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Robustness of comprehensive DNA- and RNA-based assays at diagnosis of acute myeloid leukemia using blood and bone marrow stored on filter cards

Leukemia volume 30, pages 2123–2125 (2016)Cite this article

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Molecular analyses in hematologic malignancies gained considerable importance in the last decade at diagnosis and for minimal residual disease (MRD). This is driven by improvements of applicable techniques and the detection of many new genes to be involved. The need for a comprehensive investigation of the molecular biology in these diseases has to be addressed and is increasingly offered by specialized reference laboratories. Therefore, optimization of sample processing and of pre-analytic steps before shipment is utmost important. In our study, we performed a comparison of state-of-the-art cytogenetics, FISH and molecular diagnostic assays based on standard liquid peripheral blood or bone marrow samples and on diagnostic material stored on filter cards. From both input materials, DNA and RNA were extracted and used for standard diagnostic approaches for the detection of fusion transcripts as well as for next-generation sequencing (NGS) and the detection of copy number variations by array-based comparative genomic hybridization (aCGH). Aberration-specific quantitative PCR was used for validation of findings (see Supplementary Information, Materials and methods). A flowchart of the study design is given in Supplementary Figure S1.

Although some features of filter cards, like space savings and temperature conditions, seem to be an obvious advantage in comparison with handling of frozen cell pellets, DNA or RNA, the stability of the material on filter cards had to be proven. Therefore, we extracted DNA and RNA from five patients at day 2 after dropping the sample material onto filter cards and from additionally prepared filter cards after 4 and 10 days, and after 22, 62 and 68 days, respectively. All filter cards were stored at room temperature. There was a slight decrease of DNA and RNA concentration after the storage period, however, even after more than 2 months of storage, the DNA and RNA yield remained 50% of the extraction from day 2 of storage, illustrating the integrity of DNA and RNA on filter cards during long-term storage (Supplementary Figure S4). Furthermore, panel sequencing of DNA as well as multiplex PCR on cDNA showed identical results compared with the corresponding sample extracted at day 2 of storage (Supplementary Table S5). A Swedish study already had demonstrated the RNA integrity by analyzing ß-actin expression in neonatal blood samples stored for up to 20 years on filter cards, showing no significant reduction in RNA quantity over time.1 But also storage on filter cards and subsequent analysis of cancer material have been described, like fine-needle aspiration of breast carcinoma cells,2 BCR-ABL1 detection in CML patients3, 4 or within the Iraqi pediatric AML study, where filter cards were used to transfer dried blood/bone marrow samples from Iraq to Japan for DNA- and RNA-based molecular diagnostics. The filter cards were stored for up to 6 weeks before shipment and resulted all in successful molecular diagnostics.5 However, to comprehensively mimic different climate conditions for shipment or storage further evaluation would be of interest.

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Acknowledgements

We thank all clinicians for sending samples to our laboratory for diagnostic purposes and for providing clinical information and follow-up data. In addition, we thank all the co-workers at the MLL Munich Leukemia Laboratory for approaching together many aspects in the field of leukemia diagnostics and research. We also want to thank Professor Arndt Rolfs from Centogene AG (Rostock, Germany) who provided the filter cards (CentoCard) for this study and supported the idea to apply them in leukemia.

Author contributions

MM investigated molecular mutations, analyzed the data and wrote the manuscript. SW contributed to AMLplex and quantitative PCR. RK established the extraction of nucleic acids from filter cards and performed AMLplex and quantitative PCR. NN was responsible for bioinformatics. AS contributed to cytogenetics and array CGH. WK was involved in data analyses. CH was responsible for cytogenetics. TH was responsible for cytomorphologic analysis, was the principle investigator of the study and wrote the manuscript. All authors read and contributed to the final version of the manuscript.

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  1. MLL Munich Leukemia Laboratory, Munich, Germany

    T Haferlach, S Weber, R Konietschke, N Nadarajah, A Stengel, W Kern, C Haferlach & M Meggendorfer

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  1. T Haferlach
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  2. S Weber
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Correspondence to T Haferlach.

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Competing interests

WK, CH and TH declare part ownership of the MLL Munich Leukemia Laboratory. MM, SW, RK, NN and AS are employed by the MLL Munich Leukemia Laboratory.

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Supplementary Information accompanies this paper on the Leukemia website

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Haferlach, T., Weber, S., Konietschke, R. et al. Robustness of comprehensive DNA- and RNA-based assays at diagnosis of acute myeloid leukemia using blood and bone marrow stored on filter cards. Leukemia 30, 2123–2125 (2016). https://doi.org/10.1038/leu.2016.156

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