Heparin Assay Research Articles

Various Phyllosphere and Soil Bacterial Communities of Natural Grasses and the Impact Factors in a Copper Tailings Dam.

Copper mining caused severe damage to the ecological environment of mining areas. The combination of microbe and plant remediation has an application potential in improving the absorption and transformation efficiency of heavy metals. The phyllosphere is the largest biointerface on the planet, and bacteria are the dominant microbial inhabitants of the phyllosphere, believed to be critical to plant growth and health. This study investigated the phyllospheric and soil bacteria communities using high-throughput sequencing, and endophyte infection statuses of four natural grasses by toluidine blue heparin assay. Results showed variation in phyllospheric bacterial community structure. Gammaproteobacteria were the most abundant bacterial population. Bacilli were found in the phyllosphere of Bothriochloa ischaemum and Imperata cylindrica, while Clostridia were only found in Calamagrostis epigejos. Alphaproteobacteria were the dominant bacteria in soil. In addition, bacterial communities were influenced by endophytic infection statuses. Oxalobacteraceae was associated with soil carbon and sulfur. Enterobacteriaceae had negative correlation with the ratio of soil carbon and nitrogen, and had positive correlation with Cd content. These results offer useful insights into phyllospheric bacterial community variance in four different natural grasses in a copper tailings dam.

A signal-on ratiometric fluorometric heparin assay based on the direct interaction between amino-modified carbon dots and DNA.

Amino-modified carbon dots (C-dots) with positively charged surface were prepared. They display strong blue fluorescence and are shown to act as quenchers of the green fluorescence of FAM-labeled ssDNA such as the F-probe used in this work that was immobilized on the C-dots. On the addition of highly negatively charged heparin (Hep), it will interact with the C-dots and displace the F-probe from C-dots. Once the F-probe is displaced by Hep, its green fluorescence is restored. The intrinsic blue fluorescence of the C-dots remains stable after addition of Hep. Thus, a signal-on ratiometric fluorometric assay was developed for the ultra-sensitive detection of Hep. The underlying mechanisms of quenching and recovery are discussed. Under optimized conditions, the recovery of the ratiometric fluorescence of the system composed of C-dots and quenched F-probe is proportional to the Hep concentration in the range of 0.01-2.0μg·mL-1 (= 0.00125-0.25U·mL-1). The method was successfully applied to the determination of Hep in spiked serum samples. Graphical abstract Schematic of a signal-on ratiometric fluorometric method for the ultra-sensitive detection of heparin on the basis of the displacement and fluorescence enhancement of adsorbed FAM-labeled ssDNA from amino-modified carbon dots (C-dots) by heparin.

In situ formation of SERS hot spots by a bis-quaternized perylene dye: a simple strategy for highly sensitive detection of heparin over a wide concentration range.

A simple and ultrasensitive surface-enhanced Raman scattering (SERS) assay for heparin detection has been demonstrated based on an in situ hot spot assembly method using N,N'-bis[3,3'-(trimethylammonium)propylamine]-3,4,9,10-perylenediimide (bis-quaternized perylene dye, abbreviated as BQPER). In this assay, a BQPER probe with two positive charges is employed as both Raman reporter and electrostatic bridge to cause the aggregation of citrate-capped Ag nanoparticles (Ag NPs), resulting in a strong SERS signal due to the formation of numerous hot spots in the gaps of the aggregates. The SERS intensity of BQPER decreases in the presence of heparin, because BQPER binds primarily to heparin due to its high anionic charge to mass ratio compared to that of Ag NPs. Accordingly, a novel method for the detection of heparin has been developed based on SERS of the unbound BQPER probe. Two obvious advantages of the proposed heparin assay over other reported methods are: its much wider linear concentration range (10-5-10-10 g mL-1) and lower limit of detection (0.08 ng mL-1). Besides, our proposed heparin assay has been successfully applied to the detection of heparin in serum samples, indicating its great potential in the diagnosis of heparin-related diseases. This work provides a new Raman probe for the SERS analysis of heparin and opens a different perspective for SERS-based transducer design in charged analytes.

Blood and Blood Product Conservation: Results of Strategies to Improve Clinical Outcomes in Open Heart Surgery Patients at a Tertiary Hospital

Blood product usage is a quality outcome for patients undergoing cardiac surgery. To address an increase in blood product usage since the discontinuation of aprotinin, blood conservation strategies were initiated at a tertiary hospital in Oakland, CA. Improving transfusion rates for open heart surgery patients requiring Cardiopulmonary bypass (CPB) involved multiple departments in coordination. Specific changes to conserve blood product usage included advanced CPB technology upgrades, and precise individualized heparin dose response titration assay for heparin and protamine management. Retrospective analysis of blood product usage pre-implementation, post-CPB changes and post-Hemostasis Management System (HMS) implementation was done to determine the effectiveness of the blood conservation strategies. Statistically significant decrease in packed red blood cells, fresh frozen plasma, cryoprecipitate, and platelet usage over the stepped implementation of both technologies was observed. New oxygenator and centrifugal pump technologies reduced active circuitry volume and caused less damage to blood cells. Individualizing heparin and protamine dosing to a patient using the HMS led to transfusion reductions as well. Overall trends toward reductions in hospital length of stay and intensive care unit stay, and as a result, blood product cost and total hospitalization cost are positive over the period of implementation of both CPB circuit changes and HMS implementation. Although they are multifactorial in nature, these trends provide positive enforcement to the changes implemented.

Inhaled nebulised unfractionated heparin improves lung function in moderate to very severe COPD: A pilot study

BackgroundCOPD is an inflammatory airway disease characterised by progressive airflow limitation and air trapping, leading to lung hyperinflation and exercise limitation. Acute worsening of symptoms, including dyspnea, cough and sputum production, occurs during exacerbations which are associated with significantly reduced health related quality of life, and increased morbidity and mortality. Chronic bronchial mucus production and productive cough are risk factors for exacerbations. Medicines targeting bronchoconstriction and airway inflammation are the current mainstays of COPD therapy. However, there is growing concern with an increased risk of pneumonia in patients with COPD receiving regular inhaled corticosteroids and there is therefore a need to find safer alternative treatments.Previous studies have indicated that inhalation of unfractionated heparin (UFH) treats local inflammation, mucus hypersecretion and lung injury, without systemic anticoagulation, and is safe. Therefore, our primary objective was to demonstrate that inhaled UFH significantly improves lung function (FEV1) over 21 days of treatment in patients with COPD receiving pulmonary rehabilitation and that UFH provides a novel, safe and effective way of treating this complex disease. MethodsForty patients with moderate to very severe COPD admitted to the IRCCS San Raffaele Pisana Hospital for 21 days pulmonary rehabilitation were randomised to receive nebulised inhaled UFH (75,000 or 150,000 IU BID) or placebo for 21 days. All patients also received nebulised salbutamol (1 mg) and beclomethasone dipropionate (400 μg) BID over the same period.Lung function was measured at day 0, 7, 14 and 21 of treatment and at a follow-up visit 7 days post-treatment. Exercise capacity (6MWT) and dyspnoea (Borg score) were measured before and after treatment.In pre-clinical studies, the ability of basic proteins found in COPD sputum to neutralise the anticoagulant activity of heparin was determined using the AMAX heparin assay kit. Main resultsAt both doses, UFH significantly increased FVC following 7 days of treatment and 150,000 IU BID significantly increased FEV1 (+249 ± 69 ml compared with placebo) at this time, an effect maintained to the 28 day follow-up. Clinically significant improvement in exercise capacity and dyspnoea were seen after 21 days of treatment with both doses of UFH. There were no serious adverse events or effects on systemic coagulation. Pre-clinical studies demonstrated that the basic proteins lactoferrin, platelet factor-4 (PF-4), IL-8 and polyarginine, as a model of the eosinophil cationic protein (ECP), found in COPD sputum neutralise the anticoagulant activity of heparin. ConclusionInhaled nebulised UFH is safe and provides additional clinical benefit for patients with moderate to very severe COPD through effects that are independent of its anticoagulant activity.

Highly selective and sensitive detection of heparin based on competition-modulated assembly and disassembly of fluorescent gold nanoclusters

A simple and economical fluorescence assay for heparin using glutathione-protected gold nanoclusters via competitive binding was developed.

Colorimetric assay of heparin in plasma based on the inhibition of oxidase-like activity of citrate-capped platinum nanoparticles

We report citrate-capped platinum nanoparticles (Pt NPs) as oxidase mimetics for effectively catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid), dopamine, and methylene blue in the presence of O2. To confirm oxidase-like activity of citrate-capped Pt NPs, their activity toward oxygen reduction reaction was studied using cyclic voltammetry and rotating ring-disk electrode method. The results obtained showed that Pt NP NPs can catalyze the oxidation of organic substrates to the colored product and the reduction of oxygen to water through a four-electron exchange process. Because the aggregation of Pt NPs can inhibit their oxidase-like activity and protamine can recognize heparin, we prepared the protamine-modified Pt NPs through direct adsorption on the surface of citrate-capped Pt NPs. The electrostatic attraction between heparin and protamine-stabilized Pt NPs induced nanoparticle aggregation, inhibiting their catalytic activity. Therefore, the lowest detectable heparin concentrations through UV–vis absorption and by the naked eye were estimated to be 0.3 and 60nM, respectively. Moreover, the proposed system enabled the determination of the therapeutic heparin concentration in a single drop of blood.

Thromboprophylaxis With Heparin During Orthotopic Liver Transplantation: Comparison of Hepcon HMS Plus and Anti-Xa Assays for Low-Range Heparin

The purpose of this study was to compare the agreement between two heparin assays, Hepcon HMS plus/Kaolin-ACT and Anti-Xa, and their predictive power in detecting circulating heparin levels post-reperfusion of the liver graft when compared with thromboelastogram (TEG) r time ratio in patients undergoing orthotopic liver transplantation (OLT). Prospective, observational cohort study design. Single center, university hospital. Thirty-eight consecutive adults who had undergone liver transplant. None. Paired arterial blood samples were collected before surgical incision, 5 minutes after administration of an average dose of 2,054±771 units of intravenous unfractionated heparin before caval cross-clamping, 5 minutes after portal reperfusion, 5 minutes after hepatic artery reperfusion, and 1 hour after hepatic artery reperfusion. The observations that heparin assay measurements were within the predetermined limits of agreement, strongly suggested the two heparin assays (Hepcon HMS plus and Anti-Xa assay) are interchangeable during prophylactic heparin dose therapy during OLT. Post-reperfusion, receiver operating characteristic curve analysis revealed high accuracy in measuring circulating heparin levels with both Anti-Xa and Hepcon HMS assays when compared with the TEG r time ratio assay. The point-of-care Hepcon HMS plus/Kaolin-ACT (activated clotting time) assay appeared to be a reliable alternative to the more expensive and laboratory-required Anti-Xa assay in monitoring the response to intravenous heparin in patients undergoing OLT.

Antifactor Xa levels vs. activated partial thromboplastin time for monitoring unfractionated heparin. A pilot study.

The two most common methods for monitoring unfractionated heparin (UFH) infusion are the activated partial thromboplastin time (aPTT) and the antifactor Xa heparin assay (anti-Xa). The purpose of this study is to compare the performance of an aPTT protocol vs. an anti-Xa protocol in adult patients as defined by the time to reach therapeutic range, the percentage of time the values were within the goal range and the number of times laboratory monitoring was conducted. We then analysed the discordance between paired values of anti-Xa and aPTT. This was a single-centre prospective cohort pilot study conducted from 1 September 2013 to 31 May 2014. Eighty-five patients were treated with UFH infusion, aPTT monitoring (n = 48), anti-Xa monitoring (n = 37). The number of times aPTT and anti-Xa values were ordered was (median, IQR) 14 (2-34) vs 7 (2-76); P = 0·23. The time to reach therapeutic range in hours was (mean, SD) 22 (20) aPTT vs 15 (13) anti-Xa; P = 0·08. Therapeutic range (>50-100% of the time) was achieved in only 5 (10%) patients in the aPTT group vs. 21 (57%) in the anti-Xa group; P < 0·01. Supratherapeutic values (>50-100%) were observed in 38 (78%) patients in the aPTT group vs. 14 (38%) in the anti-Xa group; P < 0·01. The discordance between aPTT and anti-Xa was evaluated using 234 paired values from 37 patients. There was discordance between anti-Xa and aPTT values 57% of the time. Two patients had bleeding complications requiring blood transfusion or discontinuation of post-pilot protocol. Utilizing an anti-Xa protocol to monitor heparin infusion showed favourable results compared with utilizing an aPTT protocol by maintaining values within the therapeutic goal range. The most common discordant pattern in our study was a disproportionate prolongation of aPTT to anti-Xa values. Patients with discordant values presenting with high aPTT to normal anti-Xa values may have an increased risk of bleeding complications.

Modulation of GAG-sulfation pattern in biohybrid hydrogels to tune growth factor release

Event Abstract Back to Event Modulation of GAG-sulfation pattern in biohybrid hydrogels to tune growth factor release Passant Atallah1, Andrea Zieris1, Uwe Freudenberg1 and Carsten Werner1, 2 1 Max Bergmann Center of Biomaterials, Leibniz Institute of Polymer Research Dresden, Germany 2 Center for Regenerative Therapies Dresden, Technische Universität Dresden, Germany Introduction: GAG based hydrogels that modulate key parameters of the extracellular matrix were previously developed showing long term delivery of signaling molecules[1]. In here, the sulfation patterns of the GAG building blocks govern the electrostatic complexation of biomolecules and thus gradual desulfation should allow for modulating the release profiles of cytokines from the hydrogels. To explore options related to this, selectively desulfated heparin derivatives were prepared, thoroughly characterized, and covalently converted with star-shaped poly (ethylene glycol) into binary polymer networks and analyzed for cytokine release and cellular response in vitro and in vivo. Methods: Different solvolytic desulfation procedures yielded N-desulfated, 6O- N- heparin, 6O-desulfated and 2O-6O-N- (completely) desulfated heparin. All the heparin derivatives were thoroughly analyzed for regioselectivity of desulfation by 1H NMR, particle size analysis by asymetrric field flow fractionation, residual sulfate content by elemental analysis and anticoagulant activity by factor Xa based heparin assay. Hydrogels with graded sulfation pattern were subsequently prepared and the impact of the variation of the net negative charge on network properties was evaluated by rheometry and swelling measurements. Vascular endothelial growth factor (VEGF 165) and fibroblast growth factor 2 (FGF-2) were non-covalently conjugated to the heparin component of the hydrogel and the initial binding affinity of cytokines and their subsequent release was analyzed by ELISA[2]. The influence of the different functionalized matrices to migration and morphology of human umbilical vein endothelial cells (HUVEC) and the modulation of wound healing in diabetic mice has been analyzed. Results and discussion: Desulfated heparin chains were systematically characterized to confirm the chemical properties of the precursors complexed with the hydrogels. Analysis of physical network properties showed the influence of the charge density as well as the sulfation site specific effect on the elasticity and network defects of the formed hydrogels. Hydrogels with variable sulfation pattern show widely tunable release profiles for FGF-2 and VEGF165. In-vitro studies showed that the increased VEGF release from desulfated heparin hydrogels resulted in more pronounced proangiogenic response of HUVECs by enhanced cell migration and tubular formation. Similarly, hydrogels transplanted in db/db mice promoted wound angiogenesis, a critical stage in the wound healing process. Conclusion: Tailoring the sulfation pattern of GAG based hydrogels enables control over cytokine delivery and therefore cellular response in vitro and in vivo. The resulting new class of cell-instructive polymer matrices with tunable GAG sulfation will be instrumental for multiple applications in biotechnology and medicine. Figure 1: Schematic design of biohybrid GAG- star PEG hydrogels with regioselective desulfation resulting in modulation of cytokine release. This work was supported by the Deutsche Forschungsgemeinschaft through grant nos. SFB-TR 67/A10

Comparison Between Activated Partial Thromboplastin Time and Anti-Xa Heparin Assays on Patients on Unfractionated Heparin Therapy: Does the Prothrombin Time Matter?

The laboratory at Veterans Affairs Medical Center–Memphis recently implemented anti-Xa heparin assays to monitor therapy for unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH). We were queried by the pharmacy department about possibly discrepant results between the new assay and the activated partial thromboplastin time (aPTT). We reviewed initial data from the anti-Xa assay and considered the possible importance of prothrombin time …

Comparison Between Activated Partial Thromboplastin Time and Anti-Xa Heparin Assays on Patients on Unfractionated Heparin Therapy

The clinical laboratory at Veterans Affairs Medical Center–Memphis recently implemented anti-Xa heparin assays to monitor therapy for unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH). The pharmacy became concerned by possible discrepant results between the new assay and the activated partial thromboplastin time (aPTT), and we reviewed early data generated by the anti-Xa assay. Consecutive anti-Xa heparin results (n = 105 from 30 patients) were collected that had sufficient …

PER977 REVERSES LOW MOLECULAR WEIGHT HEPARIN IN ADDITION TO IIA AND XA NEW ORAL ANTICOAGULANTS

There is no approved reversal agent or coagulation assay for low molecular weight heparin (LMWH). Given the significant number of bleeds on LMWH (reported as 12.7% or approximately 137,000 patients in the US annually), there is an urgent and unmet need for a reversal agent and rapid, point of care

A sensitive sequential ‘on/off’ SERS assay for heparin with wider detection window and higher reliability based on the reversed surface charge changes of functionalized Au@Ag nanoparticles

A sequential ‘on/off’ dual mode SERS assay platform for heparin with wider detection window and higher reliability is constructed based on electrostatic forces, in which the highly protonated chitosan encapsulated p-Mercaptobenzoic acid coated Au@Ag core–shell nanoparticles undergo sequential aggregation/segregation upon the additive of heparin with a limit of detection of 43.74ng/mL (5.69U/mL) and a continuous concentration range of 50–800ng/mL (6.5–104U/mL), which are lower in sensitivity and wider in detection window than the most reported assay for heparin. Remarkably, the latter declined window over a range of 350–800ng/mL in contrast, which has not reported before, is extremely important in reliable and practical assay of heparin.

Off–on phosphorescence assay of heparin via gold nanoclusters modulated with protamine

A sensitive and selective phosphorescence “turn-off–on” nanosensor to detect heparin by gold nanoclusters (Au-NCs) modulated with protamine was developed. The carboxyl groups on the surface of Au-NCs can interact with amino groups in protamine via electrostatic interactions and hydrogen bonding to induce the aggregation of Au-NCs, which results in the photoluminescence to “turn-off”. However, in the presence of heparin, protamine prefers to combine with heparin to release Au-NCs, which results in the photoluminescence to “turn-on”. The photoluminescence lifetime measurements demonstrate that the photoluminescence belongs to phosphorescence, and that the quenching progress is dynamic. The concentration of heparin can be determined by measuring the Au-NCs phosphorescence recovery. The linear response range was obtained from 0.006 to 25.0μgmL−1 with the detection limit of 6ngmL−1. Moreover, the proposed phosphorescence nanosensor was used to detect heparin in fetal bovine serum samples with satisfactory results.

A note on the biological assay of heparin

A note on the biological assay of heparin

A “turn-off” SERS assay of heparin with high selectivity based on heparin–peptide complex and Raman labelled gold nanoparticles

Stable SERS response originating from gold nanoparticles (Au NPs) is demonstrated for the first time to be suitable for detection of heparin with high selectivity. Herein we report a novel “turn-off” SERS assay for heparin based on Raman labelled Au NPs and competitive bonding between heparin and two specialized peptides, such as RKGSGRRLVKC (11-peptide) and CALNN (5-peptide). By means of coordination interactions and other non-electrostatic forces, these 5-peptide capped Au NPs are induced to undergo controllable aggregation upon the additive of 11-peptide and heparin with a concentration range (0–2.4U/mL), which correspondingly cause quantitative changes of SERS intensity of 4-MBA conjugated on Au NPs. The decreased Raman intensity obtained by subtracting two cases of additives which contain only 11-peptide and the mixture of 11-peptide and heparin, is proportional to the concentration of heparin over a range of 0.2–2.4U/mL (R2=0.99157), with a limit of detection (LOD) of 0.042U/mL (S/N=3:1). Moreover, the proposed highly selective method has potential application for rapid, separation-free detection of heparin in human blood serum samples.

Ultrasensitive fluorescence detection of heparin based on quantum dots and a functional ruthenium polypyridyl complex

A new strategy for the detection of heparin is developed by utilizing quantum dots (QDs) and a functional ruthenium polypyridyl complex [Ru(phen)2(dppz-idzo)]2+ (phen=1,10-phenanthroline, dppz-idzo=dipyrido-[3,2-a:2',3'-c] phenazine-imidazolone). The emission of CdTe QDs is found to be quenched by Ru complex due to electron transfer. Upon addition of the polyanionic heparin, it could remove the quencher (Ru complex) from the surface of QDs owing to the electrostatic and/or hydrogen bonding interactions between heparin and Ru complex, which led to significant fluorescence recovery of CdTe QDs. The fluorescence intensity enhanced with the increase of heparin and a linear relationship was observed in the range of 21–77nM for heparin detection in buffer solution and the limit of detection (LOD) is 0.38nM. Moreover, the strategy was successfully applied to detect heparin as low as 0.68nM with a linear range of 35–98nM in fetal bovine serum samples. The selectivity results of the fluorescence assay revealed that our system displayed excellent fluorescence selectivity towards heparin over its analogues, such as chondroitin 4-sulfate (Chs) or hyaluronic acid (Hya). This fluorescence “switch on” assay for heparin is label-free, convenient, sensitive and selective, which can be used to detect heparin in biological systems even with the naked eyes.

Modified Heparin Assay For The Quantitation Of Rivaroxaban

The anticoagulant rivaroxaban is not routinely monitored due to its predictable anticoagulant action. However, drug levels are clinically useful in patients requiring surgery as well as in assessing compliance. We have modified a commercial heparin assay based on inhibition of activated factor X (Xa) that was previously modified for the assay of fondaparinux (Am J Clin Pathol 2009, 132:608-612) by replacing the fondaparinux calibrators with commercial rivaroxaban calibration plasmas. Technoview rivaroxaban calibrator plasmas were purchased from Dia Pharma (West Chester, Ohio). These calibrators were substituted for the fondaparinux calibrators. The assay was run on a Siemens BCS XP, as previously described. The assay was linear in the range of 0 to 650 ng/ml. The limit of detection was 14 ng/ml, and carryover at 285 ng/ml was &lt; 1%. The intra-assay precision at 100 ng/ml was standard deviation of 1.04 ng/ml, coefficient of variation of 1.03%. The inter-assay precision at 105 ng/ml was standard deviation 3.5 ng/ml, coefficient of variation of 3.3%. The assay is unaffected by warfarin, but can be affected by other Xa inhibitors such as heparin, low molecular weight heparin, fondaparinux, and apixaban. Disclosures: No relevant conflicts of interest to declare.

Novel Point-Of-Use FXa Assay For Monitoring Anticoagulant Therapy By Oral Factor Xa Inhibitors and Heparin

Introduction A highly specific, rapid, robust and sensitive assay for Factor Xa (FXa) activity will greatly improve monitoring and optimization of anticoagulant therapy by direct FXa inhibitors and by low molecular weight (LMW) heparins. We have developed a new point-of-use diagnostic system that will provide results in less than 30 minutes, enabling rapid medical decisions based on quantitation of FXa activity and anticoagulant drugs in patients’ blood. Anticoagulant therapy with unfractionated or LMW heparins needs aggressive monitoring in pregnant women, patients with renal insufficiency, and neonates, in particular in cases of treatment-related side effects, including heparin-induced thrombocytopenia and bleeding tendency (Chest, 2008). New oral anticoagulants rivaroxaban (Xarelto®) and apixaban (Eliquis®) have been effective in reducing risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation, and in preventing and treating deep vein thrombosis. Both drugs directly inhibit FXa and have significant therapeutic potential as an alternative to warfarin. Unlike warfarin, these drugs do not require constant monitoring and dose adjustment to maintain anticoagulation within the therapeutic interval. However, excessive anticoagulation and consequent hemorrhage risks have been sufficiently common problems to warrant clinical development of specific antidotes for FXa inhibitors. Currently available anti-Xa heparin assays may not be appropriate for rivaroxaban and apixaban measurements. Additionally, recently published data indicate that new oral anticoagulants interfere with the measurement of common test parameters, expanding the need for more specific diagnostic tests (Clinical Chemistry, February 2013). This novel rapid and specific FXa assay will allow monitoring LMW heparins and the new FXa inhibitors, identify heparin-refractory patients as well as those requiring antidotes to oral Factor Xa inhibitors, in addition to directly measuring FXa activity in patients with inherited deficiency of Factor X, congenital antithrombin deficiency, and acquired FXa deficiencies. Method A diagnostic assay to measure FXa activity in human plasma using electrochemiluminescence (ECL) technology was developed at Wellstat Diagnostics, LLC. The assay utilizes a synthetic peptide containing an FXa substrate sequence that is recognized by specific antibodies (labeled with an ECL-active ruthenium chelate) only after specific cleavage by FXa protease. The resulting ECL signal is directly proportional to FXa activity within the physiological range. The assay has been adapted to also provide quantitative measurement of activity of anticoagulant drugs that inhibit FXa. In the modified format the resulting ECL signal is inversely proportional to the concentration of rivaroxaban, apixaban or heparin in plasma. Results The ECL-based assay measures activity of anticoagulant drugs on FXa in human plasma with high intra- and inter-assay reproducibility, accuracy and specificity. Using FX-deficient plasma spiked with Factor X in the range 1-100 ng/mL, we have demonstrated linear increases of ECL signal directly proportional to the concentration of spiked Factor X activated by a specific Russell Viper Venom activator. Serial dilutions of anticoagulant drugs rivaroxaban, apixaban and enoxaparin (LMW heparin) in normal donor plasma showed exponential decreases of ECL signal in the range of concentrations relevant to the therapeutic doses of all three drugs. The dynamic range of the drug detection was 15 - 500 ng/mL for rivaroxaban and apixaban, and 0.2 - 2 IU/mL of enoxaparin. The assay was specific to activity of FXa, while insensitive to variable concentrations of other clotting factors in human plasma, as well as to the presence of other drugs in plasma that don’t directly inhibit FXa. Conclusions The point-of-use FXa assay is shown to be highly specific, robust, rapid and reproducible for measurements of anticoagulant activity of apixaban, rivaroxaban and enoxaparin in human plasma. Use of this point-of-use system for monitoring anticoagulant therapy may be further extended to measure other blood clotting factors (for example, Factor IXa, VIIa, FVIII), for monitoring activity of drugs acting on these factors and for rapid diagnosis of coagulation disorders. Disclosures: No relevant conflicts of interest to declare.