This material demonstrates high activity for oxygen evolution reaction with a low overpotential of 197 millivolt in acidic electrolyte at 10 milliamperes per geometric square centimeter (mA cmgeo−2). Together, it achieves high turnover frequencies of 4.2 sUPD−1 (3.0 sBET−1) at 1.50 V vs. reversible hydrogen electrode. Furthermore, 1T-IrO2 also shows little degradation after 126 hours chronopotentiometry measurement under the high current density of 250 mA cmgeo−2 in proton exchange membrane device. Theoretical calculations reveal that the active site of Ir in 1T-IrO2 provides an optimal free energy uphill in *OH formation, leading to the enhanced performance. The discovery of this 1T-metallene oxide material will provide new opportunities for catalysis and other applications.Exploring new materials is essential in the field of material science. Especially, searching for optimal materials with utmost atomic utilization, ideal activities and desirable stability for catalytic applications requires smart design of materials’ structures. Herein, we report iridium metallene oxide: 1 T phase-iridium dioxide (IrO 2 metallene oxide as an oxygen evolution electrocatalyst in acidic electrolytes.Identifying new, active material phases provides a promising avenue in the development of efficient catalysts. Here, authors demonstrate a metastable 1T-phase IrO Electrochemical water splitting provides a promising way to make efficient energy conversion applicable, yet its biggest challenge comes from the slow kinetics of oxygen evolution reaction (OER) at the anode and the harsh working condition in the strong acidic environment10. More importantly, it is required for developing OER catalysts in acidic pH regime for their direct impact on making the proton exchange membrane (PEM) viable on an industrial scale13.The increasing worldwide energy and pollution crisis demand the highly efficient production of renewable energy2) is currently the only material that can remain stable under the harsh acidic OER condition, while its progress has been largely impeded by its unsatisfied activities and ultralow-earth abundance16. Several strategies have been reported to develop complex iridium-based oxides, such as: surface restructuration strategy15, amorphous strategy17, and doping strategy16, yet still leaving a large room for improvement. Therefore, designing IrO2 catalysts with exceptional activity and stability and high atomic utilization becomes a central issue for breaking through this trade-off.Iridium oxide  materials are highly desirable for surface-sensitive reactions in terms of cost-effectiveness due to their near-100% atom utilization, which provides the maximized density of surface active sites27. It also provides an ideal platform for catalytic optimization by virtue of favorable mass transport, fascinating mechanical behaviors, and tunable electronic properties30. As the surface energy increases with thinning the crystal thickness32, the stability of phase increases with decreasing the layer thickness. Such beneficial features may introduce insights into the role of dimensionality engineering in discovering advanced materials.Searching for an IrO33 and the strong alkaline medium is feasible for the exploration of metastable compounds34. Hence, to obtain an advanced material with high degree of crystallinity, a combination of mechanochemistry, strong alkaline environment and thermal treatment may be a reasonable attempt. However, it is a challenge to synthesize under these harsh preparation conditions. Motivated by all these possibilities, we report an iridium metallene oxide: 1 T-phase-IrO2 using a robust synthetic route referred as the “mechano-thermal” method in a strong alkaline medium. The structure of this material is characterized by a space group: P-3m1 (164) (a = b = 3.11 Å and c = 6.91 Å). 1T-IrO2 displays a substantially low overpotential of 197 millivolt (mV) at 10 milliamperes per geometric square centimeter (mA cmgeo−2) for acidic OER, with the combination effect of the optimized Ir reaction site, high number of active sites and atomic utilization. Notably high turnover frequencies (TOFs) of 4.2 sUPD−1 (3.0 sBET−1) were obtained by 1T-IrO2. More importantly, it also exhibits little activity decline for 126 h continuous test under the high current of 250 mA cmgeo−2 in PEM device. Density functional theory (DFT) calculations reveal that the binding of hydroxyl groups on the 1T-IrO2 is an endothermic process with an optimized free energy uphill closing to the equilibrium potential of 1.23 eV, which is in sharp contrast with the IrO2 of rutile phase. As a result, the typical potential-limiting step of subsequent *OH dissociation or *O formation in OER is significantly promoted.In terms of the synthetic method for discovering various materials, mechanochemical synthesis provides a unique means for the pursuit of metastable polymorphous products2 are challenging: high temperature (800 °C), a strong alkaline environment and the continuous grinding force. In this work, we successfully realized all the strict requirements in our “home-made” reactor, where the main body is made by the high-quality corundum, as the suitable material that can remain stable in an harsh environment: high temperature, continuously grinding and strong alkaline  and potassium hydroxide (KOH) were chosen as the reaction precursors. The mixture was grinded with the temperature increasing from room temperature to 800 °C with the heating rate of 10 °C min−1 and kept at 800 °C for 3 h in the mechano-thermal reactor. The detailed synthesis process can be found in the Methods section. The obtained 1T-IrO2 powder shows the dark blue color and its water dispersion in a cuvette shows the light blue color, which are totally different from those of IrO2 with rutile phase  image reveals that the 2D nanosheet is the dominant product Fig. . Transmi2 nanosheets using Cu Kα radiation (λ = 1.5406 Å). As shown in Fig. 2 [Joint Committee on Powder Diffraction Standards (JCPDS) No. 88-0288] are detected. The peaks correspond to the (0001) and (0002) diffraction planes, respectively, indicating the layered structure along the c-axis. The peak at about 12.81° is an important fingerprint to distinguish the unit-cell parameter c along c direction, which is calculated to be ~6.91 Å.The X-ray diffraction (XRD) technique was then performed to reveal the crystal structure of 1T-IrO236. A typical STEM-ADF image from monolayer region is shown in Fig. 37. Then there are only two questions about the crystal structure of this material: one is the layer stacking and the other is the atomic coordination environment surrounding Ir atoms. Figure 2, which show the identical appearance. Comparing with the simulated STEM-ADF images of AB stacking and ABC stacking, it unambiguously suggests that our 1T-IrO2 is AA stacked  results for 1T-IrO2, rutile-IrO2 and Ir metal foil are shown in Fig. 3b39. The better agreement between experimental 1T-IrO2 and the T-phase further confirms the trigonal coordination environment. The extended X-ray absorption fine structure (EXAFS) results were also collected and Fourier transformed to show the real space coordination environments as shown in Fig. 2, rutile-IrO2, and Ir metal foil. The spectrum for 1T-IrO2 is fitted and shown in Fig. 6 shell and Ir–Ir6 shell. The coordination number and bond lengths for these two shells are fitted to be 6.0/2.00 Å and 6.0/3.12 Å.The other important information is the coordination information between Ir and O atoms. We addressed this question by two different approaches. First, according to the literature there are two possible structures for oxygen coordination: one is the trigonal (T) phase and the other is the hexagonal (H) phase2 model is well consistent with the reflections observed in the experimental XRD pattern . The corresponding schematic of layered 1T-IrO2 is shown in Fig. We thus propose a structure model as shown in Fig. ern Fig. . Accordi2 since it provides a powerful way to activate the starting materials and obtain the metastable polymorphous forms34. Only the rutile-IrO2 with low crystalline was obtained when annealing the mixture of IrCl3 and KOH prepared by a simple mixing process  by analyzing the X-ray diffraction result. The thermodynamically stable rutile phase was detected, further indicating the metastable property of 1T-IrO2  was used to reveal the electronic states of 1T-IrO2. Based on the high-resolution XPS spectra in Supplementary Fig. s peaks from Ir–O binding, are clearly observed in both 1T-IrO2 and rutile-IrO2. More importantly, the Ir 4 f binding energies of 1T-IrO2 are shifted to the higher binding energy than those of rutile-IrO2  and the two reference electrocatalysts: C-IrO2 and commercial-Ir/C (C-Ir/C) were also measured for comparison. The OER tests were measured using a standard three-electrode system in 0.1 M O2-saturated HClO4 electrolyte. The reference electrode was calibrated before OER tests  of different catalysts. 1T-IrO2 exhibit the highest geometrical current density of 52.7 mA cmgeo−2, which is 9.6, 6.3 and 10.8 times high than those of Rutile-IrO2 (5.5 mA cmgeo−2), C-IrO2 (8.4 mA cmgeo−2), and C-Ir/C (4.9 mA cmgeo−2)  Fig. .Fig. 4OE2 was further evaluated by determining the mass activity and TOFs at 1.50 V vs. RHE. The mass activities of all the catalysts are calculated by normalizing the mass of iridium and the TOF values of all the catalysts are calculated by normalizing the mercury underpotential deposition (UPD)-based surface area, Brunauer-Emmett-Teller (BET) surface area or electrochemically active surface areas (ECSAs)-based surface area. The methods to determine the UPD, BET or ECSA-based surface areas of different catalysts were collected in Methods section  at 1.50 V vs. RHE. We further evaluated the OER performances of 1T-IrO2 in the scale-up experiment performed with the 1 × 1 cm2 electrodes (Methods section). The mass and TOF values of 1T-IrO2 are higher than those of C-IrO2, Rutile-IrO2 and C-Ir/C   in acidic media45. The post-reaction UPD-based surface area of 1T-IrO2 was also evaluated , while only one kind of Ir–O bond in the 1 T-phase. The relative stability of the 1 T and rutile phases can be qualitatively evaluated by the projected crystal orbital Hamilton population (pCOHP) of the three types of Ir–O bonds. The COHP plots can be employed to analyze the contribution of bonding and antibonding states in the density of states. When chemical bonds are formed in a material, the bonding orbitals are typically occupied, while the antibonding orbitals are often unoccupied. If the unoccupied antibonding orbitals are very close to the Fermi energy, they become occupied by the excited electrons at elevated temperatures due thermal excitations. Hence, this material may probably decompose at high temperatures. However, if the unoccupied antibonding orbitals are well above the Fermi energy, this suggests that the pumping of electrons into these states will require high thermal excitations. Correspondingly, the material is expected to possess excellent thermal stability. As shown in Fig. 2 is shown in Fig. 2 mainly happens on the bare (0001) surface at U < 1.76 V/RHE  surface, the corresponding limiting overpotential is 0.59 V, which agrees well with previous calculations  surfaces are either exothermic or slightly endothermic. By contrast, this step is much more endothermic on the 1T-IrO2. Therefore 1T-IrO2 provides the active site, different from rutile-IrO2, which leads to the enhanced catalytic performance. As shown in Supplementary Fig. 2 is buried under oxygen atoms, which leads to the appropriate adsorption of hydroxyl and easy desorption step. However, Ir atom on the surface of rutile-IrO2 is not buried under oxygen atoms, which leads to the strong adsorption of hydroxyl and hard desorption step. The 1T-IrO2 is thus more active than rutile-IrO2 in OER.The Gibbs free energy profile of OER on 1T-IrOons Fig. 46. By co2, has been successfully prepared by the mechano-thermal method. The optimized Ir active site in 1 T-phase and ultrathin 2D structure are the keys to the high-performance acidic OER catalysis. Computational calculations reveal that the catalytic origin of 1T-IrO2 in OER can be attributed to the optimized free energy increase of binding hydroxyl groups on Ir atoms. The present research paves out a mechano-thermal method for synthesizing 2D materials and shows great promise for sustainable energy toward real applications and fundamental insight into physical understanding.In conclusion, a metallene oxide, 1T-IrO3, 99.9%) was purchased from Alfa Aesar Co. Iridium(IV) oxide  was purchased from Aladdin Chemical Regent Co. Potassium hydroxide , sulphuric acid , and perchloric acid  were obtained by Sinopharm Chemical Reagent Co. Nafion solution (5 wt %) and mercury(II) nitrate hydrate (Hg(NO3)2·H2O, 99.99%) were obtained from Sigma–Alddrich Co. Isopropanol was purchased from Sinopharm Chemical Reagent Co. Other reagents were of analytical reagent grade without further purification. Double-distilled water was used throughout the experiment. 20% Ir on Vulcan XC-72 was purchased from Premetek Co. The PEM device was purchased from Gaossunion (TIANJIN) Photoelectric Technology CO., LTD.. A commercial Nafion N117 membrane was purchased from Alfa Aesar.Iridium trichloride .The XRD patterns of all samples were recorded by X-ray powder diffraction  by using the operation voltage of 40 kV and the current of 40 mA with Cu Kα radiation source. The TEM images of all samples were characterized by a FEI Tecnai F20 transmission electron microscope with an accelerating voltage of 200 kV and by an aberration-corrected JEOL ARM 200 F microscope operated at 200 kV. EDX was conducted by FEI Tecanai F20 transmission electron microscope. The SEM images were recorded by Zeiss G500 scanning electron microscope. The XPS results were collected by Kratos AXIS UltraDLD ultrahigh vacuum surface analysis system with Al Kα radiation (1486 eV). AFM images were captured by the atomic force microscopy . Synchrotron X-ray absorption spectroscopy data were collected at beamline 7-BM at National Synchrotron Light source II at Brookhaven National Laboratory and the corresponding data are analyzed based on the ref. 2 or 1 × 1 cm) was used as the working electrode, calomel electrode was used as the reference electrode, and carbon rod was used as the counter electrode. OER measurements were conducted in O2-saturated 0.1 M HClO4 or 0.5 M H2SO4 electrolyte. For the measurement with iR-correction, R was referred to the ohmic resistance arising from the electrolyte/contact resistance of the setup. The scan rate was 5 mV s−1 when LSV was measured. The catalysts solutions were obtained by mixing 1.5 mg catalysts  in the solution of 400 μL isopropanol and 10 μL 0.5 wt % Nafion solution with sonication to form the homogenous catalysts ink. 4 μL ink was dispersed on the GCE with drying naturally for testing. For the scale-up experiment, the catalysts inks were obtained by mixing 0.85 mg catalyst in the solution of 200 μL isopropanol and 10 μL 0.5 wt % Nafion solution with sonication to form homogenous catalysts ink. All the ink was dispersed on the 1 × 1 cm glass-carbon electrode with drying naturally for the scale-up testing. The XRD result of the 1T-IrO2 after stability test was obtained by analyzing the electrocatalyst dropping on the carbon paper after stability test. The catalytic activity and durability were measured at least three times for each catalyst. Errors are reported as relative standard deviation (RSD).All electrochemical experiments were carried out with using the CHI660E electrochemical workstation . A traditional three-electrode system was used. The glass-carbon electrode  and cathodic Pt/C  were dropped on carbon paper with the active area of 1 × 1 cm, respectively. The catalyst ink was obtained by mixing 0.85 mg 1T-IrO2 (C-IrO2) in the solution of 200 μL isopropanol and 10 μL 0.5 wt % Nafion solution with sonication to form the homogenous catalyst ink. The electrolyte is O2-saturated 0.5 M H2SO4 (3 mL min−1) and the reaction temperature is 65 °C. N117 membrane was used for PEM device.For the PEM device, the anodic 1T-IrO2, Rutile-IrO2 and C-IrO2 were calculated by the Eqs. 247  at non-Faradic potential range was obtained by measuring the capacitance of double layer at solid-liquid interface employing cyclic voltammetry (CV) with different scan rates  in a range from 1.0 and 1.1 V vs. RHE. The Cdl was calculated by the Eq. j is the difference between the anodic and cathodic currents (ja − jc) at 1.05 V vs. RHE and ν is the scan rate. Therefore, once the values of Ageo, Cs, and Cdl are determined, the value of ECSA can be calculated according to Eqs. The electrochemical double-layer capacitance . Speak is the integral area of adsorbed hydrogen in the CV curve and ν is the scan rate of 5 mV s−1.C is a value of the adsorbed capacity of hydrogen  parameterization of the generalized gradient approximation (GGA)53. The electron-ion interactions were treated within the projector augmented-wave (PAW) approximation54. A cutoff energy of 400 eV was employed for the plane-wave basis set of all calculations. A conjugate-gradient algorithm was used to relax the atoms into their instantaneous ground state positions. The structural optimizations were not stopped until the ionic relaxation reaching the convergence criteria of 1 × 10−5 eV and 0.01 eV Å−1. The 1T-IrO2 surface was modelled with a double layered (4 × 4) supercell. The rutile-IrO2 (110) surface was mimicked by a 4-layer (1 × 2) periodic slabs, in which the top two layers are relaxed in three dimensions, and the bottom two layers are restricted. The first irreducible Brillouin zone was modelled with the Gamma-centered Monkhorst-Pack scheme, where a 4 × 4 × 1 k-points and a 2 × 2 × 1 k-points were adopted for 1T-IrO2 (0001) and rutile-IrO2 (110) surfaces, respectively. The vdW-D3 method was adopted to describe the van der Waals interactions55. A vacuum of 20 Å was employed to avoid the interactions between periodic images in the direction normal to the surface. The effect of aqueous solution was considered with the implicit solvation model, in which the relative permittivity was set to 80.0056.The spin polarized DFT calculations were carried out using the Vienna Ab-initio Simulation Package (VASP version 5.4)ZPE is the zero-point energy that can be calculated by EZPE = 1/2Σhv, in which v is the vibrational frequency of a normal mode and h is the Planck constant. S is the entropy that can be calculated as57:B and νi are the Boltzmann constant and vibrational frequency, respectively. ∆GpH is the free energy contributions related to the H+ concentration, which can be calculated by: ∆59. In this work, the pH = 0.00 and T = 298.15 K were selected to represent the acidic media and room temperature. According to the computational hydrogen electrode (CHE) model proposed by Nørskov et al., the Gibbs free energy of the proton-electron pairs (H+ + e−) is equivalent to one-half of the Gibbs free energy of a hydrogen molecule57. ΔG(U) with an applied potential U can be defined as:η) of the whole OER process can be described as:equilibrium is 1.23 V and Ulimiting is the limiting potential of the OER. The limiting potential  can be obtained by the following equation: Ulimiting = ΔGmax/e, where ΔGmax is the change of Gibbs free energy in the potential-limiting step (PLS). Considering the effect of the electrochemical electrode potential and the solvation, we also used JDFTx software60 to calculate the Ulimiting at the constant electrode potential of 1.23 V along with the CANDLE implicit solvation model61 and found that both of the constant-charge and constant-potential simulation results have the same trend. The quantitative effects of electrode potential and functionals on the Ulimiting are given in Supplementary Tables The change of the Gibbs free energy (∆G) for each elementary step at the zero potential can be written as:Supplementary InformationPeer Review File


The 2014 American Heart Association/American College of Cardiology (AHA/ACC) clinical guidelines recommend cardiac troponin as a superior biomarker to creatine kinase (CK) and creatine kinase-muscle/brain (CK-MB) for the detection of acute coronary syndrome (ACS), namely myocardial infarction and unstable angina. In April 2018, our Emergency Department (ED) transitioned from using standard troponin to using high-sensitivity troponin T, and adopted a clinical guideline consistent with the AHA/ACC. The guideline recommended high-sensitivity troponin T without CK/CK-MB testing in the majority of clinical situations, limiting CK/CK-MB testing to two specific clinical cases: 1) estimated glomerular filtration rate (eGFR) value <15 mL/min, or 2) recent acute coronary syndrome (ACS) event. Per our ED’s policy, a “negative” troponin T was defined as being below the limit of detection (LOD) ; such a value obtained at least 3 hours after symptom onset “ruled out” an ACS event and did not require a repeat troponin.The goal of this retrospective study was to determine whether the guideline limiting CK-MB testing missed clinically-significant cardiac outcomes (ACS or new diagnosis of coronary artery disease [CAD]) or was associated with mortality. Pre-implementation data  was compared with post-implementation data . After guideline introduction, CK/CK-MB ordering decreased by nearly 90%, while troponin ordering increased by nearly 20%, likely due to the introduction in June 2018 of high-sensitivity troponin T, which yielded numerous intermediate/indeterminate-range results that prompted repeat testing. Fewer than 1.5% of patients with a “negative” troponin (below the LOD) and a “positive” CK-MB  had ACS or new-diagnosis CAD; patients with either diagnosis did not expire during their hospital stay or within 30 days of their index visit. CK-MB Index, which has a higher specificity than CK, only found ACS or new CAD among 0.8% of positive results. Considering both decreased CK/CK-MB and increased troponin ordering, the net annual direct cost savings in cardiac biomarker testing was extrapolated to $12,700. Had our institution not transitioned to higher cost high-sensitivity troponin ($2.054/unit) from standard troponin ($1.65/unit), and had the rate of troponin ordering increased solely proportionate to the rate of ED visit increase (2% year-over-year) rather than increase nearly 20% (likely due to the transition to high-sensitivity troponin), then the total six-month direct costs on troponin testing would have been $14,632 instead of $21,267.12, and annual direct cost savings would have been $18,945.80 instead of $12,700. The new ED clinical guideline did not result in a significant number of missed ACS or new-diagnosis CAD, and was associated with direct cost savings. These savings probably underestimate total savings, as the reduced number of “false-positive” CK-MB results likely prevented additional costs, such as hospitalization, specialty consultation, coronary calcium CT, echocardiogram, cardiac stress test, and coronary artery catheterization. The diagnosis of acute coronary syndrome (ACS), including acute myocardial infarction (AMI) and unstable angina, can be suggested through serologic testing of myoglobin, creatine kinase (CK), and creatine kinase-muscle/brain (CK-MB), CK-MB Index , and, more recently, troponin C, I, or T. Compared with CK/CK-MB and CK-MB Index, troponin levels more reliably rise in ACS. Troponin has several advantages over myoglobin, CK/CK-MB, and CK-MB Index. In AMI, myoglobin levels rise rapidly (within two hours), peak in 10 hours, and fall quickly 1-2 days). CK-MB levels rise more slowly (3-12 hours), peaking in 24 hours, and remaining elevated two-three days. Troponin levels rise within 3-12 hours, peak in 24-48 hours, and decrease over 5-14 days, providing a longer period of time in detecting AMI [ days. CKA study by Volz et al. analyzed over 11,000 patient encounters where cardiac biomarkers were ordered and found zero cases in which a patient was found to have had AMI when the CK-MB was positive and troponin negative . In 2014Troponin levels exist as a spectrum, with likelihood and severity of myocardial damage proportionate to troponin level. However, for operational purposes, organizations have adopted models that are either binary (“negative” or "positive/elevated") or with three categories: "negative," "intermediate/indeterminate," and "positive/elevated ." At ourIn April 2018, our Emergency Department (ED) created and adopted a clinical guideline consistent with the AHA/ACC guidelines, switching from standard troponin  to troponin T  without CK/CK-MB (Roche CK-MB by electrochemiluminescence immunoassay) testing in the majority of clinical situations. Per our ED’s policy, a “negative” troponin T was defined as being below LOD <6 ng/L); such a value obtained at least three hours after symptom onset “ruled out” ACS and did not require a repeat troponin. This policy was based on ED literature  ng/L; su and was In the new guideline, CK/CK-MB testing was limited to two specific clinical cases: 1) renal failure with an estimated glomerular filtration rate (eGFR) value <15 mL/min, and 2) if the patient had a known or suspected ACS within the previous two weeks. The exception for renal failure was because troponin is elevated in 100% of dialysis patients without active myocardial injury, whereas CK-MB is elevated in only 30-50% of such patients. An elevated CK-MB in a dialysis patient, therefore, more likely indicates an acute event . The excThe aims of this study were: 1) determine the percentage of patients in a large academic hospital with a largely under-served and non-English speaking population in whom the elimination of CK-MB testing would potentially have missed ACS events; and 2) determine whether implementation of the guideline to reduce CK-MB testing led to direct cost-savings. We conducted a retrospective chart review of patients with a visit between July 1, 2017 to December 31, 2017 and July 1, 2018 to December 31, 2018 to an academic, urban, tertiary-care ED with an approximate annual volume of 100,000 adult visits. In April 2018, our ED adopted the AHA/ACC guidelines for use of troponin instead of CK-MB, and in June 2018, we implemented high-sensitivity troponin T. All adult patients with joint troponin and CK-MB orders placed in the ED in these time periods were included in the sample. The final sample for retrospective chart review included patients with a “negative” troponin T  and “positive” CK-MB . Demographic information; disposition; chief complaint; past medical history; troponin and CK-MB results; whether a stress test or cardiac catheterization was conducted; and whether there were any clinically significant cardiac outcomes  were recorded during chart review. The main outcome was pre-defined as a new diagnosis of acute coronary syndrome (ACS) or coronary artery disease (CAD) during a patient’s ED/hospital visit, as determined by chart review. Patients without a history of ACS or CAD prior to the index ED/hospital visit, but whose index visit discharge summary indicated ACS or CAD, were considered to have new-onset ACS or CAD. Patients discharged from the ED were followed for 30 days post-ED discharge. Charts were reviewed for up to 30 days post-discharge from index visit to determine whether an ACS event had occurred during this timeframe; authors looked at ED and hospital discharge diagnoses to assess whether an ACS event had occurred. We excluded from analysis patients with clinically obvious ACS , as cardiac biomarker testing would not affect diagnosis or disposition. CK-MB Index was also evaluated for all patients, with a positive value being >2.5. Finally, we analyzed the absolute change in the number of troponin and CK/CK-MB lab tests ordered before and after implementation of the new ED guideline regarding CK/CK-MB testing and calculated changes in direct lab test costs to the institution.The pre-implementation period was the six-month period from July 1, 2017 through December 31, 2017, with the post-implementation period the six-month period from July 1, 2018 through December 31, 2018; troponin T was introduced in June 2018, shortly before the post-implementation period. These periods of time were coordinated to minimize any seasonal impact on testing patterns, patient volume, and house staff progression through their training. In acknowledgement that it often takes time to acclimate to new guidelines, we set the start date for post-implementation analysis three months after guideline introduction . Excluded from analysis were patients with eGFR value <15 mL/min . Per-unit cost of high-sensitivity troponin T ($2.054) and CK-MB orders ($1.054) were compared from 2017 pre-implementation period to the 2018 post-implementation period to assess for the potential direct financial savings associated with guideline implementation. As the transition from standard troponin to high-sensitivity troponin in June 2018 was planned well in advance, and independent of the CK-MB testing guidelines, we did not include in the financial estimates the costs of staffing and equipment for the new assay. A Chi-square analysis was used to determine significance for guideline adherence, as indicated by reduction in CK/CK- MB ordering.This study was deemed exempt by the Institutional Review Board as a quality improvement project to gauge compliance with and unintended consequences of transitioning to meet the standard of care for laboratory testing for ACS.During the initial six-month pre-implementation study period, 7,335 adult patients had both troponin and CK-MB orders placed in the ED, compared with 736 in the post-implementation period (90% decrease). There was a 19.1% increase in troponin ordering  had a “negative” troponin at least three hours after symptom onset and a “positive” CK-MB result. As per our ED policy , gender, and age were analyzed and not found to account for the decrease in CK/CK-MB order volume from 2017 to 2018 Table .Adoption of the new ED ACS evaluation guideline, which restricted CK-MB use in favor of a high-sensitivity troponin T strategy, was not associated with missing significant ACS events. During the six-month post-implementation period, only 6.9% of patients had a negative troponin with positive CK-MB. Of these, only 1.4% were diagnosed with either ACS or newly-diagnosed CAD; none died or had a significant complication while hospitalized. The new guidelines were also associated with a 90% reduction in the number of CK and CK-MB tests ordered. However, there was a 19.1% increase in troponins ordered, likely due to the introduction of high-sensitivity troponin two months after the CK-MB testing guidelines were implemented. Owing to this newer generation troponin T’s high sensitivity, up to one-third of samples result in intermediate/indeterminate values , in whicThis study is one of few that evaluated the safety of removing CK-MB and replacing it with troponin . Other sThis study has several limitations. First, the study was conducted at a single site and therefore might not be representative of the experiences of other institutions that have de-adopted routine CK-MB testing for ACS. Second, the study was done within a limited time frame; remote ACS events would not have been identified. Third, this study studied the behavior change of not ordering CK-MB for only a few months after introduction of the new guidelines; therefore, we do not know whether this behavior change will persist or whether providers will return to ordering CK-MB routinely. Finally, we did not determine indirect cost savings from elimination of CK-MB through the avoidance of “false positives” and subsequent downstream cascade of hospital admissions, diagnostic tests, and procedures.This study suggests that withdrawing routine-use CK/CK-MB and CK-MB Index in the evaluation of ACS in favor of an AHA/ACC-endorsed troponin-only strategy does not miss a significant number of ACS cases and is associated with direct cost savings. The cost savings we identified likely underestimate true cost savings from avoided downstream consequences of “false positive” CK-MB results .


CSF biomarkers are well-established for routine clinical use, yet a paucity of comparative assessment exists regarding CSF extraction methods during lumbar puncture. Here, we compare in detail biomarker profiles in CSF extracted using either gravity drip or aspiration.N = 36; mild cognitive impairment, N = 8; Alzheimer’s disease, N = 6).Biomarkers for β-amyloidopathy , tauopathy , or synapse pathology  were assessed between gravity or aspiration extraction methods in a sub-population of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study . Passing Bablock regression defined low beta coefficients indicating high scalability.Levels of these commonly assessed CSF biomarkers are not influenced by extraction method. Results of this study should be incorporated into new consensus guidelines for CSF collection, storage, and analysis of biomarkers. Dementia is a syndrome with a complex pathophysiology, characterised by a heterogeneous group of clinical features and pathological hallmarks  . In partFDA Tauvid [Pathological diagnosis can be achieved by regulatory approved β-amyloid (Aβ)-positron emission tomography (PET) and Tau-PET imaging methods . PET tecA Tauvid ) in adulCerebrospinal fluid (CSF), which surrounds the brain and the spinal cord, is considered as a “mirror” of the brain . CSF colCSF AD biomarker analysis, especially for inter-laboratory comparisons, has previously been hampered by variabilities across pre-analytical handling, assay design, and laboratory performance, exacerbated by the absence of consensus on how to collect, process, and store CSF . ConsequSeveral guidance papers have been published with the ultimate goal of generating a consensus on how to handle CSF sample for analysis of Aβ proteins , 22, 23.To objectively investigate potential effects of CSF collection methodology through LP when assaying AD biomarker concentrations, we provide a detailed comparison of gravity drip collection with aspiration using a polypropylene syringe during the same collection for each subject. All other pre-analytical and analytical aspects of the procedures for sample analysis were identical. We hypothesised that analyte concentrations would be unaltered by extraction method, not only for Aβ and tau proteins, but also for synapse proteins.N = 36; 70%), to have mild cognitive impairment  or to have AD  after clinical and neuropsychological assessments, conducted as previously described [APOE ε4) allele status were available for all participants. All data represented in this study are cross sectional.CSF samples from 50 participants of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study were collected using aspiration then gravity drip methods during the same LP visit. Participants were deemed either cognitively normal (CN) (escribed . Clinicatamena.com) spinal needle micro-tip , or a RapID set pencil point spinal needle  if there was difficulty with the fine needle. Initially, up to 6 mL of CSF was aspirated for routine microbiological/biochemical assessment and other concurrent studies, then 8 mL of CSF was collected by gravity drip into a 15 mL polypropylene tube , and placed onto wet ice. After gravity collection, a polypropylene syringe  was then used to aspirate 2 mL of CSF, which was then transferred to a second Greiner Bio-One188271 polypropylene tube, on wet ice. Samples were processed within 1 h by centrifugation  and the supernatant transferred to a new Greiner Bio-One188271 polypropylene tube before being aliquoted in 300 μL volumes into Nunc Cryobank polypropylene tubes . Samples were stored in liquid nitrogen vapour tanks and only thawed once, immediately before analysis. Prior to thawing, CSF was shipped on dry ice to ADx NeuroSciences and stored at − 80 °C until the biomarker analysis. The range of time taken to collect the CSF by gravity was 10–15 min and the range for aspiration was 0.5–1 min. Parameters of the sample collection and adverse incidents have been reported previously [CSF was collected by LP, in the morning, from overnight fasted participants, using protocols described in detail elsewhere . Brieflyeviously .Samples from the 50 participants were tested for six analytes in the facilities of ADx NeuroSciences: α-synuclein, Aβ1–42 (herein reported as Aβ42), Aβ1–40 (herein reported as Aβ40), total tau, BACE-1 and Ng (trunc P75)  were within the acceptance range as described on the certificate of analysis of the kit lot used. Criteria for blank value (OD < 0.100) and calibrator curves  were approved. In addition, three “in-house” QC samples  were included in each assay run for assessment of test run performance. The composition of the QC samples is based upon neat CSF obtained from a commercial source. QC1 and QC2 were samples obtained from an individual subject, while QC3 was composed of a pool of two individual CSF samples. Samples for this purpose were collected retrospectively, and no clinical diagnosis is available for these samples. After their preparation , samples were aliquoted and frozen. Before each run, an aliquot was thawed and included in the test run (duplicate testing).11C-Pittsburgh Compound B , 18F-florbetapir , or 18F-flutemetamol  tracers. The acquisition protocol for each radioligand has been detailed previously [Of the 50 samples, 49 had PET-Aβ imaging available acquired using either eviously –34. Brieeviously  and the APOE ε4 allele status), generalised linear modelling , and Kruskal-Wallis test  where appropriate. Biomarker comparisons between the two extraction methods were conducted using concordance correlations (CC), Passing Block Regression analyses, and Bland-Altman plot analyses. Paired t-tests were computed to assess possible differences between biomarker levels between extraction samples, with Box and Whisker plots demonstrating differences in biomarker means between clinical classification for both gravity drip and aspiration samples. Standardised differences between biomarker levels from aspiration and gravity drip extraction methods are presented via error bar plot  and 14.9% /Tau) for all 50 participants, concordance correlations (CC) were all greater than 0.85 Fig. . StrongeFurther testing of the concordance via the Passing Bablock method defined regression equations and plots  showed a reasonable spread of data points, with plots showing markers having only either one, two, or three points that fit outside ±1.96SD around zero Fig. . Disregap > 0.05, Table p values closer to one (p > 0.9), indicating smaller differences between extraction methods, while all ratio biomarkers achieved similar performance (p > 0.9). Similar to stratification by clinical classification, there were no statistical differences in biomarker levels found when stratifying data by PET-Aβ status  are affected by the selected biomarker or biomarker combination. Results are presented in Fig. Supplementary Table In this study, we aimed to assess the concordance in biomarker levels between gravity drip and aspiration CSF extraction methods. After investigating each of six individual and three ratio biomarkers using multiple concordance methods, it is clear that biomarker reliability is independent of CSF extraction method. For the majority of biomarkers, concordance correlation coefficients were greater than 0.95, with reduction in precision due to a few outliers and the small sample size. Overall repeatability was consistent within both the complete sample and across individual clinical groups.Assessment of biomarker outliers from both aspiration and gravity drip extracted samples showed that outlier values were independent of extraction method; i.e., the reason for the aberrant biomarker level was unrelated to extraction method. Furthermore, assessment of biomarker levels between extraction methods, stratified by either clinical classification or by PET-Aβ status, did not increase the variance in biomarker levels, strengthening the claim of stability across a range of different clinical or phenotypic populations.n = 20) [The minimal changes across extraction methods seen in most biomarkers, as compared to the variability between extraction methods for Aβ40 and Aβ42  . Some grn = 20) . The samThe method of CSF collection is an important step in the pre-analytical handling of CSF samples. While some investigators routinely use gravity drip, others use aspiration. Gravity drip has the drawback of unpredictable variation of collection times and may potentially take considerably longer than aspiration, thereby reducing feasibility in busy clinics. In dementia evaluation settings, the longer duration of CSF acquisition can be a particular problem for a patient with memory impairment or dementia since repeated reassurance and explanation may be required. Our results demonstrate that syringe aspiration does not have a significant effect on analyte concentrations and, therefore, should be acceptable and allow predictable and more rapid CSF collection.This report extends the data of Rembach et al.  by utiliThe current study is not without limitations. Firstly, regarding the extractions, all aspirations were performed subsequent to gravity drip collection as per AIBL protocol. Although reversal of collection order is thought unlikely to have any effect on biomarker levels, this notion has not been formally tested. A second limitation of this study was the relatively small number of subjects included, with varying number of samples per clinical classification. The small sample size for participants with MCI/AD may over represent the strength of the biomarker concordance between extraction methods, and future research needs to be performed with a larger sample size to specifically test this. The results of this study should therefore be interpreted in the context of these limitations.In summary, the current study provides strong evidence that key CSF protein biomarker measurements are not influenced by extraction through either the gravity drip or aspiration method, and that CSF results utilising either method are inter-changeable. Much time can potentially be saved and subject burden reduced using the syringe extraction approach compared to gravity drip. Results of this study should be incorporated into the new consensus guidelines for CSF collection, storage, and analysis of biomarkers.Additional file 1: Supplementary Table 1. Participant CSF value outliers. CN: Cognitively Normal, MCI: Mild Cognitive Impairment, AD: Alzheimer’s Disease, Box: Box and Whisker plot, BA: Bland Altman plot, Scatter: Scatter plot, APOE: Apolipoprotein E ε4 allele , Aβ; Amyloid beta, BACE1; Beta-secretase 1, MMSE; Mini mental score equivalent, CDR; Clinical dementia rating. *ID are random letters given to participants to show where specific individuals were seen to have more than one outlier. Visualisation: where outlier was seen in manuscript figure. Values were considered outliers if they were either outside the dotted lines in the Bland Altman plot, or above/below the whisker on the box and whisker plot, or sitting away from the main data group in the scatter plots. Supplementary Figure 1. Passing Bablock regression fits to biomarker data. Abbreviations: Aβ; Amyloid beta, BACE1; Beta-secretase 1. Diagonal lines represent the slope as calculated via the Passing Bablock method. Shaded grey areas represent the 95% confidence interval as calculated via bootstrap resampling. Light grey dashed line represents the identity line between bottom left and top right corners of the graph. Regression equation is shown in the top left for estimation of the conversion of a data point from aspiration to convert to the same scale as the gravity data point. For example, for any one point on the line that represents a result from a gravity drip extraction for the BACE1 biomarker, we would multiply the value by 1.02 and add the value of − 49.87; e.g., 2000 × 1.02 + (− 49.87) = 1990. Supplementary Figure 2: Box and whisker plots of median biomarker levels between extraction methods and PET-Aβ status. Abbreviations: Aβ; Amyloid beta, BACE1; Beta-secretase 1. Black boxes and points represent data from samples extracted using the aspiration method. Grey boxes and points represent data from samples extracted using the gravity drip method. Upper lines on each box represent the 3rd quartile, middle lines represent the median value and the lower lines represent the 1st quartile. All biomarker comparisons between gravity drip and aspiration by PET Aβ status were not significant .


SNCA rs3910105 genotype and RBD in Parkinson’s disease (PD) remains unclear.Rapid eye movement (REM) Rapid eye movement sleep behavior disorder (RBD) is a common non-motor symptom of PD. However, the association between the SNCA rs3910105 C carriers  and TT carriers (n = 83). They were followed up for 5 years to identify the development of RBD. To investigate the influence of cerebrospinal fluid (CSF) alpha-synuclein (α-syn) and β-amyloid 1–42 (Aβ42) in the association between rs3910105 and RBD, the patients were additionally classified into “high-level” and “low-level” groups using cutoff values for CSF α-syn and Aβ42 levels.This study used Parkinson’s Progression Markers Initiative (PPMI) data and included 270 patients with newly diagnosed PD without RBD who were divided into 42 levels than the TT group. During the 5.0-year follow-up, the rs3910105 C allele group had a higher incidence of RBD than the TT group. In the subgroup analyses, the effect of the rs3910105 C allele was not found in the “low-level” group. However, in the “high-level” group, the rs3910105 C allele independently increased the risk of RBD.At baseline, the rs3910105 C allele group had lower CSF α-syn and AβSNCA rs3910105 C allele might be a novel genetic risk factor for RBD development in PD, α-syn pathways might have a role in this association and more basic research would be needed to elucidate the mechanism in the future.The  Rapid ewith RBD . Given the brain , this obSNCA gene, is central to the pathogenesis of PD  database. The PPMI study is an ongoing international, multicenter, cohort study to identify biomarkers of progression in PD. The study protocol and methodology are available on the PPMI websitelsewhere . PatientWe downloaded data on PD patients from the PPMI database in December 2020. For this analysis, data up to 5 years of follow-up were included. We excluded patients who had RBD at the baseline visit or were functionally dependent at or were lost to follow-up in the first year following enrollment. Finally, a total of 270 patients were involved in this analysis.DNA was extracted from the whole blood of the patients according to the study protocol as described in the PPMI biologics manual. All sample were genotyped using the Illumina NeuroX array following the manufacturer’s protocol. The Genotyping Analysis Module within Genome Studio was used to analyze data .The presence of probable RBD was defined based on the RBD Screening Questionnaire (RBDSQ). The RBDSQ comprises 10 items investigating the patient’s sleep behavior. The total score is a maximum of 13 points. Only dichotomic responses (“yes” or “no”) were allowed. Dream frequency, content, and their relation to nocturnal movements and behavior were assessed by items 1–4. Item 5 evaluated the presence of self-injuries and injuries to the bed partner while asleep. Item 6 explored nocturnal vocalization, limb movements, complex movements, and objects that fall down around the bed. Items 7 and 8 assessed nocturnal awakenings. Items 9 and 10 investigated the presence of global disturbed sleep and any other neurologic disorder. As previously reported , an RBDS42 as previously described  at Penn. Commercially available sandwich type immunoassay kits  were used to analyze CSF a-syn and CSF hemoglobin levels.CSF was collected and analyzed for α-syn, total tau (t-tau), tau phosphorylated at Thr181 (p-tau), and Aβescribed , 2018. BFurther information can be found in the PPMI biologics manual (see text footnote 1).Motor symptom severity was assessed with the Movement Disorder Society–sponsored revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) Part III and Hoehn and Yahr (H&Y) scale, and non-motor symptom burden was assessed with MDS-UPDRS Part I. Global cognitive status was assessed using the Montreal Cognitive Assessment (MoCA). Domain-specific neuropsychological performance was measured using 7 standardized instruments. Verbal memory was assessed by the Hopkins Verbal Learning Test, with measures of immediate recall, delayed recognition false alarms, and delayed recognition hits. Verbal fluency was assessed by the semantic fluency test. Processing speed was assessed by the Symbol Digit Modalities Test. Visuospatial abilities were assessed by the Benton Judgment of Line Orientation Test. Executive functioning was assessed by the letter-number sequencing test. Depressive symptoms were assessed with the short version of the Geriatric Depression Scale (15-item GDS). Anxiety was assessed with Spielberger’s State-Trait Anxiety Inventory (STAI). Autonomic dysfunction was assessed with the Scales for Outcomes in Parkinson’s Disease–Autonomic (SCOPA-AUT). Sleep disturbances were assessed with the Epworth Sleepiness Scale. The Questionnaire for Impulsive Compulsive Disorders in PD was used to assess impulsive-compulsive disorders and other compulsive behaviors. Olfactory dysfunction was measured by the University of Pennsylvania Smell Identification Test (UPSIT). Clinical evaluation was performed in the “off” treatment state.t-tests continuous variables and chi-square tests or Fisher’s exact tests for categorical variables, as appropriate. We used the Kaplan-Meier method to compare the cumulative incidence of RBD between the groups. Cox proportional hazards regression models were used for an adjusted analysis of the relationship of the SNCA rs3910105 allele with RBD, and hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated. All baseline variables with p-values less than 0.2 in the univariable Cox models were included in subsequent multivariable Cox models with a backward elimination procedure . All p-values were two-sided, and a p-value of less than 0.05 was regarded as statistically significant. As for the cut off in the subgroup analysis, we used an optimum cutoff value for the CSF α-syn and Aβ42 levels using the R package “MaxStat,” which selects the cutoff value producing the maximum log-rank score. To increase stability of our findings, we also used the 25th and 50th percentile cutoff values of the CSF Aβ42 levels. All p-values were two-sided, and a p-value of less than 0.05 was regarded statistically significant.Calculations were performed with SPSS 25.0  and R version 3.6.2 . Data are presented as the mean ± standard deviation. Data normality was assessed with the use of the Shapiro-Wilk test. Baseline characteristics were compared with Student’s Among the 270 patients  included in this study, 186 (68.8%) were SNCA rs3910105 C carriers, and 84 (31.2%) were TT carriers. The baseline characteristics of the two groups are depicted in p = 0.019)  (p = 0.012) (p = 0.02)  , lower C= 0.015) , lower C= 0.012)  and lowe = 0.02)  than theSNCA rs3910105 C allele, higher MDS-UPDRS total and motor scores and lower CSF Aβ42 levels than those who did not develop RBD (p = 0.0068)  patients developed RBD, and the cumulative incidence of RBD was 18.1, 37.7, and 43.7% at the 1-, 2- and 4-year follow-ups, respectively . At baseelop RBD . There w 0.0068) .The results of the Cox regression analyses are shown in 42 could not be related to the future incidence of RBD based on the Cox regression analysis data, as shown in 42 levels were much lower in the rs3910105 C allele group than in the TT group. Since the RBD development rate was much higher in the C allele group than in the TT group, we speculated that lower baseline CSF α-syn and CSF Aβ42 might also contribute to the higher incidence of RBD. We divided the patients into two groups based on the CSF α-syn and CSF Aβ42 levels.Although the baseline levels of CSF α-syn and CSF Aβp = 0.0083)  had a significantly higher incidence of RBD than the “high-level” group  .p = 0.97) (p = 0.028)  . However= 0.028) .42 levels, the cutoff value of CSF Aβ42 in relation to RBD for producing the maximum log rank score was 385 pg/ml. When the patients were divided by this cutoff value, the “low-level” group (18/24 patients [75%]) had a significantly higher incidence of RBD than the “high-level” group  (Regarding CSF Aβ= 0.043) .p = 0.97) (p = 0.022)  . However= 0.022) .SNCA rs3910105 C allele was associated with faster development of RBD in PD patients, suggesting that the SNCA rs3910105 C allele was a novel genetic risk factor for RBD. To our knowledge, this was the first study to demonstrate the association of SNCA rs3910105 and RBD development in PD patients.This study investigated the relationship of the SNCA rs3910105 C allele with the development of RBD in patients with early PD using the PPMI cohort. Our results showed that the 42 levels  in the SNCA rs3910105 C allele group was much lower than that in the TT group. Furthermore, studies have found that CSF α-syn and Aβ42 were significantly lower in PD subjects with RBD  were lower in PD patients who later developed RBD, we did further subgroup analysis that divided the PD patients into low and high groups based on the baseline CSF pathology levels (α-syn and Aβ42), and it turned out that the “low-level” group did have a significantly higher incidence of RBD than the “high-level” group. However we found the effect of the rs3910105 C allele was not in the “low-level” group, which was possible considering the limited number of this group and a low CSF pathology baseline level that they already had, the rs3910105 C allele effect to keep the low level of CSF pathology and then increase the risk of RBD development as PD progress might not work. While in the “high-level” group, the effect was still there, the rs3910105 C allele prevalence contributed to a lower CSF α-syn and Aβ42 both at baseline and during the follow up study, thus increasing the risk of RBD during disease progress. which indirectly showed an association between CSF α-syn and Aβ42 levels and RBD incidence.Studies have shown that rs3910105 was associated with CSF Aβ2 levels , which wwith RBD . TherefoSNCA gene and is located in the CpG island of SNCA   . Howevericipants . We also = 0.44) , suggest42 levels and RBD incidence, but the Cox model did not work. We could only speculate this association may related with contributions made by rs3910105 C allele.There were some limitations to note in this study. First, the presence of RBD was based on results from the RBDSQ, while a definite diagnosis of RBD requires polysomnography (PSG). Because PSG is expensive and requires a skilled examiner, the RBDSQ is used more frequently in large cohort follow-up studies. In this context, the RBD data in the current study might have been misestimated. Second, even though using the subgroup analysis, we indirectly showed an association between baseline CSF α-syn and AβIn conclusion, our results suggest that the rs3910105 C allele is associated with an increased risk of RBD development in PD, and α-syn pathways might have a role in this association Further studies need to be conducted to validate our findings and to reveal the precise mechanisms underlying the influence of the rs3910105 C allele in the development of RBD in patients with PD.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material.The studies involving human participants were reviewed and approved by Parkinson’s Progression Markers Initiative (PPMI). The patients/participants provided their written informed consent to participate in this study.N-NY conceived and designed the project and prepared the writing of the first draft. S-SS designed and executed most of the statistical analysis and reviewed the first draft. TP executed some statistical analysis and prepared the figures and tables. Hl conceived and designed the project, make the critique, and reviewed the final draft. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Compared to the NDD− controls (379.8 ± 125.2 pg/mL), NDD+ patients showed, on average, highly significantly increased CSF αSyn , but without differences between NDD+ / EP+ and NDD+ / EP− subgroups (p = 0. 38). Moderate but highly significant association was observed between concentrations of αSyn and Tau  and pTau181 . Adjusted for diagnoses, age, and sex, subjects with more advanced neurodegeneration on neuroimaging showed significantly lower αSyn concentrations (p < 0.02). In the setting AD versus controls, the area under the receiver operating characteristic (ROC) curve was 0.804  with the sensitivity and the specificity of 0.863 and 0.618, respectively. αSyn in AD patients does not differentiate between subjects with- and without EP. Its increased average concentration reflects probably neurodegenerative process, and is not specific for any pathophysiologic mechanisms. Further studies are necessary to explain the role of CSF αSyn as a potential biomarker.Extrapyramidal symptoms (EP) are not uncommon in Alzheimer’s Disease (AD); when present, they negatively influence the course of the disorder. A large proportion of AD patients shows concomitant Lewy bodies’ pathology post mortem. Total α Synuclein (αSyn) concentrations are frequently increased in the cerebrospinal fluid (CSF) of AD patients, but are decreased in Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB). αSyn CSF concentrations in AD patients with EP (EP+) have not been reported so far. αSyn and the four Neurochemical Dementia Diagnostics (NDD) CSF biomarkers, , interpreted according to the Erlangen Score algorithm, were measured in patients with positive NDD results and presence of extrapyramidal symptoms (NDD + / EP+;  In contrast to well established and generally accepted pattern of the cerebrospinal fluid (CSF) biomarkers for the diagnosis of Alzheimer’s Disease (AD), which includes decreased values of Aβ1-42 and Aβ42/40, and increased concentrations of Tau and pTau181 . We contrasted the results with those of the control group consisting of patients without alterations in the core CSF AD biomarkers and without EP.n = 80), and the subjects with ES ≤ 1 (n = 34) were categorized as NDD− controls. Furthermore, the NDD+ subjects were split into a subgroup with EP , including: bradykinesia (in 10 subjects), muscle rigidity with cogwheel or lead-pipe muscle rigidity (in 6 subjects), concurrence of both (in 3 subjects), and asymmetric tremor (in 7 subjects), and subgroup without EP (NDD+ / EP− n = 54). Extrapyramidal symptoms did not cause pronounced motor and non-motor abnormalities and did not meet the criteria for atypical Parkinsonism. There were no episodes of psychotic productive symptoms in the study group. Neurologic conditions were assessed by two independent consultants experienced in neurogerontology.The study was approved by the ethical committee at the Medical University of Wrocław, Poland. The patients were recruited at the Diagnostic and Research Center for Alzheimer’s disease, Ścinawa, Poland. The AD diagnoses were performed according to the NIA-AA criteria , routine neuroimaging was performed with Signa HD XT 1.5 T scanner . For the evaluation of the structural alterations, Medial Temporal lobe Atrophy (MTA) scale was applied , according to the vendor's protocol. In preliminary experiments, four samples with different αSyn concentrations were pre-diluted with assay's buffer 1:4, 1:8, 1:16, and 1:32 to test for linearity and precision of the method. Resulting from this experiment, 1:8 dilution was chosen. All analyses were performed in duplicates. To test for inter-assay precision, a quality control (QC) sample of human pooled CSF was used.The number of subjects for the study was estimated under the condition of Satterthwaite's test for the populations with non-equal variances, with between-groups differences approximated according to the data in the literature  the total NDD+ group versus NDD− controls, with Helmert contrast, and (b) the NDD+ with EP (NDD+ /EP +) group versus NDD+ without EP (NDD+ / EP−) group. Correlations between continuous variables, unadjusted for other covariates, are presented as Pearson's correlation coefficients (r).If not stated otherwise, the results of the continuous variables are presented as averages ± standard deviations (SD) or 95% confidence intervals (95% CI). The results of the categorical variables are presented as percentages, and compared to one another with χAssociations between CSF αSyn concentrations and the disease categories, adjusted for explanatory variables, were analyzed with a series of linear regression models. First, a model with clinical− neurochemical categorization only was fitted (M0), subsequently supplemented with the demographic data (M1), supplemented further with the time of the disease duration and the MMSE test score (M2), and finally with the presence of the metabolic syndrome (diabetes mellitus type 2 and/or obesity) and the results of the MTA score (M3). If the numbers of the patients in the models were equal, the models were compared with the Likelihood Ratio test (LRT); otherwise the models were compared with the Akaike's Information Criterion (AIC). Taking into account the results of the models M0 − M3, in the final regression model (M4) age, sex, and MTA results were retained. Finally, interactions between the explanatory variables and the disease categorization variable were tested.The αSyn concentration cut off, optimized for the separation of NDD− and NDD+ groups, was obtained at the maximized Youden index. The area under the receiver operating characteristic (ROC) curve (AUC) was calculated with nonparametric method. Logistic regression was used to model the sensitivity and the specificity as a function of covariates, according to Coughlin et al. .A Demographic and neuropsychologic data of the patients, and the concentrations of their core CSF biomarkers, are presented in Table p = 0.14) nor presence of the metabolic syndrome (p = 0.22) differed significantly between the two groups. None of the metrics differed significantly between the NDD/EP + and the NDD/EP− patients.Briefly, NDD+ patients compared to the NDD− controls were on average significantly older, had expectedly lower concentrations of Aβ1-42 and Aβ42/40 ratio, higher Tau and pTau181 concentrations, and lower MMSE score. There were no statistically significant differences in Aβ1-40 concentrations between the two groups. The distributions of neither gender (p = 0.054) was observed.The distribution of the MTA scores is presented in Table Four dilution factors  tested in the preliminary experiments resulted in practically the same concentrations (after taking the multiplication factor into consideration), as well as neglectable differences of the intra-assay imprecision (data not shown), reconfirming the linearity and the precision of the method. Median of the ranges-to-averages of the duplicate measurements of the 114 patients CSF samples was 2.1%, with inter-quartile range: 1.0–3.6%; CV of the average results of the QC sample in the four analytical runs (i. e. the inter-assay imprecision) was 7.6%.p < 0.01). We did not observe statistically significant difference between unadjusted αSyn CSF concentrations in NDD+ / EP+ and NDD+ / EP− patients , Aβ1-40 , Aβ42/40 , Tau , and pTau181 , but not with Aβ1-42 .We observed statistically highly significant difference between unadjusted average αSyn CSF concentrations in NDD+ patients and the NDD− controls p < 0.0. We did p < 0.01), but there were no significant difference between NDD+ / EP + and NDD+/ EP− patients . Adjusted for other covariates, female gender , age , and MTA score (p < 0.02) were significantly associated with αSyn concentrations. Interestingly, keeping other covariates constant, patients with the MTA sore 3 , and the MTA score 4  had lower average αSyn concentrations than those with the MTA score 0. We did not observe such association between higher MTA scores and alterations of the concentration of Tau, conditional on other covariates (data not shown). Regression coefficients from this model are presented in Fig. In the regression models M0–M3  were considered the major categorization criterion, with the patterns of the CSF biomarkers interpreted according to the Erlangen Score algorithm , conditional on the diagnoses, age, and sex of the subjects. A simple explanation that more intense neuronal loss leaves behind less neurons capable to act as a source of αSyn is probably oversimplification, as such association was not observed for Tau. Hence, it might be speculated that the (passive) release of αSyn and Tau from degenerating neurons follows different patterns. Interestingly, Mackin et al., following their findings of the reduced αSyn CSF concentrations in AD patients with accelerated overall cognitive deterioration, also concluded that the CSF αSyn may be reduced as an effect of an overall neuronal or synaptic loss . This most probably clarifies why we observed decreased MMSE score in some of these subjects, which merely reflects their afflicted mood and mild anxiety. We also need to stress that the CSF-based categorization is not without limitations. Lumbar puncture is often regarded as an invasive procedure, and the NDD biomarkers characterize with high, but not one hundred percent, accuracy. Therefore, we believe that the diagnosis, and the classification of subjects in research studies, always requires a multi-expertise team including clinical neurochemist, neuropsychologist, neuroradiologist, and—last but not least—an experienced clinician. Certainly more studies are necessary to confirm our findings.


Cerebrospinal fluid (CSF) leaks are often the result of trauma or recent surgical procedures; however, a subset can develop from non-traumatic etiologies. Cerebrospinal fluid leaks from congenital and spontaneous encephaloceles can be clinically occult and have devastating consequences if undetected for prolonged periods of time. This report highlights a unique case of meningitis after CSF leak caused by ruptured congenital meningocele during a routine nasopharyngeal swab.A 54-year-old female with diagnosed CSF leak presented to the emergency department (ED) with acute onset of severe headache, and neck and back pain. Prior to this presentation, the patient had experienced two months of persistent headache and rhinorrhea since her coronavirus disease 2019 (COVID-19) nasopharyngeal swab. As part of her outpatient workup, an otolaryngology consultation with subsequent beta-2 transferrin testing and magnetic resonance imaging was performed and she was diagnosed with a CSF leak from ruptured congenital meningocele. On ED presentation, she was afebrile, but with mild tachycardia, leukocytosis, and meningismus. Lumbar puncture revealed acute streptococcal meningitis. This patient’s meningitis developed due to prolonged occult CSF leak after her COVID-19 nasopharyngeal swab ruptured a pre-existing congenital meningocele.Nasopharyngeal swabs are being performed much more frequently due to the COVID-19 pandemic. All front-line providers should be aware of the potential presence and rupture of congenital meningoceles in patients who have undergone recent nasopharyngeal swab when risk-stratifying for potential CSF leak and meningitis. Cerebrospinal fluid leaks (CSF-L) are rare but can occur in patients of all ages with potentially devastating consequences if not diagnosed and treated expeditiously. Most notably, the abnormal communication between the subarachnoid space and the extracranial space poses significant risk for meningitis. Meningitis, which often occurs within a year from onset of the leak, has been found to complicate almost 20% of patients with persistent leak with 10% mortality. 2Basal encephaloceles are lesions consisting of herniation of intracranial contents – meninges alone (meningoceles); brain tissue ; or a ventricle  – through a defect in the skull base. Congenital basal encephaloceles have an incidence of 1 in 35,000 live births (higher in Southeast Asia), but can also occur spontaneously. 9We describe a case of a patient who presented to the ED with acute bacterial meningitis as a complication of CSF-L caused by ruptured meningocele during a routine nasopharyngeal (NP) swab for coronavirus disease 2019 (COVID-19).A 54-year-old obese female with a history of antiphospholipid syndrome on apixaban and recently diagnosed with CSF-L presented with acutely worsened headache and new-onset neck and back pain for one hour. She had photophobia, chills, and worsening of pain with any flexion of the spine. She denied a history of trauma, drug use, ill contacts, or recent travel. Two months prior, she had a NP swab of her left nare for COVID-19 during which she described a “popping sensation and intense pain.” Since the NP swab performance, the patient had experienced waxing and waning headache, left-sided rhinorrhea, post-nasal drip, cough, and intermittent fever. After failed therapies for pneumonia and allergies, she was referred to an otolaryngologist who performed a beta-2 transferrin test on her nasal discharge, confirming a diagnosis of CSF-L. After rigid nasal endoscopy was unrevealing for a source, magnetic resonance imaging (MRI) revealed small bilateral olfactory recess meningoceles . She wasWhat do we already know about this clinical entity?Cerebrospinal fluid (CSF) leaks are relatively rare but do carry the complicating risk of secondary bacterial meningitis with a high associated mortality. Though rare, presence of encephalocele may increase risk of CSF leak as complication to nasopharyngeal (NP) swab performance.What makes this presentation of disease reportable?Incidence of basal encephaloceles is low at 1 in 35K live births. However, due to the widespread use of NP swabbing in the coronavirus disease 2019 pandemic, the potential exposure of these lesions to potential harm is great.What is the major learning point?Nasopharyngeal swab performance is not without risk, so care should be used educate performers on the appropriate technique. Onset of severe pain with persistent rhinorrhea after performance should prompt evaluation for CSF leak.How might this improve emergency medicine practice?Increased caution when performing NP swab may lead to decrease in complications, or earlier intervention for complications if recognized early.3 cells/microliters (uL) (reference range: 4.1–10.7 × 103 cells/uL) with a neutrophilic predominance. She was given intravenous (IV) dexamethasone, cefepime and vancomycin, and lumbar puncture was performed. The laboratory analysis revealed cloudy fluid, a glucose level of less than 10 milligrams per deciliter (mg/dL) (reference range: 40–70 mg/dL), protein level of 732 mg/dL (reference range: 15–45 mg/dL), and nucleated cell count of 2764/uL (reference range: 0–5 cells/mL) with 87% segmented neutrophils. The CSF Gram stain revealed Gram-positive cocci, and CSF culture grew Streptococcus salivarius. While awaiting admission, the patient developed a mild encephalopathy, which resolved over the subsequent 24 hours with continued antibiotic therapy.On ED presentation, the patient was afebrile, although mildly tachycardic. With the exception of meningismus, her exam, including neurologic exam, was normal. Her white blood cell count was 19 × 10During her hospitalization, the patient underwent bilateral endoscopic ethmoid repair with lumbar drain placement by otolaryngology and neurosurgery. Her hospital course was complicated by post-repair re-leak and development of ventriculitis. She had a ventriculostomy placed for treatment with a short course of intrathecal antibiotics in addition to a prolonged course of IV antibiotics. Once her CSF studies cleared, she underwent ventriculo-peritoneal shunt placement. Her clinical status improved, and she was ultimately discharged home with minimal neurologic sequelae.This patient had pre-existing, undiagnosed congenital meningoceles, the larger of which was iatrogenically ruptured during her NP swab for COVID-19, resulting in a CSF-L that remained undetected for two months until diagnosed by otolaryngology. While awaiting surgical repair, the patient ultimately developed meningitis requiring a lengthy hospital stay and prolonged antibiotic administration.To our knowledge, this is the first reported case of a serious complication of a NP swab, despite performance instructions mentioning potential risk of injury. Nasopharyngeal swabs are routinely used for the collection of specimens from the surface of the respiratory mucosa to aid in diagnosis of infection, most often for influenza, respiratory syncytial virus, parainfluenza, and now COVID-19. There are no specific contraindications for performing a NP swab, other than exercising caution if the patient has undergone recent nasal trauma or surgery. This case also highlights congenital and spontaneous encephaloceles as potential risk factors for CSF-L and meningitis. Most emergency providers are aware of traumatic skull fracture and encephalocele, subsequent CSF-L, and risk for meningitis. This case argues that it is prudent for emergency providers to also consider the presence of often clinically occult congenital and spontaneous encephalocele(s) when risk-stratifying a patient for CSF-Land meningitis.Congenital encephaloceles are due to a defect in the skull base usually resulting from defective ossification during development. Because of this, they are usually associated with other congenital anomalies. Providers should consider undiagnosed encephalocele in patients with a history of midline facial dysmorphisms, such as hypertelorism, broad nasal root, or cleft lip or palate, as well as optic disc anomalies. 7An additional takeaway from this case is the importance of recognizing the signs and symptoms of CSF-L to facilitate early diagnosis. The most common clinical symptoms of leak are headache, clear rhinorrhea or otorrhea, and salty post-nasal drip. 2Once a leak is confirmed, the imaging modality of choice to evaluate for dural or osseous defect is most often high-resolution computed tomography (CT), which has a reported sensitivity of 89%. 12This case demonstrates the presence of an uncommon congenital anomaly that suffered injury during testing for COVID-19, complicated further by a secondary infection resulting in bacterial meningitis. Although such anomalies are rare, the drastic increase in the performance of nasopharyngeal swabs during the COVID-19 pandemic created an increased risk of iatrogenic injury and associated complication. Emergency physicians and other front-line providers should be aware of symptoms concerning for a cerebrospinal fluid leak following NP swab, as early identification and surgical intervention may help avoid serious complications.