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A multicenter analytical performance evaluation of a multiplexed immunoarray for the simultaneous measurement of biomarkers of micronutrient deficiency, inflammation and malarial antigenemia

ABSTRACT: A lack of comparative data across laboratories is often a barrier to the uptake and adoption of new technologies. Furthermore, data generated by different immunoassay methods may be incomparable due to a lack of harmonization. In this multicenter study, we describe validation experiments conducted in a single lab and cross-lab comparisons of assay results to assess the performance characteristics of the Q-plex™ 7-plex Human Micronutrient Array (7-plex), an immunoassay that simultaneously quantifies seven biomarkers associated with micronutrient (MN) deficiencies, inflammation and malarial antigenemia using plasma or serum; alpha-1-acid glycoprotein, C-reactive protein, ferritin, histidine-rich protein 2, retinol binding protein 4, soluble transferrin receptor, and thyroglobulin. Validations included repeated testing (n = 20 separately prepared experiments on 10 assay plates) in a single lab to assess precision and linearity. Seven independent laboratories tested 76 identical heparin plasma samples collected from a cohort of pregnant women in Niger using the same 7-plex assay to assess differences in results across laboratories. In the analytical validation experiments, intra- and inter-assay coefficients of variation were acceptable at <6% and <15% respectively and assay linearity was 96% to 99% with the exception of ferritin, which had marginal performance in some tests. Cross-laboratory comparisons showed generally good agreement between laboratories in all analyte results for the panel of 76 plasma specimens, with Lin’s concordance correlation coefficient values averaging ≥0.8 for all analytes. Excluding plates that would fail routine quality control (QC) standards, the inter-assay variation was acceptable for all analytes except sTfR, which had an average inter-assay coefficient of variation of ≥20%. This initial cross-laboratory study demonstrates that the 7-plex test protocol can be implemented by users with some experience in immunoassay methods, but familiarity with the multiplexed protocol was not essential.

SUBMITTER: Brindle E

PROVIDER: S-EPMC8568126 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: ?-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate-needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world's population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and Plasmodium falciparum parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, ?-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.

Project description:Parathyroid hormone-related peptide (PTHrP) is recognized as the major causative agent of humoral hypercalcemia of malignancy (HHM). The paraneoplastic PTHrP has also been implicated in tumor progression and metastasis of many human cancers. Conventional PTHrP detection methods like immunoradiometric assay (IRMA) lack the sensitivity required to measure target peptide levels prior to the development of hypercalcemia. In general, sensitive, multiplexed peptide measurement by immunoassay represents challenges that we address in this paper. We describe here the first ultrasensitive multiplexed peptide assay to measure intact PTHrP 1-173 as well as circulating N-terminal and C-terminal peptide fragments. This versatile approach should apply to almost any collection of peptides that are long enough to present binding sites for two antibodies. To target PTHrP, we employed a microfluidic immunoarray featuring a chamber for online capture of the peptides from serum onto magnetic beads decorated with massive numbers of peptide-specific antibodies and enzyme labels. Magnetic bead-peptide conjugates were then washed and sent to a detection chamber housing an antibody-modified 8-electrode array fabricated by inkjet printing of gold nanoparticles. Limits of detection (LODs) of 150 aM (∼1000-fold lower than IRMA) in 5 μL of serum were achieved for simultaneous detection of PTHrP isoforms and peptide fragments in 30 min. Good correlation for patient samples was found with IRMA (n = 57); r(2) = 0.99 assaying PTHrP 1-86 equiv fragments. Analysis by a receiver operating characteristic (ROC) plot gave an area under the curve of 0.96, 80-83% clinical sensitivity, and 96-100% clinical specificity. Results suggest that PTHrP1-173 isoform and its short C-terminal fragments are the predominant circulating forms of PTHrP. This new ultrasensitive, multiplexed assay for PTHrP and fragments is promising for clinical diagnosis, prognosis, and therapeutic monitoring from early to advanced stage cancer patients and to examine underlying mechanisms of PTHrP overproduction.

Project description:BackgroundAnalytical approaches for the interpretation of anti-malarial clinical trials vary considerably. The aim of this study was to quantify the magnitude of the differences between efficacy estimates derived from these approaches and identify the factors underlying these differences.MethodsData from studies conducted in Africa and Thailand were compiled and the risk estimates of treatment failure, adjusted and unadjusted by genotyping, were derived by three methods (intention to treat (ITT), modified intention to treat (mITT) and per protocol (PP)) and then compared.Results29 clinical trials (15 from Africa and 14 from Thailand) with a total of 65 treatment arms (38 from Africa and 27 from Thailand) were included in the analysis. Of the 15,409 patients enrolled, 2,637 (17.1%) had incomplete follow up for the unadjusted analysis and 4,489 (33.4%) for the adjusted analysis. Estimates of treatment failure were consistently higher when derived from the ITT or PP analyses compared to the mITT approach. In the unadjusted analyses the median difference between the ITT and mITT estimates was greater in Thai studies (11.4% [range 2.1-31.8]) compared to African Studies (1.8% [range 0-11.7]). In the adjusted analyses the median difference between PP and mITT estimates was 1.7%, but ranged from 0 to 30.9%. The discrepancy between estimates was correlated significantly with the proportion of patients with incomplete follow-up; p < 0.0001. The proportion of studies with a major difference (> 5%) between adjusted PP and mITT was 28% (16/57), with the risk difference greater in African (37% 14/38) compared to Thai studies (11% 2/19). In the African studies, a major difference in the adjusted estimates was significantly more likely in studies in high transmission sites (62% 8/13) compared to studies in moderate transmission sites (24% 6/25); p = 0.035.ConclusionEstimates of anti-malarial clinical efficacy vary significantly depending on the analytical methodology from which they are derived. In order to monitor temporal and spatial trends in anti-malarial efficacy, standardized analytical tools need to be applied in a transparent and systematic manner.

Project description:BackgroundThe prevalence of micronutrient deficiencies is higher in obese individuals compared to normal-weight people, probably because of inadequate eating habits but also due to increased demands among overweight persons, which are underestimated by dietary reference intakes (DRI) intended for the general population. We therefore evaluated the dietary micronutrient intake in obese individuals compared to a reference population and DRI recommendations. Furthermore, we determined the micronutrient status in obese subjects undergoing a standardized DRI-covering low-calorie formula diet to analyze if the DRI meet the micronutrient requirements of obese individuals.MethodsIn 104 subjects baseline micronutrient intake was determined by dietary record collection. A randomly assigned subgroup of subjects (n = 32) underwent a standardized DRI-covering low-calorie formula diet over a period of three months. Pre- and post-interventional intracellular micronutrient status in buccal mucosa cells (BMC) was analyzed, as well as additional micronutrient serum concentrations in 14 of the subjects.ResultsPrior to dietetic intervention, nutrition was calorie-rich and micronutrient-poor. Baseline deficiencies in serum concentrations were observed for 25-hydroxyvitamin-D, vitamin C, selenium, iron, as well as ß-carotene, vitamin C, and lycopene in BMC. After a three-month period of formula diet even more subjects had reduced micronutrient levels of vitamin C (serum, BMC), zinc, and lycopene. There was a significant negative correlation between lipophilic serum vitamin concentrations and body fat, as well as between iron and C-reactive protein.ConclusionsThe present pilot study shows that micronutrient deficiency occurring in obese individuals is not corrected by protein-rich formula diet containing vitamins and minerals according to DRI. In contrast, micronutrient levels remain low or become even lower, which might be explained by insufficient intake, increased demand and unbalanced dispersal of lipophilic compounds in the body.Trial registrationThe study was registered at ClinicalTrials.gov (NCT01344525). The study protocol comprises only a part of the approved trial protocol.

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A multicenter analytical performance evaluation of a multiplexed immunoarray for the simultaneous measurement of biomarkers of micronutrient deficiency, inflammation and malarial antigenemia

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