Pre-analytical properties of different respiratory viruses for PCR-based detection: Comparative analysis of sampling devices and sample stabilization solutions.

After the decline of the COVID-19 pandemic, health systems were challenged by the simultaneous prevalence of different respiratory viruses causing a wide overlap in symptoms. This increased the demand for multi-virus diagnostic tests which require suitable pre-analytical workflow solutions in order to receive valid diagnostic results. In this context, the effects of specimen storage duration and temperature on the RNA/DNA copy number stability of influenza A/B, RSV A/B, SARS-CoV-2 and adenovirus were examined for four commercially available transport swab systems and saliva collection devices. The respiratory viruses were more stable in the saliva collection devices than in the transport swab systems when stored at RT or 37 °C for up to 96 h. Moreover, no differences between viral nucleic acid stability of enveloped and non-enveloped viruses were observed. The infectivity of all enveloped viruses could be inactivated by the saliva collection device from PreAnalytiX. The Norgen saliva device completely inactivated influenza A/B, while RSV A/B were partially inactivated. The non-enveloped adenovirus was inactivated by a reduction factor of 10E+ 4 in both saliva collection devices. All respiratory viruses remained infectious in the transport swab systems. Two possible transport medium additives were tested which inactivated or strongly reduced viral replication of tested enveloped viruses but had no effect on the non-enveloped adenovirus. Finally the implementation of multi-target detection procedures involving a direct amplification approach was successfully tested by spike-in of all enveloped viruses simultaneously into transport swab systems. This fast and reproducible setup presents a valuable solution for future implementations in multi-virus testing strategies.

Sensitivity assessment of workflows detecting rare circulating cell-free DNA targets: A study design proposal.

The field of liquid biopsy has seen extensive growth in recent decades, making it one of the most promising areas in molecular diagnostics. Circulating cell-free DNA (ccfDNA) especially is used as an analyte in a growing number of diagnostic assays. These assays require specified preanalytical workflows delivering ccfDNA in qualities and quantities that facilitate correct and reliable results. As each step and component used in the preanalytical process has the potential to influence the assay sensitivity and other performance characteristics, it is key to find an unbiased experimental setup to test these factors in diagnostic or research laboratories. We defined one such setup by using blood from healthy subjects and commercially available products for blood collection, spike-in material, ccfDNA isolation, and qPCR assays. As the primary read-out, we calculated the probit model-based LOD95 (limit of detection of the 95th percentile) from the qPCR assay results. In a proof of principle study we tested two different but widely used blood ccfDNA profile stabilization technologies in blood collection tubes, the Cell-Free DNA BCT and the PAXgene Blood ccfDNA Tube. We tested assays for three different EGFR gene mutations and one BRAF gene mutation. The study design revealed differences in performance between the two tested technologies for all four mutations. In conclusion, we successfully established a blueprint for a test procedure capable of verifying and validating a liquid biopsy workflow from blood collection to the analytical result.

Liquid Biopsy Preservation Solutions for Standardized Pre-Analytical Workflows-Venous Whole Blood and Plasma.

PURPOSE OF REVIEW: Liquid biopsy analyses based on circulating cell-free nucleic acids, circulating tumor cells or other diseased cells from organs, and exosomes or other microvesicles in blood offer new means for non-invasive diagnostic applications. The main goal of this review is to explain the importance of preserving whole blood specimens after blood draw for use as liquid biopsies, and to summarize preservation solutions that are currently available. RECENT FINDINGS: Despite the great potential of liquid biopsies for diagnostics and disease management, besides non-invasive prenatal testing (NIPT), only a few liquid biopsy applications are fully implemented for routine in vitro diagnostic testing. One major barrier is the lack of standardized pre-analytical workflows, including the collection of consistent quality blood specimens and the generation of good-quality plasma samples therefrom. Broader use of liquid biopsies in clinical routine applications therefore requires improved pre-analytical procedures to enable high-quality specimens to obtain unbiased analyte profiles (DNA, RNA, proteins, etc.) as they are in the patient's body. SUMMARY: A growing number of stabilizing reagents and dedicated blood collection tubes are available for the post-collection preservation of circulating cell-free DNA (ccfDNA) profiles in whole blood. In contrast, solutions for the preservation of circulating tumor cells (CTC) that enable both, enumeration and molecular analyses are rare. Solutions for extracellular vesicle (EV) populations, including exosomes, do not yet exist.

A new technology for stabilization of biomolecules in tissues for combined histological and molecular analyses.

For accurate diagnosis, prediction of outcome, and selection of appropriate therapies, the molecular characterization of human diseases requires analysis of a broad spectrum of altered biomolecules, in addition to morphological features, in affected tissues such as tumors. In a high-throughput screening approach, we have developed the PAXgene Tissue System as a novel tissue stabilization technology. Comprehensive characterization of this technology in stabilized and paraffin-embedded human tissues and comparison with snap-frozen tissues revealed excellent preservation of morphology and antigenicity, as well as outstanding integrity of nucleic acids (genomic DNA, miRNA, and mRNA) and phosphoproteins. Importantly, PAXgene-fixed, paraffin-embedded tissues provided RNA quantity and quality not only significantly better than that obtained with neutral buffered formalin, but also similar to that from snap-frozen tissue, which currently represents the gold standard for molecular analyses. The PAXgene tissue stabilization system thus opens new opportunities in a variety of molecular diagnostic and research applications in which the collection of snap-frozen tissue is not feasible for medical, logistic, or ethical reasons. Furthermore, this technology allows performing histopathological analyses together with molecular studies in a single sample, which markedly facilitates direct correlation of morphological disease phenotypes with alterations of nucleic acids and other biomolecules.

Histological assessment of PAXgene tissue fixation and stabilization reagents.

Within SPIDIA, an EC FP7 project aimed to improve pre analytic procedures, the PAXgene Tissue System (PAXgene), was designed to improve tissue quality for parallel molecular and morphological analysis. Within the SPIDIA project promising results were found in both genomic and proteomic experiments with PAXgene-fixed and paraffin embedded tissue derived biomolecules. But, for this technology to be accepted for use in both clinical and basic research, it is essential that its adequacy for preserving morphology and antigenicity is validated relative to formalin fixation. It is our aim to assess the suitability of PAXgene tissue fixation for (immuno)histological methods. Normal human tissue specimens (n = 70) were collected and divided into equal parts for fixation either with formalin or PAXgene. Sections of the obtained paraffin-embedded tissue were cut and stained. Morphological aspects of PAXgene-fixed tissue were described and also scored relative to formalin-fixed tissue. Performance of PAXgene-fixed tissue in immunohistochemical and in situ hybridization assays was also assessed relative to the corresponding formalin-fixed tissues. Morphology of PAXgene-fixed paraffin embedded tissue was well preserved and deemed adequate for diagnostics in most cases. Some antigens in PAXgene-fixed and paraffin embedded sections were detectable without the need for antigen retrieval, while others were detected using standard, formalin fixation based, immunohistochemistry protocols. Comparable results were obtained with in situ hybridization and histochemical stains. Basically all assessed histological techniques were found to be applicable to PAXgene-fixed and paraffin embedded tissue. In general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue. Compromises made in morphology can be called minor compared to the advantages in the molecular pathology possibilities.

RNA-based drug screening using automated RNA purification and real-time RT-PCR1.

A new system has been developed for RNA-based drug screening, and the feasibility of this approach has been demonstrated by the identification of new immunomodulating compounds. Peripheral blood mononuclear cells were chosen as the cellular assay system. Cells were either stimulated by TPA/ionomycin to produce T cell cytokines as asthma targets or stimulated by lipopolysaccharide to produce proinflammatory cytokines as targets for chronic obstructive pulmonary disease (COPD). The authors developed a new fully automated system for RNA purification from cells grown in 96-well plates. Gene expression was determined in 384-well plates using real-time quantitative one-tube RT-PCR. Small interdonor variation could be demonstrated. The assay system was validated with known immunosuppressants cyclosporine and dexamethasone. Screening of 800 compounds resulted in 9.5% compounds inhibiting the induction of at least 1 T cell derived cytokine and 6.8% compounds inhibiting at least 1 cytokine relevant for COPD. All these compounds were retested by analyzing remaining RNA from the 1st round of screening. The reproducibility of hits was between 56% and 74% for different cytokines. One compound selectively inhibited TNF, which was confirmed by IC(50) determination. Analyzing its effect on cells from different donors revealed little interdonor variation. In conclusion, the authors established fully automated RNA isolation and precise gene expression profiling using real-time RT-PCR for drug screening.