Determination of temporal reproducibility and variability of cancer biomarkers in serum and EDTA plasma samples using a proximity extension assay.

BACKGROUND: Proximity extension assay (PEA) is a novel antibody-based proteomic technology. Sparse data have been published concerning the matrix effect of serum vs. ethylenediamine tetraacetic acid (EDTA) plasma and the reproducibility of results obtained using PEA technology. METHODS: We analyzed samples with the PEA-based 92-plex Olink® immuno-oncology (I-O) assay. To estimate the matrix effect, we analyzed paired serum and EDTA plasma samples from 12 patients with biliary tract cancer. To evaluate the reproducibility, we used data from 7 studies, where 6-8 serum samples from patients with pancreatic cancer were used as bridging samples on 3 versions of the panel over a 2.5-years period. RESULTS: For the study of serum vs. plasma, 80 proteins were evaluable. The mean serum to EDTA plasma ratio ranged from 0.41-3.01. For 36 proteins, the serum and plasma values were not comparable due to high variability of the ratio, poor correlation, or possible concentration effect. For the bridging samples, the mean intra-study inter-assay coefficient of variation (CV) ranged from 11.3% to 26.1%. The mean inter-study CV was 42.0% before normalization and 26.2% after normalization. Inter-study results were well correlated (r ≥ 0.93), especially for studies using the same version of the panel (r ≥ 0.99). CONCLUSION: For 44 of 92 proteins included in the Olink® I-O panel, the variation between results obtained using serum and EDTA plasma was constant and results were well correlated. Furthermore, samples could be stored for several years and used on different versions of the same PEA panel without it effecting results.

Comparison of multiplex platforms for cytokine assessments and their potential use for biomarker profiling in multiple sclerosis.

BACKGROUND: The levels of pro and anti-inflammatory cytokines can be altered in different autoimmune pathologies, such as multiple sclerosis (MS). It is likely that cytokines in bodily fluids can provide a good reflection of ongoing disease patho-physiology. In this study we aimed to validate multiplex cytokine platforms and evaluate whether these cytokines are differentially expressed in MS. METHODS: Assay validation for simultaneous quantification of IL-1ß, IL-6, IL-8 and TNF-α in serum and CSF were performed using both the Luminex-xMAP (Luminex) and Meso Scale Discovery (MSD) platforms. Next, the relation of the pro-inflammatory cytokine 4-plex with disease progression, symptoms and subtypes was studied in paired serum and CSF of MS patients (n=56), and compared with healthy controls (n=203), with the use of the MSD-platform. RESULTS: The MSD-platform showed overall better assay characteristics such as, sensitivity, recovery and linearity compared to the Luminex for the 4-plex cytokines in CSF and serum. IL-6, IL-8 and TNF-α (p<0.001) levels were significantly increased in MS serum compared to healthy controls. Moreover, serum IL-1ß levels correlated with expanded disability status scale (EDSS) scores (r=-0.34, p<0.05). Additionally, IL-6 and IL-8 CSF levels were both significantly decreased in MS patients compared to non-inflammatory neurological disease controls. Noteworthy, higher IL-8 CSF levels than IL-8 serum levels were observed for MS patients, indicating intrathecal activation of macrophages in MS. CONCLUSION: We have demonstrated that the pro-inflammatory 4-plex kit of the MSD-platform shows better assay characteristics in comparison with Luminex kit for quantification of these cytokines in serum and CSF. Overall, the increased levels of IL-6, IL-8 and TNF-α in serum of MS patients compared to healthy controls, support the use of multiple cytokines for future MS biomarker and disease progression research.

Cytokine profile in gingival crevicular fluid and plasma of patients with aggressive periodontitis.

OBJECTIVE: This study aimed to determine the content of cytokines in gingival crevicular fluid (GCF) as well as in plasma of Sudanese patients with aggressive periodontitis (AgP) and healthy controls (HC). MATERIALS AND METHODS: Nineteen AgP patients and 19 HC were included. The mean probing pocket depth and clinical attachment level of the GCF sampled sites in patients were both ≥5 mm. The GCF and plasma levels of 27 cytokines were determined using 27-multiplex fluorescent bead-based immunoassays. Ratios were calculated among cytokines of the T-helper cell subsets Th1 and Th2. Descriptive statistics, the Mann-Whitney U-test and Spearman's rho rank correlation coefficient analysis were used. RESULTS: Interferon-γ was the only cytokine found in significantly lower levels in GCF of patients compared with HC. Levels of interleukin (IL)-10, IL-13, IL-1Ra, monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted (RANTES), granulocyte-colony-stimulating factor (G-CSF), and granulocyte-macrophage-CSF (GM-CSF) were significantly lower in plasma of AgP compared with HC. The ratios of Th1:Th2 in GCF and Treg:Th17 in plasma were significantly lower in AgP. CONCLUSIONS: The lower levels of cytokines detected systemically in plasma of AgP patients may have an impact on the immune response. The lower ratio of Th1:Th2 cytokines in GCF samples of AgP patients suggests a role for Th2 at the local site of disease.

Evaluation of a Novel Multiplex Platform for Simultaneous Detection of IgG Antibodies Against the 4 Main SARS-CoV-2 Antigens.

BACKGROUND: Numerous serology assays are available for detection of SARS-CoV-2 antibodies but are limited in that only 1 or 2 target antigen(s) can be tested at a time. Here, we describe a novel multiplex assay that simultaneously detects and quantifies IgG antibodies to SARS-CoV-2 antigens, spike (S), nucleocapsid (N), receptor-binding domain (RBD), and N-terminal domain (NTD) in a single well. METHODS: Sensitivity was determined using samples (n = 124) from confirmed SARS-CoV-2 RT-PCR positive individuals. Prepandemic (n = 100) and non-COVID respiratory infection positive samples (n = 100) were used to evaluate specificity. Samples were analyzed using COVID-19 IgG multiplex serology assay from Meso Scale Discovery (MSD) and using commercial platforms from Abbott, EUROIMMUN, and Siemens. RESULTS: At >14 days post-PCR, MSD assay displayed >98.0% sensitivity [S 100% (95% CI 98.0%-100.0%); N 98.0% (95% CI 97.2%-98.9%); RBD 94.1% (95% CI 92.6%-95.6%); NTD 98.0% (95% CI, 97.2%-98.9%)] and 99% specificity (95% CI 99.3%-99.7%) for antibodies to all 4 antigens. Parallel assessment of antibodies to more than 1 antigen improved the sensitivity to 100% (95% CI 98.0%-100.0%) while maintaining 98% (95% CI 97.6%-98.4%) specificity regardless of the combinations used. When AU/mL concentrations of IgG antibodies from the MSD assay were compared against the corresponding IgG signals acquired from the single target commercial assays, the following correlations were observed: Abbott (vs MSD N, R2 = 0.73), Siemens (vs MSD RBD, R2 = 0.92), and EUROIMMUN (vs MSD S, R2 = 0.82). CONCLUSION: MSD assay offers an accurate and a comprehensive assessment of SARS-CoV-2 antibodies with higher sensitivity and equivalent specificity compared to the commercial IgG serology assays.

Ability of different assay platforms to measure renal biomarker concentrations during ischaemia-reperfusion acute kidney injury in dogs.

Several protein biomarkers have been shown to be useful for the early diagnosis of acute kidney injury (AKI) in animals and people. Multiplex assays for measurement of a panel of renal biomarkers in canine samples have recently become available. This study compared the use of two such assays, versus previously validated ELISAs, to measure five biomarkers in canine samples during ischaemia-reperfusion (IR) AKI. Blood and urine was collected from six male anaesthetised greyhounds that underwent 1-h of renal ischaemia (severe hypotension induced by acute haemorrhage) and 2-h of reperfusion (intravenous fluid resuscitation). Histology confirmed presence of acute tubular injury at 2 h of reperfusion. Concentrations of clusterin, cystatin C, kidney-injury molecule 1 (KIM-1), monocyte chemoattractant protein 1, and neutrophil gelatinase-associated lipocalin (NGAL) at baseline and following IR, measured by two different multiplex assays and previously-validated single analyte immunoassays, were compared. Only NGAL was significantly elevated following IR with all assays investigated. Whether concentrations of the other four biomarkers were significantly increased following IR depended on the assay used. Concentrations of cystatin C and KIM-1 measured with the multiplex assays were of a vast magnitude lower than those measured with the corresponding single analyte ELISAs. We conclude that further validation is required before these assays can reliably be used to measure AKI biomarkers in canine samples.

Size-Resolved Single Entity Collision Biosensing for Dual Quantification of MicroRNAs in a Single Run.

Limited to the accuracy of size resolution, single entity collision biosensing (SECBS) for multiplex immunoassays remains challenging, because it is difficult to get the true value of nanoparticle (NP) sizes based on the current intensity due to the complex movement of NPs on the electrode surface. Considering that the size-dependent movement of NPs meanwhile will generate a characteristic current shape, in this work, the huge difference in the current rise time of 5 and 15 nm Pt NPs colliding on an Au ultramicroelectrode (d = 30 µm) was originally used to develop a size-resolved SECBS for multiplex immunoassays of miRNAs. The limit concentration that can be detected was 0.5 fM. Compared with conventional electrochemical biosensors for multiplex immunoassays, for the size-resolved SECBS, one does not need to worry about potential overlapping. Therefore, the proposed method demonstrates a promising potential for the application of SECBS in multiplex immunoassays.

Cytokine changes in fatal cases of paraquat poisoning.

Cytokine-mediated inflammation is involved in the pathophysiology of paraquat toxicity. Nevertheless, few human studies have examined fluctuations in circulating cytokine levels. Blood samples were obtained from 21 patients with paraquat poisoning and compared to those of 18 healthy controls. All paraquat patients received a standard detoxification protocol composed of hemoperfusion, pulse therapies of methylprednisolone and cyclophosphamide, followed by dexamethasone therapy. Nonsurvivors not only had higher scores for the severity index of paraquat poisoning (P=0.004) but also presented with higher white blood cell counts (P=0.046) than survivors. Multiplex immunoassays revealed higher circulating levels of interleukin 2 (IL-2), interleukin 9 (IL-9), interleukin 10 (IL-10) and macrophage inflammatory protein-1 beta (MIP-1ß) in survivors than in healthy controls. Furthermore, the circulating levels of interleukin 1 beta (IL-1ß), IL-2, interleukin 5 (IL-5), interleukin 8 (IL-8), IL-9, IL-10, interleukin 12 (IL-12 p70), interleukin 17A (IL-17A), eotaxin, granulocyte colony-stimulating factor (G-CSF), monocyte chemoattractant protein-1 (MCP-1), interferon gamma-induced protein 10 (IP-10) and MIP-1ß were higher in nonsurvivors than in healthy controls. Finally, the circulating levels of IL-1ß and MCP-1 were higher in nonsurvivors than in survivors. Therefore, the observation of cytokine-mediated inflammation is in line with the detoxification protocol because glucocorticoids and cyclophosphamide are potent anti-inflammatory agents. Additionally, circulating levels of IL-1ß and MCP-1 could serve as promising prognostic markers for patients with paraquat poisoning.

Contribution of Multiplex Immunoassays to Rheumatoid Arthritis Management: From Biomarker Discovery to Personalized Medicine.

Rheumatoid arthritis (RA) is a multifactorial, inflammatory and progressive autoimmune disease that affects approximately 1% of the population worldwide. RA primarily involves the joints and causes local inflammation and cartilage destruction. Immediate and effective therapies are crucial to control inflammation and prevent deterioration, functional disability and unfavourable progression in RA patients. Thus, early diagnosis is critical to prevent joint damage and physical disability, increasing the chance of achieving remission. A large number of biomarkers have been investigated in RA, although only a few have made it through the discovery and validation phases and reached the clinic. The single biomarker approach mostly used in clinical laboratories is not sufficiently accurate due to its low sensitivity and specificity. Multiplex immunoassays could provide a more complete picture of the disease and the pathways involved. In this review, we discuss the latest proposed protein biomarkers and the advantages of using protein panels for the clinical management of RA. Simultaneous analysis of multiple proteins could yield biomarker signatures of RA subtypes to enable patients to benefit from personalized medicine.

Altered protein levels in bone marrow lesions of hip osteoarthritis: Analysis by proteomics and multiplex immunoassays.

AIM: To assess tissue level changes of proteome and cytokine profiles of subchondral bone in hip osteoarthritis (OA) affected by bone marrow lesions (BMLs). We compared significant protein level differences in osteoarthritic bone with BMLs to control bone without bone marrow lesions. METHODS: Subchondral bone biopsies were taken from femoral heads of end-stage osteoarthritis patients with (BML, n = 21) and without (CON, n = 9) BMLs. Proteins were extracted through a standardized Trizol protocol and used in the subsequent analyses. Angiogenesis and bone markers were assessed using multiplex immunoassays (Luminex). Liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to detect significant differences in proteome and peptide profiles between BML and CON. RESULTS: Multiplex immunoassays revealed increased tissue contents of vascular endothelial growth factors (VEGF-A/C/D), endothelin-1, angiopoietin-2 and interleukin-6 (IL-6) in bone with BMLs compared to control bone, whereas osteoprotegerin levels were reduced. Mass spectrometry demonstrated pronounced increase in the levels of hemoglobin (73-fold), serum albumin (30-fold), alpha-1-antitrypsin (9-fold), apolipoprotein A1 (4.7-fold), pre-laminin-A/C (3.7-fold) and collagen-alpha1-XII (3-fold) in BMLs, while aggrecan core protein (ACAN) and hyaluronan and proteoglycan link protein 1 (HAPL1) decreased 37- and 29-fold respectively. CONCLUSION: Reduced osteoprotegerin, ACAN and HAPL1 are consistent with osteoclastic activation and high remodeling activity in BMLs. The pronounced increase in angiogenesis markers, hemoglobin and serum albumin support the presence of increased vascularity in subchondral bone affected by BMLs in OA. VEGFs and IL-6 are known nociceptive modulators, and increased levels are in keeping with pain being a clinical feature frequently associated with BMLs.

Immune response proteins as predictive biomarkers of doxorubicin-induced cardiotoxicity in breast cancer patients.

Cancer treatment with doxorubicin (DOX) can induce cumulative dose-dependent cardiotoxicity. Currently, there are no specific biomarkers that can identify patients at risk during the initial doses of chemotherapy. The aim of this study was to examine plasma cytokines/chemokines and potential cardiovascular biomarkers for the prediction of DOX-induced cardiotoxicity. Plasma samples were collected before (T0), and after the first (T1) and the second (T2) cycles of DOX-based chemotherapy of 27 breast cancer patients, including five patients who presented with >10% decline of left ventricular ejection fraction (LVEF), five patients with LVEF decline of 5-10%, and 17 patients who maintained normal LVEF at the end of chemotherapy (240 mg/m2 cumulative dose of DOX from four cycles of treatment). Multiplex immunoassays were used to screen plasma samples for 40 distinct chemokines, nine matrix metalloproteinases, 33 potential markers of cardiovascular diseases, and the fourth-generation cardiac troponin T assay. The results showed that the patients with abnormal decline of LVEF (>10%) had lower levels of CXCL6 and sICAM-1 and higher levels of CCL23 and CCL27 at T0; higher levels of CCL23 and lower levels of CXCL5, CCL26, CXCL6, GM-CSF, CXCL1, IFN-γ, IL-2, IL-8, CXCL11, CXCL9, CCL17, and CCL25 at T1; and higher levels of MIF and CCL23 at T2 than the patients who maintained normal LVEF. Patients with LVEF decline of 5-10% had lower plasma levels of CXCL1, CCL3, GDF-15, and haptoglobin at T0; lower levels of IL-16, FABP3, and myoglobin at T1; and lower levels of myoglobin and CCL23 at T2 as compared to the patients who maintained normal LVEF. This pilot study identified potential biomarkers that may help predict which patients are vulnerable to DOX-induced cardiotoxicity although further validation is needed in a larger cohort of patients. Impact statement Drug-induced cardiotoxicity is one of the major concerns in drug development and clinical practice. It is critical to detect potential cardiotoxicity early before onset of symptomatic cardiac dysfunction or heart failure. Currently there are no qualified clinical biomarkers for the prediction of cardiotoxicity caused by cancer treatment such as doxorubicin (DOX). By using multiplex immunoassays, we identified proteins with significantly changed plasma levels in a group of breast cancer patients who were treated with DOX-based chemotherapy and produced cardiotoxicity. These proteins were associated with immune response and were identified before DOX treatment or at early doses of treatment, thus they could be potential predictive biomarkers of cardiotoxicity although further validation is required to warrant their clinical values.

Multiparameter flow cytometry: advances in high resolution analysis.

Over the past 40 years, flow cytometry has emerged as a leading, application-rich technology that supports high-resolution characterization of individual cells which function in complex cellular networks such as the immune system. This brief overview highlights advances in multiparameter flow cytometric technologies and reagent applications for characterization and functional analysis of cells modulating the immune network. These advances significantly support high-throughput and high-content analyses and enable an integrated understanding of the cellular and molecular interactions that underlie complex biological systems.