others (65.2–6520; 50.7–5070; 54.5–5450 ; 59.8–5980 nmol/L for D, M, NorMN and 3-MT, respectively). Overall imprecision and bias did not exceed 15%. No significant matrix effect was observed. Correlation between the two assays was good except for epinephrine. Epinephrine concentrations measured by LC-EC method were slightly higher than values obtained with LC-HRMS method but without impact on clinical decision. This LC-HRMS assay provides a new tool for simultaneous quantitative catecholamine determination and was successfully applied in routine for the screening or follow up of pheochromocytoma, paraganglioma and neuroblastoma. LC-HRMS method offers significant advantages compared to LC-EC with good sensitivity, an unambiguous analyte determination and high sample throughput.

Porous thin-film molecularly imprinted polymer device for simultaneous determination of phenol, alkylphenol and chlorophenol compounds in water

A porous water-compatible molecularly imprinted polymer (MIP) coating using catechol as a pseudo-template and a water-soluble functional monomer (4-vinyl benzoic acid) with ethylene glycol dimethacrylate as the crosslinker was developed for extraction of phenols from environmental water samples. The MIP devices were combined with ultra high performance liquid chromatography with a photodiode array detector (UHPLC-PDA) suitable for the simultaneous determination of trace levels of phenolic compounds with a wide range of polarities —phenol, alkylphenols and chlorophenols— in seawater and produced water. Parameters that influence extraction efficiency (salinity, pH, polymer mass, desorption solvent, and desorption time) were optimized to give method detection limits (LOD) ranging from 0.1 to 2 μg L−1 and linearity (R2>0.99) over at least three orders of magnitude for the hydrophobic phenols (e.g., 0.5–1000 μg L−1 for 2,4,6-trichlorophenol) and ~2 orders of magnitude for the light phenols (e.g., 10–120 μg L−1 for phenol, 5–120 μg L−1 for methylphenols and 2-chlorophenol, 0.5–120 μg L−1 3-methyl-4-chlorophenol and 2,4-dichlorophenol). The recoveries from authentic spiked samples ranged from 85 to 100% with %RSDs of 0.2–14% for seawater and 81–107% with %RSD of 0.1–11% for produced water. The resulting MIP-based extraction requires no pre-conditioning of the sorbent, and because the required sample size is small and sample manipulation is limited, the method is easy to multiplex for high throughput sample processing.

Discrimination of phenethylamine regioisomers and structural analogues by Raman spectroscopy.

In this study, the Raman spectra of 21 phenethylamines were obtained using far-red excitation (785nm). The distinguishing ability of Raman for phenethylamines, especially for phenethylamine regioisomers and structural analogues, was investigated. Here, the evaluation of a cross section of Raman spectra demonstrated that all types of phenethylamines were distinguishable, even for certain structural analogues with high spectrum similarity. Raman exhibited high distinguishing ability for phenethylamine regioisomers that differ in the substitution position of halogen, methoxy, alkyl, or other substituted groups; as well as for structural analogues containing different groups, such as furanyl, 2,3-dihydrofuranyl, halogen, and alkyl substituted at the same position. The Raman spectra for homologues with differences in only a methyl group were found to be highly similar; however, their spectra demonstrated small but detectable differences. Four analogue mixtures and 59 seized samples were also analyzed to study the practical use of the Raman method in forensic field. 95% of the seized samples were correctly identified, which significantly validated the ability of Raman method in identifying the correct isomers. Accordingly, this study provides a non-destructive, high-throughput and minimal sample preparation technique for the discrimination of phenethylamines.

Evaluation of 16S rRNA Hypervariable Regions for Bioweapon Species Detection by Massively Parallel Sequencing.

Molecular detection and classification of the bacterial groups in a sample are relevant in several areas, including medical research and forensics. Sanger sequencing of the 16S rRNA gene is considered the gold standard for microbial phylogenetic analysis. However, the development of massively parallel sequencing (MPS) offers enhanced sensitivity and specificity for microbiological analyses. In addition, 16S rRNA target amplification followed by MPS facilitates the combined use of multiple markers/regions, better discrimination of sample background, and higher sample throughput. We designed a novel set of 16S rRNA gene primers for detection of bacterial species associated with clinical, bioweapon, and biohazards microorganisms via alignment of 364 sequences representing 19 bacterial species and strains relevant to medical and forensics applications. In silico results indicated that the hypervariable regions (V1V2), (V4V5), and (V6V7V8) support the resolution of a selected group of bacteria. Interspecies and intraspecies comparisons showed 74.23%–85.51% and 94.48%–99.98% sequencing variation among species and strains, respectively. Sequence reads from a simulated scenario of bacterial species mapped to each of the three hypervariable regions of the respective species with different affinities. The minimum limit of detection was achieved using two different MPS platforms. This protocol can be used to detect or monitor as low as 2,000 genome equivalents of bacterial species associated with clinical, bioweapon, and biohazard microorganisms and potentially can distinguish natural outbreaks of pathogenic microorganisms from those occurring by intentional release.

Transcriptomics of the Rooibos (Aspalathus linearis) Species Complex.

Rooibos (Aspalathus linearis), widely known as a herbal tea, is endemic to the Cape Floristic Region of South Africa (SA). It produces a wide range of phenolic compounds that have been associated with diverse health promoting properties of the plant. The species comprises several growth forms that differ in their morphology and biochemical composition, only one of which is cultivated and used commercially. Here, we established methodologies for non-invasive transcriptome research of wild-growing South African plant species, including (1) harvesting and transport of plant material suitable for RNA sequencing; (2) inexpensive, high-throughput biochemical sample screening; (3) extraction of high-quality RNA from recalcitrant, polysaccharide- and polyphenol rich plant material; and (4) biocomputational analysis of Illumina sequencing data, together with the evaluation of programs for transcriptome assembly (Trinity, IDBA-Trans, SOAPdenovo-Trans, CLC), protein prediction, as well as functional and taxonomic transcript annotation. In the process, we established a biochemically characterized sample pool from 44 distinct rooibos ecotypes (1–5 harvests) and generated four in-depth annotated transcriptomes (each comprising on average ≈86,000 transcripts) from rooibos plants that represent distinct growth forms and differ in their biochemical profiles. These resources will serve future rooibos research and plant breeding endeavours.

Identification of novel testing matrices for African swine fever surveillance.

African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 107 genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.

High-throughput quantitation of serological ceramides/dihydroceramides by LC/MS/MS: Pregnancy baseline biomarkers and potential metabolic messengers

Ceramides and dihydroceramides are sphingolipids that present in abundance at the cellular membrane of eukaryotes. Although their metabolic dysregulation has been implicated in many diseases, our knowledge about circulating ceramide changes during the pregnancy remains limited. In this study, we present the development and validation of a high-throughput liquid chromatography-tandem mass spectrometric method for simultaneous quantification of 16 ceramides and 10 dihydroceramides in human serum within 5 min. by using stable isotope-labeled ceramides as internal standards. This method employs a protein precipitation method for high throughput sample preparation, reverse phase isocratic elusion for chromatographic separation, and Multiple Reaction Monitoring for mass spectrometric detection. To qualify for clinical applications, our assay has been validated against the FDA guidelines for Lower Limit of Quantitation (1 nM), linearity (R2>0.99), precision (imprecision<15 %), accuracy (inaccuracy<15 %), extraction recovery (>90 %), stability (>85 %), and carryover (<0.01 %). With enhanced sensitivity and specificity from this method, we have, for the first time, determined the serological levels of ceramides and dihydroceramides to reveal unique temporal gestational patterns. Our approach could have value in providing insights into disorders of pregnancy.

Evaluation of on-line solid-phase extraction capillary electrophoresis–mass spectrometry with a nanoliter valve for the analysis of peptide biomarkers

On-line solid-phase extraction capillary electrophoresis-mass spectrometry (SPE-CE-MS) is a powerful technique for high throughput sample clean-up and analyte preconcentration, separation, detection, and characterization. The most typical design due to its simplicity and low cost is unidirectional SPE-CE-MS. However, in this configuration, the sample volumes introduced by pressure depend on the dimensions of the separation capillary and some matrix components could be irreversibly adsorbed in its inner walls. Furthermore, in many cases, the requirements of on-line preconcentration are incompatible with the background electrolyte necessary for an efficient separation and sensitive MS detection. Here, we present SPE-CE-MS with a nanoliter valve (nvSPE-CE-MS) to overcome these drawbacks while keeping the design simple. The nvSPE-CE-MS system is operated with a single CE instrument and two capillaries for independent and orthogonal SPE preconcentration and CE separation, which are interfaced through an external and electrically isolated valve with a 20 nL sample loop. The instrumental setup is proved for the analysis of opioid and amyloid beta peptide biomarkers in standards and plasma samples. NvSPE-CE-MS allowed decreasing the limits of detection (LODs) 200 times with regard to CE-MS. Compared to unidirectional SPE-CE-MS, peak efficiencies were better and repeatabilities similar, but total analysis times longer and LODs for standards slightly higher due to the heart-cut operation and the limited volume of the valve loop. This small difference on the LODs for standards was compensated for plasma samples by the improved tolerance of nvSPE-CE-MS to complex sample matrices. In view of these results, the presented setup can be regarded as a promising versatile alternative to avoid complicated matrix samples entering the separation capillary in SPE-CE-MS.

Magnetic solid-phase extraction of gingerols in ginger containing products

In this study, a graphene oxide/magnetite (GO-Fe3O4) nanocomposite was synthesized and used as a sorbent in the magnetic solid-phase extraction (MSPE) of gingerols from fresh ginger rhizomes, ginger extracts, commercial tea samples, ginger candies, thermogenic supplements, and tonic water. An MSPE method was developed, and the main influencing parameters in the sample preparation process were investigated. After GO-Fe3O4 based MSPE, 6-gingerol, 8-gingerol, and 10-gingerol were determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The whole GO-Fe3O4-MSPE-LC-MS/MS method proved high selectivity and consistent analytical confidence. The limits of detection (LOD) ranged between 2 and 3 μg L-1. Intra-day and inter-day RSDs fluctuated between 1.7 - 13.4% and 0.4–10.9%, respectively. Weighted calibration revealed good linearity within the studied range (5–200 μg L−1) and guaranteed appropriate accuracy (relative residues < 25%). MSPE with GO-Fe3O4 demonstrated to be a practical, fast, efficient, high-throughput, and environmental-friendly sample preparation technique for determining of gingerols in commercial products, and its hyphenation with LC-MS/MS analysis yield a valuable analytical tool for the confident quality control of commercial ginger-containing products.

Stereoselective quantification of phase 1 and 2 metabolites of clomiphene in human plasma and urine

Clomiphene citrate is first line therapy of female infertility but is also frequently abused by athletes. Human biotransformation of clomiphene results in numerous phase 1 and phase 2 metabolites. The involvement of the polymorphic cytochrome P450 2D6 leads to a high inter-individual variability. To comprehensively investigate clomiphene metabolism in vivo we established a highly sensitive and specific UPLC-MS/MS method for the stereoselective quantification of clomiphene and its phase 1 and phase 2 metabolites in plasma and urine.Reference compounds and stable isotope labelled internal standards were synthesized in-house. High-throughput sample preparation was done by protein precipitation. Analytes were separated by UPLC on a C18 column (1.8 μm, 2.1 * 100 mm) using a gradient of 0.1% formic acid in acetonitrile in 0.1% aqueous formic acid and detected by positive ESI-MS/MS in MRM mode. The lower limit of quantification was below 1 nM for all analytes. The method was validated according to recent guidelines. However, due to absorption effects during sampling the quantification of metabolites in urine was limited to phase 2 metabolites. The method was successfully applied to determine the pharmacokinetic of (E)- and (Z)-clomiphene and 14 metabolites following a single dose of 100 mg clomiphene citrate in 3 healthy subjects and proofed to be an essential tool to comprehensively investigate the human biotransformation of clomiphene.

One-Step High-Throughput Telomerase Activity Measurement of Cell Populations, Single Cells, and Single-Enzyme Complexes.

Telomerase, a key enzyme involved in telomere homeostasis, is a major player involved in or required for sustained cell proliferation. It is expressed in ∼90% tumor but rarely in normal somatic cells. Therefore, telomerase serves as a diagnostic marker and therapeutic target of cancers. Although many methods are available for measuring telomerase activity, a convenient, fast, sensitive, and reliable method is still lacking for routine use in both clinics and research. Here, we present a single-enzyme sensitivity telomere repeat amplification protocol for quantifying telomerase activity. With multiple optimizations, the protocol pushes the ultimate detection limit down to a single telomerase complex, enabling measurement of telomerase activity of not only multiple cancerous/normal cell samples but also single cancer cells alone or even in the presence of 8000 normal cells. Implemented in a one-step mix-and-run format, the protocol offers a most sensitive, fast, accurate, and reproducible quantification of telomerase activity with linearity ranging from 20,000 HeLa cancer cells to a single telomerase complex. It requires minimal manual operation and experimental skill and is convenient for either low or high throughput of samples. We expect that the protocol should provide practical routine analyses of telomerase in both research and clinical applications. As an example, we demonstrate how telomerase activity evolves at the single-cell level and partitions in cell division in early mouse embryo development.

Determination of chloride in crude oil using isotope dilution GC–MS: A comparative study

Optimisation and validation of a novel isotope dilution gas chromatography mass spectrometry (GC–MS) method for determination of inorganic chloride (Cl−) in crude oils is presented. The use of stable isotope dilution calibration approach decreases losses and biases and it is considered a higher order measurement approach. Samples were diluted with a 1:1 blend of isopropyl alcohol : toluene with 5% water, mixed with 37Cl− internal standard, water extracted and derivatized with triethyloxonium tetrafluoroborate at room temperature. This reagent converted Cl− into stable ethyl chloride (EtCl) which was detected by headspace GC–MS. A limit of quantitation of 100 µg kg−1 and limit of detection of 30 µg kg−1 were observed in crude oil samples when initial dilution was not required. The performance of this novel method was compared with combustion ion chromatography, ion chromatography with conductivity detection after microwave-induced combustion, and ASTM D3230 method on a set of medium and heavy crude oil samples. Despite the challenges of the measurement, a reasonable level of agreement was observed among methods over the range of 0.38 ± 0.02 to 993 ± 103 mg kg−1 of Cl−. Presented headspace GC–MS method proved to be suitable for analysis of all types of crude oils samples with an excellent reproducibility (RSD < 6%) and high sample throughput (7 min GC–MS run time).

Simplified LC-MS Method for Analysis of Sterols in Biological Samples.

We developed a simple and robust liquid chromatographic/mass spectrometric method (LC-MS) for the quantitative analysis of 10 sterols from the late part of cholesterol synthesis (zymosterol, dehydrolathosterol, 7-dehydrodesmosterol, desmosterol, zymostenol, lathosterol, FFMAS, TMAS, lanosterol, and dihydrolanosterol) from cultured human hepatocytes in a single chromatographic run using a pentafluorophenyl (PFP) stationary phase. The method also avails on a minimized sample preparation procedure in order to obtain a relatively high sample throughput. The method was validated on 10 sterol standards that were detected in a single chromatographic LC-MS run without derivatization. Our developed method can be used in research or clinical applications for disease-related detection of accumulated cholesterol intermediates. Disorders in the late part of cholesterol synthesis lead to severe malformation in human patients. The developed method enables a simple, sensitive, and fast quantification of sterols, without the need of extended knowledge of the LC-MS technique, and represents a new analytical tool in the rising field of cholesterolomics.

Automated sample preparation for the detection and confirmation of hypoxia-inducible factor stabilizers in urine.

As hypoxia-inducible factor stabilizers (HIFs) can artificially enhance an athlete's erythropoiesis, the World Anti-Doping Agency prohibits their use at all times. Every urine sample for doping control analysis has to be evaluated for the presence of HIFs and therefore sensitive methods that allow high sample throughput are needed. Samples suspicious for the presence of HIFs need to be confirmed following the identification criteria established by the World Anti-Doping Agency. Previous work has shown the advantages of using turbulent flow online solid-phase extraction (SPE) procedures to reduce matrix effects and retention time shifts. Furthermore, the use of online SPE allows for automation and high sample throughput. Both an initial testing procedure (ITP) and a confirmation method were developed and validated, using online SPE liquid chromatography-tandem mass spectrometry (LC-MS/MS), with limits of detection between 0.1 ng/ml (or possibly lower) and 4 ng/ml (or higher for GSK360a) and limits of identification between 0.1 ng/ml (or possibly lower) and 1.17 ng/ml. The ITP only takes 6.5 min per sample. To the best of our knowledge, these are the first ITP and confirmation methods that include more than three HIFs without the need for manual sample preparation.

Electrochemical flow injection analysis for the rapid determination of reducing sugars in potatoes

Non-enzymatic electrochemical sensors for the monitoring of reducing sugars in foods has great potential as a rapid in-situ detection method. This development involved the assembly of a nanoporous platinum structure on a screen-printed carbon electrode (SPCE). The modified electrode was then employed as an amperometric sensing element in a flow injection analysis (FIA) manifold. The system was successfully applied to the rapid detection of reducing sugars in potatoes, without the need for sample preparation. Optimal signals were achieved in phosphate buffer (pH 7.4) at a flow rate of 0.5 mL min−1 and an applied potential of 0.6 V. Experimental results demonstrated the sensor’s long-term stability and high selectivity for reducing sugars. This method provides high sample throughput due to a rapid response time of less than five seconds. Reducing sugar values determined were in good agreement with those recorded using a commercially available enzymatic assay kit.

Assessing Antigen-Specific Cellular Immune Responses upon HIV /SIV Plasmid DNA Vaccination in the Nonhuman Primate Model.

Reliable detection and quantification of antigen-specific T cells are critical for assessing the immunogenicity of vaccine candidates. In this chapter, we describe the use of ELISpot and flow cytometry-based assays for efficient detection, mapping, and functional characterization of memory T lymphocytes in different tissues of rhesus macaques immunized with plasmid DNA. Flow cytometric assays provide a large amount of information, both phenotypic and functional, about individual cells, while the ELISpot is well suited for high throughput sample screening.

M6A-Atlas: a comprehensive knowledgebase for unraveling the N6-methyladenosine (m6A) epitranscriptome.

N 6-Methyladenosine (m6A) is the most prevalent RNA modification on mRNAs and lncRNAs. It plays a pivotal role during various biological processes and disease pathogenesis. We present here a comprehensive knowledgebase, m6A-Atlas, for unraveling the m6A epitranscriptome. Compared to existing databases, m6A-Atlas features a high-confidence collection of 442 162 reliable m6A sites identified from seven base-resolution technologies and the quantitative (rather than binary) epitranscriptome profiles estimated from 1363 high-throughput sequencing samples. It also offers novel features, such as; the conservation of m6A sites among seven vertebrate species (including human, mouse and chimp), the m6A epitranscriptomes of 10 virus species (including HIV, KSHV and DENV), the putative biological functions of individual m6A sites predicted from epitranscriptome data, and the potential pathogenesis of m6A sites inferred from disease-associated genetic mutations that can directly destroy m6A directing sequence motifs. A user-friendly graphical user interface was constructed to support the query, visualization and sharing of the m6A epitranscriptomes annotated with sites specifying their interaction with post-transcriptional machinery (RBP-binding, microRNA interaction and splicing sites) and interactively display the landscape of multiple RNA modifications. These resources provide fresh opportunities for unraveling the m6A epitranscriptomes. m6A-Atlas is freely accessible at: www.xjtlu.edu.cn/biologicalsciences/atlas.

Sensitive and Specific Detection of SARS-CoV-2 Antibodies Using a High-Throughput, Fully Automated Liquid-Handling Robotic System.

As of July 22, 2020, more than 14.7 million infections of SARS-CoV-2, the virus responsible for Coronavirus Disease 2019 (COVID-19), have been confirmed globally. Serological assays are essential for community screening, assessing infection prevalence, aiding identification of infected patients, and enacting appropriate treatment and quarantine protocols in the battle against this rapidly expanding pandemic. Antibody detection by agglutination–PCR (ADAP) is a pure solution phase immunoassay that generates a PCR amplifiable signal when patient antibodies agglutinate DNA-barcoded antigen probes into a dense immune complex. Here, we present an ultrasensitive and high-throughput automated liquid biopsy assay based on the Hamilton Microlab ADAP STAR automated liquid-handling platform, which was developed and validated for the qualitative detection of total antibodies against spike protein 1 (S1) of SARS-CoV-2 that uses as little as 4 µL of serum. To assess the clinical performance of the ADAP assay, 57 PCR-confirmed COVID-19 patients and 223 control patients were tested. The assay showed a sensitivity of 98% (56/57) and a specificity of 99.55% (222/223). Notably, the SARS-CoV-2–negative control patients included individuals with other common coronaviral infections, such as CoV-NL63 and CoV-HKU, which did not cross-react. In addition to high performance, the hands-free automated workstation enabled high-throughput sample processing to reduce screening workload while helping to minimize analyst contact with biohazardous samples. Therefore, the ADAP STAR liquid-handling workstation can be used as a valuable tool to address the COVID-19 global pandemic.

Bioorthogonal Non-canonical Amino Acid Tagging Combined With Flow Cytometry for Determination of Activity in Aquatic Microorganisms.

In this study, we have combined bioorthogonal non-canonical amino acid tagging (BONCAT) and flow cytometry (FCM) analysis, and we demonstrate the applicability of the method for marine prokaryotes. Enumeration of active marine bacteria was performed by combining the DNA stain SYBR Green with detection of protein production with BONCAT. After optimization of incubation condition and substrate concentration on monoculture of Escherichia coli, we applied and modified the method to natural marine samples. We found that between 10 and 30% of prokaryotes in natural communities were active. The method is replicable, fast, and allow high sample throughput when using FCM. We conclude that the combination of BONCAT and FCM is an alternative to current methods for quantifying active populations in aquatic environments.

Abstract 386: Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology

Abstract Background: The tumor microenvironment is composed of a complicated network of different cell types and activation states, the exact composition of which is important for immuno-oncology research efforts to correlate treatment options and outcome. The number of biomarkers necessary to sufficiently profile the various cellular phenotypes and elucidate contextual relationships in situ is larger than can be accommodated by standard immunohistochemistry (IHC) approaches. Furthermore, conventional IHC requires more sections, potentially exhausting tissue sample availability. To overcome these limitations, Ultivue has developed InSituPlex® technology and UltiMapper™ assays that enable biomarker multiplexing in tissue samples with an automated workflow to support high sample throughput. Methods: An 8-plex immune-profiling panel using the InSituPlex approach was designed and tested to detect and classify T cells, macrophages, and tumor cell populations along with PD-L1 checkpoint expression in multiple tumor types. This assay was designed, developed, and tested using UltiMapper reagents and corresponding protocols. The assay was run on multiple FFPE tissue samples from breast cancer, NSCLC, and colorectal cancer (CRC) cases. Each sample was stained for the following 8 markers: CD3, CD4, CD8, CD68, FoxP3, PD-1, PD-L1, and pan-CK. Staining was performed in a single step using the Leica Biosystems BOND RX autostainer. Whole slide imaging (WSI) was performed in two steps on the ZEISS Axio Scan.Z1 slide scanner, and image analysis was performed using HALO software from Indica Labs. Results: The 8-plex immune-profiling panel demonstrated specific biomarker staining and was verified for staining reproducibility. The 8-plex workflow was compatible across multiple tumor tissue types. The samples were characterized by cell analysis and phenotyped based on their expression of specific biomarkers. The following cell phenotypes were detected: T cells (CD3+), cytotoxic T cells (CD3+/CD8+), regulatory T cells (CD4+/FOXP3+), exhausted T cells (CD3+/PD-1+), macrophages (CD68+), and tumor cells (CK+) with a dynamic range of PD-L1 expression levels. Conclusion: The 8-plex panel revealed important immunophenotypes across multiple sample types. The assay was automated and optimized for a high-throughput workflow. By testing across multiple tissue types, this assay showcases the potential for broad application across additional cancer types. The ability to interrogate an 8-plex I/O panel on a single slide with WSI presents an attractive approach for the next generation of IHC, enabling profiling within the tumor microenvironment and sensitive detection of PD-L1 expression in cancer tissues. Funded by NCI Contract No. 75N910D00024 Author affiliations for CGR staff: Cancer Genomics Research Laboratory, Frederick National Laboratory for Cancer Research, Frederick, MD 21702 Citation Format: Alexis Wong, Mara Koerner, Katir K. Patel, Anna Potier, Stephanie Hennek, Mustapha Abubakar, Mary E. Olanich, Yelena G. Golubeva, Karun Mutreja, Scott Lawrence. Design and application of an 8-plex multiplex immunofluorescence panel for deep phenotypic profiling of the tumor microenvironment using InSituPlex technology [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 386.