Heparin Assay Research Articles

Anti-factor Xa as the preferred assay to monitor heparin for the treatment of pulmonary embolism.

The mainstay of acute pulmonary embolism (PE) treatment is anticoagulation. Timely anticoagulation correlates with decreased PE-associated mortality, but the ability to achieve a therapeutic activated partial thromboplastin time (aPTT) with unfractionated heparin (UFH) remains limited. Although some institutions have switched to a more accurate and reproducible test to assess for heparin's effectiveness, the anti-factor Xa (antiXa) assay, data correlating a timely therapeutic antiXa to PE-associated clinical outcomes remains scarce. We evaluated time to a therapeutic antiXa using intravenous heparin after PE response team (PERT) activation and assessed clinical outcomes including bleeding and recurrent thromboembolic events. This was a retrospective cohort study at NYU Langone Health. All adult patients ≥18 years with a confirmed PE started on IV UFH with >2 antiXa levels were included. Patients were excluded if they received thrombolysis or alternative anticoagulation. The primary endpoint was the time to a therapeutic antiXa level of 0.3-0.7 units/mL. Secondary outcomes included recurrent thromboembolism, bleeding and PE-associated mortality within 3 months. A total of 330 patients with a PERT consult were identified with 192 patients included. The majority of PEs were classified as sub massive (64.6%) with 87% of patients receiving a bolus of 80 units/kg of UFH prior to starting an infusion at 18 units/kg/hour. The median time to the first therapeutic antiXa was 9.13 hours with 93% of the cohort sustaining therapeutic anticoagulation at 48 hours. Recurrent thromboembolism, bleeding and mortality occurred in 1%, 5% and 6.2%, respectively. Upon univariate analysis, a first antiXa <0.3 units/ml was associated with an increased risk of mortality [27.78% (5/18) vs 8.05% (14/174), p = 0.021]. We observed a low incidence of recurrent thromboembolism or PE-associated mortality utilizing an antiXa titrated UFH protocol. The use of an antiXa based heparin assay to guide heparin dosing and monitoring allows for timely and sustained therapeutic anticoagulation for treatment of PE.

Serotonin Release Assay: Functional Assay for Heparin- and Vaccine-Induced (Immune) Thrombotic Thrombocytopenia.

The serotonin release assay (SRA) has been the gold-standard assay for detection of heparin-dependent platelet-activating antibodies and integral for the diagnosis for heparin-induced thrombotic thrombocytopenia (HIT). In 2021, a thrombotic thrombocytopenic syndrome was reported after adenoviral vector COVID-19 vaccination. This vaccine-induced thrombotic thrombocytopenic syndrome (VITT) proved to be a severe immune platelet activation syndrome manifested by unusual thrombosis, thrombocytopenia, very elevated plasma D-dimer, and a high mortality even with aggressive therapy (anticoagulation and plasma exchange). While the platelet-activating antibodies in both HIT and VITT are directed toward platelet factor 4 (PF4), important differences have been found. These differences have required modifications to the SRA to improve detection of functional VITT antibodies. Functional platelet activation assays remain essential in the diagnostic workup of HIT and VITT. Here we detail the application of SRA for the assessment of HIT and VITT antibodies.

Accuracy of a Single, Heparin-Calibrated Anti-Xa Assay for the Measurement of Rivaroxaban, Apixaban, and Edoxaban Drug Concentrations: A Prospective Cross-Sectional Study.

BackgroundApplying a single anti-Xa assay, calibrated to unfractionated heparin to measure rivaroxaban, apixaban, and edoxaban would simplify laboratory procedures and save healthcare costs.AimWe hypothesized that a heparin-calibrated anti-Xa assay would accurately measure rivaroxaban, apixaban, and edoxaban drug concentrations and correctly predict clinically relevant drug levels.MethodsThis analysis is part of the Simple-Xa study, a prospective multicenter cross-sectional study conducted in clinical practice. Patients treated with rivaroxaban, apixaban, or edoxaban were included. Anti-Xa activity was measured using the Siemens INNOVANCE® Heparin assay. Drug concentrations were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cut-off levels were determined in a derivation dataset (50% of patients) and sensitivities and specificities were calculated in a verification dataset (50% of patients).ResultsOverall, 845 patients were available for analysis. Correlation coefficients (rs) between the heparin-calibrated anti-Xa assay and drug concentrations were 0.97 (95% CI 0.97, 0.98) for rivaroxaban, 0.96 (0.96, 0.97) for apixaban, and 0.96 (0.94, 0.99) for edoxaban. The area under the receiver operating characteristics curve (ROC) was 0.99 for all clinically relevant drug concentrations. In the verification dataset, the sensitivity was 94.2% (95% CI 90.8–96.6) for 30 μg L–1, 95.8% (92.4–98.0) for 50 μg L–1, and 98.7% (95.5–99.9) for 100 μg L–1. Specificities were 86.3% (79.2–91.7), 89.8% (84.5–93.7), and 88.7% (84.2–92.2), respectively.ConclusionIn a large prospective study in clinical practice, a strong correlation of heparin-calibrated anti-Xa measurements with LC-MS/MS results was observed and clinically relevant drug concentrations were predicted correctly.

An indicator displacement assay-based optical chemosensor for heparin with a dual-readout and a reversible molecular logic gate operation based on the pyranine/methyl viologen

We have reported an optical indicator displacement assay (IDA) for heparin with a UV–vis absorbance and fluorescence dual-readout based on pyranine/methyl viologen (MV2+). Upon introducing heparin, pyranine/MV2+ shows a clearly observable increase in UV–vis absorbance and a turn-on of the fluorescence signal. We have demonstrated that the ionic nature of buffers significantly affects the pyranine displacement and the zwitterionic HEPES was most suitable for heparin sensing. After careful screening of experimental conditions, the pyranine/MV2+-based optical chemosensor exhibits a fast, sensitive, and selective response toward heparin. It shows dynamic linear concentration of heparin in the ranges of 0.1–40 U·mL−1 and 0.01–20 U·mL−1 for the absorptive and fluorescent measurements, respectively, which both cover the clinically relevant levels of heparin. As with the animal experiments, the optical chemosensor has been demonstrated to be selective and effective for heparin level qualification in rat plasma. The chemosensor is readily accessible, cost-effective, and reliable, which holds a great promise for potential application on clinical and biological studies. Furthermore, this IDA system can serve as an IMPLICATION logic gate with a reversible and switchable logical manner.

Dengue virus 3 genotype I shows natural changes in heparan sulphate binding sites, cell interactions, and neurovirulence in a mouse model.

Dengue virus (DENV) is the most prevalent pathogen of the Flaviviridae family. Due to the considerable increase in DENV incidence and spread, symptoms such as CNS involvement have increased. Heparan sulphate (HS) was the first molecule identified as an adhesion factor for DENV in mammalian cells. Viral phenotypes with different HS interactions are associated with various clinical symptoms, including neurological alterations. Here, using in silico analyses, in vitro studies, and the in vivo mouse model, we characterized two natural circulating DENV3 genotype I (GI) lineage 1 (L1) in Brazil-DENV3 MG-20 (from Minas Gerais) and DENV3 PV_BR (from Rondônia) that present divergent neurovirulent profiles and sensitivity to sulphated molecules. We identified substitutions at the viral envelope (E) in positions 62 and 123 as likely responsible for the differences in neurovirulence. The E62K and E123Q substitutions in DENV3 MG-20 and DENV3 PV_BR, respectively, greatly influenced in silico electrostatic density and heparin docking results. In vivo, mice inoculated with DENV3 MG-20 died, but not those infected with DENV3 PV_BR. The clinical symptoms, such as paralysis of the lower limbs and meningoencephalitis, and histopathology, also differed between the inoculated groups. In vitro heparin and heparinases assays further demonstrated the biological impact of these substitutions. Other characteristics that have been previously associated with alterations in cell tropism and neurovirulence, such as changes in the size of lysis plaques and differences in cytopathic effects in glioblastoma cells, were also observed.

Upconversion-based dual-mode optical nanosensor for highly sensitive and colorimetric evaluation of heparin in serum

Heparin is an important anticoagulant in clinics, which requires regular detection and dose adjustment. Fluorimetry is a powerful tool considering the advantages of fast response and high sensitivity while most fluorescent sensors suffer from spontaneous fluorescence for excitation under short-wavelength light, the occurrence of false positives, complex and time-consuming preparation, as well as expensive detection instruments. Herein, we developed upconversion nanoparticles (UCNPs) based nanosensor for fluorescent and colorimetric detection of heparin. The nanosensor exhibited sensitive detection for heparin with the low limit of detection (LOD) of 0.1 nM in fluorescent mode and 0.3 nM in colorimetric mode. In addition, we used a smartphone-sensing platform for quantitative detection of heparin with the LOD of 2 nM. Depending on long-wavelength excitation and fluorescent-colorimetric dual-response detection signal, the designed nanosensor could eliminate the auto-fluorescence, which effectively increased the accuracy of the sensor, thus extending the applications for bed-side heparin assay and related medical safety.

A turn‐on fluorescence assay for heparin based on DNA‐templated gold nanoclusters via ET

AbstractA novel fluorescence turn‐on assay for heparin has been developed based on DNA‐templated gold nanoclusters (AuNCs) and Cytochrome c (Cyt c). In the design, poly A (A15) ssDNA was selected as a template for fabricating AuNCs. AuNCs possess strong fluorescence emission at 471 nm. Upon addition of Cyt c, the fluorescence of AuNCs was quenched effectively by electron transfer (ET) between AuNCs and the heme cofactor of Cyt c. In the presence of heparin, the much higher electrostatic binding of heparin to Cyt c caused Cyt c to be away from AuNCs, leading to a recovery of fluorescence intensity of AuNCs. The proposed method was found to be simple, fast, and sensitive for heparin detection, with a linear range from 0.5 μg/mL to 12 μg/mL, and a detection limit of 0.15 μg/mL. Furthermore, this work was successfully applied to detection of heparin in fetal bovine serum samples, with good recoveries from 93.53% to 103.6%.

Ultrafine fluorene-pyridine oligoelectrolyte nanoparticles for supersensitive fluorescence sensing of heparin and protamine.

A new fluorene-pyridine oligoelectrolyte (OFP) is rationally proposed and readily synthesized via a simple one-pot Sonogashira approach. Hence, an unexpectedly small cationic oligomer nanosensor (i.e. OFPNPs, ∼ 1.2 nm in diameter) was conveniently fabricated owing to the enhanced flexibility endowed by the meta-substituted pyridyl unit. Inspiringly, this facile nanoplatform with low cytotoxicity favors the ultrasensitive fluorescence assay for heparin and protamine with a detection limit (LOD, S/N = 3) as low as 1.2 ng mL-1 and 0.5 ng mL-1, respectively, involving heparin-induced aggregation of OFPNPs through electrostatic interaction or competitive rebinding of protamine to heparin.

Anti‐factor IIa (FIIa) heparin assay for patients on direct factor Xa (FXa) inhibitors

BackgroundDirect factor Xa (FXa) inhibitors are increasingly prescribed for outpatients, and those transitioning to unfractionated heparin (UFH) for hospital admission are monitored via an anti‐FXa assay. Because of assay interference, UFH results would often be critically elevated, confounding dosing. ObjectivesAn anti‐factor IIa (FIIa) UFH assay was evaluated for clinical use. MethodsThe BIOPHEN ANTI‐IIa (Aniara Diagnostica) assay and anti‐FXa INNOVANCE Heparin assay (Siemens Healthcare Diagnostics Products GmbH) were compared on the Siemens BCS XP system. Samples included UFH controls and calibrators and specimens from patients transitioning from apixaban or rivaroxaban to UFH. Method comparison, linearity, recovery, precision, and interference by direct FXa inhibitors were evaluated. The effect of the BIOPHEN ANTI‐IIa assay on the rate of critically high UFH results was retrospectively reviewed 4 months after implementation. ResultsAccuracy studies using 0.24 and 0.50 IU/mL UFH yielded means and standard deviations of 0.26 ± 0.01 and 0.58 ± 0.01 IU/mL, respectively. Within‐run and between‐run coefficients of variation were 4.6% and 15.5% for the low control, and 1.8% and 10.6% for the high control. The method comparison slope was 0.9965 (r2 = 0.9468). The linear range was 0.1 to 1.3 IU/mL. The assay measured UFH in the presence of 192 ng/mL apixaban or 158 ng/mL rivaroxaban. Introduction of the assay for clinical use reduced the monthly percentage of critically high results from 9.4% to 3.8% for admitted heparinized patients who recently discontinued apixaban or rivaroxaban. ConclusionsThe BIOPHEN ANTI‐IIa assay is suitable for patients transitioning off apixaban or rivaroxaban.

Can an anti-Xa assay for unfractionated heparin be used to assess the presence of rivaroxaban in critical situations?

ObjectiveAlthough rivaroxaban has recently become widely used for thrombosis treatment, it is difficult for clinicians to make clinical decisions in critical situations, such as emergent surgery or interventions, because a specific anti-Xa assay is not available in many laboratories. This study assessed the relationships between rivaroxaban-specific anti-factor Xa activity (AXA) and unfractionated heparin (UFH)-specific AXA and determined the cutoff level for UFH-specific AXA in critical situations for patients undergoing rivaroxaban therapy. MethodsThirty-eight blood samples were collected from patients with cancer-associated thrombosis receiving rivaroxaban therapy. All samples were assessed using both rivaroxaban-specific and UFH-specific anti-Xa assays. Routine coagulation studies, including prothrombin time (PT) and activated partial thromboplastin time, were also conducted on the samples. ResultsA positive dose-dependent correlation between rivaroxaban-specific and UFH-specific AXA was evident (R = 0.97; P < .0001). Rivaroxaban-specific AXA was also positively correlated with PT (R = 0.95; P < .0001) but only weakly with activated partial thromboplastin time (R = 0.67; P < .0001). Patients with plasma rivaroxaban concentrations <100 ng/mL were found to have UFH-specific AXA <1.41 IU/mL and PT <17.3 seconds, with sensitivities of 100% and 93.3% and specificities of 87.0% and 95.7%, respectively. ConclusionsOur study demonstrates that UFH-calibrated AXA correlates strongly with plasma rivaroxaban concentration. This assay appears to be sensitive to the presence of rivaroxaban, which may be advantageous in the setting of assessing drug levels for critical events. These findings suggest that if a rivaroxaban-specific anti-Xa assay is unavailable, the chromogenic anti-Xa assay for UFH may be useful to assess the anticoagulant effects of rivaroxaban.

Establishment and Qualification of A Competitive Enzyme-Linked Immunosorbent Assay for the Measurement of Heparin in Plasma Products

Abstract Heparin is used in plasma fractionation processes to prevent activation of coagulation factors. Consequently, intermediates and final products have to be measured for their heparin content. Established chromogenic heparin assays rely on measurement of the accelerated inhibitory action of antithrombin against thrombin or activated factor X. These assays, however, show limitations, especially at the low sample dilutions required to provide adequate sensitivity. These limitations, potentially biasing results, originate from the specific sample matrix including proteases or protease inhibitors or from the test sample’s color. To overcome these shortcomings, a commercially available competitive enzyme-linked immunosorbent assay (ELISA) was established, described so far to measure heparin in plasma only. Briefly, heparin contained in the sample competed with the binding of a peroxidase-labelled heparin binding protein to the wells of a heparin-coated plate. The six-point calibration curve ranging from 0.03 to 7.14 IU heparin/mL showed acceptable accuracy and reproducibility. Test samples were diluted at least 1/6 in normal human citrated plasma. Spike-recovery was carried out by adding heparin to obtain a concentration of 0.26 IU/mL in the test dilution of the respective sample. Acceptable recovery was found in all sample types investigated (final immunoglobulin G product and process intermediates, albumin and α1-proteinase inhibitor), while intra-and inter-run precision data showed relative standard deviations lower than 10%. The ELISA’s limit of quantification was 0.18 IU/mL, thus demonstrating required sensitivity. In summary, the competitive heparin ELISA allowed accurate, precise and sensitive heparin measurement in specific plasma protein matrices, which interfere with chromogenic heparin assays.

Colorimetric and fluorometric aggregation-based heparin assay by using gold nanoclusters and gold nanoparticles.

An optical nanoprobe consisting of gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) is described for ultrasensitive detection of heparin (Hep). Polyethyleneimine (PEI) induces the aggregation of AuNPs which results in a color change from wine red (peak at 520nm) to blue (peak at 610nm). In parallel, the fluorescence of AuNCs (with excitation/emission maxima at 370/610nm) is weakened. However, in the presence of Hep (which is strongly negatively charged), it will electrostatically bind to positively charged PEI and then will prevent aggregation. Hence, the color changes from blue (aggregated) to red (non-aggregated). In parallel, fluorescence remains unchanged. Hep can be quantified by using the nanoprobe in the range of 4-220ng·mL-1, with the detection limits as low as 1.6 (colorimetry) and 3.4ng·mL-1 (fluorometry). The assay was applied to the detection of Hep in (spiked) human serum with satisfactory results. Graphical abstractSchematic illustration for colorimetric and fluorometric determination of heparin based on the use of a nanoprobe consisting of gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) with polyethyleneimine (PEI) as the mediator.

Factor XII Deficiency in ECMO Patients

Background: Extracorporeal membrane oxygenation (ECMO) for cardiopulmonary support of critically ill patients is used frequently in the pediatric and adult population. Although a lifesaving modality, it is burdened with high morbidity and mortality as a result of hematologic complications (Dalton, et al. Am J Respir Crit Care Med. 2017). Bleeding and thrombosis are related to contact of blood and its cellular components with the non-biologic surface of the extracorporeal circuit used that results in a massive inflammatory and clotting response. Factor XII deficiency is not associated with bleeding, but results in a significant prolongation of conventional coagulation assays making them unreliable for monitoring. Here we discuss 3 cases of Factor XII deficiency and the implications it has on monitoring anticoagulation in patients on ECMO. Laboratory characteristics for all 3 patients are outlined in Table 1. Case 1: Newborn full-term male with persistent pulmonary hypertension (PPHN) and meconium aspiration syndrome (MAS) with respiratory failure was placed on ECMO support on day of life 1. Patient received several units of cryoprecipitate and fresh frozen plasma (FFP) to correct deficiencies throughout his 31 day ECMO course. Patient did not have any bleeding or thrombotic complications, however he showed no improvement in lung function and decision was made to discontinue ECMO support. Case 2: Newborn full-term male with severe hypoxic ischemic encephalopathy (HIE) and MAS with secondary PPHN required ECMO support on day of life 1. His Factor XII level normalized with replacement by FFP and was decannulated after 11 days of ECMO support. This patient was discharged home with family in stable condition at 5 weeks of age. Case 3: 20-year-old female with history of recurrent astrocytoma, chronic lung disease, hydrocephalus with ventriculoperitoneal (VP) shunt, and tracheostomy presented with multifocal pneumonia and suspected sepsis. Despite fluid resuscitation and ventilatory management patient continued to have hypotension and hypoxia and thus was placed on ECMO. Prior to cannulation, patient was noted to have a coagulopathy. In addition, Factor II was low at 57% and corrected with one unit of FFP; which was thought to be related to consumptive process. On ECMO day 8, she had worsening hypotension despite vasopressor support and fixed and dilated pupils; suspected to have thromboembolic stroke and thus decision was made to withdraw life support. Discussion: Adequate anticoagulation in patients with Factor XII deficiency requiring ECMO support presents a challenging task. Patients with Factor XII deficiency generally do not show symptoms of a bleeding disorder, which may lead to misinterpretation of coagulation assays (Kokoye, et al. Thromb Res 2016). Increased contact activation inside the ECMO cannula causes activation of platelets, consumption of Factor XII, and formation of FXIIa-antithrombin complexes that may contribute to increased risk for thrombus formation (Kokoye, et al. Thromb Res 2016 and Bachler, et al. J Thromb Thrombolysis 2019). All patients had Factor XII levels &lt;40% on initial testing, which is sufficient to cause severe changes to the aPTT. A circulating anticoagulant aPTT was completed to assess for presence of lupus anticoagulants in 2 of the patients, which has been known to falsely elevate the aPTT and was negative (Bachler, et al. J Thromb Thrombolysis 2019). All patients received FFP in an attempt to correct deficiency and facilitate the use of aPTT and ACTs for monitoring. However, this did not prove to be successful in 2/3 patients despite receiving adult plasma. Hepzyme TEGs showed improvement in R-time post-FFP in 2 patients, but in 1 there remained evidence of factor deficiency. It is clear that traditional coagulation assays, including aPTT and ACT, become unreliable and the R-time on TEG becomes difficult to dissect heparin versus coagulation factor effect. We recommend attempting to correct factor deficiency with FFP to potentially decrease risk of thrombotic complications, and choosing an alternative laboratory monitoring assay for heparin, such as Anti-Xa levels, which may more accurately correspond to the anticoagulation status in this population. Currently, we are running a modified thrombin generation assay in these Factor XII deficient patients, pre/post-FFP, to identify if there is a decrease in thrombin generation post-FXII/FFP supplementation. Table 1 Disclosures Chitlur: Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda/Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioveritiv/Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL-Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Research Funding; Octapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

International Survey for Strategies Used for Oral Anticoagulation Reversal in Children

Background: Vitamin K antagonists (VKA) have been the main stay of oral anticoagulation (OAC) in pediatrics. However, VKA have multiple challenges. The pharmacological properties of direct oral anticoagulants (DOACs) suggest that they may have advantages particularly for children. The off-label use of DOACs is however on the rise within the pediatric population. The increasingly broad and varied use of DOACs, lack of published clinical guidance, and limited data on reversal strategies have created the imperative to identify strategies for OAC reversal in pediatrics. We conducted an online survey for strategies used for oral anticoagulants reversal in pediatrics. Study Design: Institutional review board approval was obtained and an online survey was developed using the RedCap. The survey was electronically distributed by International Society of Hemostasis and Thrombosis (ISTH) to its Pediatric/Neonatal Thrombosis and Hemostasis Subcommittee group members. The survey questions asked approach to common hypothetical clinical scenarios for OAC reversal.The data were analysed descriptively. Results: There were 76 respondents, majority from academic free-standing Children'sHospitals. Seventy-two percent reported having a hemostasis-thrombosis/anticoagulation service but only 29 % have a dedicated anticoagulation pharmacist. Approximately 40% do not have a formal protocol in place for VKA reversal. For a supra-therapeutic INR (INR &gt; 5) in a non-bleeding patient, 95% opted to manage by omitting the next dose of VKA while 18 % opted to give oral vitamin K alone or comitantly. For clinically relevant non-major bleeding, majority indicated using Vitamin K; oral (51%) or IV (37). For major bleeding on VKA, majority use either a combination of 4F-PCC and IV Vitamin K or plasma and IV Vitamin K (44/76 and 26/76 respectively). The presence of bleeding seemed to be the major driver for the choice of route (enteral versus parenteral) for Vitamin K for VKA reversal. Thirty-six of the 76 respondents indicated using DOACs; 94% used FXa inhibitors and 1/3 use dabigatran in their clinical practice. For non-urgent DOAC reversal, 97% indicated omitting the next dose. For non-major bleeding on DOAC, majority (29/36) indicated omitting the next dose/doses, some chose 4F-PCC (8/36) and only a few indicated use of specific reversal agents (3/36 and 1/36 for Dabigatran and Andexanet respectively). For major bleeding while on DOACs, the use of specific reversal agents (11/35, 6/35 for Andexanet and idarucizumab respectively) followed by 4F-PCC (9/35) was the major intervention indicated. Dilute thrombin time and partial thromboplastin time were the most commonly utilized tests to measure residual dabigatran activity. For Factor Xa inhibitors routine heparin assay rather than DOAC calibrated anti-Xa activity is utilized by most of the responders to assess presence of the plasma drug activity. Conclusion: Practices for oral anticoagulants reversal vary substantially in the pediatric population. Plasma is still used for urgent VKA reversal in many pediatric centers. The off-label use of DOACs in children is on the rise. Our results highlight the need for further studies to standardize OAC reversal in children. Disclosures Gupta: Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda-Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Research Funding; Novartis: Honoraria, Speakers Bureau; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Sarode:Portola: Honoraria; Octaphrarma: Consultancy; CSL Behring: Consultancy; Siemens: Research Funding. OffLabel Disclosure: The pharmacological properties of direct oral anticoagulants (DOACs) suggest that they may have advantages particularly for children. They are currently not approved in children. The off-label use of DOACs is however on the rise within the pediatric population.

A colorimetric heparin assay based on the inhibition of the oxidase mimicking activity of cerium oxide nanoparticles.

A colorimetric method is described for the sensitive detection of heparin (Hep). It is based on the finding that Hep can effectively inhibit the oxidase mimicking activity of cerium oxide nanoparticles (nanoceria). In the presence of Hep, the catalytic activity of nanoceria toward the oxidation of the chromogenic substrate 3,3',5,5'-tetramethylbenzidine by oxygen is strongly decreased. The inhibition mechanism is attributed to the fact that Hep is adsorbed on the surface of the nanoceria. Under optimal condition, the absorbance (measured at 652nm) decreases with increasing Hep concentrations in the range from 30 to 700nM. The detection limit is 20nM. The method was applied to the determination of Hep in medical injection sample and serum sample with satisfactory results. Graphical abstract Schematic presentation of the inhibition of oxidase-like activity of nanoceria by heparin. This allows the sensitive detection of heparin in medical injection sample and serum sample with satisfactory results.

Evaluation of Antifactor-Xa Heparin Assay and Activated Partial Thromboplastin Time Values in Patients on Therapeutic Continuous Infusion Unfractionated Heparin Therapy

Clinical uncertainty exists regarding which assay should be designated as the standard monitoring coagulation test for intravenous unfractionated heparin (UFH). Several studies have compared the use of activated partial thromboplastin time (aPTT) and antifactor-Xa (anti-Xa) and have come out with varying results. The correlation between these 2 tests varied, markedly from strong to weak. Some have demonstrated that monitoring with anti-Xa heparin assay leads to fewer dose adjustments, resulting in fewer laboratory tests, while others have not. In the current study, we evaluated the correlation between aPTT and anti-Xa values to guide clinical management of UFH, with the intention to develop a new correlation nomogram.

Colorimetric sensing strategy for heparin assay based on PDDA-induced aggregation of gold nanoparticles.

Herein, we report a highly sensitive colorimetric sensor for the detection of heparin based on its anti-aggregation effect of the PDDA–gold nanoparticle colloidal system. PDDA-induced non-crosslinking aggregation of gold nanoparticles is firstly investigated and the phenomenon of heparin mediated color recovery is then observed, which can be used to indicate the concentration of heparin. This method is proved to be highly sensitive and selective. Moreover, it has been successfully applied to determine human blood serum samples, the results of which demonstrate great potential practical utility with simple operations.

A cellulose-based photoacoustic sensor to measure heparin concentration and activity in human blood samples

Heparin is an indispensable drug in anticoagulation therapy but with a narrow therapeutic window, which dictates regular testing and dose adjustment. However, current monitoring tools have a long turnaround time or are operator intensive. In this work, we describe a cellulose-based photoacoustic sensor for heparin. The sensors have a turnaround time of 6 min for whole blood samples and 3 min for plasma samples regardless of heparin concentration. These sensors have a limit of detection of 0.28 U/ml heparin in human plasma and 0.29 U/ml in whole blood with a linear response (Pearson's r = 0.99) from 0 to 2 U/ml heparin in plasma and blood samples. The relative standard deviation was < 12.5% in plasma and < 17.5% in whole blood. This approach was validated with heparin-spiked whole human blood and had a linear correlation with the activated partial thromboplastin time (aPTT) (r = 0.99). We then studied 16 sets of clinical samples—these had a linear correlation with the activated clotting time (ACT) (Pearson's r = 0.86, P < 0.0001). The photoacoustic signal was also validated against the cumulative heparin dose (Pearson's r = 0.71, P < 0.0001). This approach could have applications in bed-side heparin assays for continuous heparin monitoring.

An Analysis of Risk Factors for Hemolysis in Children on Extracorporeal Membrane Oxygenation.

Hemolysis is a known complication of pediatric extracorporeal membrane oxygenation associated with renal failure and mortality. We sought to identify predictors of hemolysis in pediatric extracorporeal membrane oxygenation patients and determine its influence on outcomes. Retrospective, single-center study. Urban, quaternary care center pediatric and neonatal ICU. Ninety-six patients requiring extracorporeal membrane oxygenation. None. Daily measurements of plasma-free hemoglobin were obtained while patients were on extracorporeal membrane oxygenation. Patients with a prior extracorporeal membrane oxygenation run, on extracorporeal membrane oxygenation for less than 24 hours, or without complete medical records were excluded from the study. Ninety-six patients met inclusion criteria, of which, 25 patients (26%) had plasma-free hemoglobin greater than 30 mg/dL. Of those patients, 15 of 25(60%) had plasma-free hemoglobin greater than 50 mg/dL, and 21 of 25(84%) occurred during the first 7 days on extracorporeal membrane oxygenation. Compared with patients without hemolysis, those with hemolysis were younger (0.2 mo [0.06-3.2 mo] vs 8.2 mo [0.6-86 mo]; p < 0.001), had a higher pericannulation international normalized ratio (3.9 [3.5-5.5] vs 2.6 [1.8-3.7]; p = 0.003), lower pericannulation platelet count (33 × 10/μL [22-42 × 10/μL] vs 61 × 10/μL [38-86 × 10/μL]; p < 0.001), and had a less negative inlet pressure (-3.5 mm Hg [-14 to 11.5 mm Hg] vs -19 mm Hg [-47 to 0 mm Hg]; p = 0.01). A greater proportion of patients with hemolysis had a heparin assay less than 0.2 mg/dL (50% vs 17%; p = 0.001) and had fluid removal via slow continuous ultrafiltration (32% vs 6%; p < 0.001). Patients with hemolysis had increased risk of in-hospital mortality (odds ratio 10.0; 95% CI 3.4-32; p < 0.001). On multivariable analysis, continuous ultrafiltration (odds ratio, 8.0; 95% CI, 1.9-42; p = 0.007) and pericannulation international normalized ratio greater than 3.5 (odds ratio, 7.2; 95% CI, 2.3-26; p = 0.001) were significantly associated with hemolysis. Hemolysis is a common complication of pediatric extracorporeal membrane oxygenation. We found that patients with hemolysis (plasma-free hemoglobin > 30 mg/dL) had a 10-fold increase in in-hospital mortality. In our study cohort, hemolysis was associated with continuous ultrafiltration use, but not continuous renal replacement therapy. Additionally, our results suggest that the degree of coagulopathy (international normalized ratio > 3.5) at the time of cannulation influences hemolysis. Additional prospective studies are necessary to define further strategies to prevent hemolysis and improve outcomes in pediatric extracorporeal membrane oxygenation patients.

A ratiometric fluorometric heparin assay based on the use of CdTe and polyethyleneimine-coated carbon quantum dots.

CdTe quantum dots (QDs) were integrated with polyethyleneimine-coated carbon dots (PEI-CDs) to form a dually emitting probe for heparin. The red fluorescence of theCdTe QDs is quenched by thePEI-CDs due to electrostatic interactions. In the presence of heparin, the blue fluorescence of PEI-CDs remains unaffected, while its quenching effect on the fluorescence of CdTe QDs is strongly reduced. A ratiometric fluorometric assay was worked out. The ratio of the fluorescences at 595 and 436nm serves as the analytical signal. Response is linear in the concentration range of 50-600ng·mL-1 (0.1-1.2U·mL-1) ofheparin. The limit of detection is 20ng·mL-1 (0.04U·mL-1). This makes the method a valuable tool for heparin monitoring during postoperative and long-term care. This assay is relatively free from the interference by other analogues which commonly co-exist with heparin in samples, and it is more robust than single-wavelength based assays. Graphical abstract In the presence of heparin, the fluorescence of polyethyleneimine-coated carbon dots (PEI-CDs) at 436nm remains unaffected, while its quenching effect on the fluorescence of CdTe at 595nm is strongly reduced.