Intraplatform Reproducibility and Technical Precision of Gene Expression Profiling in Four Laboratories Investigating 112 Leukemia Samples: The DACH Study.

Abstract

Gene expression profiling has the potential to offer consistent objective diagnostic test results once a standardized protocol is established. We investigated the robustness, precision, and reproducibility of this technology and present data that complements the Microarray Innovations in LEukemia study (MILE study). In four laboratories, located in Germany (D), Austria (A), and Switzerland (CH) (DACH study), replicates of 112 patient samples were analyzed using the AmpliChip Leukemia research test. Patient samples were centrally collected and diagnosed in daily routine at the Munich Leukemia Laboratory and represented 8 distinct classes of acute and chronic leukemias, with non-leukemia as control group. After purification of the mononuclear cells by Ficoll density centrifugation, 4 × 5 million cells were frozen in lysis buffer and stored at −80°C. Equipped with identical instruments, software, and reagents, study operators were trained on the microarray sample preparation protocol using total RNA from commercially available cell lines. Upon receipt of the frozen lysates each of the four laboratories purified the total RNA from the 112 technical quadruplicates. 99.3% (445/448) of the sample preparations were successfully performed. On average, 8.4 μg, 7.2 μg, 7.4 μg, or 7.5 μg of total RNA, respectively, were isolated from the mononuclear cells from the four laboratories. In three samples less than 1.0 μg of total RNA was obtained and thus the preparation failed. Bland-Altman plots of agreement showed that any two centers were unlikely to have more than an 8.3 μg difference in yield of total RNA from the same sample. On average there was between 0.1 μg to 1.2 μg difference in total RNA yield from the same sample. Further processing of the 445 samples resulted in 437 (98.2%) successfully performed in vitro transcription reactions, i.e. obtained cRNA yield of >8.0 μg. On average there was between 0.4 μg to 7.4 μg difference in cRNA yield from the same sample. After hybridization to microarrays on average, 46.1%, 48.6%, 46.5%, and 47.3% of probe sets were detected as present with mean scaling factors of 4.3, 2.9, 3.9, and 3.7, respectively. The mean values and standard deviations of distributions of the coefficient of variation (CV) within each site over all the probe sets of the quantile normalized signals on the chip were 27.2% (StdDev: 12.3%), 27.0% (StdDev: 12.3%), 27.3% (StdDev: 12.3%), 26.9% (StdDev: 12.4%), respectively. Furthermore, in unsupervised hierarchical cluster and principal component analyses replicates from the same patient always clustered closely together, with no indications of association between gene expression profiles due to different operators or laboratories. In conclusion, we demonstrated that microarray analysis can be performed with remarkably high inter-laboratory reproducibility and with comparable quality and high technical precision across laboratories.