Systemic Profile of Cytokines in Arteriovenous Fistula Patients and Their Associations with Maturation Failure

Background: Systemic cytokines are elevated in chronic kidney disease (CKD) and hemodialysis patients compared to the general population. However, whether cytokine levels interfere with vascular remodeling increasing the risk of arteriovenous fistula (AVF) failure remains unknown. Methods: This is a case-control study of 64 patients who underwent surgery for AVF creation (32 with AVF maturation failure and 32 matching controls with successful maturation). A total of 74 cytokines, including chemokines, interferons, interleukins, and growth factors, were measured in preoperative plasma samples using multiplex assays. Sixty-two patients were included in the statistical analyses. Associations with AVF failure were assessed using paired comparisons and conditional logistic regressions accounting for paired strata. Results: Seven cytokines were significantly higher in patients with AVF maturation failure than in matching controls (G-CSF, IL-6, MDC, RANTES, SDF-1α/β, TGFα, and TPO). Of these, G-CSF (odds ratio 1.71 [1.05-2.79] per 10 pg/mL), MDC (1.60 [1.08-2.38] per 100 pg/mL), RANTES (1.55 [1.10-2.17] per 100 pg/mL), SDF-1α/β (1.18 [1.04-1.33] per 1000 pg/mL), and TGFα (OR 1.39 [1.003-1.92] per 1 pg/mL) showed an incremental association by logistic regression. Conclusions: This study identified a profile of plasma cytokines associated with adverse maturation outcomes in AVFs. These findings may open the doors for future therapeutics and markers for risk stratification.

A cell-based multiplex immunoassay platform using fluorescent protein-barcoded reporter cell lines

Multiplex immunoassays with acellular antigens are well-established based on solid-phase platforms such as the Luminex® technology. Cell barcoding by amine-reactive fluorescent dyes enables analogous cell-based multiplex assays, but requires multiple labeling reactions and quality checks prior to every assay. Here we describe generation of stable, fluorescent protein-barcoded reporter cell lines suitable for multiplex screening of antibody to membrane proteins. The utility of this cell-based system, with the potential of a 256-plex cell panel, is demonstrated by flow cytometry deconvolution of barcoded cell panels expressing influenza A hemagglutinin trimers, or native human CCR2 or CCR5 multi-span proteins and their epitope-defining mutants. This platform will prove useful for characterizing immunity and discovering antibodies to membrane-associated proteins.

Responsive Janus Structural Color Hydrogel Micromotors for Label-Free Multiplex Assays

Micromotors with self-propelling ability demonstrate great values in highly sensitive analysis. Developing novel micromotors to achieve label-free multiplex assay is particularly intriguing in terms of detection efficiency. Herein, structural color micromotors (SCMs) were developed and employed for this purpose. The SCMs were derived from phase separation of droplet templates and exhibited a Janus structure with two distinct sections, including one with structural colors and the other providing catalytic self-propelling functions. Besides, the SCMs were functionalized with ion-responsive aptamers, through which the interaction between the ions and aptamers resulted in the shift of the intrinsic color of the SCMs. It was demonstrated that the SCMs could realize multiplex label-free detection of ions based on their optical coding capacity and responsive behaviors. Moreover, the detection sensitivity was greatly improved benefiting from the autonomous motion of the SCMs which enhanced the ion-aptamer interactions. We anticipate that the SCMs can significantly promote the development of multiplex assay and biomedical fields.

Plasma C-C Chemokine Concentrations in Intermediate Age-Related Macular Degeneration

Purpose: To determine the relationship between plasma concentrations of the C-C chemokines CCL2, CCL3, CCL4, and CCL5 and intermediate age-related macular degeneration (iAMD) patients compared with control inidividuals to further define the inflammatory pathways associated with age-related macular degeneration.Methods: The concentrations of CCL2, CCL3, CCL4, and CCL5 were measured using multiplex assays in plasma collected from 210 patients with iAMD and 102 control individuals with no macular degeneration as defined by multi-modal imaging. Non-inflammatory data included in the analysis were: age, sex, family history of AMD, history of smoking, body mass index, presence of reticular pseudo-drusen and cardiovascular disease. Median concentrations as well as a cutoff value for each chemokine were compared between the two groups.Results: The median concentrations of CCL2 and CCL4 did not differ between control and iAMD groups, however, CCL2 was elevated in iAMD when a cutoff comparison was used (p < 0.05). Median CCL3 and CCL5 concentrations were significantly decreased in the macular degeneration group compared with controls (p < 0.001) as well as when a cutoff value comparison was used. CCL3 and CCL5 were negatively correlated in cases and positively correlated in controls.Conclusions: Plasma CCL3 and CCL5 concentrations were significantly decreased and CCL2 concentrations were increased in patients with iAMD compared with controls, suggesting a role for C-C chemokines in the systemic inflammatory processes associated with disease development.

Stringent Base Specific and Optimization-Free Multiplex Mediator Probe ddPCR for the Quantification of Point Mutations in Circulating Tumor DNA

There is an increasing demand for optimization-free multiplex assays to rapidly establish comprehensive target panels for cancer monitoring by liquid biopsy. We present the mediator probe (MP) PCR for the quantification of the seven most frequent point mutations and corresponding wild types (KRAS and BRAF) in colorectal carcinoma. Standardized parameters for the digital assay were derived using design of experiments. Without further optimization, the limit of detection (LoD) was determined through spiking experiments with synthetic mutant DNA in human genomic DNA. The limit of blank (LoB) was measured in cfDNA plasma eluates from healthy volunteers. The 2-plex and 4-plex MP ddPCR assays showed a LoB of 0 copies/mL except for 4-plex KRAS G13D (9.82 copies/mL) and 4-plex BRAF V600E (16.29 copies/mL) and allele frequencies of 0.004% ≤ LoD ≤ 0.38% with R2 ≥ 0.98. The quantification of point mutations in patient plasma eluates (18 patients) during follow-up using the 4-plex MP ddPCR showed a comparable performance to the reference assays. The presented multiplex assays need no laborious optimization, as they use the same concentrations and cycling conditions for all targets. This facilitates assay certification, allows a fast and flexible design process, and is thus easily adaptable for individual patient monitoring.

PSX-A-8 Late-Breaking: Development of multiplex assays for equine cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα

Equine-specific assays to quantify cytokine concentrations are limited and often have a restricted range such that physiological concentrations of many cytokines are below detectable limits of the assay. We aimed to develop custom multiplex assays for equine interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) α using the Meso Scale Discovery U-PLEX platform. Equine-specific ELISA kits containing unlabeled and biotinylated polyclonal antibodies and the specific recombinant equine cytokine were purchased for each cytokine. Each biotinylated antibody was coupled to a linker specific for a unique spot within each well of the U-PLEX plates. The unlabeled antibodies were conjugated with electrochemiluminescent labels to serve as detection antibodies. Each cytokine assay was optimized individually prior to optimization in multiplex. Two preliminary experiments were performed: 1) multiplexed conjugation of equine IL-10 and TNFα to the U-PLEX plates; and 2) multiplexed conjugation of equine IL-1β, IL-4, IL-6, and IL-8 to the U-PLEX plates. Standard curves were run at concentrations ranging from 0 to 5,000 pg/mL for TNFα and IL-8, to 12,500 pg/mL for IL-10, to 25,000 pg/mL for IL-1β and IL-6, and to 50,000 pg/mL for IL-4. The minimum average concentrations measured by the standard curves were 0.065, 0.006, 0.017, 0.00013, 0.196, and 0.050 pg/mL for IL-1β, IL-4, IL-6, IL-8, IL-10, and TNFα, respectively. Test samples of equine serum (n = 5) and bronchoalveolar fluid (n = 3) before and after exercise and of ConA-stimulated equine peripheral mononuclear cell supernatants (positive control) were analyzed for each multiplexed assay. With the exception of serum from one horse, all samples ran within detectable limits of each assay. This preliminary work indicates the U-PLEX platform is a viable option to simultaneously quantify concentrations of multiple equine cytokines, allowing for expansion of research efforts focused on understanding immune responses in the horse.

The identification and semi-quantitative assessment of gastrointestinal nematodes in faecal samples using multiplex real-time PCR assays

Background The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods, followed by larval culture (LC) techniques. Such an approach is laborious, time-consuming, requires a skilled expert, and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies. Methods Two multiplex real-time polymerase chain reaction (PCR) assays for specific detection of five main and one invasive GIN species, including an internal amplification control to avoid false-negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi-quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms, and results were compared to those from faecal egg counts (FEC) using the concentration McMaster technique and LC. Results Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing three false-negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp. Conclusions Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment. Graphical Abstract

The Evaluation of Gastrointestinal Nematodes in Faecal Samples Using Multiplex Real-Time PCR Assays

Background: The diagnosis of gastrointestinal nematode (GIN) infections in ruminants is routinely based on morphological/morphometric analysis of parasite specimens recovered by coprological methods and followed by larval culture techniques. Such an approach is laborious, time-consuming, requires a skilled expert and moreover suffers from certain limitations. Molecular tools are able to overcome the majority of these issues, providing accurate identification of nematode species and, therefore, may be valuable in sustainable parasite control strategies.Methods: Two multiplex real-time PCR assays for specific detection of six main GIN species, including an internal amplification control to avoid false negative results, were designed targeting SSU rRNA and COI genetic markers, as well as established ITS1/2 sequences. The assays were optimized for analysis of DNA extracted directly from sheep faeces and verified for Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus colubriformis, Nematodirus battus, Chabertia ovina, and Ashworthius sidemi. Semi‑quantitative evaluation of infection intensity was enabled using a plasmid construct and a dilution series of sheep faeces with a known number of nematode eggs. Assays were tested on 44 individually collected faecal samples from three farms and results were compared to those from faecal egg counts (FEC) using the Concentration McMaster technique, and larval cultures (LC).Results: Multiplex real-time PCR assays showed great specificity to target nematodes. During the analysis of faecal samples, the assays proved to have higher sensitivity in strongylid-type egg detection over FEC by revealing 3 false negative samples, while showing moderate agreement in evaluation of infection intensity. The multiplex assays further clarified GIN species identification compared to LC, which had confused determination of Teladorsagia spp. for Trichostrongylus spp.Conclusions: Our multiplex assays proved to be a rapid and accurate approach enabling simultaneous and reliable GIN species identification from faeces and semi-quantitative estimation of the number of eggs present. This approach increases diagnostic value and may add a high degree of precision to evaluation of anthelmintic efficacy, where it is important to identify species surviving after treatment.

A multiplex assay for the detection of antibodies to relevant swine pathogens in serum

Livestock industry supports the livelihood of around 1.3 billion people in the world, with swine industry contributing with 30 % of total livestock production worldwide. To maintain and guarantee this production, a pivotal point according to the OIE is addressing potential biohazards. To control them, permanent sero-surveillance is crucial to achieve more focused veterinary public health intervention and prevention strategies, to break the chains of transmission, and to enable fast responses against outbreaks. Within this context, multiplex assays are powerful tools with the potential to simplify surveillance programs, since they reduce time, labour, and variability within analysis. In the present work, we developed a multiplex bead-based assay for the detection of specific antibodies to six relevant pathogens affecting swine: ASFV, CSFV, PRRSV, SIV, TB, and HEV. The most immunogenic target antigen of each pathogen was selected as the target protein to coat different microsphere regions in order to develop this multiplex assay. A total of 1544 serum samples from experimental infections as well as field samples were included in the analysis. The 6plex assay exhibited credible diagnostic parameters with sensitivities ranging from 87.0 % to 97.5 % and specificities ranging from 87.9 % to 100.0 %, demonstrating it to be a potential high throughput tool for surveillance of infectious diseases in swine.