Measurement of microRNA with isothermal DNA amplification on fully automated immunoassay analyzers.

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.

Improvement and multicenter evaluation of the analytical performance of an automated chemiluminescent immunoassay for alpha fetoprotein.

BACKGROUND: A new ARCHITECT® alpha fetoprotein (AFP) assay was developed to improve the linearity at the upper end of the calibration curve and to enhance other performance characteristics. In addition, this reformulation eliminated the possibility of falsely depressed samples at high AFP concentrations. The purpose of this study was to evaluate its analytical performance at multiple sites. METHODS: The assay configuration, the diluent formulation, and the manufacturing process were redesigned. Analytical performance was evaluated at Abbott Laboratories, Sapporo Medical University, VU University Medical Center, and Johns Hopkins University. RESULTS: The limit of quantitation of the assay was 1.00-1.30 ng/mL. Total precision (%CV) across the assay range varied between 1.41 and 3.52. The assay was linear from 1.19 to 2535 ng/mL, and the range of the assay was expanded from 200 ng/mL to 2000 ng/mL. Comparison of this assay with the on-market ARCHITECT, AxSYM, ADVIA Centaur, DxI, AIA-1800, and E 170 systems yielded regression slopes of 0.91-1.08 and correlation coefficients of =0.99 for serum samples. No falsely depressed results were observed in 174 serum samples with AFP concentrations of 2018-1,196,856 ng/mL and in a spiked sample containing up to 10 mg/mL of purified AFP. CONCLUSIONS: The new AFP assay has improved an issue of the on-market ARCHITECT AFP assay and demonstrated excellent assay performance.