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BACKGROUND The implementation of new biomarkers into clinical practice is one of the most important areas in medical research. Besides their clinical impact, novel in vitro diagnostic markers promise to have a substantial effect on healthcare costs. Although numerous publications report the discovery of biomarkers, only a fraction of those markers are routinely used. One key challenge is a measurement system that is compatible with clinical workflows. METHODS We designed a new immunoassay for microRNA (miRNA) quantification. The assay combines streptavidin-linked microparticles, a biotinylated catcher oligonucleotide complementary to a single miRNA species, and finally, a monoclonal antibody to DNA/RNA heterohybrids labeled with acridinium ester. Importantly, our assay runs on standard immunoassay analyzers. After a technical validation of the assay, we evaluated the clinical performance on 4 Alzheimer disease miRNAs. RESULTS Our assay has an analytical specificity of 99.4% and is at the same time sensitive (concentrations in the range of 1 pmol/L miRNA can be reliably profiled). Because the novel approach did not require amplification steps, we obtained high reproducibility for up to 40 biological replicates. Importantly, our assay prototype exhibited a time to result of <3 h. With human blood samples, the assay was able to measure 4 miRNAs that can detect Alzheimer disease with a diagnostic accuracy of 82% and showed a Pearson correlation >0.994 with the gold standard qRT-PCR. CONCLUSIONS Our miRNA immunoassay allowed the measurement of miRNA signatures with sufficient analytical sensitivity and high specificity on commonly available laboratory equipment.

BackgroundThe assessment of circulating tumor cells (CTCs) has been shown to enable monitoring of treatment response and early detection of metastatic breast cancer (MBC) recurrence. The aim of this study was to compare a well-established CTC detection method based on immunomagnetic isolation with a new, filtration-based platform.MethodsIn this prospective study, two 7.5 ml blood draws were obtained from 60 MBC patients and CTC enumeration was assessed using both the CellSearch® and the newly developed filtration-based platform. We analyzed the correlation of CTC-positivity between both methods and their ability to predict prognosis. Overall survival (OS) was calculated and Kaplan-Meier curves were estimated with thresholds of ≥1 and ≥5 detected CTCs.ResultsThe CTC positivity rate of the CellSearch® system was 56.7% and of the filtration-based platform 66.7%. There was a high correlation of CTC enumeration obtained with both methods. The OS for patients without detected CTCs, regardless of the method used, was significantly higher compared to patients with one or more CTCs (p < 0.001). The median OS of patients with no CTCs vs. ≥ 1 CTC assessed by CellSearch® was 1.83 years (95% CI: 1.63–2.02) vs. 0.74 years (95% CI: 0.51–1.52). If CTCs were detected by the filtration-based method the median OS times were 1.88 years (95% CI: 1.74–2.03) vs. 0.59 years (95% CI: 0.38–0.80).ConclusionsThe newly established EpCAM independently filtration-based system is a suitable method to determine CTC counts for MBC patients. Our study confirms CTCs as being strong predictors of prognosis in our population of MBC patients.

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