Methods, precision, and analytical sensitivity of a novel low-plasma-volume assay of fibrinolytic capacity utilizing the euglobulin fraction.

INTRODUCTION: Fibrinolysis is a critical aspect of the hemostatic system, with assessment of fibrinolytic potential being critical to predict bleeding and clotting risk. We describe the method for a novel low-plasma-volume assay of fibrinolytic capacity utilizing the euglobulin fraction (the "modified mini-euglobulin clot lysis assay [ECLA]"), its analytic sensitivity to alterations in key fibrinolytic substrates/regulators, and its initial applications in acute and convalescent disease cohorts. METHODS: The modified mini-ECLA requires 50 µL of plasma, a maximal read time of 3 h (with most results available within 60 min), and is entirely performed in a 96-well microplate. Assay measurements were obtained in a variety of commercial control and deficient plasmas representing clinically relevant hypo- and hyperfibrinolytic states, and in three distinct adolescent cohorts with acute or convalescent illness: critically ill, following endotracheal intubation; acute COVID-19-related illness; and ambulatory, 3 months following a venous thromboembolic event. RESULTS: In 100% and 75% deficient plasmas, hypofibrinolysis for plasminogen-deficient, fibrinolysis for alpha-2-antiplasmin-deficient, and hyperfibrinolysis for plasminogen activator inhibitor-1-deficient plasmas were observed. CONCLUSION: The modified mini-ECLA Clot Lysis Time Ratio ("CLTR") demonstrated moderate-strength correlations with the Clot Formation and Lysis (CloFAL) assay, is analytically sensitive to altered fibrinolytic states in vitro, and correlates with clinical outcomes in preliminarily-studied patient populations.

What can the plasma proteome tell us about platelets and (vice versa)?

Multi-omics approaches are being used increasingly to study physiological and pathophysiologic processes. Proteomics specifically focuses on the study of proteins as functional elements and key contributors to, and markers of the phenotype, as well as targets for diagnostic and therapeutic approaches. Depending on the condition, the plasma proteome can mirror the platelet proteome, and hence play an important role in elucidating both physiologic and pathologic processes. In fact, both plasma and platelet protein signatures have been shown to be important in the setting of thrombosis-prone disease states such as atherosclerosis and cancer. Plasma and platelet proteomes are increasingly being studied as a part of a single entity, as is the case with patient-centric sample collection approaches such as capillary blood. Future studies should cut across the plasma and platelet proteome silos, taking advantage of the vast knowledge available when they are considered as part of the same studies, rather than studied as distinct entities.

Platelets are key cellular elements of blood with plasma constituting the liquid component. Both platelets and proteins found in plasma rapidly work in unity to prevent/limit blood loss in response to blood vessel damage. Proteomics is the analysis of the entire protein complement of a cell, tissue, or organism under a specific, defined set of conditions. Of note, research to date has shown that platelet and plasma proteomes share many common proteins. In some disease scenarios, plasma proteomes can be used to identify platelet function or dysfunction, while in other scenarios, platelet-specific proteins are needed for physiological assessment. Thus, it may be beneficial to simultaneously study the plasma and platelet proteomes, thereby exploiting the considerable wealth of information provided under such circumstances.

Bacterial Whole Genome Sequencing on the Illumina iSeq 100 for Clinical and Public Health Laboratories.

Bacterial whole-genome sequencing (WGS) provides clinical and public health laboratories an unprecedented level of information on species identification, antimicrobial resistance, and epidemiologic typing. However, multiple barriers to widespread adoption still exist. This research describes bacterial WGS using the Illumina iSeq 100 instrument to overcome some of these barriers. Using an in-house, high-quality single-nucleotide polymorphism analysis pipeline and a commercial whole-genome multilocus sequence typing program, the sequencing of Acinetobacter baumannii, Burkholderia cepacia, Clostridioides difficile, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus isolates was validated. The genome coverage range was 17× to 149×, with a mean of 59×. The limit of detection for single-nucleotide polymorphisms was 30×. Overall platform base calling accuracy was >99.999%. Reproducibility and repeatability of base calling inferred from whole-genome multilocus sequence typing was species dependent and ranged from >97% similarity for P. aeruginosa to >99.9% similarity for S. aureus. Resistance gene and multilocus sequence typing allele identification was 100% concordant with expected results. A simple, modified library preparation reduces the per-sample cost by half to give overall theoretical sample costs ranging from approximately $50 to $100 for library preparation and sequencing. The iSeq 100 provides a cost-effective and easy-to-use platform for clinical and public health laboratories to sequence bacterial isolates for a wide range of potential applications.