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Nucleic acid extraction (NAE) plays a crucial role for diagnostic testing procedures. For decades, dried blood spots (DBS) have been used for serology, drug monitoring, and molecular studies. However, extracting nucleic acids from DBS remains a significant challenge, especially when attempting to implement these applications to the point-of-care (POC). To address this issue, we have developed a paper-based NAE method using cellulose filter papers (DBSFP) that operates without the need for electricity (at room temperature). Our method allows for NAE in less than 7 min, and it involves grade 3 filter paper pre-treated with 8% (v/v) igepal surfactant, 1 min washing step with 1× PBS, and 5 min incubation at room temperature in 1× TE buffer. The performance of the methodology was assessed with loop-mediated isothermal amplification (LAMP), targeting the human reference gene beta-actin and the kelch 13 gene from P. falciparum. The developed method was evaluated against FTA cards and magnetic bead-based purification, using time-to-positive (min) for comparative analysis. Furthermore, we optimised our approach to take advantage of the dual functionality of the paper-based extraction, allowing for elution (eluted disk) as well as direct placement of the disk in the LAMP reaction (in situ disk). This flexibility extends to eukaryotic cells, bacterial cells, and viral particles. We successfully validated the method for RNA/DNA detection and demonstrated its compatibility with whole blood stored in anticoagulants. Additionally, we studied the compatibility of DBSFP with colorimetric and lateral flow detection, showcasing its potential for POC applications. Across various tested matrices, targets, and experimental conditions, our results were comparable to those obtained using gold standard methods, highlighting the versatility of our methodology. In summary, this manuscript presents a cost-effective solution for NAE from DBS, enabling molecular testing in virtually any POC setting. When combined with LAMP, our approach provides sample-to-result detection in under 35 minutes.

Critically ill COVID-19 patients are characterized by a severely dysregulated cytokine profile and elevated neutrophil counts, which are thought to contribute to disease severity. However, to date it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the tightly regulated cell death program of neutrophils. We show that in a subpopulation of neutrophils, canonical apoptosis was skewed towards rapidly occurring necroptosis. This phenotype was characterized by abrogated caspase-8 activity and increased RIPK1 levels, favoring execution of necroptosis via the RIPK1-RIPK3-MLKL axis, as further confirmed in COVID-19 biopsies. Moreover, reduction of sFas-L levels in COVID-19 patients and hence decreased signaling to Fas directly increased RIPK1 levels and correlated with disease severity. Our results suggest an important role for Fas signaling in the regulation of cell death program ambiguity via the ripoptosome in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome.