AMH immunoassay: A multi center evaluation of the precision of a novel fully automated AMH assay under routine conditions Johan Schiettecatte1; Murat Öktem2; Arne Thies3; Martine Cohen-Bacrie4; Ellen Anckaert1; Cyrill Müller5; Deniz Topcu2; Arndt Gröning3, Sabine Engelmann6; Christoph Milczynski5 1. UZ Brussels, Laarbeeklaan 101, 1090 Brussels, Belgium, 2. Duzen Laboratories Group, Tunus Cad. No. 95 Kavaklidere, Ankara, Turkey, 3. MVZ wagnerstibbe für Laboratoriumsmedizin und Pathologie GmbH, Georgstraße 50, 30159 Hannover, Germany, 4. Laboratoire Eylau Saint Didier, 55, rue Saint Didier, 75116 Paris, France, 5. Limbach Laboratory, Medical Care Center, Im Breitspiel 15, 69126 Heidelberg, Germany, 6. Roche Diagnostics GmbH, Sandhoferstr. 116, 68305 Mannheim Background 21-day precision according to CLSI-EP5: AMH immunoassay The CLSI EP5 precision performance experiment (21 days) was performed on cobas e 601, cobas e 411 and MODULAR® ANALYTICS E170. The experiment included PC AMH 1, PC AMH 2 as control material and 5 serum pools with different levels. Anti-Müllerian-Hormone (AMH) is an established marker for assessing the ovarian reserve and optimization of in-vitro fertilization treatment. Current existing manual AMH assays exhibit limitations with respect to throughput and reliability of results. Recently a fully automated AMH assay has been developed on the Elecsys®/cobas e electrochemiluminescence immunoassay platform (product launch in preparation). The objective of the technical performance study was to evaluate the precision of this highthroughput assay under routine conditions. Repeatability of AMH immunoassay according to CLSI-EP5 e 411 e 601 E170 2.0 1.8 Study design and methods applied 1.6 1.4 1.2 CV (%) The precision performance of AMH immunoassay was evaluated in five different routine laboratories across Europe. The evaluation of the immunoassay was performed on a total of two cobas e 601 instruments, three cobas e 411 instruments and one MODULAR® ANALYTICS E170 and compared to the respective routinely used manual method. Each laboratory evaluated the performance of the immunoassay independently. The study design encompassed a within-run-precision experiment and the evaluation of the repeatability and intermediate precision, following the procedure recommended according to CLSI-EP5. For conducting the experiments, each laboratory was provided with an identical set of spiked sample and control material covering major parts of the measuring range (sample concentration between 0.24 ng/mL up to 18.5 ng/mL). 1.0 0.8 0.6 0.4 0.2 0.0 Figure Figure 2: 2: Repeatability Repeatability precision precisionperformance performance (CV (CV(%)) [%])ofofAMH AMHimmunoassay immunoassayon onRoche Roche ® ® ANALYTICS ANALYTICS E170,cobas cobas e 411 cobas e 601. analyzers such such as as MODULAR MODULAR E170, e 411 andand cobas e 601. Within-run-precision: AMH immunoassay The within-run-precision experiment was performed by measuring 21 replicates in one run using 5 different serum level pools. On cobas e 601 and MODULAR® ANALYTICS E170 randomly assigned measuring cells were used to determine the within-run-precision coefficient of variation (CV) [%]. Intermediate precision of AMH immunoassay according to CLSI-EP5 e 601 Within-run-precision AMH immunoassay Within-run-precision AMH immunoassay E170 4.5 4.0 3.0 3.5 CV (%) CV (%) e 411 5.0 3.5 2.5 2.0 3.0 2.5 2.0 1.5 1.5 1.0 1.0 Instrument 0.5 19.17 Laboratory 3 Laboratory 3 12.75 Laboratory 3 Laboratory 3 e 601 2.52 Laboratory 3 0.73 Laboratory 2 0.24 Laboratory 1 Laboratory 2 19.17 Laboratory 1 Laboratory 2 12.75 Laboratory 1 Laboratory 2 e 411 2.52 Laboratory 1 Laboratory 2 0.73 Laboratory 1 Laboratory 5 0.24 Laboratory 3 Laboratory 5 19.17 Laboratory 4 Laboratory 3 12.75 Laboratory 4 Laboratory 5 Laboratory 4 Laboratory 3 2.52 Laboratory 5 Laboratory 4 Laboratory 3 Laboratory 5 0.73 0.24 Mean (ng/mL) Laboratory 4 Laboratory 3 0.5 Laboratory Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 0.24 ng/mL 0.72 ng/mL 1.1 ng/mL 2.4 ng/mL 5.5 ng/mL 12.5 ng/mL 18.8 ng/mL 0.0 Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. Lab. 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 1 2 2 0.24 ng/mL 0.72 ng/mL 1.1 ng/mL 2.4 ng/mL 5.5 ng/mL 12.5 ng/mL 18.8 ng/mL Figure 3: 3: Intermediate Intermediate precision precision performance performance (CV (CV (%)) [%]) ofofAMH AMHimmunoassay immunoassayon onRoche Roche Figure ® 411 and cobas e 601. analyzers such such as analyzers as MODULAR MODULAR®ANALYTICS ANALYTICSE170, E170,cobas cobase e 411 and cobas e 601. E170 Figure Within-run-precision performance on Roche Rocheanalyzers analyzerssuch such Figure 1: Within-run-precision performance of of AMH AMH immunoassay on ®® as ANALYTICS ANALYTICSE170, E170,cobas cobasee411 411and andcobas cobase e601. 601. as cobas cobas ee601 601and andMODULAR MODULAR The AMH immunoassay’s precision performance following the CLSI-EP5 protocol resulted in a maximum day-to-day variability CV of 3.7% and part-to-part variability CV of 2.1%. The repeatability was below CV 1.8%. At all testing sites the intermediate-precision exhibited CVs below 4.4%. The within-run-precision CV observed on Roche routine analyzers using AMH immunoassay was below 3.4 % in all five laboratories. 5-day precision according to CLSI-EP5 scheme: comparison between AMH immunoassay and ELISA methods under routine conditions 6 4 2 0 -2 Gobal mean (ng/mL) of sample 0.29 0.92 0.93 3.00 4.51 13.96 Laboratory 5 Laboratory 3 Laboratory 1 Laboratory 2 manual method 1 Laboratory 2 Laboratory 1 Laboratory 5 E170 Laboratory 3 e 601 Laboratory 3 e 411 Laboratory 4 Laboratory 5 Laboratory 3 manual method 2 Laboratory 4 Laboratory 1 Laboratory 2 manual method 1 Laboratory 2 E170 Laboratory 3 Laboratory 5 Laboratory 1 e 601 Laboratory 3 e 411 Laboratory 4 Laboratory 5 manual method 2 Laboratory 4 Laboratory 3 Laboratory 1 Laboratory 2 manual method 1 Laboratory 2 Laboratory 1 Laboratory 5 E170 Laboratory 3 e 601 Laboratory 3 e 411 Laboratory 4 Laboratory 5 manual method 2 Laboratory 4 Laboratory 3 Laboratory 2 manual method 1 Laboratory 1 Laboratory 2 Laboratory 1 Laboratory 5 E170 Laboratory 3 e 601 Laboratory 3 e 411 Laboratory 4 manual method 2 Laboratory 4 Laboratory 5 Laboratory 3 Laboratory 2 manual method 1 Laboratory 1 E170 Laboratory 3 Laboratory 2 Laboratory 1 e 601 Laboratory 5 e 411 Laboratory 4 Laboratory 3 manual method 2 Laboratory 4 Laboratory 5 Laboratory 3 Laboratory 2 manual method 1 Laboratory 1 E170 Laboratory 3 Laboratory 2 e 601 Laboratory 1 Laboratory 5 Laboratory 3 e 411 Laboratory 4 manual method 2 Laboratory 4 Laboratory 5 Laboratory 3 manual method 1 Laboratory 2 Laboratory 1 E170 Laboratory 3 Laboratory 2 e 601 Instrument/ Method Laboratory 1 Laboratory Laboratory 5 -6 Laboratory 3 -4 e 411 Laboratory 4 Difference to global mean (ng/mL) Precision data were collected over a time period of 5 days at all sites, using PreciControl AMH level 1 and 2 and five serum level pools as sample material. Two different ELISA assays were compared to the AMH assay. For the ELISA each day two independent measurements with each two aliquots of control and serum level pool material were performed to imitate the CLSI guidance. 17.96 Figure 5 days measuring 7 sample material concentrations with with ELISA methods (black) and AMH immunoassay (blue) (blue). Figure4:4:Summary Summaryofofresults resultsobtained obtainedduring during 5 days measuring 7 sample material concentrations ELISA methods (black) and AMH immunoassay Under routine conditions the AMH immunoassay’s repeatability CV was below 5.1%, the intermediate precision CV did not exceed 6.9%. For the both ELISA methods the repeatability was up to 53.1% (method 1) and 150.1% (method 2), the intermediate precision CV was up to 63.8% (method 1) and 185.3% (method 2). Conclusion The fully automated AMH immunoassay demonstrated a within-run-precision CV of below 3.4% for native and spiked sample material across all five laboratories. The assay’s precision performance following the 21-day-CLSI-EP5 protocol resulted in an intermediate precision CV below 4.4%. The repeatability precision did not exceed 1.8%. At all testing sites the maximum day-to-day variability CV was 3.7%, the part-to-part variability CV below 2.1%. In a comparison with commercially available ELISA tests, the automated AMH immunoassay exhibited a manyfold better precision performance than the routinely used manual methods. The availability of the fully automated AMH immunoassay run on routine Elecsys®/cobas e instruments will represent a fast and precise alternative to manual AMH assay testing.
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