Microfluidic Chain Reaction: Conditional, Structurally Programmed Propagation of Capillary Flow Events

Chain reactions are characterized by initiation, propagation and termination, are stochastic at microscopic scales and underlie vital chemical (e.g. combustion engines), nuclear and biotechnological (e.g. polymerase chain reaction) applications.1-5 At macroscopic scales, chain reactions are deterministic and limited to applications for entertainment and art such as falling domino and Rube Goldberg machines. Appositely, the microfluidic lab-on-a-chips (also called a micro total analysis system),6,7 was envisioned as an integrated chip, akin to microelectronic integrated circuits, yet in practice remain dependent on cumbersome peripherals, connections, and a computer for automation.8-11 Capillary microfluidics integrate energy supply and flow control onto a single chip by using capillary phenomena, but programmability remains rudimentary with at most a handful (eight) operations possible.12-19 Here we introduce the microfluidic chain reaction (MCR) as the conditional, structurally programmed propagation of capillary flow events. Monolithic chips integrating a MCR are 3D printed, and powered by the free-energy of a paper pump, autonomously execute liquid handling algorithms step-by-step. With MCR, we automated (i) the sequential release of 300 aliquots across chained, interconnected chips, (ii) a protocol for SARS-CoV-2 antibodies detection in saliva, and (iii) a thrombin generation assay by continuous subsampling and analysis of coagulation-activated plasma with parallel operations including timers, iterative cycles of synchronous flow and stop-flow operations. MCRs are untethered from and unencumbered by peripherals, encode programs structurally in situ, and can form frugal, versatile, bona fide lab-on-a-chip with wide-ranging applications in liquid handling and point-of-care diagnostics.

Dynamic Hemostasis and Fibrinolysis Assays in Intensive Care COVID-19 Patients and Association with Thrombosis and Bleeding—A Systematic Review and a Cohort Study

Patients admitted to the intensive care unit (ICU) with coronavirus disease 2019 (COVID-19), the infectious pathology caused by severe acute respiratory syndrome coronavirus 2, have a high risk of thrombosis, though the precise mechanisms behind this remain unclarified. A systematic literature search in PubMed and EMBASE identified 18 prospective studies applying dynamic coagulation assays in ICU COVID-19 patients. Overall, these studies revealed normal or slightly reduced primary hemostasis, prolonged clot initiation, but increased clot firmness. Thrombin generation assay parameters generally were equivalent to the control groups or within reference range. Fibrinolysis assays showed increased clot resistance. Only six studies related their findings to clinical outcome. We also prospectively included 51 COVID-19 patients admitted to the ICU. Blood samples were examined on day 1, 3–4, and 7–8 with platelet function tests, rotational thromboelastometry (ROTEM), in vivo and ex vivo thrombin generation, and clot lysis assay. Data on thrombosis, bleeding, and mortality were recorded during 30 days. Primary hemostasis was comparable to healthy controls, but COVID-19 patients had longer ROTEM-clotting times and higher maximum clot firmness than healthy controls. Ex vivo thrombin generation was similar to that of healthy controls while in vivo thrombin generation markers, thrombin–antithrombin (TAT) complex, and prothrombin fragment 1 + 2 (F1 + 2) were higher in ICU COVID-19 patients than in healthy controls. Impaired fibrinolysis was present at all time points. TAT complex and F1 + 2 levels were significantly higher in patients developing thrombosis (n = 16) than in those without. In conclusion, only few previous studies employed dynamic hemostasis assays in COVID-19 ICU-patients and failed to reveal a clear association with development of thrombosis. In ICU COVID-19 patients, we confirmed normal platelet aggregation, while in vivo thrombin generation was increased and fibrinolysis decreased. Thrombosis may be driven by increased thrombin formation in vivo.

Thrombin generation assay for hemostasis assessment in trans- latitudinal Arctic voyage

Введение. Существуют данные об активации системы гемостаза в виде формирования гиперкоагуляции при вахтовой работе в циркумполярных территориях. Новым перспективным методом, способным оценить как гипер-, так и гипокоагуляционные состояния, является тест генерации тромбина (ТГТ). Цель исследования: анализ динамики параметров ТГТ в условиях трансширотного морского арктического рейса. Материалы и методы. Выполнено проспективное клинико-лабораторное исследование во время трансширотной экспедиции «ТрансАрктика-2019». Отбор образцов плазмы для ТГТ у 52 членов экспедиции выполнен в нулевой точке (г. Архангельск, 64°33’ с. ш., 40°32’ в. д.) до выхода судна в рейс и в самой высокой точке экспедиции (остров Хейса, архипелаг Франца–Иосифа, 80°34’ с. ш., 57°41’ в. д.) на 18–20-е сутки арктического рейса. Параметры кинетики тромбина определены на анализаторе Ceveron- alpha с TGA-модулем (Technoclone, Австрия). Результаты. В высокой точке транширотного рейса время лаг-фазы (tLag) и время достижения пика тромбина (tPeak) были значимо меньше, чем в исходной нулевой точке, что указывало на состояние гиперкоагуляции. Значения эндогенного потенциала тромбина (AUC) у членов экспедиции были существенно больше в высокой точке экспедиции. Заключение. Нахождение в высоких широтах возможно связано с формированием состояния гиперкоагуляции. Background. Data are exist about hemostasis activation in the form of hypercoagulation during rotational work in circumpolar territories. Thrombin generation assay (TGA) is a new perspective method for assessing both hyper- and hypocoagulation. Objectives: to analyze the dynamics of TGA parameters in trans- latitudinal Arctic sea voyage. Patients/Methods. A prospective clinical and laboratory study was carried out in 52 expedition members during the trans- latitudinal expedition “TransArktika-2019”. Plasma samples for TGA were taken at the zero point (Arkhangelsk, 64°33’ N, 40°32’ E) prior to the ship’s voyage and at the highest point of the expedition (Heiss Island, Franz Josef Land, 80°34’ N, 57°41’ E) on 18–20 days of the voyage. Thrombin kinetic parameters were determined on a Ceveron-alpha analyzer with a TGA-module (Technoclone, Austria). Results. At the highest point of the trans-latitudinal voyage, the lag phase (tLag) and the time to peak thrombin (tPeak) were statistically less than at the initial zero point that pointed to hypercoagulation. The endogenous thrombin potential (AUC) values were significantly higher at the highest point. Conclusions. Location in high latitudes is possibly associated with the formation of hypercoagulation.

Automated Thrombin Generation Assay for Rivaroxaban, Apixaban, and Edoxaban Measurements

Background: The thrombin generation assay (TG) is a promising approach to measure the degree of anticoagulation in patients treated with direct oral anticoagulants (DOAC). A strong association with plasma drug concentrations would be a meaningful argument for the potential use to monitor DOAC.Objectives: We aimed to study the correlation of TG with rivaroxaban, apixaban, and edoxaban drug concentrations in a large, prospective multicenter cross-sectional study.Methods: Five-hundred and fifty-nine patients were included in nine tertiary hospitals. The Technothrombin® TG was conducted in addition to an anti-Xa assay; LC-MS/MS was performed as the reference standard.Results: Correlation (rs) between thrombin generation measurements and drug concentrations was −0.72 for peak thrombin generation (95% confidence interval, CI, −0.77, −0.66), −0.55 for area under the curve (AUC; 95% CI −0.61, −0.48), and 0.80 for lag time (95% CI 0.75, 0.84). In contrast, rs was 0.96 with results of the anti-Xa activity (95% CI 0.95–0.97). Sensitivity with regard to the clinically relevant cut-off value of 50 μgL−1 was 49% in case of peak thrombin generation (95% CI, 44, 55), 29% in case of AUC (95% CI, 24, 34), and 64% in case of lag time (95% CI, 58, 69). Sensitivity of the anti-Xa assay was 95% (95% CI, 92, 97).Conclusions: The correlation of thrombin generation measurements with DOAC drug concentrations was weak, and clinically relevant drug levels were not predicted correctly. Our results do not support an application of TG in the monitoring of DOAC.

Association between the use of female hormones and the thrombin generation: Cross-sectional analysis of the Longitudinal Study on Adult Health (ELSA-Brasil)

Introduction: The thrombin generation assay (TGA) assesses the risk of developing thrombotic events. The aim of this study was to investigate the association between the use of oral contraceptives (OCs) and hormone therapy (HT) with the TGA and resistance to activated protein C (APC). Methods: Cross-sectional study with women from the Longitudinal Study of Adult Health (ELSA-Brasil). TGA was performed by the CAT method. Results: There was an association between all parameters of the TGA and the use of OCs and an association of lag time and peak time with the use of HT. Conclusion: The TGA allows us to assess the hypercoagulability status and may be useful in the management of female hormones users.