Factors Affecting the Discrepancy Between Coagulation Times on Extracorporeal Circulation Using Unfractionated Heparin in Children and Young Adults.

In unfractionated heparin (UFH) monitoring during extracorporeal circulation, the traditional measures of activated clotting time (ACT) or activated partial thromboplastin time (APTT) may diverge, confounding anticoagulant adjustments. We aimed to explore the factors explaining this discrepancy in children and young adults. This retrospective observational study, conducted at an urban regional tertiary hospital, included consecutive pediatric patients who received UFH during extracorporeal circulation (continuous kidney replacement therapy or extracorporeal membrane oxygenation) between April 2017 and March 2021. After patients whose ACT and APTT were not measured simultaneously or who were also taking other anticoagulants were excluded, we analyzed 94 samples from 23 patients. To explain the discrepancy between ACT and APTT, regression equations were created using a generalized linear model (family = gamma, link = logarithmic) with ACT as the response variable. Other explanatory variables included age, platelet count, and antithrombin. Compared to APTT alone as an explanatory variable, the Akaike information criterion and pseudo-coefficient of determination improved from 855 to 625 and from 0.01 to 0.42, respectively, when these explanatory variables were used. In conclusion, we identified several factors that may explain some of the discrepancy between ACT and APTT in the routinely measured tests. Evaluation of these factors may aid in appropriate adjustments in anticoagulation therapy.

Early unfractionated heparin treatment in patients with STEMI - trial design and rationale.

The early unfractionated heparin (UFH) treatment in patients with ST-elevation myocardial infarction (STEMI) is a single-center, open-label, randomized controlled trial. The study population are patients with STEMI that undergo primary percutaneous coronary intervention (PPCI). The trial was designed to investigate whether early administration of unfractionated heparin immediately after diagnosis of STEMI is beneficial in terms of patency of infarct-related coronary artery (IRA) when compared to established UFH administration at the time of coronary intervention. The patients will be randomized in 1:1 fashion in one of the two groups. The primary efficacy endpoint of the study is Thrombolysis in myocardial infarction (TIMI) flow grades 2 and 3 on diagnostic coronary angiography. Secondary outcome measures are: TIMI flow after PPCI, progression to cardiogenic shock, 30-day mortality, ST-segment resolution, highest Troponin I and Troponin I values at 24 hours. The safety outcome is bleeding complications. The study of early heparin administration in patients with STEMI will address whether pretreatment with UFH can increase the rate of spontaneous reperfusion of infarct-related coronary artery.

Differential Solvent DEEP-STD NMR and MD Simulations Enable the Determinants of the Molecular Recognition of Heparin Oligosaccharides by Antithrombin to Be Disentangled.

The interaction of heparin with antithrombin (AT) involves a specific sequence corresponding to the pentasaccharide GlcNAc/NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S (AGA*IA). Recent studies have revealed that two AGA*IA-containing hexasaccharides, which differ in the sulfation degree of the iduronic acid unit, exhibit similar binding to AT, albeit with different affinities. However, the lack of experimental data concerning the molecular contacts between these ligands and the amino acids within the protein-binding site prevents a detailed description of the complexes. Differential epitope mapping (DEEP)-STD NMR, in combination with MD simulations, enables the experimental observation and comparison of two heparin pentasaccharides interacting with AT, revealing slightly different bound orientations and distinct affinities of both glycans for AT. We demonstrate the effectiveness of the differential solvent DEEP-STD NMR approach in determining the presence of polar residues in the recognition sites of glycosaminoglycan-binding proteins.

How We Interpret Thrombosis with Thrombocytopenia Syndrome?

Platelets play an important role in hemostasis, and a low platelet count usually increases the risk of bleeding. Conditions in which thrombosis occurs despite low platelet counts are referred to as thrombosis with thrombocytopenia syndrome, including heparin-induced thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia, paroxysmal nocturnal hemoglobinuria, antiphospholipid syndrome, thrombotic microangiopathy (TMA), and disseminated intravascular coagulation. TMA includes thrombotic thrombocytopenic purpura, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (HUS), and atypical HUS. Patients with these pathologies present with thrombosis and consumptive thrombocytopenia associated with the activation of platelets and the coagulation system. Treatment varies from disease to disease, and many diseases have direct impacts on mortality and organ prognosis if therapeutic interventions are not promptly implemented. Underlying diseases and the results of physical examinations and general laboratory tests as part of a thorough workup for patients should promptly lead to therapeutic intervention before definitive diagnosis. For some diseases, the diagnosis and initial treatment must proceed in parallel. Utilization of not only laboratory tests but also various scoring systems is important for validating therapeutic interventions based on clinical information.

Improving Compliance with a Nurse-Driven Protocol for Unfractionated Heparin Infusions in Patients with Venous Thromboembolism.

BACKGROUND: Unfractionated heparin (UFH) is a high-risk medication that can cause bleeding and/or thrombotic complications if not managed appropriately. Between January and July 2019, our institution experienced a high number of patient safety events related to UFH infusion for the treatment of venous thromboembolism (VTE). PURPOSE: The aim of this quality improvement (QI) initiative was to prevent these safety events by improving compliance with our institution's nurse-driven VTE UFH infusion protocol. METHODS: Baseline data for patients on the VTE UFH protocol were collected to identify improvement opportunities. Compliance with eight standards of care related to the VTE UFH infusion protocol was measured. Time to first therapeutic activated partial thromboplastin time (aPTT) was recorded to assess the benefit of improved compliance. INTERVENTIONS: Institutional policy updates were made to clarify the management of UFH infusions and documentation in the electronic health record. A multidisciplinary workgroup implemented order set changes, nursing communication orders, UFH infusion reports, and a nursing education module to promote compliance with the protocol. RESULTS: The overall rate of compliance with the VTE UFH infusion protocol increased from 79.4% at baseline to 85.2% following implementation of the QI initiative, and the median time to first therapeutic aPTT decreased from 831.5 minutes to 808 minutes over the same period. CONCLUSIONS: A multidisciplinary initiative to address improvement opportunities in a nurse-driven UFH protocol for VTE treatment increased compliance with the protocol and decreased the time to first therapeutic aPTT.

Comparing FGFR-3 and TS-HDS Seropositive Small Fiber Neuropathy: Unique Patient Features, Symptoms, Laboratory, and Nerve Conduction Study Findings.

OBJECTIVES: Small fiber neuropathy presents a significant diagnostic and therapeutic challenge. To solve this challenge, efforts have been made to identify autoantibodies associated with this condition. Previous literature has often considered tri-sulfated heparin disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR3) as a singular seropositive group and/or focused primarily on symptomatic associations. METHODS: One hundred seventy-two small fiber neuropathy patients with a Washington University Sensory Neuropathy panel were selected for TS-HDS seropositivity, FGFR-3 seropositivity, and seronegative controls. Data were collected to on the demographic, symptomatic, and laboratory profiles of each subgroup. RESULTS: Percent female (P = 0.0043), frequency of neuropathic pain symptoms (P = 0.0074), and erythrocyte sedimentation rate (P = 0.0293), vitamin D (P < 0.0001), and vitamin B12 (P = 0.0033) differed between the groups. Skin biopsy was more frequently normal within both the FGFR-3 and the TS-HDS cohort (P = 0.0253). CONCLUSIONS: TS-HDS and FGFR-3 display a distinct phenotype from both controls and one another. Immunoglobulin M (IgM) against FGFR-3 and IgM against TS-HDS may be individually valuable markers for the development of distinct clinical phenotypes.

Effectiveness of IVIG on Non-Length-Dependent Skin Biopsies in Small Fiber Neuropathy With Plexin D1, Trisulfated Heparin Disaccharide, and Fibroblast Growth Factor Receptor 3 Autoantibodies.

OBJECTIVES: To demonstrate treatment efficacy on composite and non-length-dependent (NLD) punch biopsy specimens from intravenous immunoglobulin (IVIG) in pure small-fiber neuropathy (SFN) with trisulfated heparin disaccharide (TS-HDS), fibroblast growth factor-3 (FGFR-3), or Plexin D1 antibodies. SFN has an increasing prevalence, and over 30% of cases may be immune-mediated. TS-HDS, FGFR-3, and Plexin D1 autoantibodies have been shown to be present in 44%-55% of cryptogenic SFN cases, suggesting an immune mechanism. Reports have shown IVIG to be effective for this condition, but some controversy exists based on length-dependent (LD) post-IVIG treatment data in a recent trial. METHODS: In a retrospective review, all pure SFN cases tested for the 3 antibodies from January 2021 to May 2022 were tabulated, and patients who underwent IVIG treatment were separated and analyzed for changes in epidermal nerve fiber density (ENFD) on skin biopsy, as well as SFN-specific questionnaire and pain scores. RESULTS: Ninety-one patients with pure SFN had antibody testing. Sixty of these (66%) were seropositive, and 31 (34%) were seronegative. Seventeen seropositive patients (13 female patients, 4 male patients, 6 FGFR-3, 2 TS-HDS, 4 Plexin D1, 2 with all 3 antibodies, 1 with FGFR-3 and Plexin D1, 1 with FGFR-3 and TS-HDS, and 1 with TS-HDS and Plexin D1) underwent IVIG treatment. Of these, 2 patients stopped treatment due to side effects, and the remaining 15 completed at least 6 months of IVIG. Of these, 12 had a post-IVIG skin biopsy, and of these, 11 (92%) had a 55.1% improved mean composite ENFD (P = 0.01). NLD-ENFD specimens improved by 42.3% (P = 0.02), and LD-ENFD specimens improved by 99.7% (P = 0.01). Composite ENFD in Plexin D1-SFN patients improved by 139% (P = 0.04). In addition, 14 patients had questionnaires pre-IVIG/post-IVIG, and average pain decreased by 2.7 (P = 0.002). CONCLUSIONS: IVIG shows disease-modifying effect in immune SFN with novel antibodies, especially Plexin D1-SFN, as well as significantly improved pain. NLD-ENFD should be examined as well as LD-ENFD to see this effect. Further randomized controlled trials looking at NLD-ENFD as well as LD-ENFD improvement, along with pain and SFN-specific questionnaires, are needed to confirm these findings.

An evaluation of a Stenotrophomonas maltophilia outbreak due to commercial arterial blood gas collection kit.

BACKGROUND: Stenotrophomonas maltophilia is a gram-negative bacterium that can cause hospital infections and outbreaks within hospitals. This study aimed to evaluate an outbreak of Stenotrophomonas maltophilia, caused by ready-to-use commercial syringes containing liquid lithium and heparin for arterial blood gas collection in a university hospital. METHODS: Upon detecting an increase in Stenotrophomonas maltophilia growth in blood cultures between 15.09.2021 and 19.11.2021, an outbreak analysis and a case-control study (52 patients for the case group, 56 patients for the control group) were performed considering risk factors for bacteremia. Samples from possible foci for bacteremia were also cultured. Growing bacteria were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The genetic linkage and clonal relationship isolates were investigated with pulsed-field gel electrophoresis (PFGE) in the reference laboratory. RESULTS: In the case-control study, the odds ratio for the central venous catheter [3.38 (95% confidence interval [CI]: 1.444, 8.705 ; p = 0.006)], for surgery [3.387 (95% confidence interval [CI]: 1.370, 8.373 ; p = 0.008)] and for arterial blood gas collection history [18.584 (95% confidence interval [CI]:4.086, 84.197; p < 0.001)] were identified as significant risk factors. Stenotrophomonas maltophilia growth was found in ready-to-use commercial syringes used for arterial blood gas collection. Molecular analysis showed that the growths in the samples taken from commercial syringes and the growths from blood cultures were the same. It was decided that the epidemic occurred because the method for sterilization of heparinized liquid preparations were not suitable. After discontinuing the use of the kits with this lot number, the outbreak was brought under control. CONCLUSIONS: According to our results, disposable or sterile medical equipment should be included as a risk factor in outbreak analyses. The method by which injectors containing liquids, such as heparin, are sterilized should be reviewed. Our study also revealed the importance of the cooperation of the infection control team with the microbiology laboratory.

Marine-Derived Sulfated Glycans Inhibit the Interaction of Heparin with Adhesion Proteins of Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a notable pathogen behind respiratory infections, employs specialized proteins to adhere to the respiratory epithelium, an essential process for initiating infection. The role of glycosaminoglycans, especially heparan sulfate, is critical in facilitating pathogen-host interactions, presenting a strategic target for therapeutic intervention. In this study, we assembled a glycan library comprising heparin, its oligosaccharide derivatives, and a variety of marine-derived sulfated glycans to screen the potential inhibitors for the pathogen-host interactions. By using Surface Plasmon Resonance spectroscopy, we evaluated the library's efficacy in inhibiting the interaction between M. pneumoniae adhesion proteins and heparin. Our findings offer a promising avenue for developing novel therapeutic strategies against M. pneumoniae infections.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Differential Use of Glycoprotein IIb/IIIa Inhibitors with Bivalirudin in Patients with STEMI Undergoing PCI: A Systematic Review and Meta-Analysis.

AIM: The efficacy and safety of bivalirudin when used concurrently with glycoprotein IIb/IIIa inhibitors (GPI) is uncertain. In this systematic review and meta-analysis, we aimed to evaluate the efficacy and safety of bivalirudin versus heparin in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI) and to explore the impact of differential use (greater and balanced) of GPI. METHODS: Online databases were queried from inception to March 2023 to identify eight randomized controlled trials (n = 22,483) for inclusion. The primary outcomes included all-cause mortality, major bleeding, major adverse cardiovascular events (MACE), and net adverse clinical events (NACE). Secondary efficacy endpoints included cardiac death, reinfarction, stent thrombosis (ST), and stroke. Data were pooled using a random-effects model to derive risk ratios (RRs) and 95% confidence intervals (CIs). RESULTS: When compared to heparin, bivalirudin was associated with a significant reduction in all-cause mortality (RR 0.83; 95% CI 0.72-0.97; P = 0.02), major bleeding (RR 0.73; 95% CI 0.57-0.93; P = 0.01), cardiac death (RR 0.79; 95% CI 0.66-0.94; P = 0.01), and NACE (RR 0.80; 95% CI 0.72-0.89; P < 0.0001). However, while the bivalirudin arm showed an increased likelihood of ST in the greater GPI subgroup (RR 1.70; 95% CI 1.13-2.56; P = 0.01), it was associated with a decreased likelihood of ST in the balanced GPI subgroup (RR 0.40; 95% CI 0.24-0.65; P = 0.0003). CONCLUSION: Overall, our findings suggest that bivalirudin may be a more efficacious intervention than heparin for reducing certain adverse events in patients with STEMI undergoing primary PCI.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.

Bivalirudin as a substitute for heparin in neurointervention for patients with heparin-induced thrombocytopenia.

OBJECTIVES: Heparin-induced thrombocytopenia is a known complication of heparin exposure with potentially life-threatening sequelae. Direct thrombin inhibitors can be substituted for heparin in patients with heparin-induced thrombocytopenia that require anticoagulation. However, the use of direct thrombin inhibitors as a substitute for heparin has not been widely reported in the neuroendovascular literature. MATERIALS AND METHODS: Here we report the first use of the direct thrombin inhibitor bivalirudin in a neuroendovascular procedure as a substitute for heparin in a patient with a ruptured pseudoaneurysm and heparin-induced thrombocytopenia, and review the literature on the use of bivalirudin and argatroban for such patients. RESULTS: Bivalirudin was safely and effectively used in the case reported, with no thrombotic or hemorrhagic complications. Our literature review revealed a paucity of studies on the use of heparin alternatives, including bivalirudin, in neuroendovascular procedures in patients with heparin-induced thrombocytopenia. CONCLUSIONS: Heparin-induced thrombocytopenia is an important iatrogenic disease process in patients undergoing neuroendovascular procedures, and developing protocols to diagnose and manage heparin-induced thrombocytopenia is important for healthcare systems. While further research needs to be done to establish the full range of anticoagulation options to substitute for heparin, our case indicates bivalirudin as a potential candidate.

Exploring single-molecule interactions: heparin and FGF-1 proteins through solid-state nanopores.

Detection and characterization of protein-protein interactions are essential for many cellular processes, such as cell growth, tissue repair, drug delivery, and other physiological functions. In our research, we have utilized emerging solid-state nanopore sensing technology, which is highly sensitive to better understand heparin and fibroblast growth factor 1 (FGF-1) protein interactions at a single-molecule level without any modifications. Understanding the structure and behavior of heparin-FGF-1 complexes at the single-molecule level is very important. An abnormality in their formation can lead to life-threatening conditions like tumor growth, fibrosis, and neurological disorders. Using a controlled dielectric breakdown pore fabrication approach, we have characterized individual heparin and FGF-1 (one of the 22 known FGFs in humans) proteins through the fabrication of 17 ± 1 nm nanopores. Compared to heparin, the positively charged heparin-binding domains of some FGF-1 proteins translocationally react with the pore walls, giving rise to a distinguishable second peak with higher current blockade. Additionally, we have confirmed that the dynamic FGF-1 is stabilized upon binding with heparin-FGF-1 at the single-molecule level. The larger current blockades from the complexes relative to individual heparin and the FGF-1 recorded during the translocation ensure the binding of heparin-FGF-1 proteins, forming binding complexes with higher excluded volumes. Taken together, we demonstrate that solid-state nanopores can be employed to investigate the properties of individual proteins and their complex interactions, potentially paving the way for innovative medical therapies and advancements.

Heparin-Modified Superparamagnetic Iron Oxide Nanoparticles Suppress Lithium Chloride/Pilocarpine-Induced Temporal Lobe Epilepsy in Rats through Attenuation of Inflammation and Oxidative Stress.

The development of antiepileptic drugs is still a long process. In this study, heparin-modified superparamagnetic iron oxide nanoparticles (UFH-SPIONs) were prepared, and their antiepileptic effect and underlying mechanism were investigated. UFH-SPIONs are stable, homogeneous nanosystems with antioxidant enzyme activity that are able to cross the blood-brain barrier (BBB) and enriched in hippocampal epileptogenic foci. The pretreatment with UFH-SPIONs effectively prolonged the onset of seizures and reduced seizure severity after lithium/pilocarpine (LP)-induced seizures in rats. The pretreatment with UFH-SPIONs significantly decreased the expression of inflammatory factors in hippocampal tissues, including IL-6, IL-1ß, and TNF-α. LP-induced oxidative stress in hippocampal tissues was in turn reduced upon pretreatment with UFH-SPIONs, as evidenced by an increase in the levels of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) and a decrease in the level of lipid peroxidation (MDA). Moreover, the LP-induced upregulation of apoptotic cells was decreased upon pretreatment with UFH-SPIONs. Together, these observations suggest that the pretreatment with UFH-SPIONs ameliorates LP-induced seizures and downregulates the inflammatory response and oxidative stress, which exerts neuronal protection during epilepsy.

d-type peptides based fluorescent probes for "turn on" sensing of heparin.

Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes.

Storage of plasma-derived exosomes: evaluation of anticoagulant use and preserving temperatures.

Exosomes carry large cargo of proteins, lipids, and nucleic acids, serving as versatile biomarkers for disease diagnosis and vehicles for drug delivery. However, up to date, no well recognized standard procedures for exosome storage were available for clinical application. This study aimed to determine the optimal storage conditions and the anticoagulants for plasma-derived exosome isolation. Fresh whole blood samples were collected from healthy participants and preserved in four different anticoagulants including sodium citrate (SC1/4), sodium citrate (SC1/9), lithium heparin (LH), or Ethylenediamine tetraacetic acid (EDTA), respectively. Exosomes were extracted from the plasma by differential ultracentrifugation and stored at three different temperatures, 4°C, -20°C or - 80°C for a duration ranging from one week to six months. All plasma samples for storage conditions comparison were pretreated with LH anticoagulant. Exosome features including morphological characteristics, pariticles size diameter, and surface protein profiles (TSG101, CD63, CD81, CD9, CALNEXIN) were assessed by transmission electron microscopy, Nanoparticle Tracking Analysis, and Western Blotting, respectively. Exosomes preserved in LH and SC1/4 group tended to remain intact microstructure with highly abundant protein biomarkers. Exosomes stored at 4°C for short time were prone to be more stable compared to thos at -80°C. Exosomes stored in plasma were superior in terms of ultrastructure, size diameter and surface protein expression to those stored in PBS. In conclusion, plasma-dervied exosome characteristics strictly depend on the anticoagulants and storage temperature and duration.

What is the context? Effective isolation of exosomes is a prerequisite for subsequent investigation into its involvemnt in disease development as well as potentialtherapeutic applications.Anticoagulants, storage temperature and durations might change the microscopical structure, integrity and also the stability of plasma-derived exosomes. However, no internationally recognized standard of exosome storage procedure was available for clinical use.What is new? Our finding evaluated the effect of anticoagulants and storage on plasma exosome characteristics.Exosomes isolated from plasma preserved with Li-heparin and sodium citrate (1/4) showed better physical properties and surface marker protein expression.Isolated exosomes appeared more stable in a short time for 4°C compared to −80°C. Storage of exosomes in plasma showed better physical properties and surface marker protein expression than in PBS.What is the impact? Our findings inform the significance of standardizing procedure of exosome isolation and preservation.