Longitudinal analysis of 20 Years of external quality assurance schemes for PCR/NAAT-based bacterial genome detection in diagnostic testing.

Background: Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers. Objectives: The study assesses the evolving diagnostic performance trends over 2 decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains. Results: The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance. Conclusion: Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.

Longitudinal analysis of external quality assessment of immunoassay-based steroid hormone measurement indicates potential for improvement in standardization.

As hormonal disorders are linked to several diseases, the accurate quantitation of steroid hormone levels in serum is crucial in order to provide patients with a reliable diagnosis. Mass spectrometry-based methods are regarded as having the highest level of specificity and sensitivity. However, immunoassays are more commonly used in routine diagnostics to measure steroid levels as they are more cost effective and straightforward to conduct. This study analyzes the external quality assessment results for the measurement of testosterone, progesterone and 17ß-estradiol in serum using immunoassays between early 2020 and May 2022. As reference measurement procedures are available for the three steroid hormones, the manufacturer-specific biases were normalized to the reference measurement values. The manufacturer-specific coefficients of variation were predominantly inconspicuous, below 20% for the three hormones when outliers are disregarded, however there were large differences between the various manufacturer collectives. For some collectives, the median bias to the respective reference measurement value was repeatedly greater than ±35%, which is the acceptance limit defined by the German Medical Association. In the case of testosterone and progesterone determination, some collectives tended to consistently over- or underestimate analyte concentrations compared to the reference measurement value, however, for 17ß-estradiol determination, both positive and negative biases were observed. This insufficient level of accuracy suggests that cross-reactivity continues to be a fundamental challenge when antibody detection is used to quantify steroids with a high structural similarity. Distinct improvements in standardization are required to provide accurate analysis and thus, reliable clinical interpretations. The increased accuracy of the AX immunoassay for testosterone measurement, as observed in the INSTAND EQAs between 2020 and 2022, could be the result of a recalibration of the assay and raises hope for further improvement of standardization of immunoassay-based steroid hormone analyses in the coming years.

cGMP modulates hemin-mediated platelet death.

BACKGROUND AND AIMS: Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. METHODS: Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. RESULTS: Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. CONCLUSION: We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

The Subtilisin-Like Protease Furin Regulates Hemin-Dependent Ectodomain Shedding of Glycoprotein VI.

INTRODUCTION: Hemolysis results in release of free hemoglobin and hemin liberation from erythrocytes. Hemin has been described to induce platelet activation and to trigger thrombosis. METHODS: We evaluated the effect of hemin on platelet function and surface expression of the platelet collagen receptor glycoprotein VI (GPVI). Isolated platelets were stimulated with increasing concentrations of hemin. RESULTS: We found that hemin strongly enhanced platelet activation, aggregation, and aggregate formation on immobilized collagen under flow. In contrast, we found that surface expression of GPVI was significantly reduced upon hemin stimulation with high hemin concentrations indicating that hemin-induced loss of surface GPVI does not hinder platelet aggregation. Loss of hemin-induced surface expression of GPVI was caused by shedding of the ectodomain of GPVI as verified by immunoblotting and is independent of the GPVI or CLEC-2 mediated ITAM (immunoreceptor-tyrosine-based-activation-motif) signaling pathway as inhibitor studies revealed. Hemin-induced GPVI shedding was independent of metalloproteinases such as ADAM10 or ADAM17, which were previously described to regulate GPVI degradation. Similarly, concentration-dependent shedding of CD62P was also induced by hemin. Unexpectedly, we found that the subtilisin-like proprotein convertase furin controls hemin-dependent GPVI shedding as shown by inhibitor studies using the specific furin inhibitors SSM3 and Hexa-D-arginine. In the presence of SSM3 and Hexa-D-arginine, hemin-associated GPVI degradation was substantially reduced. Further, SSM3 inhibited hemin-induced but not CRP-XL-induced platelet aggregation and thrombus formation, indicating that furin controls specifically hemin-associated platelet functions. CONCLUSION: In summary, we describe a novel mechanism of hemin-dependent GPVI shedding and platelet function mediated by furin.

ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

Platelet-Derived PCSK9 Is Associated with LDL Metabolism and Modulates Atherothrombotic Mechanisms in Coronary Artery Disease.

Platelets play a significant role in atherothrombosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is critically involved in the regulation of LDL metabolism and interacts with platelet function. The effect of PCSK9 in platelet function is poorly understood. The authors of this article sought to characterize platelets as a major source of PCSK9 and PCSK9's role in atherothrombosis. In a large cohort of patients with coronary artery disease (CAD), platelet count, platelet reactivity, and platelet-derived PCSK9 release were analyzed. The role of platelet PCSK9 on platelet and monocyte function was investigated in vitro. Platelet count and hyper-reactivity correlated with plasma LDL in CAD. The circulating platelets express on their surface and release substantial amounts of PCSK9. Release of PCSK9 augmented platelet-dependent thrombosis, monocyte migration, and differentiation into macrophages/foam cells. Platelets and PCSK9 accumulated in tissue derived from atherosclerotic carotid arteries in areas of macrophages. PCSK9 inhibition reduced platelet activation and platelet-dependent thrombo-inflammation. The authors identified platelets as a source of PCSK9 in CAD, which may have an impact on LDL metabolism. Furthermore, platelet-derived PCSK9 contributes to atherothrombosis, and inhibition of PCSK9 attenuates thrombo-inflammation, which may contribute to the reported beneficial clinical effects.

Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation.

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.