Overland Park Research Articles

Decreased Thrombin Generation Potential in Lymphoma Patients Is Associated with Increased D-Dimer, CRP, Vwf and TNF-α. Interrelationship between Thrombogenesis and Inflammation

Introduction: The prevalence of thrombosis in lymphoma patients is reportedly high and ranges from 3 - 10%, and further increased at advanced stages of the disease especially in hgNHL. Inflammation and other vascular factors contribute to the pathogenesis of these thrombotic complications. Biomarkers of hemostatic activation, vascular dysfunction and inflammation are elevated in lymphoma. We have previously reported that thrombin generation biomarkers such as D-Dimer, thrombin anti-thrombin complex (TAT) and prothrombin fragment (F1.2) along with inflammatory biomarkers are increased in cancer patients. Interestingly, despite an increase in thrombin generation markers, thrombin generation potential in these patients is decreased. This study was designed to compare the thrombin generation potential and its relevance to the generation of various biomarkers of hemostatic activation process and inflammatory responses. Methods: Citrated blood samples from 90 patients with confirmed diagnosis of non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL) and Chronic lymphocytic leukemia/Small lymphocytic lymphoma (CLL/SLL) were collected from the Clinic of Hematology Unit, University of Belgrade, Belgrade, Serbia. 50 samples of normal human plasma (NHP) was obtained from George King Biomedical (Overland park, KS). NHP was prepared for referencing purposes. Thrombin generation studies were carried out using a commercially available a kinetic fluorogenic substrate method (calibrated automated thrombogram; CAT). Thrombin generation parameters such as peak thrombin (PT), lag time (LT) and area under the curve (AUC) were compiled. Plasma levels of D-Dimer, CRP, vWF and TNF- α were measured by using commercially available Sandwich ELISA assays. Results were compiled in terms of mean ± SD. Correlation analysis carried out by using Prism, Graphpad. Results: On a cumulative basis, lymphoma patients showed an increase in lag time (2.9 1.17) in comparison to the NHP (2.7). The peak thrombin levels was decreased (119.0 46.2) in the lymphoma patients in comparison to NHP (159.7 31.1). The AUC was decreased (670.2 290.01) in the lymphoma patients in comparison to NHP (700.9 ). When the lymphoma patients were sub grouped, peak thrombin levels were similar in HL (129.9), NHL (114.7) and CLL (127.4) and were decreased compared to NHP (159.7). However the AUC was increased in the HL (769), decreased in NHL (642.4) and was comparable for CLL (715.4) compared to NHP (700.9). Variations in lag time were noted in the three groups. The lymphoma patients also exhibited elevated levels of D-Dimer (878 1205 ng/ml) in comparison to NHP (280 ng/ml), CRP (15.2 26.6 ug/ml) in comparison to NHP (0.9 ug/ml), vWF (271.8 189.7 %) in comparison to NHP (122%) and TNF-α (16.4 20.64 pg/ml) in comparison to NHP (4.2 pg/ml). Interestingly, when the lymphoma group were subdivided into various groups, the D-Dimer level was highest in HL following the order HL>NHL>CLL. CRP and vWF levels were highest in HL and comparable in both NHL and CLL. Wide scatter in TNF-α data was noted with CLL exhibiting highest levels with the ranked order CLL>NHL>HL. The composite data on thrombin generation parameters and biomarkers is shown on the table 1A. In the HL group D-Dimer and CRP correlated well with AUC and PT, in this group vWF also showed a good correlation with PT. In the NHL group, CRP correlated well with LT. In the CLL group none of the biomarkers correlated well with the thrombin generation parameters. On a cumulative basis, D-Dimer, CRP, vWF and TNF-α had no significant relevance to thrombin generation parameters. The composite regression coefficience data is shown on table 1B. Conclusion: Lymphoma patients represent a heterogenous group in which both the hypercoagulable state and inflammatory responses simultaneously occur as evident by increase in D-Dimer and inflammatory biomarkers. While the overall thrombin generation potential in the plasma of these patients is decreased, thrombin is constantly generated in some of these patient leading to the formation of fibrin. The observed decrease in thrombin generation potential in these patients may be due to the consumption of prothrombin and other coagulation factors. Wide variations among these lymphoma groups are noted. Profiling of thrombin generation and inflammatory biomarkers may be helpful in thrombotic risk stratification of lymphoma patients and their anti-thrombotic management. Disclosures No relevant conflicts of interest to declare.

Biomarkers of Hemostatic Activation and Inflammation Are Associated with Altered Coagulation Parameters in Sepsis Patients

Background : Sepsis is characterized by a simultaneous activation of inflammation and hemostasis in response to microbial infection. This systemic inflammatory response is due to the release of pro-inflammatory cytokines, pro-coagulants and adhesion molecules from immune cells and/or damaged endothelial tissue. Simultaneous activation of coagulation and fibrinolysis leads to consumption coagulopathy and severe vascular dysfunction. Profiling of biomarkers of hemostatic activation and inflammation along with the measurement of coagulation parameters has provided useful data in the understanding of the pathogenesis of sepsis. This study was designed to profile biomarkers of hemostatic activation, inflammation and endothelial dysfunction along with the measurement of coagulation parameters in a defined clinically confirmed sepsis population in conjunction with an IRB approved clinical trial. Materials & Methods: Citrated blood samples were collected from sepsis patients with suspected or confirmed infection, and organ dysfunction as defined by a SOFA ³ baseline. Plasma samples from septic shock patients were collected in citrated tubes within 72 hours of ICU admission under an IRB approved protocol in conjunction with an ongoing trial at the University of Utah and Veteran's Affair FFC Health Care System VAMC. Normal controls were comprised of commercially available 25 male and 25 female citrated plasma samples (George King Biomedical, Overland Park, Kansas City). Such biomarkers as CRP, PAI-1, D-Dimer, vWF and microparticle tissue factor complex (MP-TF) were measured using a commercially available sandwich ELISA methods. Nitric oxide (NO) levels were measured using a commercially available Griess reaction based colorimetric method. PT/INR, aPTT and fibrinogen measurements were based on clot based assays. All results were compiled as mean ± SD and SEM. Correlation analysis was carried out to determine relevance between different parameters. Results: Most of the biomarkers of hemostatic activation and inflammation were elevated in patients with sepsis as shown on table 1a, CRP (66 fold) and D-Dimer (23 fold) showed the most pronounced increase in comparison to the control. Other parameters also showed increase levels including MP-TF (5.3 fold), PAI-1 (3.5 fold), vWF (3.1 fold) and NO (3.0 fold). Clotting parameters such as PT/INR (2.0 fold), aPTT (2.5 fold) and fibrinogen (2.0 fold) were also significantly elevated in the sepsis patients. These differences were significant (p value ≤0.0009) for all of the parameters except for NO (p value 0.0937) and fibrinogen (p value 0.4694). As shown on table 1b, there was no correlation between various biomarkers and fibrinogen in the sepsis patients. Summary & Conclusion: In comparison to the control group, the sepsis patients showed wide variations in all of the parameters investigated in this study. The marked prolongation of PT and aPTT are suggestive of both the extrinsic and intrinsic pathway defects and consumption of clotting factors. The aPTT data showed wider scatter in comparison to PT data. The fibrinogen levels were also elevated and nearly 1/3 of the patients showed >1000 mg/dL levels. The markedly higher level of CRP in the sepsis group are indicative of severe inflammatory response. Marked elevation of D-Dimer is indicative of endogenous fibrin formation and its consumption consistent with activation of secondary fibrinolysis. MP-TF, vWF and PAI-1 were also increased in the sepsis patients suggesting marked endothelial dysfunction. This is consistent with increased NO levels which may be due to induction of iNOS in the endothelial lining of sepsis patients. These results further underscore the multifactorial pathophysiology of sepsis which results in the dysregulation of hemostasis, upregulation of inflammatory responses and generalized endothelialopathy. Profiling of the biomarkers included in this study and coagulation parameters may be helpful in the risk stratification and clinical management of patients with sepsis and related disorders. Disclosures No relevant conflicts of interest to declare.

Prothrombinase Induced Clotting Time Is More Sensitive then aPTT and PT and Can be Used for the Monitoring of Anti-Xa Agents in Whole Blood and Plasma

Introduction: Currently there are four commercially available oral anti-Xa agents namely apixaban, betrixaban, edoxaban and rivaroxaban available for indication specific clinical use. All agents used are at a fixed dosage and their use may be associated with potential hemorrhagic complications. Although factor Xa inhibitory effect is considered to be a surrogate marker for the biologic action of these drugs we have demonstrated that factor Xa inhibitory profile of apixaban, betrixaban, edoxaban, and rivaroxaban does not fully reflect their biologic spectrum (https://doi.org/10.1177/1076029619847524). Furthermore the prothrombin time (PT) and activated partial thromboplatin time (aPTT) methods are of limited sensitivity and are dependant on several other enodogenous factors. Prothrombinase induced clotting time (PiCT), (Pentaharm, Basel, Switzerland) is a sensitive test for the global monitoring of anticoagulant drugs including heparins and parenteral oral anti-Xa and anti-IIa agents. The test is based on the RVV venom activation of endogenous FVa which forms prothrombinase complex with phospholipids (PL), FXa and calcium. The clot-based end point is proportional to the activities of FXa and FIIa. This study is designed to compare the relative responses of PT, aPTT and PiCT test in normal human blood, retrieved plasma and agents supplemented plasma with the oral anti-Xa agents to demonstrate the relative sensitivity of this assay in comparison to the PT and aPTT. Materials and Methods: Active pharmaceutical ingredient versions of apixaban, betrixaban, edoxaban and rivaroxaban were obtained from commercial sources. All agents were prepared at a stock concentration of 100ug/ml in saline and diluted to a working solution of 10ug/ml. Serial dilutions were prepared in various matrices. Citrated plasma from normal individuals (n=50) was obtained from a commercial source (George King Biomedical, Overland Park, Kansas). Whole blood and plasma samples (n=20) were supplemented at a concentration of 0-1000 ng/ml. Whole blood samples were also centrifuged to obtain retrieved plasma. These samples were analyzed using a single stage PiCT, aPTT and PT. Whole blood and retrieved plasma studies were carried out by using ST4 (Diagnostica Stago). PiCT were measured by using Pefakit PiCT. aPTT measurements were made by using Tcoag, TriniCLOT aPTT (Diagnostica Stago, Paris, France). For PT, HemosIL TM (Instrumentation Laboratory, MA, USA) was used. All results were compiled in individual groups as mean + SD. Results: All of these drugs produced a concentration dependent anticoagulant effects in the PiCT, aPTT and PT tests in both the whole blood and plasma systems. PiCT consistently showed much higher sensitivity in comparison to other tests. When compared at 1000ng/ml the anticoagulant effects of these drugs were stronger in plasma systems as shown in the table. PiCT consistently showed higher sensitivity in comparison to PT and aPTT in the whole blood, retrieved plasma and agent supplemented plasma. Edoxaban showed the strongest anticoagulant activity measured by PiCT in comparison to the other agents. Matrix based variations in the PiCT results were observed. Interestingly, the retrieved plasma from whole blood showed weaker anticoagulant effects in comparison to the directly supplemented plasma systems. Discussion: In comparison to PT and aPTT, the PiCT test was found to be the most sensitive in the three matrices studied. In the whole blood and plasma-based systems PiCT test showed linearity and high sensitivity for all of the four anti-Xa agents. In the PiCT test, consistently drug supplemented plasma showed the highest response in comparison to retrieved plasma and whole blood, suggesting differential binding of these drugs to cells. These results indicate that the PiCT test can be reliably used for the monitoring of anti-Xa agents. PiCT test can also be performed on currently available optical and mechanical instrument used for clotting studies. Owing to rapid turnaround time, high sensitivity, and lower cost PiCT can be used for the routine monitoring of oral anti-Xa agents. Table Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

Abstracts from the 151st Annual Meeting of the Kansas Academy of Science and the 95th Annual Meeting of the Kansas Entomological Society, Johnson County Community College, Overland Park, Kansas, March 29-30, 2019

Abstracts from the 151st Annual Meeting of the Kansas Academy of Science and the 95th Annual Meeting of the Kansas Entomological Society, Johnson County Community College, Overland Park, Kansas, March 29-30, 2019

Drug Diversion in the Anesthesia Profession: How Can Anesthesia Patient Safety Foundation Help Everyone Be Safe? Report of a Meeting Sponsored by the Anesthesia Patient Safety Foundation.

Diversion in the workplace can adversely impact the safety of healthcare professionals and patients. The Anesthesia Patient Safety Foundation (APSF) believes that substance use disorder, diversion in the workplace, and their potential adverse effects on patient safety need to be addressed through open discussion, education, research, policy, and possible other interventions. To make progress in this area, the APSF convened a conference entitled "Drug Diversion in the Anesthesia Profession: How Can APSF Help Everyone Be Safe?" in Phoenix, Arizona, on September 7, 2017 (Supplemental Digital Content, Document, http://links.lww.com/AA/C616). It was comoderated by the authors.

Decreased Thrombin Generation Potential Is Associated with Increased Thrombin Generation Markers in Sepsis Associated Coagulopathy

Introduction: Sepsis associated coagulopathy (SAC) is commonly seen in patients which eventually leads to dysfunctional hemostasis and disseminated intravascular coagulation (DIC). Thrombin generation plays an important role in the overall pathophysiology of this process. Previous studies have reported an increase in thrombin generation markers such as thrombin antithrombin complex (TAT) and prothrombin fragment (F1.2). (Hoppensteadt et al. Clin Appl Thromb Hemost. 2014 Mar;20(2):129-35). Sepsis eventually results in consumption coagulopathy in which some of the clotting factors are consumed. The purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sepsis associated coagulopathy patients and demonstrate their relevance to thrombin generation markers.Materials and Methods: Baseline citrated blood samples were prospectively collected from 49 patients with sepsis and suspected DIC. DIC scores were determined according to the ISTH scoring system (PTINR, fibrinogen, D-dimer and platelet count). Healthy control (n=50) represented citrated plasma obtained from a commercial supplier (George King Biomediacl, Overland Park, KS). Thrombin generation studies were carried out using a commercially available florigenic substrate methods with thrombin calibrator and PPP reagent (calibrated automated thrombogram; CAT). Such parameters as peak thrombin, lag time and area under the curve were compiled. TAT and F1.2 were measured using commercially available ELISA methods (Seimens, Indianpolis, IN). Functional antithrombin levels were measured using a chromogenic substrate method. All results were calculated in terms of mean ± SD. Applicable statistical methods were used to correlate the thrombin generation parameters with thrombin generation markers and antithrombin.Results: The peak thrombin levels were lower (82±40nM) in the SAC patients in comparison to higher levels observed in the normal plasma (133±10nM). The AUC was lower (561 ±280) in the SAC group in comparison to the normal (624±18). The SAC group showed much longer lag time (4.1±2.1) in comparison to the normal (2.1 ± 2.2). Wide variations in the results were observed in these parameters in the SAC group. The compsoite data is compiled in the table. the F1.2 levels in the DIC group were much higher (570±48 pmol) in comparison to the normal (210±25 pmol). The TAT levels also increased in the SAC group (27.9 ±5.1 ng/ml) in comparison to the normal (2.8 ±0.8 ng/ml). The functional antithrombin levels were decreased in the SAC group (64 ±11%). No correlation was observed between thrombin generation parameters and thrombin generation markers.Conclusion: These results validate the earlier observations that thrombin generation such as F1.2 and TAT are decreased in patients with SAC. However thrombin generation parameters are significantly elevated in this group in comparison to normals. This may be due to the consumption of prothrombin due to the activation of the coagulation system. The SAC group also showed wide variations in both the thrombin generation parameters and the F1.2 and TAT. These variations may be due to the differences in the endogenous pathophysiology state in the SAC patients. The decreased functional AT levels observed in the SAC group are due to the formation of the complex between generated thrombin and antithrombin. Simultaneous profiling of thrombin generation and thrombin generation markers may be helpful in the risk stratification of these patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Time series analysis of soybean response to varying atmospheric conditions for precision agriculture

Four time-lapse cameras, Bushnell Nature View HD Camera (Bushnell, Overland Park, KS, USA) were installed in a soybean field to track the response of soybean plants to changing weather conditions. The purpose was to confirm if visible spectroscopy can provide useful data for tracking the condition of crops and, if so, whether game and trail time-lapse cameras can serve as reliable crop sensing and monitoring devices. Using the installed cameras, images were taken at 30-min intervals between July 22 and August 1, 2015. Using the RGBExcel software application developed in-house, image data from the R (red), G (green), and B (blue) bands were exported to Microsoft Excel for further processing and analysis. Daytime adjusted green red index data for the plant, based on the R and G data, were plotted against time of image acquisition and also regressed with selected weather parameters. The former showed a rise-and-fall trend with daily peaks around 13:00, while the latter showed a decreasing order of correlation with weather variables as follows: log of solar radiation > log of soil surface temperature > log of air temperature > log of soil temperature at 50-mm depth > log of relative humidity. Despite some low correlations, the potential for using game and trail cameras with time-lapse capability to track changes in crop vegetation response under varying conditions is established. The resulting data can be used to develop models that can aid precision agriculture applications. This can be further explored in future studies.

US Emergency Medical Services Fellows.

This was a cross-sectional survey of EMS fellows in ACGME-accredited programs in conjunction with the first EMS In-Training Examination (EMSITE) between April and June 2016. Fellows completed a 14-question survey composed of multiple-choice and free-response questions. Basic frequency statistics were performed on their responses. Fifty fellows from 35 ACGME-accredited programs completed the survey. The response rate was 100%. Forty-eight (96%) fellows reported previous training in emergency medicine. Twenty (40%) were undergoing fellowship training at the same institution as their prior residency training. Twenty-five (50%) fellows performed direct patient care aboard a helicopter during their fellowship. Thirty-three (66%) fellows had a dedicated physician response vehicle for fellows. All fellows reported using the National Association of EMS Physicians (NAEMSP; Overland Park, Kansas USA) textbooks as their primary reference. Fellows felt most prepared for the Clinical Aspects questions and least prepared for Quality Management and Research questions on the board exam. These data provide insight into the characteristics of EMS fellows in ACGME-accredited programs. ClemencyB, Martin-GillC, RallN, PatelD, MyersJ. US Emergency Medical Services fellows. Prehosp Disaster Med. 2018;33(3):339-341.

Trespassing across America: One Man’s Epic, Never-Done-Before (and Sort of Illegal) Hike across the Heartland by Ken Ilgunas

Reviewed by: Trespassing across America: One Man’s Epic, Never-Done-Before (and Sort of Illegal) Hike across the Heartland by Ken Ilgunas Patrick Dobson Trespassing across America: One Man’s Epic, Never-Done-Before (and Sort of Illegal) Hike across the Heartland. By Ken Ilgunas. New York: Blue Rider Press, 2016. 264 pp. Map, illustrations, bibliography. $16.00 paper. In 1995 I walked from Kansas City to Helena, Montana, and canoed home on the Missouri River. Great Plains denizens, particularly those of modest or little means, proved to be the most generous and open, albeit hard-headed and sometimes backward-looking, people I’ve met in my lifetime. Ken Ilgunas now reports Great Plains dwellers’ character has changed little in the intervening years. Trespassing across America is, on the surface, the story of Ilgunas’s quest to follow the controversial Keystone XL pipeline through the heart of the Great Plains from the Alberta tar sands to the Valero refinery in Port Arthur, Texas. To pursue his journey, he hikes the pipeline route as closely as he can. Finding roads often far from the pipeline path, he hops fences, at first with reservation and then unabashedly. Meanwhile, locals open their homes to him and warn him that landowners let rifles do the talking. He finds, however, that landowners forgive more than they reprimand (or shoot) and almost everyone offers more than Ilgunas asks. While informative about the various aspects of the pipeline controversy, Ilgunas’s story is an exquisite travel memoir celebrating individual effort and Great Plains social diversity. It shows us the people of the Great Plains in all their wealth and poverty, pride and tatters. It walks the line between love of the people he meets and criticism of their (and our society’s) small-world views. Trespassing is as much celebration of Great Plains life as it is a cautionary tale. Ilgunas demonstrates acuity in several levels of narration. Relating the history and background of the XL pipeline and the intricacies of climate science, he contemplates the intersection between human and nature. He weaves technology and Great Plains homespun wisdom into the 1,915-mile trip. Detailing the individuals with whom he forms relationships, he also shows cultural differences between provinces and states. He also informs us enough of his own travails, successes, and humor to make him an apt and welcome travel partner. Ilgunas finds that for all their generosity, strength, and individual spirit, many Great Plains dwellers sacrifice “local” thinking for corporate-brewed nostrums, reactionary political talking points, and groupthink. He finds many reject climate science and sidestep social good for jobs propaganda, pointing out the pipeline promises to create only about 30 full-time positions along its route. “What happens to our country’s core will affect the rest of the body,” Ilgunas writes (79). He shows us the healthy and diseased aspects of Great Plains life, and hopes the negatives don’t constitute an incurable infection. Ilgunas begins and ends Trespassing an opponent of the pipeline. But this is no polemic. He enlightens us about the complications of the Keystone XL debate, the interests involved, and the pipeline’s implications for global warming. He holds little hope for a solution to the increasing greenhouse gas emissions the pipeline will enable. But he believes that in the long term, Americans, and perhaps the world, will figure their way out of a tangled and urgent conundrum—How do people addicted to oil maintain their habit and keep from smothering themselves? [End Page 209] Patrick Dobson Johnson County Community College Overland Park, Kansas Copyright © 2018 Center for Great Plains Studies, University of Nebraska–Lincoln

An Interview with Andrea Broomfield, the First Recipient of the VanArsdel Prize

An Interview with Andrea Broomfield, the First Recipient of the VanArsdel Prize Jordan Osterman (bio) Click for larger view View full resolution Photo credit: Bret Gustafson, 2015 [End Page 425] Andrea Broomfield, PhD, is a professor of English at Johnson County Community College in Overland Park, Kansas, and has been a longtime member of the Research Society for Victorian Periodicals. In 1990, she was the recipient of the first VanArsdel Essay Prize. A mentee of the late Sally Mitchell, Broomfield transitioned into her current focus on food writing and historicism more than fifteen years ago. Throughout that time, she has done extensive research and writing, especially on Victorian food and dining. She was the co-editor with Sally Mitchell of Prose by Victorian Women: An Anthology. She is also the author of Food and Cooking in Victorian England: A History and Kansas City: A Food Biography. Her publications in Victorian Periodicals Review have included "Interdisciplinary Work and Periodicals Connections: An Issue in Honor of Sally H. Mitchell" and "Rushing Dinner to the Table: The Englishwoman's Domestic Magazine and Industrialization's Effect on Middle-Class Food and Cooking, 1852–1860." Jordan Osterman: What has it been like for you to be involved with RSVP over the years? Andrea Broomfield: If it wasn't for Sally Mitchell, my route as a food historian would have taken a very different path, maybe not a path at all. I was brought up in RSVP; it made me who I am. I didn't have to go through the anxiety and fear that a lot of young grad students have about scholarship because I was so welcomed into this group. There's no way I would have been able to build my network and confidence, to understand the ropes of academe, if not for RSVP. [End Page 426] After first being introduced to the society, my network continued to grow. When I won the VanArsdel Prize in 1990, I did not merely fly down to Waco, Texas, where the RSVP Conference was held that year, to pick up a plaque. Instead, I was invited by many scholars in the organization to play an active part in the conference. Sally was instrumental in introducing me around, but I was tremendously grateful that I could meet people on my own and begin to develop professional relationships. George Worth, Maria Frawley, Barbara Quinn Schmidt, Dick Fulton, and Louis James were just some of the RSVP members I met in Waco and began to develop important connections with. While I was never able to meet Rosemary VanArsdel in person, she and I began a letter exchange and occasionally talked on the phone. She wrote reference letters for me and opened doors. This type of generosity and collegiality was overwhelming to someone who had always assumed that academe was a cutthroat business where you had to watch out for potential sabotage. Maybe I had read too many David Lodge novels! The next thing I knew, I was graduating and starting my first job. Once I became a professor, RSVP members still nurtured me, giving me leads and helping me. I feel like I didn't contribute very much to RSVP, but its members contributed a lot to me. I owe them a very big debt of gratitude. What makes this society so special is that it started as a group of people with a desperate need for information about periodicals, but much of that information didn't exist. A group of people had to come together with a mission of trying to attribute hundreds of anonymous articles to authors and create a theory for how to read and understand this journalistic material, whether the author was known or was stubbornly anonymous. The mission these founders had was straightforward but pretty profound. The society still retains so many vestiges of that mission: why periodicals matter and how they should and shouldn't be used. The organization has grown, but it still has an intimacy. RSVP has a unique story about how and why it started, and it welcomes members and embraces newcomers. It doesn't make people feel excluded. Jordan Osterman: You wrote in your tribute to Sally Mitchell (in...

Persistent Prothrombotic State in Atrial Fibrillation Despite Use of Novel Oral Anti-Coagulants

Background: Oral anticoagulants such as warfarin have been conventionally used for the management of atrial fibrillation (AF). Despite the effectiveness of warfarin, its use in AF patients requiring anticoagulation is suboptimal with an even greater underuse seen in elderly patients who are at higher risk of stroke. New oral anticoagulants such as rivaroxaban(R), apixaban (A), dabigitran (D) and edoxaban (E) have been approved to manage thrombotic and cardiovascular disorders including AF. The newer anticoagulants do not require continuous monitoring like warfarin and are much more convenient for patients with AF.Objective: To profile the baseline level of circulating thrombotic biomarkers plasminogen activator inhibitor (PAI-1), von Willebrand Factor (vWF), microparticle bound tissue factor (MP-TF) and prothrombin fragment 1.2 (F1.2) in patients with AF. Additionally, the effect of novel oral anticoagulants (R, A, D and E) on the levels of thrombotic biomarkers in patients with AF is assessed.Materials and Methods: Citrated blood samples were drawn from 30 patients (21 male and 9 female; mean age 59.1) prior to ablation surgery for AF at Loyola University Medical Center in Maywood, Illinois. Citrated blood was spun at 3000 rpm to obtain platelet poor plasma. Normal plasma samples from healthy controls (24 male and 24 female; mean age 33) were purchased from a commercial source (George King Biomedical, Overland Park, KS). The plasma samples were analyzed using a biochip array (Randox, London, UK) for metabolic syndrome biomarkers including PAI-1 and ELISA kits for vWF, MP-TF (Hyphen BioMed, Nueville-Sur-Oise, France) and prothrombin F1.2 (Siemens, Newark, DE).Results: Compared to the control group, circulating levels of vWF, MP-TF and PAI-1 were statistically increased in patients with AF (P<0.0001, P<0.0001, and P=0.0014, respectively). Circulating levels of prothrombin F1.2 showed no difference between the AF and the control group (P=0.2696). AF patients (n=30) were divided into two groups based on their usage (Group 1, n=17) and non-usage (Group 2, n=10) of any novel oral anticoagulant (R, A, D and E). Three patients on warfarin were excluded from this section of data analysis. A statistical increase in vWF (P=0.0018) and MP-TF (P=0.0039) remained in those taking novel oral anticoagulants compared to group 2. No difference was seen in PAI-1 (P=0.333) or F1.2 (P=0.31) between groups 1 and 2. Percent change from the normal mean was also calculated for PAI-1, vWF, MP-TF and F1.2 in group 1 (78%, 61.1%, 142%, 8.9%, respectively) and group 2 (113.6%, 16.9%, 31.7%, 41.4%, respectively) as seen in table 1.Discussion: Elevated levels of PAI-1, vWF and MP-TF seen in AF patients compared to normal provide insight into an additional risk of thrombogenesis associated with AF which is not targeted by current anticoagulant medications. Most AF patients are assessed using a stroke risk stratification scale (CHA2DS2VASc) to determine if anti-coagulants should be used to prevent stroke associated with AF. Patients in group 1 had a mean CHA2DS2VASc score of 1.15 compared to 2.18 see in group 2. This data supports studies which suggest that including levels of prothrombotic biomarkers to current risk stratification scales could be more effective in assessing the risk of stroke of patients with AF. This data also suggests that although very effective in lowering prothrombin F1.2 levels in AF, the newer anticoagulants, R,A,D and E still leave additional prothrombotic biomarkers unaffected. These unaffected biomarkers could be the potential target of future drug therapies which could lower the risk of stroke in patients with AF even more than the use of newer anticoagulants alone. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Platelet Activation in End-Stage Renal Disease Is Mediated By Extracellular Nucleosomes That Originate from White Blood Cells

IntroductionExtracellular nucleosomes in plasma (PNs) are complexes of DNA and histones that are released during cell death and inflammatory responses. End-stage renal disease (ESRD) represents a complex syndrome where inflammation, endothelial dysfunction, and hemostatic aberrations contribute to the observed vascular manifestations. The purpose of this investigation is to profile PNs in patients with ESRD and to demonstrate their relevance to blood cells and platelet activation products.MethodsPre-dialysis plasma samples from patients undergoing maintenance hemodialysis (n = 90) at Loyola University outpatient dialysis unit were collected under an approved IRB protocol. Plasma samples from healthy individuals (n = 50) were purchased from a biobank as a control (George King Biomedical, Overland Park, Kansas). Complete blood count profiles, including white blood cells (WBCs), red blood cells (RBCs), and platelets were obtained from the patients’ medical records. The levels of PNs in ESRD patients and healthy volunteers were measured using the Cell Death Detection ELISA PLUS assay (Roche Diagnostics, Mannheim, Germany). MP-TF levels were measured using the Zymuphen MP-TF kit (Hyphen BioMed, Neuville-sur-Oise, France). PDGF levels were measured using the Human PDGF-BB Quantikine ELISA Kit (R&D Systems, Minneapolis, Minnesota). Human PF4 levels were measured using the Zymutest PF4 ELISA Kit (Hyphen BioMed, Neuville-sur-Oise, France). All individual results were tabulated and analyzed using the statistical software GraphPad Prism 7. The Mann-Whitney test for non-parametric data was utilized in comparing ESRD to control groups. PN levels were correlated with cell counts and platelet activation factors using the non-parametric Spearman correlation. Individual cell counts were also correlated with platelet activating factors using the same method.ResultsIn the ESRD patients, the average hematocrit was 31.7 ± 4.4 %, the average WBC count was 6.5 ± 4.0 K/uL, and the average platelet count was 179.4 ± 66.3 K/uL. The levels of PNs in the ESRD patients (15.5 ± 14.1 Arbitrary Units (AU)) were markedly higher in comparison to that of the controls (6.74 ± 13.7 AU; p < 0.0001). Similarly, MP-TF levels were significantly elevated in ESRD patients (3.00 ± 1.42 pg/mL) compared to normal (0.363 ± 0.263 pg/mL; p < 0.0001). PF4 levels were also significantly elevated in ESRD patients (95.3 ± 35.3 ng/mL) compared to normal (27.4 ± 19.8 ng/mL; p < 0.0001). While PDGF levels were higher in the ESRD group (116.0 ± 172.5 pg/mL) in comparison to the controls (82.7 ± 113.5 pg/mL), this increase was not statistically significant (p = 0.405). A positive correlation was observed between PNs and WBCs (p = 0.024; r = 0.244). PN levels did not show a correlation with RBC (p = 0.448; r = 0.083) and platelet levels (p = 0.545; r = 0.066). Furthermore, there was no correlation between PNs and MP-TF (p = 0.501; r = 0.077), PDGF (p = 0.314; r = 0.110) and PF4 (p = 0.524; r = -0.070) in the ESRD patients. However, the platelet count showed a positive correlation with PDGF (p = 0.044; r = 0.218) and MP-TF (p = 0.042; r = 0.237). Similarly, the WBC count showed a positive correlation with the platelet count (p < 0.0001; r = 0.476) and PDGF (p = 0.016; r = 0.260).ConclusionThis study clearly demonstrates that extracellular nucleosomes are elevated in the plasma of patients with ESRD. The fact that the PN levels correlated with the number of circulating white blood cells suggest that the PNs originate from these cells. Since the ESRD patients exhibited platelet activation, as evident by the observed increase in PDGF, MP-TF and PF4, it is plausible that this activation is mediated by PNs originating from the WBCs. As observed by the positive correlation between WBCs, PDGF, and platelet count, this study underscores the potential role of nucleosomes originating from WBCs in the activation of platelets. These results are consistent with previously reported observations that extracellular histones can induce platelet activation in a TLR2 and TLR4 dependent manner (http://www.ncbi.nlm.nih.gov/pubmed/21673343). DisclosuresNo relevant conflicts of interest to declare.

Effect of erythropoietin stimulating agents on vascular endothelial growth factor levels in patients with end stage renal disease.

End stage renal disease (ESRD) is a debilitating disease that not only impacts quality of life, but also increases the risk of cardiovascular disease, stroke, and overall mortality. By improving our understanding of the molecular patterns seen in progression of chronic renal disease (CRD), we may be able to slow down and possibly even prevent progression renal failure. The vascular endothelial growth factor (VEGF) has been implicated as a major contributor to CRD disease progression, thus our aim was to profile the VEGF levels in patients with ESRD and to determine the effects of the erythropoietin stimulating agents (ESAs). Under institution review board (IRB) approval, plasma samples were collected from 77 patients prior to hemodialysis and heparin administration. Normal human plasma samples (female & male, 18-35 years old) were purchased from George King Biomedical Inc (Overland Park, KS). All samples were stored at -80 °C. Inflammatory biochips were purchased from RANDOX (Co. Antrim, Northern Ireland) to test VEGF on 77 ESRD and 48 normal samples. The VEGF was significantly elevated in ESRD vs. normal (P<0.0001), ESRD+ESA vs. normal (P<0.0001), and ESRD-ESA vs. normal (P<0.0001). No difference was seen between ESRD+ESA and ESRD-ESA. Hemoglobin and free iron were significantly elevated in ESRD-ESA compared to ESRD+ESA (PHemoglobin=0.0007, PIron<0.0001). Our finding that VEGF was significantly elevated in ESRD vs. normal, aligns with the literature and suggests that VEGF may in fact play a key role in CRD progression. However, further studies to evaluate VEGF isomer levels are needed. While we saw lack of difference in VEGF levels between ESRD+ESA and ESRD-ESA, this may be due to the treatment algorithm used. Further investigation is warranted to determine whether the ESAs and hemoglobin levels show any influence on or crosstalk with the VEGF levels.

Taking Care of the Squeaky Wheel: Joint Replacement Long-term Issues

•We provide history and physical examination priorities for patients with postarthroplasty issues. •Intrinsic causes of joint pain postarthroplasty that may initiate patient complaints are discussed. •Origins of pain extrinsic to the joint prosthesis that may cause patient complaints are explored. •We provide tips for managing the postarthroplasty patient with joint issues. Laurel Short, MSN, FNP-C, is a Physical Medicine and Rehabilitation Nurse Practitioner in Overland Park, KS. She can be reached at . Department Editor Laurel Halloran, PhD, APRN, would like to hear your ideas for future columns, can be reached at .

Prevalence of metabolic syndrome in patients undergoing total joint arthroplasty and relevance of biomarkers.

Metabolic syndrome (MetS) is a collection of clinical conditions, including central obesity, hypertension, glucose intolerance and dyslipidemia. The long-term inflammatory and metabolic dysfunction associated with MetS may contribute to osteoarthritic processes leading up to total joint arthroplasty (TJA). The purpose of this study was to investigate levels of metabolic biomarkers and the prevalence of MetS in patients undergoing TJA. Under IRB approval, citrated plasma samples were collected from 41 patients undergoing total hip and knee arthroplasty (THA/TKA) preoperatively and day 1 postoperatively. Control group consisted of 25 healthy human plasma samples (female and male, 18-35 years old) purchased from George King Biomedical Inc. (Overland Park, KS, USA). Samples were profiled for c-peptide, ferritin, IL-6, insulin, resistin, TNF-α, IL-1a, leptin, and PAI-1 using metabolic biochips purchased from RANDOX Co. (Antrim, Northern Ireland). NCEP/ATP III guidelines were used to evaluate which patients met MetS criteria. Levels of IL-6, resistin, TNF-a, IL-1a, leptin, and PAI-1 were significantly elevated in patients undergoing TJA compared to normal. C-peptide and insulin were both decreased in TJA compared to normal. No significance was found when comparing TJA to normal for ferritin. TNFα was significantly lower in TJA+MetS compared to TJA-MetS, while other biomarkers showed no difference in TJA±MetS populations. Insulin & c-peptide both showed a significant decrease in TJA-MetS compared to normal, but levels in TJA+MetS patients were not significantly different from controls. Resistin showed significant increases in TJA+MetS vs. normal, but not in TJA-MetS vs. normal. Overall, the differing metabolic profile seen in patients undergoing TJA suggest ongoing metabolic dysfunction. Insulin and c-peptide patterns among the different test groups hint toward a complex and dysfunctional metabolic process involved, with leptin and underlying insulin resistance playing a role. Increased resistin in TJA+MetS, but not in TJA-MetS, compared to normal, suggests that while elevated resistin levels may be associated with the osteoarthritic process, levels are further attenuated by MetS, which is highly prevalent in this population. Increased TNFα in TJA-MetS compared to TJA+MetS may be an artifact of differing sample populations or a true complication of the complex pathophysiology and medical regimen seen in patients with both OA and MetS. The lack of difference seen in the remaining biomarkers suggest that having MetS as a comorbidity does not contribute to the elevated levels seen in patients undergoing TJA.

Evaluation of Confirmation Lights on Red Light Running Violations by Time-into-Red Phase

Red light running (RLR) crashes at urban signalized intersections continue to be a serious safety concern for many communities across the United States. Recent national crash data indicated that 676 fatalities were caused by RLR in 2009, which represents approximately 10% of all intersection crashes and 2% of all roadway fatalities. This research studied confirmation lights and their effect on RLR violations’ time into red. Two sets of confirmation lights were installed at two busy urban intersections in Overland Park, Kansas. With the use of a binomial test of proportions, the study found that there was a significant increase in violations within the first second of the red indication 1 month after deployment. Three months after deployment, there was a decrease in violations over 1 s, but this decrease was statistically not significant. Although there was an observed increase in violation rate, the increase did not translate into violations occurring later in the red cycle. In the 3-month after-period, there was a decrease in the violation rate but an increase in violations within the first second of the red indication and a decrease in violations between 1.0 and 3.0 s. The results for this period were statistically not significant and which suggest that the confirmation light had little effect on a violation’s time into the red. In all periods after deployment, there were no violations occurring after 3 s into the red. This finding means that drivers did not use the confirmation lights to traverse the intersection during the all-red cycle.

23.4% Saline Decreases Brain Tissue Volume in Severe Hepatic Encephalopathy as Assessed by a Quantitative CT Marker.

Cerebral edema is common in severe hepatic encephalopathy and may be life threatening. Bolus 23.4% hypertonic saline improves surveillance neuromonitoring scores, although its mechanism of action is not clearly established. We investigated the hypothesis that bolus hypertonic saline decreases cerebral edema in severe hepatic encephalopathy utilizing a quantitative technique to measure brain and cerebrospinal fluid volume changes. Retrospective analysis of serial CT scans, and clinical data for a case-control series were performed. ICUs of a tertiary care hospital. Patients with severe hepatic encephalopathy treated with 23.4% hypertonic saline and control patients who did not receive 23.4% hypertonic saline. 23.4% hypertonic saline bolus administration. We used clinically obtained CT scans to measure volumes of the ventricles, intracranial cerebrospinal fluid, and brain using a previously validated semiautomated technique (Analyze Direct, Overland Park, KS). Volumes before and after 23.4% hypertonic saline were compared with Wilcoxon signed rank test. Associations among total cerebrospinal fluid volume, ventricular volume, serum sodium, and Glasgow Coma Scale scores were assessed using Spearman rank correlation test. Eleven patients with 18 administrations of 23.4% hypertonic saline met inclusion criteria. Total cerebrospinal fluid (median, 47.6 mL [35.1-69.4 mL] to 61.9 mL [47.7-87.0 mL]; p < 0.001) and ventricular volumes (median, 8.0 mL [6.9-9.5 mL] to 9.2 mL [7.8-11.9 mL]; p = 0.002) increased and Glasgow Coma Scale scores improved (median, 4 [3-6] to 7 [6-9]; p = 0.008) after 23.4% hypertonic saline. In contrast, total cerebrospinal fluid and ventricular volumes decreased in untreated control patients. Serum sodium increase was associated with increase in total cerebrospinal fluid volume (r = 0.83, p < 0.001), and change in total cerebrospinal fluid volume was associated with ventricular volume change (r = 0.86; p < 0.001). Total cerebrospinal fluid and ventricular volumes increased after 23.4% hypertonic saline, consistent with a reduction in brain tissue volume. Total cerebrospinal fluid and ventricular volume change may be useful quantitative measures to assess cerebral edema in severe hepatic encephalopathy.

How I Do It: 3D FUE: A New Dimension for Better, Easier FUE

1. Timothy Carman, MD, FISHRS (tcarmanmd{at}mac.com) 1. La Jolla, California, USA 2. Tseng-Kuo Shiao, MD, FISHRS (drtksmail{at}gmail.com) 1. Overland Park, Kansas, USA Most of us would agree FUE harvesting is generally a more labor intensive manner of harvesting when compared with

Biomarkers of Hemostatic Dysregulation, Inflammation, and Infection in Patients Diagnosed with Sepsis Associated Coagulopathy

Introduction: Sepsis associated coagulopathy (SAC) is one of the major pathophysiological mechanisms of sepsis and has been shown to greatly increase mortality in septic patients. SAC is characterized by the inappropriate activation of the coagulation cascade, leading to the formation of microthrombi and the potential for multiple organ failure. Additionally, the excessive consumption of platelets and coagulation factors can create a risk of excessive bleeding. SAC is a complex syndrome, involving coagulation factors, inflammatory cytokines, and several other notable factors of varied origin. In this study, our aim was to evaluate the levels of endocan, pentraxin, and procalcitonin in individuals with SAC diagnosed according to the ISTH criteria in comparison to healthy individuals. Endocan is a soluble, circulating proteoglycan that normally binds to LFA-1 and inhibits leukocyte diapedesis. It is produced by vascular endothelial cells as well as the lung and kidney and has been proposed as a marker of endothelial dysfunction and disease severity in patients with sepsis. Endocan may be useful in the evaluation and tracking of SAC as a marker of endothelial damage caused by excessive inflammation and coagulation. Pentraxin (PTX3) is a protein structurally similar to CRP that is produced by several cell types in response to inflammatory signals and is known to be a marker of inflammation in a number of disease processes. In addition to being the precursor of the hormone calcitonin, procalcitonin is produced by a variety of tissues and may correlate with degree of infection and response to treatment in individuals with sepsis. Taken together, endocan, pentraxin, and procalcitonin may be useful in the evaluation and monitoring of SAC as they are representative of endothelial damage, inflammation, and systemic infection, respectively.Materials and Methods: Blood from 50 patients with SAC and 33 normal individuals obtained from a commercial source (George King Biomedical, Overland Park, KS) were evaluated. Levels of pentraxin, procalcitonin, endocan, and coagulation factors VII, IX, and X were measured using commercially available ELISA kits from Stago (Parsippany, NJ), Lunginnov (Lillie, France) and R&D Systems (Minneapolis, MN). All results are compiled as group mean and expressed as average mean + SD. These values are compared with normal and results were computed as percent increase or decrease.Results: The levels of coagulation factors VII and X were found to be reduced in patients with SAC compared to normal individuals (p < 0.05); the level of factor IX was statistically unchanged. The reduction in factor X was relatively modest, less than a 20% reduction compared to normal, while the reduction in factor VII was more marked, with a greater than 40% reduction compared to normal. The levels of pentraxin, proxalcitonin, and endocan were all found to be significantly elevated in blood from SAC patients compared to blood from normal healthy individuals (p < 0.05). All three markers exhibited a greater than 100% average increase when compared to normal.Discussion: These results indicate that endocan, pentraxin, procalcitonin, and factor VII are all candidates for further investigation in the identification of a more comprehensive molecular profile of SAC and in the development of diagnostic or prognostic tests. The significant degree of change observed in each marker from normal provides a baseline for future studies of these markers in SAC patients. Although these factors individually are not specific markers of SAC, each is a marker for a specific system that is dysregulated in SAC; endocan for endothelial damage, pentraxin for inflammation, procalcitonin for infection, and factor VII for coagulation. Taken together, these biomarkers may be useful in the diagnosis and monitoring of SAC. DisclosuresNo relevant conflicts of interest to declare.

Pre-Existence of Prothrombotic State in Patients with Atrial Fibrillation Despite Therapy with New and Traditional Anti-Coagulant Drugs

Background: Oral anticoagulants such as warfarin (W) have been conventionally used for the management of atrial fibrillation (AF). Despite the effectiveness of W, its use in AF patients requiring anticoagulation is suboptimal with an even greater underuse seen in elderly patients who are at higher risk of stroke. New oral anticoagulants such as rivaroxaban (R) and apixaban (A) have been approved to manage thrombotic and cardiovascular disorders including AF. The newer anticoagulants do not require continuous monitoring like W does and are much more convenient for patients with AF.Objective: To profile the baseline level of circulating thrombogenic biomarkers von Willebrand Factor (vWF), prothrombin fragment 1.2 (F1+2), microparticle bound tissue factor (MP-TF) and plasminogen activator inhibitor (PAI-1) in patients with AF. Additionally, the effect of both newer (R and A) and traditional (W) anticoagulants on the levels of thrombogenic biomarkers in patients with AF will be assessed.Materials: Citrated blood was drawn from thirty AF patients prior to ablation surgery and spun at 3000 rpm to obtain platelet poor plasma. Normal plasma samples from healthy controls were purchased from a commercial source (George King Biomedical, Overland Park, KS). The plasma samples were analyzed using a biochip array (Randox, London, UK) for metabolic syndrome biomarkers including PAI-1 and ELISA kits for vWF, MP-TF (Hyphen BioMed, Nueville-Sur-Oise, France) and prothrombin F1+2 (Siemens, Newark, DE).Results: Circulating levels of vWF, MP-TF and PAI-1 were statistically increased in patients with AF compared to normal (P<0.0001, P<0.0001, and P=0.0014, respectively). Circulating levels of prothrombin F1+2 showed no difference between the AF and normal group (P=0.2696). AF patients (n=30) were divided into two groups based on their usage (Group 1, n=21) and non-usage (Group 2, n=9) of any anticoagulant. Furthermore, those on anticoagulants were divided based on their use of newer (R and A, Group 3, n=16) or traditional (W, Group 4, n=4) anticoagulants. A statistical increase in vWF (P<0.0001), MP-TF (P<0.0001) and PAI-1 (P=0.011) remained in Group 1 compared to normal while a statistical increase in prothrombin F1+2 (P=0.0343) and PAI-1 (P=0.0040) were noted in Group 2 compared to normal. vWF (P=0.0036) and MP-TF (P=0.0059) were elevated in Group 1 compared to Group 2 while prothrombin F1+2 (P=0.0697) and PAI-1 (P=0.4548) showed no difference between the two groups. Furthermore, there was no statistical difference in the level of any thrombogenic biomarker in AF patients between Group 3 (R and A) and Group 4 (W). (Table 1)Discussion: Elevated levels of vWF, MP-TF and PAI-1 seen in AF patients compared to normal provide insight into an additional risk of thrombogenesis associated with AF which is not targeted by current anticoagulant medications. Most patients are assessed using a stroke risk stratification scale (CHA2DS2VASc, CHADS2, CHADS-VASC, or CHADS) to determine if anti-coagulants should be used to prevent stroke associated with AF. Of the 30 patients examined in this study, 8/9 (89%) patients who were not on anticoagulants had a stroke risk stratification score of 0 while 20/21(95%) patients who were on anticoagulants had a score of >1. This data supports studies which suggest that adding levels of prothrombotic biomarkers to current risk stratification scales could be more effective in assessing the risk of stroke of patients with AF. This data also suggests that although very effective in lowering prothrombin F1+2 levels in AF, the newer anticoagulants, R and A, and the traditional anticoagulant, W, still leave additional prothrombotic biomarkers unaffected. These unaffected biomarkers could be the potential target of future drug therapies which could lower the risk of stroke in patients with AF even more than the use of newer/traditional anticoagulants alone. Table 1Biomarkers of Thrombogenesis in AF and Normal GroupsGroupvWF (concentration %)Prothrombin F1+2 (pmol/L)MP-TF (pg/mL)PAI-1 (ng/mL)Normal4140 ± 919 n=46106.1 ± 52.7 n=500.38 ± 0.25 n=483.21 ± 4.13 n=25AFGroup 1 Anticoagulant n=216616 ± 1173107.9 ± 61.20.93 ± 0.715.69 ± 4.15Group 2 Non-Anticoagulant n=94788 ± 1338162.5 ± 93.30.51 ± 0.136.49 ± 3.14Group 3 New Anticoagulants (R and A) n=166721 ± 1127103.5 ± 37.60.76 ± 0.345.68 ± 4.53Group 4 Traditional Anticoagulants (W) n=46387 ± 158075.2 ± 52.60.94 ± 0.335.63 ± 3.52 DisclosuresNo relevant conflicts of interest to declare.