Abstract
Multiplexing arrays increase the throughput and decrease sample requirements for studies employing multiple biomarkers. The goal of this project was to examine the performance of Multiplex arrays for measuring multiple protein biomarkers in saliva and serum. Specimens from the OsteoPerio ancillary study of the Women’s Health Initiative Observational Study were used. Participants required the presence of at least 6 teeth and were excluded based on active cancer and certain bone issues but were not selected on any specific condition. Quality control (QC) samples were created from pooled serum and saliva. Twenty protein markers were measured on five multiplexing array panels. Sample pretreatment conditions were optimized for each panel. Recovery, lower limit of quantification (LLOQ) and imprecision were determined for each analyte. Statistical adjustment at the plate level was used to reduce imprecision estimates and increase the number of usable observations. Sample pre-treatment improved recovery estimates for many analytes. The LLOQ for each analyte agreed with manufacturer specifications except for MMP-1 and MMP-2 which were significantly higher than reported. Following batch adjustment, 17 of 20 biomarkers in serum and 9 of 20 biomarkers in saliva demonstrated acceptable precision, defined as <20% coefficient of variation (<25% at LLOQ). The percentage of cohort samples having levels within the reportable range for each analyte varied from 10% to 100%. The ratio of levels in saliva to serum varied from 1∶100 to 28∶1. Correlations between saliva and serum were of moderate positive magnitude and significant for CRP, MMP-2, insulin, adiponectin, GM-CSF and IL-5. Multiplex arrays exhibit high levels of analytical imprecision, particularly at the batch level. Careful sample pre-treatment can enhance recovery and reduce imprecision. Following statistical adjustments to reduce batch effects, we identified biomarkers that are of acceptable quality in serum and to a lesser degree in saliva using Multiplex arrays.
Highlights
Accurate and reliable measurement of inflammatory biomarkers is critical to assessing inflammatory mechanisms involved in many diseases including periodontal disease
In this report we describe the ability of these assays to provide reliable measurements of inflammatory cytokines and other biomarkers in homologous samples of serum and saliva collected from participants in the Buffalo OsteoPerio Study, ancillary to the national Women’s Health Initiative Observational Study
Upon encountering significant plate-to-plate variations in the means of samples and quality control (QC) materials, we examined whether demographic variables were randomly distributed across the plates and no significant differences were found
Summary
Accurate and reliable measurement of inflammatory biomarkers is critical to assessing inflammatory mechanisms involved in many diseases including periodontal disease. Saliva presents specific measurement challenges due to its viscosity, differences in matrix, and molecular content It is not known how comparable the content of saliva is to the widely used serum in screening for biological changes indicative of disease onset or progression. High-throughput measures of analytes in saliva and serum offer a novel and convenient method for comparing and assessing the role of biomarkers in oral and systemic compartments. These methods need to be efficient with respect to cost and sample volume requirements while being accurate and reproducible in characterizing ‘‘health’’ and ‘‘disease.’’
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