PreAnalytix, a joint venture between Qiagen and BD, has released two CE-marked in vitro diagnostic products that are compliant with the new Regulation (EU) 2017/746 on in vitro diagnostic medical devices (IVDR) and are part of a preanalytical workflow for collecting, stabilizing, transporting, and storing blood for circulating cell-free DNA (ccfDNA)-based assays.
Diagnostic laboratories in the EU can utilize the IVDR-compliant PAXgene Blood ccfDNA workflow in a variety of noninvasive ccfDNA-based diagnostic applications. Daniel Grölz, scientific associate director of research and development at PreAnalytix, said that compliance with the new IVDR rules, published in 2017 and in full effect as of May, will help to address the challenges associated with collecting and analyzing ccfDNA and will assist with the more widespread adoption of ccfDNA-based diagnostics.
Analysis of ccfDNA from whole blood has multiple applications, such as non-invasive prenatal testing (NIPT) for genomic abnormalities, as well as cancer monitoring and screening. However, there are many challenges associated with the isolation of ccfDNA as an analyte. Usually, ccfDNA is highly fragmented and present in concentrations as low as one to 50 nanograms of DNA per milliliter of plasma. Additionally, blood cells begin releasing their genomic DNA (gDNA) via cell lysis or apoptosis after blood is drawn, thereby increasing the level of unwanted background DNA.
“Patient specimens can significantly alter after collection from the body, for example, during transport, storage, and processing,” said Uwe Oelmüller, management committee co-chair at PreAnalytix. “This is the major source for medical laboratory test errors.” Standardization and protocol harmonization of the preanalytical workflow through compliance with standards such as IVDR or International Organization for Standardization (ISO) stipulations can help to reduce these errors and produce reliable and reproducible diagnostic ccfDNA test results, he said. The PAXgene Blood ccfDNA workflow consists of a blood collection tube, the PAXgene Blood ccfDNA Tube, and IVD-designated purification kits for automated or manual extraction of ccfDNA from plasma and gDNA from the cellular fraction remaining after centrifugation and plasma removal (see figure 1). It was developed, verified, and validated following the ISO standards for ccfDNA and gDNA examination from whole blood (ISO 20186-2:2019 and ISO 20186-3:2019).
The new European in vitro diagnostic regulations prominently stipulate that preanalytical variables that could have an impact on sample quality are specified and verified before diagnostic products reach the market, Oelmüller explained. For example, Annex II, Part 6 states that, for product verification and validation, the description of the specimen type shall include “stability such as storage, where applicable specimen transport conditions and, with a view to time-critical analysis methods, information on the timeframe between taking the specimen and its analysis and storage conditions such as duration, temperature limits and freeze/thaw cycles.” This emphasizes the importance of understanding the entire preanalytical workflow for a sample to use it for verification and validation, Oelmüller said. These preanalytical variables are also covered in the ISO standards for ccfDNA and gDNA examination from whole blood.
As part of PreAnalytix’s efforts to achieve compliance with IVDR 2017/746, the impact of preanalytical parameters on ccfDNA and gDNA yield, quality, and stability was verified on samples that were collected, stored, and transported using the PAXgene Blood ccfDNA Tube. This testing evaluated the effects of variables including the draw volume, mixing through inversion, endogenous interferants, blood transport (see figure 2), plasma processing conditions, short- and long-term storage, and compatibility with extraction protocols for manual and automated workflow (see figure 3). Long-term eluate storage is under evaluation in an ongoing study.
To use the PAXgene Blood ccfDNA workflow, 10 milliliters of whole blood are collected into the PAXgene Blood ccfDNA Tube. The PAXgene Tube is a plastic evacuated tube developed using BD Vacutainer technology containing a blood stabilizing reagent that helps prevent blood coagulation, minimizes cell lysis, and is free of crosslinking reagents such as formaldehyde or formaldehyde-releasing substances. An analysis from a PreAnalytix study published in PLOS One found that, as the PAXgene Blood ccfDNA Tubes do not chemically modify DNA, the stabilizing reagent has the potential to increase assay sensitivity in PCR applications in comparison to sample fixation using crosslinking substances. ccfDNA can be isolated from plasma generated from PAXgene Blood ccfDNA Tubes using automation with the QIAsymphony PAXgene Blood ccfDNA Kit on the Qiagen QIAsymphony SP instrument, or manually with the Qiagen QIAamp DSP Circulating Nucleic Acid Kit. The nucleated cellular fraction or buffy coat remaining after removal of the plasma can be used for gDNA isolation through automation using the Qiagen QIAsymphony DSP DNA Mini and Midi Kits, or manually with the Qiagen QIAamp DSP DNA Blood Mini Kit.