Limits...
An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase.

Kohlmann A, Kipps TJ, Rassenti LZ, Downing JR, Shurtleff SA, Mills KI, Gilkes AF, Hofmann WK, Basso G, Dell'orto MC, Foà R, Chiaretti S, De Vos J, Rauhut S, Papenhausen PR, Hernández JM, Lumbreras E, Yeoh AE, Koay ES, Li R, Liu WM, Williams PM, Wieczorek L, Haferlach T - Br. J. Haematol. (2008)

Bottom Line: Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies.Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study.Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

View Article: PubMed Central - PubMed

Affiliation: Roche Molecular Systems, Inc., Department of Genomics and Oncology, Pleasanton, CA, USA. alexander.kohlmann@roche.com

ABSTRACT
Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

Show MeSH

Related in: MedlinePlus

Unsupervised principal component analysis (PCA). A total of 204 experiments are included in the three-dimensional PCA and each sphere represents the gene expression profile for a cell line or leukaemia sample. The signal used is DQN1. The first three principal components (PC) account for 41·0% of variation of the data (PC1 = 18·1%, PC2 = 14·9%, PC3 = 8·0%). The analysis is based on all probe sets represented on the HG-U133 Plus 2.0 microarray without any filtering process (n = 54 613). Outliers are marked with arrows. (A) The same sample types are represented by the same colour spheres. Distinct manufacturing batch numbers of the cell lines are given in Appendix SI. (B) Samples processed within the same centre are represented by the same colour spheres.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2654477&req=5

fig01: Unsupervised principal component analysis (PCA). A total of 204 experiments are included in the three-dimensional PCA and each sphere represents the gene expression profile for a cell line or leukaemia sample. The signal used is DQN1. The first three principal components (PC) account for 41·0% of variation of the data (PC1 = 18·1%, PC2 = 14·9%, PC3 = 8·0%). The analysis is based on all probe sets represented on the HG-U133 Plus 2.0 microarray without any filtering process (n = 54 613). Outliers are marked with arrows. (A) The same sample types are represented by the same colour spheres. Distinct manufacturing batch numbers of the cell lines are given in Appendix SI. (B) Samples processed within the same centre are represented by the same colour spheres.

Mentions: As shown in an unsupervised Principal Component Analysis (PCA), the individual gene expression profiles grouped closely together with their corresponding biological sample types based on the underlying similarity, but not according to the centre where the microarray experiments were performed (Fig 1). The arrows in Fig 1 indicate that the four leukaemia sample preparations from Centre 9 (N17-20), as well as one HepG2 preparation from Centre 3 (N18) were outliers in the PCA. Large differences in gene expression profiles were also observed with respect to the manufacturing batches for MCF-7 total RNA, but overall, a high level of reproducibility between laboratories was seen when a standardized protocol for microarray analysis was followed by trained operators. According to the unsupervised PCA plots, replicated gene expression profiles of the HepG2 cell line were more biologically homogeneous and not as influenced by manufacturing batch numbers, as seen for MCF-7 cell line replicates. Therefore, replicated profiles of the HepG2 cell line were chosen to further investigate the intra- and inter-laboratory correlations. All centres generated highly reproducible gene expression profiles for this cell line, as shown in the box plot analysis of r2 values from all pairwise comparisons within each centre for the sample type HepG2 (Fig 2A), where mean r2 values range from 0·973 to 0·988. The slightly higher variability at Centre 11 might be explained by a higher number of operators and replicate analyses than in other centres. Figure 2B shows the intra-site repeatability of microarray data based on quantitative signal values and qualitative detection calls. The number of generally detected genes for each sample type at each centre varied from 24 627–27 075 for HepG2 and 25 841–28 953 for MCF-7. The coefficient of variation (CV) of the quantitative signal values between the intra-site replicates was calculated using the generally detected subset of genes for each sample type HepG2 and MCF-7 at each laboratory. The distribution of the replicate CV measures across the set of detected genes is displayed in a series of box plots. The different laboratories demonstrated similar replicate CV median values of 1·962–3·234% for HepG2 and 1·869–2·864% for MCF-7.


An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase.

Kohlmann A, Kipps TJ, Rassenti LZ, Downing JR, Shurtleff SA, Mills KI, Gilkes AF, Hofmann WK, Basso G, Dell'orto MC, Foà R, Chiaretti S, De Vos J, Rauhut S, Papenhausen PR, Hernández JM, Lumbreras E, Yeoh AE, Koay ES, Li R, Liu WM, Williams PM, Wieczorek L, Haferlach T - Br. J. Haematol. (2008)

Unsupervised principal component analysis (PCA). A total of 204 experiments are included in the three-dimensional PCA and each sphere represents the gene expression profile for a cell line or leukaemia sample. The signal used is DQN1. The first three principal components (PC) account for 41·0% of variation of the data (PC1 = 18·1%, PC2 = 14·9%, PC3 = 8·0%). The analysis is based on all probe sets represented on the HG-U133 Plus 2.0 microarray without any filtering process (n = 54 613). Outliers are marked with arrows. (A) The same sample types are represented by the same colour spheres. Distinct manufacturing batch numbers of the cell lines are given in Appendix SI. (B) Samples processed within the same centre are represented by the same colour spheres.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2654477&req=5

fig01: Unsupervised principal component analysis (PCA). A total of 204 experiments are included in the three-dimensional PCA and each sphere represents the gene expression profile for a cell line or leukaemia sample. The signal used is DQN1. The first three principal components (PC) account for 41·0% of variation of the data (PC1 = 18·1%, PC2 = 14·9%, PC3 = 8·0%). The analysis is based on all probe sets represented on the HG-U133 Plus 2.0 microarray without any filtering process (n = 54 613). Outliers are marked with arrows. (A) The same sample types are represented by the same colour spheres. Distinct manufacturing batch numbers of the cell lines are given in Appendix SI. (B) Samples processed within the same centre are represented by the same colour spheres.
Mentions: As shown in an unsupervised Principal Component Analysis (PCA), the individual gene expression profiles grouped closely together with their corresponding biological sample types based on the underlying similarity, but not according to the centre where the microarray experiments were performed (Fig 1). The arrows in Fig 1 indicate that the four leukaemia sample preparations from Centre 9 (N17-20), as well as one HepG2 preparation from Centre 3 (N18) were outliers in the PCA. Large differences in gene expression profiles were also observed with respect to the manufacturing batches for MCF-7 total RNA, but overall, a high level of reproducibility between laboratories was seen when a standardized protocol for microarray analysis was followed by trained operators. According to the unsupervised PCA plots, replicated gene expression profiles of the HepG2 cell line were more biologically homogeneous and not as influenced by manufacturing batch numbers, as seen for MCF-7 cell line replicates. Therefore, replicated profiles of the HepG2 cell line were chosen to further investigate the intra- and inter-laboratory correlations. All centres generated highly reproducible gene expression profiles for this cell line, as shown in the box plot analysis of r2 values from all pairwise comparisons within each centre for the sample type HepG2 (Fig 2A), where mean r2 values range from 0·973 to 0·988. The slightly higher variability at Centre 11 might be explained by a higher number of operators and replicate analyses than in other centres. Figure 2B shows the intra-site repeatability of microarray data based on quantitative signal values and qualitative detection calls. The number of generally detected genes for each sample type at each centre varied from 24 627–27 075 for HepG2 and 25 841–28 953 for MCF-7. The coefficient of variation (CV) of the quantitative signal values between the intra-site replicates was calculated using the generally detected subset of genes for each sample type HepG2 and MCF-7 at each laboratory. The distribution of the replicate CV measures across the set of detected genes is displayed in a series of box plots. The different laboratories demonstrated similar replicate CV median values of 1·962–3·234% for HepG2 and 1·869–2·864% for MCF-7.

Bottom Line: Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies.Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study.Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

View Article: PubMed Central - PubMed

Affiliation: Roche Molecular Systems, Inc., Department of Genomics and Oncology, Pleasanton, CA, USA. alexander.kohlmann@roche.com

ABSTRACT
Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.

Show MeSH
Related in: MedlinePlus