Comparison of animal and human blood for in vitro dynamic thrombogenicity testing of biomaterials.

BACKGROUND: To determine suitable alternatives to human blood for in vitro dynamic thrombogenicity testing of biomaterials, four different animal blood sources (ovine, bovine, and porcine blood from live donors, and abattoir porcine blood) were compared to fresh human blood. METHODS: To account for blood coagulability differences between individual donors and species, each blood pool was heparinized to a donor-specific concentration immediately before testing in a dynamic flow loop system. The target heparin level was established using a static thrombosis pre-test. For dynamic testing, whole blood was recirculated at room temperature for 1 h at 200 ml/min through a flow loop containing a single test material. Four materials with varying thrombotic potentials were investigated: latex (positive control), polytetrafluoroethylene (PTFE) (negative control), silicone (intermediate thrombotic potential), and high-density polyethylene (HDPE) (historically thromboresistant). Thrombus weight and surface area coverage on the test materials were quantified, along with platelet count reduction in the blood. RESULTS: While donor-specific heparin levels varied substantially from 0.6 U/ml to 7.0 U/ml among the different blood sources, each source was able to differentiate between the thrombogenic latex and the thromboresistant PTFE and HDPE materials (p < 0.05). However, only donor ovine and bovine blood were sensitive enough to differentiate an increased response for the intermediate thrombotic silicone material compared to PTFE and HDPE. CONCLUSIONS: These results demonstrated that multiple animal blood sources (particularly donor ovine and bovine blood) may be suitable alternatives to fresh human blood for dynamic thrombogenicity testing when appropriate control materials and donor-specific anticoagulation levels are used.

Clinical Features and Treatment Response in Immune-Mediated Small Fiber Neuropathy with Trisulfated Heparin Disaccharide or Fibroblast Growth Factor Receptor 3 Antibodies.

OBJECTIVE: Novel antibodies to trisulfated heparin disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR-3) have been recently described in otherwise cryptogenic small fiber neuropathy (SFN) cases. Our goal was to further describe clinical features in such cases and to analyze treatment responses. METHODS: In a retrospective analysis, 40 cases of cryptogenic SFN in a university neuropathy clinic were identified. Of these, TS-HDS and FGFR-3 cases were identified, and clinical features and treatment responses were analyzed. RESULTS: In this cohort, 95% were women, and 55% had either TS-HDS or FGFR-3 antibodies (77% of these had TS-HDS). Of the seropositive group, 41% had a nonlength dependent epidermal nerve fiber density on skin punch biopsy (OR = 1.80). In the seropositive group, 82% had neuropathic pain as their primary symptom (OR = 1.73). Also 32% of seropositive patients reported widespread pain (OR = 1.63). 63% of seropositive cases presented acutely (OR = 11.0). In the seropositive group, 23% had an initial erroneous diagnosis (OR = 1.47). Eight seropositive patients improved on intravenous immunoglobulin treatment, with a 42% reduction in pain scores (P = 0.02), a 44% reduction in the Utah Neuropathy Score, and improved epidermal nerve fiber density post-treatment. CONCLUSIONS: TS-HDS and FGFR-3 antibodies may be present in a high proportion of cryptogenic SFN cases with acute onset, nonlength dependent pathology, and primary neuropathic and widespread pain. They are often misdiagnosed as other conditions including fibromyalgia. These cases may be responsive to immune treatment, especially with intravenous immunoglobulin.

TS-HDS and FGFR3 antibodies in small fiber neuropathy and Dysautonomia.

INTRODUCTION: The specificity of trisulfated heparin disaccharide/fibroblast growth factor receptor 3 (TS-HDS/FGFR3) antibodies in the diagnosis of autoimmune small fiber neuropathy (SFN) is unclear. METHODS: This was a retrospective study of patients evaluated for SFN and dysautonomia in the Brigham and Women's Faulkner Hospital Autonomic Laboratory in 2019-2020. Associations were assessed between TS-HDS/FGFR3 antibodies and SFN markers, including epidermal nerve fiber density (ENFD), sweat gland nerve fiber density (SGNFD), and autonomic dysfunction assessed by Valsalva maneuver, deep breathing, sudomotor, and tilt testing. RESULTS: Of 322 patients; 28% had elevated anti-TS-HDS, 17% had elevated anti-FGFR3, 96% had autonomic dysfunction, 71% had abnormal ENFD, and 49% had abnormal SGNFD. TS-HDS/FGFR3 antibodies were present in patients with autonomic dysfunction irrespective of whether they had normal or abnormal skin biopsies unless ENFD/SGNFD were combined for anti-FGFR3 seropositivity. DISCUSSION: TS-HDS/FGFR3 antibodies are present in patients with evidence of autonomic dysfunction. Further studies are needed to document the clinical value of these antibodies in assessment of immune mediated dysautonomia.

Comparative metabolomics studies of blood collected in streck and heparin tubes from lung cancer patients.

Metabolomics analysis of blood from patients (n = 42) undergoing surgery for suspected lung cancer was performed in this study. Venous and arterial blood was collected in both Streck and Heparin tubes. A total of 96 metabolites were detected, affected by sex (n = 56), collection tube (n = 33), and blood location (n = 8). These metabolites belonged to a wide array of compound classes including lipids, acids, pharmaceutical agents, signalling molecules, vitamins, among others. Phospholipids and carboxylic acids accounted for 28% of all detected compounds. Out of the 33 compounds significantly affected by collection tube, 18 compounds were higher in the Streck tubes, including allantoin and ketoleucine, and 15 were higher in the Heparin tubes, including LysoPC(P-16:0), PS 40:6, and chenodeoxycholic acid glycine conjugate. Based on our results, it is recommended that replicate blood samples from each patient should be collected in different types of blood collection tubes for a broader range of the metabolome. Several metabolites were found at higher concentrations in cancer patients such as lactic acid in Squamous Cell Carcinoma, and lysoPCs in Adenocarcinoma and Acinar Cell Carcinoma, which may be used to detect early onset and/or to monitor the progress of the cancer patients.

A Systemwide Approach for Navigating the Dilemma of Oral Factor Xa Inhibitor Interference With Unfractionated Heparin Anti-Factor Xa Concentrations.

BACKGROUND: Oral factor Xa inhibitors are known to significantly increase heparin anti-Xa concentrations, which leads to inaccuracies when monitoring intravenous unfractionated heparin (IV UFH). Guidance for managing this laboratory interference is lacking, creating substantial uncertainty in clinical practice. OBJECTIVE: To describe a strategy used by a large academic institution for managing the controversy of laboratory interference in the setting of oral factor Xa inhibitor use and provide effectiveness and safety data for this approach. METHODS: In December 2016, a new Heparin IV Direct Oral Anticoagulant (DOAC) Interference PowerPlan (a comprehensive order set) was made available in the electronic health record (Cerner, North Kansas City, MO) throughout the health system. We retrospectively examined 169 patients with events reported in the error reporting system, RISKMASTER, and evaluated reports with and without the use of the PowerPlan. Effectiveness was determined through evaluation of thrombosis. The Naranjo criteria for causality were applied to assess thrombotic events. RESULTS: Of 56 events that were reported with apixaban when the PowerPlan was not ordered, 4 (7%) thrombotic events occurred within 7 days of UFH initiation. One out of the 4 events (25%) that occurred when the PowerPlan was not appropriately initiated was considered probable using the Naranjo Scale. Three additional events (75%) were possible using the Naranjo Scale. CONCLUSION AND RELEVANCE: The Heparin IV DOAC Interference PowerPlan appears to be conducive to positive patient outcomes when evaluating voluntary reported events and may assist clinicians with managing the therapeutic dilemma of this laboratory interference.

A rolling circle amplification based platform for ultrasensitive detection of heparin.

Heparin has a variety of pharmacological uses, including applications for anti-tumor metastasis, anti-inflammatory and anti-viral activities and is widely used as a clinical anticoagulant. Due to its widespread applications in the clinical procedures, monitoring heparin levels is critically important to ensure the safe use of heparin and to prevent overdose and complications, such as hemorrhage and thrombocytopenia. However, traditional heparin detection relies on the measurements of the activated clotting time or activated partial thromboplastin time, which are not sufficiently reliable or accurate measurements for certain clinical settings. In this work, we describe a dumbbell probe-aided strategy for ultrasensitive and isothermal detection of heparin based on a uniquely strong protamine-heparin interaction and rolling circle amplification driven signal amplification. The detection limit for heparin is 12.5 ng mL-1 (0.83 nM), which is much lower than the therapeutic level of heparin in cardiovascular surgery (17-67 µM) and in postoperative and long-term treatment (1.7-10 µM). Additionally, the proposed sensing platform works well for heparin monitoring in human plasma samples. This simple and ultrasensitive heparin biosensor has potential application in diagnostics, therapeutics, and in biological research.

Early detection of deep vein thrombosis in patients with coronavirus disease 2019: who to screen and who not to with Doppler ultrasound?

PURPOSE: Aim of the study is to evaluate the incidence of DVT in COVID-19 patients and its correlation with the severity of the disease and with clinical and laboratory findings. METHODS: 234 symptomatic patients with COVID-19, diagnosed according to the World Health Organization guidelines, were included in the study. The severity of the disease was classified as moderate, severe and critical. Doppler ultrasound (DUS) was performed in all patients. DUS findings, clinical, laboratory's and therapeutic variables were investigated by contingency tables, Pearson chi square test and by Student t test and Fisher's exact test. ROC curve analysis was applied to study significant continuous variables. RESULTS: Overall incidence of DVT was 10.7% (25/234): 1.6% (1/60) among moderate cases, 13.8% (24/174) in severely and critically ill patients. Prolonged bedrest and intensive care unit admission were significantly associated with the presence of DVT (19.7%). Fraction of inspired oxygen, P/F ratio, respiratory rate, heparin administration, D-dimer, IL-6, ferritin and CRP showed correlation with DVT. CONCLUSION: DUS may be considered a useful and valid tool for early identification of DVT. In less severely affected patients, DUS as screening of DVT might be unnecessary. High rate of DVT found in severe patients and its correlation with respiratory parameters and some significant laboratory findings suggests that these can be used as a screening tool for patients who should be getting DUS.

Heparin concentration in cell salvage during heparinization: a pilot study.

Cell salvage is frequently used to avoid unnecessary allogeneic blood transfusions, which results in a reduction in blood transfusion volume and cost. The aspirated blood is washed with normal saline and centrifuged to recover only blood cells, salvaged blood is then made. In cardiovascular surgery, heparin is used to maintain activated clotting time over 400 seconds. Some practitioners believe that heparin remains in the salvaged blood. Therefore, we hypothesized that salvaged blood during cardiovascular surgery includes heparin. A pilot study was conducted to evaluate our hypothesis using three different salvage systems. This study was a prospective, observational, pilot study, with patients aged 20-85 years old who were scheduled for cardiovascular surgery from May 2018 to October 2018. The intent of this study was to evaluate whether salvaged blood with three different devices includes large enough quantities of heparin to influence activated clotting time in cardiovascular surgery. Between May and October 2018, 12 samples during heparinization were collected, and 12 samples of salvaged blood from 3 devices were collected after administrating protamine. The heparin concentration of the 24 samples was measured. All heparin concentrations in salvage blood sample from two devices was below the limit of measurement (0.10 IU/mL). Slightly measurable heparin was detected in salvaged blood sample from one device (mean 0.15 IU/mL). Salvaged blood during cardiovascular surgery intervention does not contain enough heparin to influence activated clotting time.

Conversion From Activated Clotting Time to Anti-Xa Heparin Activity Assay for Heparin Monitoring During Extracorporeal Membrane Oxygenation.

OBJECTIVES: Anticoagulation with unfractionated heparin remains the most common therapy used to prevent circuit thrombosis during extracorporeal membrane oxygenation, but no consensus exists on the optimal method or targets for heparin monitoring. From 2015 to 2018, we switched from monitoring heparin during extracorporeal membrane oxygenation using activated clotting times to anti-Xa heparin activity assays. This study describes the transition from activated clotting time to anti-Xa heparin activity assay monitoring and the associated clinical changes. DESIGN: Retrospective analysis at single institution. SETTING: Referral Children's Hospital. PATIENTS: A total of 145 pediatric patients over 152 extracorporeal membrane oxygenation runs using 206 extracorporeal membrane oxygenation circuits. INTERVENTIONS: Anticoagulation protocol quality improvement. MEASUREMENTS AND MAIN RESULTS: From 2015 to 2018, heparin monitoring during extracorporeal membrane oxygenation changed from hourly activated clotting time to anti-Xa heparin activity assay every 6 hours with an associated 75% reduction in the circuit changes per extracorporeal membrane oxygenation day. Over the 4 years, patients with an average anti-Xa heparin activity assay of at least 0.25 U/mL showed a 59% reduction in circuit changes per extracorporeal membrane oxygenation day compared with less than 0.15 U/mL. In addition to its association with reduced circuit changes, anti-Xa heparin activity assay monitoring was also associated with reduced heparin dose changes per day from 11 ± 4 to 2 ± 1 (p < 0.001), smaller heparin dose changes (less variation in dose), and reduced diagnostic phlebotomy volumes from 41 ± 6 to 25 ± 11 mL/day (p < 0.001). The number of patients with reported bleeding decreased from 69% using activated clotting time to 51% (p = 0.03). Transfusion rates did not change. CONCLUSIONS: Over 4 years, we replaced the activated clotting time assay with the anti-Xa heparin activity assay for heparin monitoring during extracorporeal membrane oxygenation. Minimum anti-Xa heparin activity assay levels of 0.25 U/mL were associated with reduced circuit changes. Further studies are needed to determine the optimum anti-Xa heparin activity assay therapeutic range during extracorporeal membrane oxygenation.

Rethinking the one-size-fits-most approach to venous thromboembolism prophylaxis after radical cystectomy.

OBJECTIVES: Empirically dosed enoxaparin is routinely given in the postoperative period for venous thromboembolism (VTE) prophylaxis after radical cystectomy (RC). Patient-specific factors may alter its pharmacokinetics, and it is unclear whether this leads to levels sufficient for antithrombosis. We sought to evaluate variability of anti-factor Xa levels in a cohort of RC patients receiving perioperative enoxaparin prophylaxis. MATERIAL AND METHODS: Patients undergoing RC at a single institution were placed on a postoperative pathway that included enoxaparin. An anti-factor Xa level was drawn 2 to 4 hours after the third dose. The target range for prophylaxis was 0.3 IU/ml to 0.5 IU/ml. RESULTS: The primary outcome was anti-factor Xa level. Demographics, operative time, hospital course, and 30-days post-operative VTE were compared by anti-factor Xa level group using univariate and multivariable analyses. Between January 2018 and 2019, 107 RC patients remained on pathway and were included in our analysis. Sixty-five (61%) were below target range for VTE prophylaxis.  A single VTE event (0.9%) occurred in a subprophylactic individual. The subprophylactic group had a significantly higher body mass index (P < 0.01) than those within target range. CONCLUSIONS: Higher body mass index was associated with subprophylactic enoxaparin dosing after RC. Nearly two-thirds of patients had below target anti-factor Xa levels. This suggests that dosing could be further individualized, but given the low incidence of VTE, implications of dose-adjusted prophylaxis on VTE prevention remain unknown.

UHPLC-MS/MS method for iohexol determination in human EDTA and lithium-heparin plasma, human urine and in goat- and pig EDTA plasma.

Aim: Iohexol plasma clearance is used as an indicator of kidney function in clinical and preclinical settings. To investigate the pharmacokinetic profile of iohexol, a rapid, simple method for measurement of iohexol in different matrices and species was needed. Materials & methods: Iohexol was separated on an Accucore C18 column (Thermo Fisher Scientific, CA, USA). Detection was performed on a Thermo Scientific Quantiva tandem quadrupole mass spectrometer. The method was validated according to the requirements for bioanalytical methods issued by the US FDA and European Medicines Agency. Conclusion: We developed and validated a fast and efficient analytical method, suitable for analyzing iohexol in human EDTA plasma, human lithium-heparin plasma, human urine and goat- and pig EDTA plasma, using only one calibration line prepared in human EDTA plasma.

High affinity heparin detection by multivalent supramolecular polymers through aggregation induced emission.

Amphiphilic di-cationic cyanostilbene derivatives with aggregation induced emission (AIE) features formed luminescent supramolecular polymers in aqueous media. They bind bio-polyanion heparin, a known anticoagulant, in a multivalent fashion through the formation of co-assemblies with highly enhanced greenish-yellow emission. Heparin detection in nanomolar concentrations in buffer and in medically relevant concentrations in human serum and plasma was achieved.

Micellization-induced amplified fluorescence response for highly sensitive detection of heparin in serum.

Fluorescence-based assays should be feasible in aqueous media for effectively detecting the biological factors. However, numerous sensors have limited signal transductions and low fluorescence quantum yields due to the ingerently reduced excited state energy of fluorophores in aqueous solution, which reduces their sensitivity. This necessitates a smart sensing approach with an amplified fluorescence response for analytes in aqueous solution. Herein, a new building block which self-assembles in aqueous media, giving a micellar sturcuture with the hydrophobic π-extended conjugated system at the core and hydrophilic groups at the periphery, was devised for the first time. We demonstrated that the aggregated fluorophores in a micelle induce amplified fluorescence quenching, in which the excited electron efficiently migrates through π-extended conjugated system in a micelle, as in a polymeric system. Such feature differentiates this sensing approach from the numerous fluorescence-based tools previously developed for sensitive detection. This new system exhibited highly sensitive signal transduction for specific analytes even under actual bioanalytical conditions.

Highly Sensitive and Selective Detection of Heparin in Serum Based on a Long-Wavelength Tetraphenylethylene-Cyanopyridine Aggregation-Induced Emission Luminogen.

A positively charged aggregation-induced emission luminogen (AIEgen), TPE-P+, was constructed by linking a pyridyl cation to tetraphenylethylene (TPE) via a cyanoethylene bond. TPE-P+ can realize the identification of heparin (Hep) by aggregating with negatively charged Hep via electrostatic interactions. Upon addition of Hep, TPE-P+ exhibited 36-fold fluorescence enhancement in less than 5 s, exhibiting quick and sensitive response to Hep with a low detection limit down to 4 nM. Among various biological substances, even Hep analogs like chondroitin 4-sulfate and hyaluronic acid, TPE-P+ showed the most significant fluorescence enhancement to Hep only, demonstrating its excellent selectivity for Hep. In particular, with long-wavelength emission near 600 nm and large stocks shift (∼160 nm), TPE-P+ enabled minimization of autofluorescence interference from a complex biological matrix and provided more accurate results. Finally, TPE-P+ was successfully applied for sensitive and selective detection of Hep in serum. Notably, there existed a good linear relationship in a serum assay when the Hep concentration ranging from 0 to 4 µM (R2 = 0.9934) covered the clinical dosage level during both cardiovascular surgery and long-term care, suggesting the potential clinical practice for quantifying Hep in serum. Moreover, TPE-P+-Hep complex can be further disaggregated by protamine (PRTM) due to the stronger affinity between Hep and PRTM, thereby leading to further detection of PRTM effectively. Last, but not least, the "off-on-off" system designed for both Hep and PRTM detection proved to be reversible.

One-pot facile synthesis of CuNCs/RGO nanocomposite for the sensitive detection of heparin in human serum samples.

A simple and facile one-pot approach for the synthesis of copper nanoclusters decorated reduced graphene oxide (CuNCs/RGO) nanocomposite was proposed, in which the CuNCs attached to the surface of the reduced glutathione (GSH) functionalized RGO through ligand exchange via their thiol functionalities. The synthesized nanocomposite was verified by structural characterizations, and the further investigation of density functional theory (DFT) indicated that Cu3R2 cluster (R = C10H16O6N3S) with the lowest energy was the most stable structure in GSH-capped CuNCs. Although the CuNCs/RGO nanocomposite exhibited rather weak fluorescence, with the addition of heparin (Hep), the significant enhancement of fluorescence at 595 nm was achieved, which was developed to detect Hep in human serum samples with high selectivity and sensitivity. The mechanisms of fluorescence quenching of CuNCs/RGO nanocomposite and the sensing of Hep were discussed. The linear range was 0.1-10 µM with the detection limit of 26 nM in buffer solution containing 2% human serum sample, and satisfactory recovery in the range of 96.6%-104% was obtained, suggesting that the proposed method could applied to the detection of Hep in human serum samples.

Potential and Limitations of the New P2Y12 Inhibitor, Cangrelor, in Preventing Heparin-Induced Platelet Aggregation During Cardiac Surgery: An In Vitro Study.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) can put cardiac surgery patients at a high risk of lethal complications. If anti-PF4/heparin antibodies (anti-PF4/Hep Abs) are present, 2 strategies exist to prevent intraoperative aggregation during bypass surgery: first, using an alternative anticoagulant, and second, using heparin combined with an antiaggregant. The new P2Y12 inhibitor, cangrelor, could be an attractive candidate for the latter strategy; several authors have reported its successful use. The present in vitro study evaluated cangrelor's ability to inhibit heparin-induced platelet aggregation in the presence of anti-PF4/Hep Abs. METHODS: Platelet-poor plasma (PPP) from 30 patients with functional anti-PF4/Hep Abs was mixed with platelet-rich plasma (PRP) from 5 healthy donors.Light transmission aggregometry was used to measure platelet aggregation after adding 0.5 IU·mL of heparin (HIT) to the plasma, and this was compared with samples spiked with normal saline (control) and samples spiked with cangrelor 500 ng·mL and heparin 0.5 IU·mL (treatment). Friedman test with post hoc Dunn-Bonferroni test was used for between-group comparisons. RESULTS: Heparin 0.5 IU·mL triggered aggregation in 22 of 44 PPP-PRP mixtures, with a median aggregation of 86% (interquartile range [IQR], 69-91). The median aggregation of these 22 positive samples' respective control tests was 22% (IQR, 16-30) (P < .001). Median aggregation in the cangrelor-treated samples was 29% (IQR, 19-54) and significantly lower than the HIT samples (P < .001). Cangrelor inhibited heparin-induced aggregation by a median of 91% (IQR, 52-100). Cangrelor only reduced heparin-induced aggregation by >95% in 10 of the 22 positive samples (45%). Cangrelor inhibited heparin-induced aggregation by <50% in 5 of the 22 positive samples (22%) and by <10% in 3 samples (14%). CONCLUSIONS: This in vitro study found that cangrelor was an unreliable inhibitor of heparin-induced aggregation in the presence of anti-PF4/Hep Abs. We conclude that cangrelor should not be used as a standard antiaggregant for cardiac patients affected by HIT during surgery. Unless cangrelor's efficacy in a particular patient has been confirmed in a presurgery aggregation test, other strategies should be chosen.

Sperm Micromotors for Cargo Delivery through Flowing Blood.

Micromotors are recognized as promising candidates for untethered micromanipulation and targeted cargo delivery in complex biological environments. However, their feasibility in the circulatory system has been limited due to the low thrust force exhibited by many of the reported synthetic micromotors, which is not sufficient to overcome the high flow and complex composition of blood. Here we present a hybrid sperm micromotor that can actively swim against flowing blood (continuous and pulsatile) and perform the function of heparin cargo delivery. In this biohybrid system, the sperm flagellum provides a high propulsion force while the synthetic microstructure serves for magnetic guidance and cargo transport. Moreover, single sperm micromotors can assemble into a train-like carrier after magnetization, allowing the transport of multiple sperm or medical cargoes to the area of interest, serving as potential anticoagulant agents to treat blood clots or other diseases in the circulatory system.