Multiplex

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  • 文章类型: Journal Article
    COVID-19大流行,由SARS-CoV-2病毒引起,是历史上最严重的呼吸道疾病爆发之一。COVID-19的临床症状可能与流感相似,尽管它们可能会危及生命,特别是在老年人和免疫功能低下的人群中。连同核酸检测,血清学检测对于SARS-CoV-2感染的诊断至关重要,但对于研究流行病学至关重要,血清监测,以及疫苗研发。多重免疫测定技术具有特别的优势,因为它们可以同时测量来自单个样品的多个分析物。xMAP技术是一种多重分析平台,可同时从同一样品中测量多达500种分析物。它已被证明是研究对各种SARS-CoV-2抗原的免疫反应的重要工具,以及测量宿主蛋白生物标志物水平作为COVID-19的预后指标。在这一章中,我们描述了几项关键研究,其中使用xMAP技术对COVID-19患者的SARS-COV-2抗体反应和宿主蛋白表达进行多重分析.
    The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has been one of the most severe outbreaks of respiratory illness in history. The clinical symptoms of COVID-19 may be similar to flu, although they can be life-threatening, particularly in the elderly and immunocompromised population. Together with nucleic acid detection, serological testing has been essential for the diagnosis of SARS-CoV-2 infection but has been critically important for studying the epidemiology, serosurveillance, and for vaccine research and development. Multiplexed immunoassay technologies have a particular advantage as they can simultaneously measure multiple analytes from a single sample. xMAP technology is a multiplex analysis platform that can measure up to 500 analytes at the same time from the same sample. It has been shown to be an important tool for studying immune response to the various SARS-CoV-2 antigens, as well as for measuring host protein biomarker levels as prognostic indicators of COVID-19. In this chapter, we describe several key studies where xMAP technology was used for multiplexed analysis of SARS-COV-2 antibody responses and host protein expression in COVID-19 patients.
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  • 文章类型: Journal Article
    Ovarian cancer (OC) is the most lethal genital malignancy in women. We aimed to develop and validate new proteomic-based models for non-invasive diagnosis of OC. We also compared them to the modified Risk of Ovarian Malignancy Algorithm (ROMA-50), the Copenhagen Index (CPH-I) and our earlier Proteomic Model 2017. Biomarkers were assessed using bead-based multiplex technology (Luminex®) in 356 women (250 with malignant and 106 with benign ovarian tumors) from five European centers. The training cohort included 279 women from three centers, and the validation cohort 77 women from two other centers. Of six previously studied serum proteins (CA125, HE4, osteopontin [OPN], prolactin, leptin, and macrophage migration inhibitory factor [MIF]), four contributed significantly to the Proteomic Model 2021 (CA125, OPN, prolactin, MIF), while leptin and HE4 were omitted by the algorithm. The Proteomic Model 2021 revealed a c-index of 0.98 (95% CI 0.96, 0.99) in the training cohort; however, in the validation cohort it only achieved a c-index of 0.82 (95% CI 0.72, 0.91). Adding patient age to the Proteomic Model 2021 constituted the Combined Model 2021, with a c-index of 0.99 (95% CI 0.97, 1) in the training cohort and a c-index of 0.86 (95% CI 0.78, 0.95) in the validation cohort. The Full Combined Model 2021 (all six proteins with age) yielded a c-index of 0.98 (95% CI 0.97, 0.99) in the training cohort and a c-index of 0.89 (95% CI 0.81, 0.97) in the validation cohort. The validation of our previous Proteomic Model 2017, as well as the ROMA-50 and CPH-I revealed a c-index of 0.9 (95% CI 0.82, 0.97), 0.54 (95% CI 0.38, 0.69) and 0.92 (95% CI 0.85, 0.98), respectively. In postmenopausal women, the three newly developed models all achieved a specificity of 1.00, a positive predictive value (PPV) of 1.00, and a sensitivity of >0.9. Performance in women under 50 years of age (c-index below 0.6) or with normal CA125 (c-index close to 0.5) was poor. CA125 and OPN had the best discriminating power as single markers. In summary, the CPH-I, the two combined 2021 Models, and the Proteomic Model 2017 showed satisfactory diagnostic accuracies, with no clear superiority of either model. Notably, although combining values of only four proteins with age, the Combined Model 2021 performed comparably to the Full Combined Model 2021. The models confirmed their exceptional diagnostic performance in women aged ≥50. All models outperformed the ROMA-50.
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  • 文章类型: Journal Article
    目的:移植肾功能恶化与细胞衰老加速有关。海洋n-3多不饱和脂肪酸(PUFA)具有良好的特性,可以抵消衰老相关分泌表型(SASP)分泌组引起的细胞衰老发育和损伤。我们的目的是研究补充海洋n-3PUFA对肾移植受者SASP分泌组的潜在影响。
    方法:Omega-3脂肪酸在肾移植试验中的探索性亚研究。
    方法:在挪威进行的这项研究中,纳入了移植后8周具有功能性肾移植(定义为估计肾小球滤过率>30mL/min/1.73m2)的成人肾移植受者。
    方法:干预包括每天2.6g海洋n-3PUFA或橄榄油(安慰剂),持续44周。结果是血浆和尿液中SASP组分的预定组。
    结果:共有132名患者参加了Omega-3脂肪酸肾移植试验,66例患者被分配接受研究药物或安慰剂治疗.海洋n-3PUFA的干预与粒细胞集落刺激因子的血浆水平降低有关,白细胞介素1α,巨噬细胞炎性蛋白1α,基质金属蛋白酶(MMP)-1、MMP-13与干预对照组比较。
    结论:事后分析。
    结论:结果表明,补充海洋n-3PUFA对血浆SASP成分粒细胞集落刺激因子具有缓解作用,白细胞介素1α,巨噬细胞炎性蛋白1α,肾移植受者的MMP-1和MMP-13。肾脏移植受者处于维持阶段的未来研究,结合肾移植活检中细胞衰老标志物的评估,需要进一步阐明海洋n-3PUFA的潜在抗衰老作用。该试验注册为NCT01744067。
    OBJECTIVE: Deterioration of kidney graft function is associated with accelerated cellular senescence. Marine n-3 polyunsaturated fatty acids (PUFAs) have favorable properties that may counteract cellular senescence development and damage caused by the senescence-associated secretory phenotype (SASP) secretome. Our objective was to investigate the potential effects of marine n-3 PUFA supplementation on the SASP secretome in kidney transplant recipients.
    METHODS: Exploratory substudy of the Omega-3 Fatty Acids in Renal Transplantation trial.
    METHODS: Adult kidney transplant recipients with a functional kidney graft (defined as having an estimated glomerular filtration rate of >30 mL/min/1.73 m2) 8 weeks after engraftment were included in this study conducted in Norway.
    METHODS: The intervention consisted of 2.6 g of a marine n-3 PUFA or olive oil (placebo) daily for 44 weeks. The outcome was a predefined panel of SASP components in the plasma and urine.
    RESULTS: A total of 132 patients were enrolled in the Omega-3 Fatty Acids in Renal Transplantation trial, and 66 patients were allocated to receive either the study drug or placebo. The intervention with the marine n-3 PUFA was associated with reduced plasma levels of granulocyte colony-stimulating factor, interleukin 1α, macrophage inflammatory protein 1α, matrix metalloproteinase (MMP)-1, and MMP-13 compared with the intervention in the control group.
    CONCLUSIONS: Post hoc analysis.
    CONCLUSIONS: The results suggest that marine n-3 PUFA supplementation has mitigating effects on the plasma SASP components granulocyte colony-stimulating factor, interleukin 1α, macrophage inflammatory protein 1α, MMP-1, and MMP-13 in kidney transplant recipients. Future studies with kidney transplant recipients in maintenance phase, combined with an evaluation of cellular senescence markers in kidney transplant biopsies, are needed to further elucidate the potential antisenescent effect of marine n-3 PUFAs. This trial is registered as NCT01744067.
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  • 文章类型: Journal Article
    背景:膀胱内卡介苗(BCG),一种作为非特异性免疫系统兴奋剂的减毒活结核疫苗,是中度或高危非肌肉浸润性膀胱癌(NMIBC)患者最有效的辅助治疗方法。然而,到目前为止,没有可靠的测试可以预测BCG治疗的反应.在这项研究中,我们评估了OncuriaTM的性能,膀胱癌检测测试,预测对膀胱内BCG的反应。
    方法:在使用膀胱内BCG治疗前,从64名中度或高风险NMIBC患者的前瞻性收集队列中获得的尿液样本中评估了OncuriaTM数据。OncuriaTM测试,在独立的临床实验室中进行了10项癌症相关生物标志物的测量.测试了鉴定BCG对肿瘤复发无效的患者的测试能力。使用监督学习和交叉验证分析得出预测模型。使用ROC曲线评估模型性能。
    结果:治疗前MMP9,VEGFA,CA9,SDC1,PAI1,APOE,A1AT,发生疾病复发的患者ANG和MMP10升高。治疗结果的组合预测模型达到AUROC0.89[95%CI:0.80-0.99],胜过任何单一的生物标志物,测试灵敏度为81.8%,特异性为84.9%。危险比分析显示,尿液中ANG水平较高的患者,CA9和MMP10具有显著较高的疾病复发风险。
    结论:监测与癌症相关的生物标志物组的尿水平能够区分对膀胱内BCG治疗无反应的患者。随着进一步的研究,多重OncuriaTM检测可能适用于考虑膀胱内BCG治疗的膀胱癌患者的临床评估。
    BACKGROUND: Intravesical Bacillus Calmette-Guerin (BCG), a live attenuated tuberculosis vaccine that acts as a non-specific immune system stimulant, is the most effective adjuvant treatment for patients with intermediate or high-risk non-muscle-invasive bladder cancer (NMIBC). However, to date, there are no reliable tests that are predictive of BCG treatment response. In this study, we evaluated the performance of OncuriaTM, a bladder cancer detection test, to predict response to intravesical BCG.
    METHODS: OncuriaTM data was evaluated in voided urine samples obtained from a prospectively collected cohort of 64 subjects with intermediate or high risk NMIBC prior to treatment with intravesical BCG. The OncuriaTM test, which measures 10 cancer-associated biomarkers was performed in an independent clinical laboratory. The ability of the test to identify those patients in whom BCG is ineffective against tumor recurrence was tested. Predictive models were derived using supervised learning and cross-validation analyses. Model performance was assessed using ROC curves.
    RESULTS: Pre-treatment urinary concentrations of MMP9, VEGFA, CA9, SDC1, PAI1, APOE, A1AT, ANG and MMP10 were increased in patients who developed disease recurrence. A combinatorial predictive model of treatment outcome achieved an AUROC 0.89 [95% CI: 0.80-0.99], outperforming any single biomarker, with a test sensitivity of 81.8% and a specificity of 84.9%. Hazard ratio analysis revealed that patients with higher urinary levels of ANG, CA9 and MMP10 had a significantly higher risk of disease recurrence.
    CONCLUSIONS: Monitoring the urinary levels of a cancer-associated biomarker panel enabled the discrimination of patients who did not respond to intravesical BCG therapy. With further study, the multiplex OncuriaTM test may be applicable for the clinical evaluation of bladder cancer patients considering intravesical BCG treatment.
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  • 文章类型: Journal Article
    背景:特发性肺纤维化(IPF)是一种慢性,导致呼吸衰竭和死亡的进行性纤维化肺病。尽管人们对这种疾病的病因有了更多的了解,仍然无法准确预测个体患者的病程。本研究旨在评估血清细胞因子/趋化因子作为预测IPF患者预后的潜在生物标志物。
    方法:采用了使用两个独立队列的多机构前瞻性两阶段发现和验证设计。对于发现分析,来自100名IPF患者和32名健康对照的血清样本使用无偏检查,48种细胞因子/趋化因子的多重免疫测定。在IPF患者和对照组之间比较血清细胞因子/趋化因子值;在IPF患者中评估多重测量与生存时间之间的关联。在验证分析中,我们在另外81例IPF患者的血清样本中检测了发现分析中鉴定的细胞因子/趋化因子,以验证这些细胞因子/趋化因子预测生存期的能力.还进行IPF衍生的肺样品的免疫组织化学评估以确定该新型生物标志物在何处表达。
    结果:在发现队列中,与对照组相比,IPF患者血清中的18种细胞因子/趋化因子显着升高。白细胞介素-1受体α(IL-1Rα),白细胞介素-8(IL-8),巨噬细胞炎性蛋白1α(MIP-1α),皮肤T细胞吸引趋化因子(CTACK)与存活相关:IL-1Rα,危险比(HR)=每10个单位1.04,95%置信区间(95%CI)1.01-1.07;IL-8,HR=1.04,95%CI1.01-1.08;MIP-1α,HR=1.19,95%CI1.00-1.36;和CTACK,HR=每100单位1.12,95%CI1.02-1.21。仅对CTACK进行复制分析,因为先前报道其它是间质性肺病的潜在生物标志物。在验证队列中,CTACK与生存率相关:HR=1.14/100单位,95%CI1.01-1.28。免疫组织化学显示CTACK和CC趋化因子受体10(CTACK的配体)在IPF患者的气道和II型肺泡上皮细胞中表达,但在对照组中未表达。
    结论:CTACK是IPF的一种新的预后生物标志物。试验登记无(因为没有医疗干预)。
    BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disease that leads to respiratory failure and death. Although there is a greater understanding of the etiology of this disease, accurately predicting the disease course in individual patients is still not possible. This study aimed to evaluate serum cytokines/chemokines as potential biomarkers that can predict outcomes in IPF patients.
    METHODS: A multi-institutional prospective two-stage discovery and validation design using two independent cohorts was adopted. For the discovery analysis, serum samples from 100 IPF patients and 32 healthy controls were examined using an unbiased, multiplex immunoassay of 48 cytokines/chemokines. The serum cytokine/chemokine values were compared between IPF patients and controls; the association between multiplex measurements and survival time was evaluated in IPF patients. In the validation analysis, the cytokines/chemokines identified in the discovery analysis were examined in serum samples from another 81 IPF patients to verify the ability of these cytokines/chemokines to predict survival. Immunohistochemical assessment of IPF-derived lung samples was also performed to determine where this novel biomarker is expressed.
    RESULTS: In the discovery cohort, 18 cytokines/chemokines were significantly elevated in sera from IPF patients compared with those from controls. Interleukin-1 receptor alpha (IL-1Rα), interleukin-8 (IL-8), macrophage inflammatory protein 1 alpha (MIP-1α), and cutaneous T-cell-attracting chemokine (CTACK) were associated with survival: IL-1Rα, hazard ratio (HR) = 1.04 per 10 units, 95% confidence interval (95% CI) 1.01-1.07; IL-8, HR = 1.04, 95% CI 1.01-1.08; MIP-1α, HR = 1.19, 95% CI 1.00-1.36; and CTACK, HR = 1.12 per 100 units, 95% CI 1.02-1.21. A replication analysis was performed only for CTACK because others were previously reported to be potential biomarkers of interstitial lung diseases. In the validation cohort, CTACK was associated with survival: HR = 1.14 per 100 units, 95% CI 1.01-1.28. Immunohistochemistry revealed the expression of CTACK and CC chemokine receptor 10 (a ligand of CTACK) in airway and type II alveolar epithelial cells of IPF patients but not in those of controls.
    CONCLUSIONS: CTACK is a novel prognostic biomarker of IPF. Trial registration None (because of no healthcare intervention).
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  • 文章类型: Journal Article
    Background: To support the rapid development of an antibody cocktail against Ebola virus and avoid unnecessary exposure to infectious environments, an automatic and fast turnover triplex assay was developed using Simoa® (Quanterix Corporation, MA, USA). Materials & methods: A robust triplex assay was developed and validated for simultaneous quantification of the antibody cocktail against Ebola virus in cynomolgus serum. Results: The assay had a quantitation range of 78.1-5000 ng/ml. The intra- and interassay precisions (%CV) were within 11.4 and 13.9%, and the accuracies (%RE) were within -10.8 to 6.8%, respectively. Cross-reactivity was evaluated, and the results met the acceptance criteria. Conclusion: The assay was successfully applied to a pharmacokinetics study following a single-dose intravenous administration of 10 mg/kg the antibody cocktail against Ebola virus to cynomolgus monkeys.
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  • 文章类型: Journal Article
    Aim: Accumulating evidence suggests that neural inflammation plays an important role in psychiatric disorders. We aimed to identify inflammatory cytokines involved in the pathophysiology of such disorders by quantifying them in cerebrospinal fluid (CSF) samples from a large sample of patients with major psychiatric disorders and healthy controls. Methods: The subjects included 94 patients with schizophrenia, 68 with bipolar disorder, 104 with major depressive disorder, and 118 healthy controls, matched for age, sex, and ethnicity (Japanese). Lumbar puncture was performed to collect these CSF samples. A multiplex immunoassay was then performed to measure CSF cytokine levels using magnetic on-bead antibody conjugation for 19 inflammatory cytokines. Results: CSF interferon-β level was significantly higher in total psychiatric patients than in healthy controls (corrected p = 0.000029). In diagnostic group comparisons, CSF interferon-β level was significantly higher in patients with schizophrenia, or bipolar disorder (corrected p = 0.000047 or 0.0034) than in healthy controls. Conclusion: We present novel evidence that CSF IFN-β level showed prominent statistical differences between psychiatric groups and healthy controls. This suggests IFN-β as the most important player among the 19 cytokines tested here in the inflammation-related pathophysiology of major psychiatric disorders.
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  • 文章类型: Comparative Study
    Case-control study for the evaluation of innovative test formats for second-tier testing for the serodiagnosis of Lyme borreliosis (LB). A head-to-head comparison was performed with the test systems ViraStripe, SeraSpot, ViraChip, and recomBead. Serum samples from 62 patients (21 erythema migrans, 33 Lyme neuroborreliosis, 8 late LB) and 91 controls (including 29 potentially cross-reacting sera) were tested. For ViraChip and recomBead, optimised interpretation criteria were developed for both IgG and IgM. The most important modification for the proposed interpretation criteria for ViraChip is the interpretation of strong (> 2.5-fold above cutoff) singular IgG reactions against VlsE as positive. This significantly improves sensitivity (32 to 85%, p < 0.0001) without significant changes in specificity (borderline reactions interpreted as negative). By application of our modified rules, specificity of ViraChip IgM is significantly increased (89 to 97%, p < 0.05; borderline results included to negatives), and sensitivities of recomBead IgG and IgM are also significantly improved (69 to 87%, p < 0.01, and 57 to 74%, p < 0.01, respectively; borderline results included to positives). Further improvement of sensitivity by the rating of strong singular IgG reactions against VlsE as positive can also be shown for recomBead. IgG/IgM result combinations must be interpreted as a function of the assumed disease stage, and the best combinations differ for the various assays. Application of our proposed interpretation criteria significantly improve the discriminatory abilities of two assays; however, this must be confirmed with other data sets. Recommendations from Scientific Societies should be updated as may be necessary.
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  • 文章类型: Journal Article
    The HIV/HCV/HBsAg Triplex consists in manually performed, visually interpreted, lateral flow, immunochromatographic rapid diagnostic test simultaneously detecting in 15min human immunodeficiency virus (HIV)-1 and HIV-2 and hepatitis C virus (HCV)- specific antibodies (Ab) (IgG and IgM) and hepatitis B virus (HBV) surface antigen (HBsAg) in serum, plasma and whole blood.
    A hospital-based cross-sectional study was conducted on a prospective panel of serum samples from adult inpatients included from routine analysis irrespectively of age and sex, including 250 sera positive for HIV-1-specific Ab, 250 for HCV-specific Ab, 250 for HBsAg and 250 sera negative for HIV- and HCV- Ab and HBsAg, and from 110 HIV-2-infected patients living in Ivory Coast, according to the results obtained by the reference chemiluminiscent microparticle immunoassay (CMIA) Abbott Architect i2000SR analyzer (Abbott Diagnostic, Chicago, IL, USA). Among HCV-seropositive sera, 187 were positive for HCV RNA (chronic infection), whereas 63 were negative (resolved infection), respectively. Serum samples were further tested blindly by HIV/HCV/HBsAg Triplex according to manufacturers\' recommendations.
    HIV/HCV/HBsAg Triplex showed very high sensitivity and specificity, as well as excellent concordance with CMIA Abbott results, as shown in the Table. Lower sensitivity was observed only in individuals who had cleared their HCV infection (presence of HCV-specific Ab in absence of HCV RNA). The mean lower limit of HBsAg detection was 2.38±0.63 IU/ml. Erythrocytes-spiked serum samples gave similar results than serum samples.
    Advantages of HIV/HCV/HBsAg Triplex for HIV-1, HIV-2, HCV and HBV include the requirement for less overall specimen volume, fewer finger-sticks if capillary whole blood is used, cost savings through lower cost per virus tested, improved patient flow with results for multiple viruses available at the same time, overall service delivery efficiencies with less time required per infected patient; and patient benefits from fewer visits and lower cost associated with each clinic attendance. The screening of chronic HIV, HCV and HBV by multiplex HIV-1/HIV-2/HCV/HBsAg Triplex may improve the \"cascade of screening\" and quite possibly linkage-to-care with reduced cost.
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  • 文章类型: Journal Article
    OBJECTIVE: The aim of this study was to analyze the differences in inflammatory and catabolic mediators expressed in peri-implantitis compared to periodontitis lesions after non-surgical therapy. Peri-implantitis is associated with a faster rate of bone loss when compared with periodontitis, and peri-implant non-surgical therapy is ineffective to cure peri-implantitis. This may be due to persistent inflammation in peri-implantitis tissues after initial mechanical treatment.
    METHODS: Eleven patients with peri-implantitis and 10 with severe chronic periodontitis received non-surgical therapy. They were included at re-evaluation (8 weeks) if they presented pocket depth ≥6 mm with bleeding on probing, and the indication for open flap debridement surgery. Connective tissues were harvested during surgery from diseased sites. Healthy gingiva were harvested during third molar extraction in a third group of healthy patients (n=10). Explants were incubated for 24 hours in media culture and the release of cytokines, chemokines, growth factors, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand (RANKL), matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase (TIMP) in the conditioned media was analyzed by an exploratory multiplex immunoassay. When difference was found in the conditioned media, an immunohistochemistry was performed to compare expression in the tissues.
    RESULTS: Connective tissues from non-stabilized peri-implantitis exhibited a distinct cytokine profile compared to periodontitis lesions that did not respond to initial therapy. Indeed, TIMP-2 was significantly increased in media from peri-implantitis (P≤.05). In addition, the in situ expression of TIMP-2, interleukin-10 and RANKL was also significantly increased in peri-implantitis tissues (P≤.05). However, the ratio of RANKL/osteoprotegerin-positive cells did not vary (P≥.05).
    CONCLUSIONS: This study suggests that peri-implantitis and periodontitis connective tissues exhibit differences in response to non-surgical treatment, which may contribute to a different pattern of disease evolution.
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