Wearable devices play an increasing role in the rehabilitation of patients with movement disorders. Although information about muscular activation is highly interesting, no approach exists that allows reliable collection of this information when the sensor is applied autonomously by the patient. This paper aims to demonstrate the proof-of-principle of an innovative sEMG sensor system, which can be used intuitively by patients while detecting their muscular activation with sufficient accuracy. The sEMG sensor system utilizes a multichannel approach based on 16 sEMG leads arranged circularly around the limb. Its design enables a stable contact between the skin surface and the system’s dry electrodes, fulfills the SENIAM recommendations regarding the electrode size and inter-electrode distance and facilitates a high temporal resolution. The proof-of-principle was demonstrated by elbow flexion/extension movements of 10 subjects, proving that it has root mean square values and a signal-to-noise ratio comparable to commercial systems based on pre-gelled electrodes. Furthermore, it can be easily placed and removed by patients with reduced arm function and without detailed knowledge about the exact positioning of the sEMG electrodes. With its features, the demonstration of the sEMG sensor system’s proof-of-principle positions it as a wearable device that has the potential to monitor muscular activation in home and community settings. Wearable sensors have increasingly been incorporated and used in the rehabilitation of patients who suffer from diseases with associated movement restrictions. The advantage here is that wearable sensors provide real-world data on patients’ movement performance. Wearable sensors are used in various contexts, both for monitoring purposes at home and in community settings. Applications focus on the assessment of treatment efficacy, early detection of disorders, prevention and home rehabilitation ,2 as welBesides physiological and biochemical sensing, the detection of motion with wearable sensors is particularly prevalent. Here, accelerometers or inertial measurement units  are mainThere are certainly many reasons why the translation of sEMG into clinical application is limited . In the In order to enable autonomous use of the sEMG sensor by the patient in rehabilitation settings, the sensor design has to meet specific requirements. These concern on the one hand patients’ ability to apply the sensor by themselves and on the other hand the minimization of the risk of a misplacement of the sensor. With regard to the first requirement, it should be noted that the functionality of the upper extremity may be considerably limited, especially in patients with movement restrictions due to neurological diseases. To ensure usability by patients with limited arm function, the sEMG sensor should be attachable with one hand. This is achieved with many of the active sEMG sensors currently used in rehabilitation ,22,23,24The second challenge sEMG faces when monitoring exercises and everyday movements is that the signal has to be recorded in non-isometric conditions. This not only creates special requirements for signal processing , it alsoTo summarize, the autonomous use of sEMG sensors by the patient in the context of rehabilitation is still a problem. Three particular challenges are to ensure easy handling of the device, if possible with one hand, sufficiently correct positioning of the sensor relative to the muscle and to avoid motion artifacts. Presently, there is no sEMG sensor or sensor system available that meets the requirements for usability and signal quality. This paper aims to demonstrate the proof-of-principle of an innovative sEMG sensor system, which can be used intuitively and independently by patients but yet allows the accurate and reliable measurement of muscular activation. The sEMG sensor system introduced here, fulfills the SENIAM recommendations for sEMG recording and it will be shown that it is not sensitive to incorrect positioning. Therefore, it enables the autonomous application by patients while allowing a reliable detection of the muscular activation of different muscles during everyday tasks and therapeutic exercises.The sEMG sensor system was designed to be a simple-to-use and size-adaptive device. Its properties should allow for the user to independently put on and take off the device even in the presence of reduced arm function. Since laypersons cannot ensure correct positioning of the sEMG electrodes, a multichannel approach was chosen. The multichannel approach assumes that if the electrodes are randomly positioned by the user, the likelihood that at least one sEMG channel is sufficiently correct positioned increases. Therefore, the sEMG sensor system consists of a total of 8 sEMG sensor modules, which are arranged in rotationally symmetrical ring form A. Each sTo ensure that the sEMG sensor system can be positioned and operated by any patient autonomously, the sEMG sensor modules are connected by extension springs A. When tA data cable and power supply runs through all sensor modules A. It is The sEMG sensor system can be effortlessly released by pressing the rotary knob. The spring force of the extension springs pushes the sEMG sensor modules apart and the elastic bands unroll from the rotary knob. This increases the circumference of the armband and it can be easily removed from the limb. Each individual sEMG sensor module has a size of 15 mm × 25 mm × 50 mm (height × width × depth) and is equipped with seven dry cap-electrodes. The cap-electrodes are gold plated and have a hemispherical shape. The active surface of the electrodes has a diameter of 8 mm and protrudes above the case of the sEMG sensor module by 2 mm A. The shSince a correct localization of the sEMG electrodes cannot be ensured when used autonomously by the patient, each sEMG sensor module provides two sEMG channels. These are obtained by three active electrodes arranged along the center axis of the sensor module A buildinTo reduce motion artifacts and noise, the sEMG signals are pre-processed directly at the electrodes. For this purpose, each sEMG sensor module contains its own circuit B. An impA special challenge is represented by the ground electrode, since all interferences that occur at the ground electrode are reflected in each sEMG channel. This is especially the case for motion artifacts. For this reason, each sEMG sensor module is equipped with four additional cap-electrodes, which are symmetrically positioned in a rectangular arrangement on the outside of the active electrodes array A. Like tRotational symmetry and the high number of sEMG channels increase the likelihood, that at least some sEMG channels are correctly positioned and aligned to the muscles for which muscular activation is to be determined. Since the sEMG sensor system provides 16 bipolar sEMG channels, it is necessary to determine those channels that best represent correct muscle localization. Identification is done by the microcontroller . Only thThe first step in identifying the relevant channels is to determine the number of muscles from which the muscular activation is to be recorded. Afterwards, an arbitrary calibration movement has to be performed by the subject. All 16 sEMG signals, derived during the calibration movement, are band pass filtered , full wave rectified and smoothed with a moving average 80 ms sliding window to obtain the envelope of the sEMG signal. The envelopes resulting from each of the 16 sEMG channels are divided into epochs of 80 ms duration. Afterwards, the root mean square (RMS) value of all envelope samples belonging to a single epoch is obtained .The assumption that an appropriate electrode location relative to the excited muscle is characterized by a high and stable RMS value throughout the movement, suggests a hierarchic approach to identify the relevant channels. Therefore, for each epoch the 16 sEMG channels are ordered hierarchically starting with the highest RMS value. The sEMG channel that reached the highest RMS value in most epochs, was identified as the first relevant channel . In ordeIt is obvious that the described procedure is not suitable to assign a specific muscle to each sEMG channel. The detection of 16 sEMG channels was motivated by the fact that the sEMG sensor system should be applied independently by the subjects and that a greater number of derived sEMG channels, increases the likelihood that at least a few of them are correctly positioned relative to the muscles or muscle groups involved in the movement. Therefore, the approach presented here is rather intended to identify one, maximal three sEMG channels to which the activation of individual muscles can be reliably assigned. The number of channels to be considered as relevant ones depends on the number of muscles from which a sEMG signal is to be derived or can realistically be derived. When the relevant channels have been determined by means of the algorithm for identification of the relevant sEMG channels, the microcontroller reads only these channels at the A/D converter and transfers them to the external storage device according to the data transmission protocol. This reduction of the sEMG channels from 16 to one or three enables the realization of high sampling rates and wireless data transmission for further processing.The proof-of-principle of the sEMG sensor system was demonstrated using the example of a voluntarily performed elbow extension/flexion movement. The measurement protocol comprised two parts. First, the subjects were asked to position the sEMG sensor system by themselves at the level of the biceps on their dominant arm. Thus, the localization of the sEMG channels around the upper arm at the level of the biceps was arbitrary. No skin preparation was done before the assessment. The position of the sEMG sensor system was photographically documented at the start of each measurement. After putting on the sEMG sensor system and waiting for 60 s, the sEMG signals were recorded with the arm at rest over a period of 20 s. Based on this static measurement, the noise level of the sEMG sensor system was determined separately for each channel. Afterwards the subjects were strapped to a pulley machine (5 kg load) so that the axis of the elbow coincided with the central axis of a deflection pulley. In this way, a constant torque was ensured over the entire range of motion . SubjectFrom the 16 recorded sEMG channels, the first relevant sEMG channel was determined for each positioning of the device according to the algorithm described in ® Blue Sensor N, Ambu GmbH, Bad Nauheim, Germany). The electrodes were placed on the biceps by an experienced user following the recommendations of SENIAM. After preparation, the noise level at rest as well as the biceps sEMG during five trials with five repetitions of elbow flexion and extension movements each was measured.The second part of the protocol involved the use of a commercial sEMG system  using self-adhesive Ag-AgCl bar shaped pre-gelled surface electrode pairs with an inter-electrode distance of 2 cm , full wave rectified and smoothed with a moving average 80 ms sliding window to obtain the envelope of the sEMG signal. Mean plus 10-fold standard deviation of the sEMG envelope was chosen as a threshold to determine the phases of muscular activation in the sEMG signal. The sEMG envelope’s RMS was calculated during phases of muscular activation in order to quantify signal energy. In addition, the signal-to-noise-ratio (SNR) was determined by relating the RMS of the sEMG envelope during the phases of muscular activation to the RMS of the sEMG envelope resulting from the measurement at rest.The measurements were performed by 10 healthy subjects . Exclusion criteria included upper limb disorders and left-handed subjects. In the case of the sEMG sensor system this results in 50 (5 positions × 10 subjects) different positions of the device, combined with a 50-fold determination of the first relevant channel, 50 RMS values for the phases of biceps activation in the first relevant SEMG channel and a 50-fold determination of the SNR of the system. The study was approved by the Human Ethics Committee of the RWTH Aachen University and all subjects gave written informed consent prior to the study.All subjects included in the study were able to put on and take off the sEMG sensor system independently. The position at which they located the armband corresponded to the SENIAM recommendations for sensor location on the biceps . The combination of extension springs, elastic band and rotary knob resulted in parallel alignment of the sEMG sensor modules with the bony axis of the upper arm in all cases. During the execution of the elbow flexion/extension movements, movement artifacts in the sEMG signals occurred only rarely, even at high movement speeds. The contact of the electrodes to the skin surface was maintained even if the form of soft tissue in the upper arm changed during the contraction and relaxation of the biceps. This issue was particularly important in the case of athletic subjects. None of the subjects complained about discomfort from the device during the measurements. p = 2.6 × 10−7 when using a one-sided, paired Student’s t-test. As was to be expected, the RMS values varied considerably between the different subjects. However, with the exception of subjects no. 2 and no. 8, all other subjects showed a high RMS value in the first relevant channel that was matched by a high RMS value when using the commercial system (pre-gelled electrodes) , in phases of muscular activation the RMS values of the first relevant channel of the sEMG sensor system  were generally higher . This dictrodes) . When coctrodes) . This isctrodes) . This dip = 0.003. When the sEMG sensor system using dry electrodes was used, the SNR obtained was between 71.15 dB and 4.08 dB, whereas it was between 67.93 dB and 10.87 dB when using the system based on pre-gelled electrodes. Similarly to the RMS, there are large inter-individual differences in SNR between the individual subjects, whereby, with two exceptions (subjects no. 3 and no. 4), a high individual SNR value in the first relevant channel of the sensor system is always accompanied by a high SNR value when detected with pre-gelled electrodes and vice versa (p = 0.009) when using the commercial sEMG system (pre-gelled electrodes) (mean STD 3.50 ± 3.49 dB) (ce versa . Intra-i3.49 dB) .The main objective of this paper was the demonstration of the proof-of-principle of a sEMG sensor system, which can be used intuitively and autonomously by the patients and still allow a reliable and accurate detection of muscular activity, which is comparable to recordings complying with the SENIAM recommendations. These characteristics as well as the fact that the design of the sEMG sensor system ensures that it can be applied easily with one hand, provide the sEMG sensor system with great advantages compared to other armband designs mentioned in the introduction and even textile applications. The introduced device has a rotationally symmetric design of eight sEMG sensor modules, which provide a total of 16 bipolar sEMG channels. The gold coated, dry, cap electrodes have a diameter of 8 mm and an inter-electrode distance of 20 mm. Thus, the minimum requirements of the SENIAM recommendations for the electrode arrangement are fulfilled by the device . The choIn a total of 50 trials on 10 healthy volunteers it was shown that the device can be put on and taken off independently by the subject, even if one arm was not used to support the donning and removal process. Thus, it was demonstrated that the device can also be used by persons with  paralysis of one limb. This is an essential requirement for applications in the field of rehabilitation. However, the most important prerequisite for autonomous usage by the patient is to ensure sufficiently correct localization of the sEMG electrodes relative to the muscle from which the sEMG signal is to be derived. Incorrect localization of the electrodes affects the signal amplitude as well as the signal dynamics and carries the risk of crosstalk from adjacent muscles ,27,28. TAs was to be expected, the RMS values as well as the SNR of both methods vary considerably among the individual subjects. This is most likely due to subject specific differences such as skin impedance, distance between muscle and electrode or percentage of the maximum voluntary contraction (MVC) required to lift the 5 kg weight. Furthermore, an influence of a subject specific predisposition to crosstalk cannot be excluded. All these subject specific influences have effects on the RMS of the sEMG signal and therefore on the SNR, too . In addiThe proof-of-principle is given using the example of biceps activation during an elbow flexion/extension movement. However, the presented principle can be scaled up and down without any problems. Thus, by just adding more sEMG sensor modules, extremities with a larger circumference and more muscles can be analyzed. At the same time, an extension in distal/proximal direction is possible by enlarging the modules in longitudinal direction. The addition of further sEMG sensor systems enables the simultaneous examination of different segments of an extremity B as wellAlthough there is a significant group difference in the RMS values as well as in the SNR, the respective values are in a similar range. This means that the signal quality achieved with the sEMG sensor system is comparable to that of a commercial system using pre-gelled electrodes. According to Sinderby, the signal quality of the sEMG signal is considered poor if the SNR falls below a threshold of 15 dB . In the Surprisingly, the RMS values of sEMG signals obtained by the sEMG sensor system were significantly higher than RMS values of sEMG signals derived with the commercial system using pre-gelled electrodes. There is no direct explanation for this, since dry electrodes are usually associated with a higher electrode impedance ,18. MotiThe sEMG sensor system introduced has demonstrated, in experimental validation, its proof-of-principle that it is able to reliably obtain high quality signals even when applied by unexperienced users without detailed knowledge about the exact positioning of the sEMG electrodes and with  paralysis of one limb. Electrode size and inter-electrode distance in each sensor module correspond to the SENIAM recommendations. The use of extension springs and elastic bands leads to a stable contact between the skin surface and the electrode and, together with the rotational symmetry of the device, limits movement artifacts even in movements with high acceleration. The chosen multi-channel approach ensures that the electrodes of a few of the 16 sEMG leads are positioned relative to the muscle with sufficient accuracy. To detect the relevant sEMG channels, a simple identification algorithm was implemented on the sensor system’s microcontroller. After identification of the relevant channels, only their data are processed in order to achieve a high temporal resolution of up to 2000 Hz. Despite the progress made, there is still room for improvement. First and foremost, it would be desirable to increase the density of the sEMG leads in relation to the circumference of the sEMG sensor system’s circular form. This would allow a more precise positioning of the electrodes relative to the activated muscle and thus contribute to both an improvement in SNR as well as a reduction of the risk of cross-talk. For this purpose, the use of electrode arrays would be appropriate, but it must be ensured that they can be independently utilized by patients with disabilities. Secondly, the experimental evaluation of the sEMG sensor system should be extended to more complex movements involving more muscles. It is to be expected that, under such conditions, the algorithm for identifying the relevant sEMG channels will need to be refined, including for the identification of malfunctioning electrodes. The mechanical and electrical components of the sEMG sensor system allow for these modifications and it would even be possible to use two sEMG sensor systems to obtain information from several segments of the limb B. Despit


LRRK2) gene mutations constitute an ideal population for discovering prodromal biomarkers of Parkinson’s disease (PD). In this study, we aim to identify CSF candidate risk biomarkers of PD in individuals with LRRK2 mutation carriers.Asymptomatic carriers of leucine-rich repeat kinase 2 (n = 23) and asymptomatic (n = 51) LRRK2 mutation carriers, subjects with a clinical diagnosis of PD (n = 60) and age-matched healthy controls (n = 34). General linear models corrected for age and gender were performed to assess differences in CSF biomarkers between the groups. Markers that varied significantly between the groups were then analyzed using backward-elimination logistic regression analysis to identify an ideal biomarkers panel of prodromal PD.We measured the levels of CSF total- (t-), oligomeric (o-) and phosphorylated S129 (pS129-) α-syn, total-tau (tTau), phosphorylated threonine 181 tau (pTau), amyloid-beta 40 (Aβ-40), amyloid-beta-42 (Aβ-42) and 40 inflammatory chemokines in symptomatic (LRRK2 mutation carriers from both symptomatic PD and healthy controls. Assessing the discriminative power using receiver operating curve analysis, an area under the curve > 0.80 was generated.Discriminant function analysis revealed low levels of CSF t-α-syn, high levels of CSF o-α-syn and TNF-α best discriminated asymptomatic The current study suggests that CSF t-, o-α-syn and TNF-α are candidate risk biomarkers for the detection of PD at the prodromal stage. Our findings also highlight the dynamic interrelationships between CSF proteins and the importance of using a biomarkers’ panel approach for an accurate and timely diagnosis of PD. While the exact involvement of LRRK2 in PD pathogenesis remains only partially understood, converging evidence suggests a role for LRRK2 in modulating inflammation [LRRK2 mutations and inflammation.Our understanding of the genetic basis of Parkinson’s disease (PD) has increased tremendously over the past 20 years. Mutations in the gene encoding alpha-synuclein (α-syn) were the first to be associated with genetic PD. Another monogenic causative factor in PD patients is ammation , 5, it iLRRK2 mutations have an elevated risk of developing PD and they therefore represent a useful population in which to identify biomarkers of prodromal PD [LRRK2 mutation carriers [LRRK2 mutations: 23 symptomatic individuals and 51 asymptomatic mutation carriers. In parallel, we included 60 patients with sporadic (i.e. idiopathic) PD (sPD) and 43 healthy control subjects (first-degree relatives of LRRK2 mutation carriers (Ctrl)).Several research groups, including ours, have explored the potential of CSF alpha-synuclein (α-syn) forms as diagnostic or progression biomarkers for PD. Total α-syn (t-α-syn) levels were reported to be lower in PD, whereas oligomeric (o-α-syn) and phosphorylated Ser129-α-syn (pS129-α-syn) appear to be elevated –9. CSF cromal PD . Howevercarriers –16. In tLRRK2 mutations were assessed in the current study. Twenty-three patients were clinically diagnosed with PD, whereas 51 patients were healthy, asymptomatic LRRK2 mutation carriers when enrolled in the study. These families have been extensively described in previous reports [LRRK2 mutation carriers who were not carrying LRRK2 mutations. PD clinical diagnoses were made by experienced senior clinicians based on guidelines described by Gelb and colleagues [LRRK2 mutations. Patients with an age at onset of ≤50 years were confirmed to be negative for known pathogenic mutations in Parkin and PINK1. All family members of LRRK2 patients were examined for clinical features of PD by movement disorder specialists and found to be asymptomatic, although a few had mild premotor signs and an increased Unified Parkinson’s Disease Rating Scale (UPDRS) score (three cases scored > 10 on UPDRS-III) [LRRK2-mutant PD patients were taking levodopa, and some were taking dopamine agonists and/or monoamine oxidase-B (MAO-B) inhibitors.Patient selection criteria and the method of CSF collection were as described in previous publications , 17. In  reports . In addilleagues  and diseDRS-III) . The LRRTo collect CSF, lumbar punctures were performed in overnight fasted patients between 8 and 10 am. CSF samples were aliquoted in 1.2–1.5 ml low-binding tubes, and one vial was sent for routine laboratory analysis , whereas the majority of the vials were frozen fewer than 15 min after collection following centrifugation at 2000 g at 4 °C then sub-aliquoted and stored at − 80 °C until further analysis. All patients provided signed informed consent, and the study was approved by the Regional Committee for Medical and Health Research Ethics.3, pH 9.6 (50 μl/well) by overnight incubation at 4 °C, while 0.2 μg/ml of our mouse conformation-specific antibody, Syn-O2, was used to capture o-α-syn. After incubation with 100 μl/well of blocking buffer (PBS-T containing 2.25% gelatin) for 2 h at 37 °C, 50 μl/well of the CSF samples  along with serial dilutions of recombinant human t-, pS129-or o-α-syn (50 μl) were dispensed in each well, and the plate was incubated at 37 °C for 2.5 h. After washing with PBS-T, 50 μl/well of 11D12  [All immunoassays used to measure the different species of α-syn were developed in-house and described in previous reports , 22, 23 CSF Aβ42, Aβ40, total tau (tTau), and phosphorylated threonine 181 tau (pTau) were measured using MILLIPLEX® MAP Human Amyloid Beta Tau Magnetic Bead Panel (Luminex xMAP) run on the Bio-Plex® 3D instrument  according to manufacturer’s instructions. The kit allows simultaneous quantification of Aβ40, Aβ42, tTau, and pTau.A magnetic human chemokine bioplex assay  was used to measure 40 chemokines from human CSF samples . Bioplex assays was run according to the manufacturer’s instructions using the recommended 1 in 2 dilution of CSF. Plates were read using the Bio-Plex® 3D suspension array system; the next generation multiplexing platform based on xMAP technology. Enclosed standards were used to generate an 8-point standard curve to which a 5-parameter logistic curve was fitted and were used to quantify unknown concentrations using BioPlex manger software. The coefficient of variance between duplicates was mostly < 10%, as was the variance between standard curves run on separate plates. Only chemokines with robust readings above background were considered for further analysis.IBM SPSS software  was used for the statistical analyses of the data, whereas R 3.6.1 was used for plotting the box and whisker plots.LRRK2 mutation carriers and sPD groups were combined for discriminant function analysis. Discriminant function analysis evaluates canonical discriminant functions based on combinations of the selected markers which contribute maximally to group separation and assesses how well these canonical discriminant functions discriminate the diagnostic groups. CSF biomarker data were Z-transformed prior to discriminant function analysis.Demographic and clinical characteristics were compared between study groups using chi-square tests, analysis of variance with post hoc Bonferroni tests or Kruskal- Wallis tests followed by Mann-Whitney U tests, where appropriate. All datasets were tested for normality and the presence of outliers. As data were considered inappropriate for parametric analyses, Spearman rank-order correlation coefficients were used to examine correlations within the study group. For all CSF biomarkers that showed robust readings above background , differences between the diagnostic groups were assessed using general linear models (GLMs) corrected for age and gender with post-hoc Bonferroni corrections for multiple comparisons. Only biomarkers that varied significantly among the study groups were included in the next Discriminant Function analyses. Since our main objective was to identify predictive markers for the early detection of PD, both symptomatic We also performed multivariate logistic regression analyses with backward stepwise selection . The control group was entered as a reference category and t-, o- and pSer129-α-syn, TNF-α and IL-16 were enrolled as predictors. For the resulting models, we report AUC, sensitivity, specificity and OR (95% CI) of the individual model.LRRK2 mutations analyzed had a manifest PD and were carrying either the most common LRRK2 mutation, G2019S, or a different LRRK2 mutation, N1437H. The symptomatic LRRK2 mutation carriers had a mean age of 60 ± 11 years. 51 subjects of the 74 subjects (70%) had no symptoms of PD at the time of CSF sample collection and these cases had a mean age of 57 ± 14 years. There was no significant difference in disease duration between the symptomatic LRRK2 mutation carriers and the sPD patients. Moreover, no difference was found between groups with regard to the routine CSF levels of white-red cell count as well as the total protein, albumin, and glucose levels, including the plasma glucose level. Controlling for age and gender did not significantly alter the results.Demographics, clinical characteristics and CSF biomarkers levels of the study groups are summarized in Table P < 0.01, adjusted for age and gender; Table n = 43), levels of CSF t-α-syn were significantly lower in sPD group (median and IQR = 573 (466.5–710.5), n = 60)  pg/ml, n = 51)  pg/ml, n = 23)   . Examination of CSF levels of pS129-α-syn revealed a trend of an increase in sPD group (median and IQR = 139 (114.25–163) pg/ml, n = 60) compared to Ctrl (median and IQR = 116 (103–145) pg/ml, n = 43), however the difference did not reach statistical significance.  (P < 0.05) compared with Ctrl (median and IQR 6.5 = (4.94–8) pg/ml, n = 28). More interestingly, analysis showed a significant increase of TNF-α in asymptomatic LRRK2 mutation carriers (median and IQR = 5.5 (4.2–7.4) pg/ml (P < 0.05), n = 24) compared to Ctrl (median and IQR = 3.3 (2.6–5.5) pg/ml (P < 0.05), n = 28). The inflammatory profiles of symptomatic LRRK2 mutation carriers and sPD cases were not significantly different (data not shown).To determine whether inflammatory biomarkers were altered among the different study groups, a panel of 40 proinflammatory cytokines, chemokines, and growth factors were assessed in CSF samples from all study participants. Of the 40 markers measured, 18 showed robust readings above background and were further analyzed . GLM Bonferroni corrected analysis revealed that of the remaining 18, only IL-16 and TNF-α were significantly different. A univariate, post-hoc analysis corrected for age and gender as covariates demonstrated that IL-16 levels were significantly lower in sPD group (median and IQR = 5.1 (4–6.6) pg/ml, r = 0.39, P < 0.01). We also noted an inverse correlation between t-α-syn and o-α-syn  that was only present in asymptomatic LRRK2 mutation carriers. When we explored correlations between α-syn species and the AD biomarkers, we found that t-α-syn positively correlated with Aβ-40 and Aβ-42 in the asymptomatic LRRK2 group . No other correlations were noted.By use of Spearman correlations, we evaluated associations between different CSF α-syn forms and AD core biomarkers  in the sPD group. Similarly, a weak correlation of t-α-syn and age was noted in PD group . While pS129-α-syn positively correlated with age in PD group , aging was also associated with increased levels of CSF TNF-α in both PD and asymptomatic carriers groups .Correlational analyses of CSF levels of α-syn species with clinical parameters  are shown in Supplementary Table LRRK2 mutation carriers, PD, and Ctrl groups . The discrimination plot of the two canonical discriminant functions for discrimination of the three groups is presented in Fig. r = − 0.694*), o-α-syn (r = − 0.499*) and pS129-α-syn (r = 0.390*) and discriminated both asymptomatic LRRK2 mutation carriers and PD groups from Ctrl group; we will subsequently refer to this function as the Disease function. Canonical discriminant function 2 strongly correlated with TNF-α (r = 0.678*) and IL-16 (r = − 0.554*) and further discriminated asymptomatic LRRK2 mutation carriers group from Ctrl group; we will subsequently refer to this function as the Prodromal function. Asymptomatic LRRK2 mutation carriers’ centroid is located at the intersection of both the Disease axis and the Prodromal disorders axis.In an attempt to identify the optimal panel that can serve as predictive markers for PD at the prodromal stage, we performed a discriminant function analysis of biomarkers that were significantly different between the groups. Canonical discriminant function classification results are presented in Table LRRK2 mutation carriers and Ctrl, and [LRRK2 mutation carriers group from the Ctrl group: low levels of t-α-syn , high levels of o-α-syn , and high levels of TNF-α  indicate that individuals are at higher risk developing PD. Examining PD and Ctrl groups only, we found that low levels of t-α-syn , high levels of o-α-syn , high levels of pS129-α-syn , low levels of IL-16  differentiates the PD group from the Ctrl group. Receiver operating characteristic curves for both models are illustrated in Fig. We employed backward-elimination multiple logistic regression analyses to identify optimal biomarker panels for bilateral comparisons between:  asymptomLRRK2 mutations are well-described as a cause of genetic familial parkinsonism, and drugs inhibiting LRRK2 kinase activity are already in clinical trials [LRRK2 mutation carriers are at high risk of developing PD, asymptomatic individuals with LRRK2 mutations are an excellent group for discovery of biomarkers of prodromal PD based on the premise that they are highly likely to develop PD in future.l trials . HoweverLRRK2 mutation carriers, i.e. PD subjects at the prodromal phase, from healthy controls. As these biomarkers in combination had greater discriminant power than those in isolation, these findings emphasize the value of combining multiple markers for early detection of PD.In the current study, we measured 47 different candidate biomarkers and found evidence that low levels of CSF t-α-syn, and high levels of CSF o-α-syn and TNF-α potentially differentiate asymptomatic LRRK2 mutation carriers displayed significantly higher CSF o-α-syn than Ctrls. These findings support the hypothesis that α-syn oligomerization is an early event in the pathophysiology of PD and add further weight to evaluation of CSF o-α-syn as a candidate biomarker for detection of prodromal PD. A previous study showed a similar trend, although the results were not significant [In line with previous cross-sectional studies assessing the diagnostic power of CSF α-syn forms , 9, we rnificant . One pos, 2016 study, where CSF t-α-syn levels were lowered only in idiopathic (i.e. sporadic) PD group in contrast to other diagnostic groups; however, the differences in CSF tTau, pTau, Aβ-40 or Aβ-42 levels didn’t reach statistical significance. Immunoassays employed to measure AD biomarkers in both studies used Luminex xMAP but employed different antibodies, which may underlie different results. If this is the case, this would have important implications for the design of future biomarkers studies.Our results partially contradict with Vilas et al.LRRK2 mutation carriers compared to Ctrls. In a recent 11-year study in over 4 million Norwegians, an inhaled asthma medication with anti-TNF activity, a brain-penetrant drug, was associated with lower levels of t-α-syn and decreased the risk of PD [LRRK2 mutation carriers showed significantly lower CSF t-α-syn and higher TNF-α and o-α-syn. However, the control subjects in our study were significantly younger than sPD, symptomatic and asymptomatic LRRK2 mutation carriers (p < 0.05), a notable finding given that previous studies have demonstrated an age-dependent increase in CSF TNF-α levels [LRRK2 mutation carriers compared with Ctrl subjects when CSF TNF-α values were analyzed after age adjustment  have incomplete penetrance, meaning that not all of the asymptomatic LRRK2 mutation carriers in our cohort would develop PD. This may explain the overlap between asymptomatic LRRK2 mutation carriers and both Ctrl and sPD groups  and asymptomatic LRRK2 mutation carriers (n = 25). However, in this study, none of the markers, including TNF-α, distinguished between the two groups but CSF TNF-α levels combined with 5 other cytokines significantly differentiated these cases from sPD (n = 29) and symptomatic LRRK2 mutation (G2019S) carriers. The discrepancy seen in our results and the above mentioned study could be due to the differences in the inclusion and exclusion criteria of the subjects, and/or time difference in processing or storing the samples.In a recent study by Halliday and colleagues, using CSF and serum samples from the Michael J. Fox Foundation LRRK2 cohort consortium , 28 cytoLRRK2 mutations carriers and, although they were confirmed negative for LRRK2 mutations, including another standard control group shall be considered in further studies. Previous studies have described heterogeneous pathologies in LRRK2 mutation carriers, where different brain areas and methods were studied and used [LRRK2 mutations necessitates the need for cohorts where neuropathological examination of LRRK2 mutation carriers is conducted both to provide neuropathological confirmation of clinical diagnoses, and to better define and validate differential profiles of biomarkers.Admittedly, the size of our cohort is relatively small, particularly considering the number of subjects in which all the biomarkers’ measurements were available. Furthermore, the only control group in this study was composed of first-degree relatives of and used –33. SuchLRRK2 mutation carriers highlights the power of those biomarkers at providing an early detection of PD. Future studies are necessary to confirm the potential sensitivity and specificity of combing α-syn species with inflammatory biomarkers as predictive and perhaps progression biomarkers of PD. Comparison of CSF and blood levels of the current biomarkers in larger cohorts with longitudinal follow-up of LRRK2 mutation carriers, and other “at risk” groups, are of great importance. Such studies are crucial to select biomarkers that could identify individuals at high risk to convert to PD, who would then be the target group for the development of preventive treatments.Our study, by demonstrating lower CSF t-α-syn levels, higher o-α-syn and TNF-α in asymptomatic Additional file 1: Supplementary Table 1. Associations between CSF biomarkers. No correlation between CSF biomarkers were present in symptomatic LRRK2 mutation carriers group alone. Both sPD and symptomatic LRRK2 mutation carriers groups were combined as one PD group. Associations between CSF biomarkers were assessed with Spearman correlation coefficients. Data shown as r. Significance: *** p < 0.001; ** p < 0.01; * p < 0.05 Aβ1–42, amyloid β1–42; Ctrl, Healthy controls; o-α-syn, oligomeric α-synuclein; pSer129-α-synuclein, phosphorylated α-synuclein protein at serine 129; pTau, tau phosphorylated at threonine 181; PD, Parkinson’s disease patients; tTau, total tau protein; and t-α-syn, total α-synuclein. Supplementary Table 2. Associations between CSF α-syn species and clinical parameters. Associations between CSF biomarkers were assessed with Spearman correlation coefficients. Data shown as r. Significance: *** p < 0.001; ** p < 0.01; * p < 0.05. Both sPD and symptomatic LRRK2 mutation carriers groups were combined as one PD group. Aβ1–42, amyloid β1–42; Ctrl, healthy controls; H&Y, Hoehn and Yahr scale; MoCA, Montreal Cognitive Assessment; NA, not applicable; o-α-syn, oligomeric α-synuclein; pSer129-α-synuclein, phosphorylated α-synuclein protein at serine 129; pTau, tau phosphorylated at threonine 181; sPD, sporadic PD; TNF-α, tumor necrosis factor-alpha; tTau, total tau protein; t-α-syn, total α-synuclein; UPDRS-III, Unified Parkinson’s Disease Rating Scale. Supplementary Table 3. Discriminant loadings for each individual predictor. The correlation coefficient represents the relative contribution for each predictor to group separation. IL-16, interlukin-16; o-α-syn, α-synuclein oligomers; pS129-α-syn, phosphorylated Ser 129 α-synuclein; t-α-syn, total α-synuclein; TNF- α, tumor necrosis factor- α.


The Barriers Questionnaire II (BQ-II) was developed to assess barriers to effective pain management. In this study, we aimed to assess the reliability and validity of the newly developed Japanese version of the BQ-II (JBQ-II).This study used a cross-sectional design. The study was conducted an ambulatory infusion center for cancer in a general hospital in Tokyo, Japan. Participants were 120 Japanese patients with cancer and 21 Japanese health professionals with experience in pain management. Cronbach’s alpha coefficient was used to calculate reliability. Test–retest reliability was assessed with Spearman’s intra-class correlation coefficient (ICC). Construct, criterion-related, and discriminant validity were assessed using information about pain management, daily life, mental health, and subjective health.P < 0.01). Factor analysis showed the JBQ-II had a virtually identical structure to the BQ-II, and path analysis supported the JBQ-II constructs. The JBQ-II was weakly correlated with poor mental state . Patients’ JBQ-II scores were significantly higher than health professionals’ scores.The Cronbach’s alpha was 0.90 for the JBQ-II, and all ICCs exceeded 0.70 (The JBQ-II is a valid and reliable measure of patient-related barriers to pain management among Japanese adult patients with cancer. Pain is a symptom commonly experienced by patients with cancer . HoweverThe Barriers Questionnaire (BQ) and the Barriers Questionnaire II (BQ-II) were developed to determine patient-related barriers to cancer pain management. These instruments have been translated and used in many countries –13. HoweThis study used a cross-sectional design. Approval was obtained from the institutional review boards of Keio University (No. 2012–10) and St. Luke’s International Hospital (No. 12-R066). Participation in this study was voluntary. All participants provided informed consent before participating.Patients with cancer were recruited from an ambulatory infusion center for cancer in a general hospital in Tokyo, Japan. Eligibility criteria were patients: a) aged 20 years or older; b) able to speak and read Japanese; c) with a diagnosis of metastatic or advanced cancer; (d) with an Eastern Cooperative Oncology Group performance status of 2 or less; and (e) with a pain rating of 1 or higher  in the previous week. Clinical staff identified eligible patients. An impartial researcher described the study to potential participants and obtained consent.Health professional participants were recruited from the same hospital. The hospital manager was responsible for recruiting these participants. Inclusion criteria were professionals with a valid license as a physician, nurse, or pharmacist, and at least 3 years of experience in providing pain management. A participation request form and an anonymous questionnaire were sent to identified health professionals who met the inclusion criteria. Health professionals who submitted completed questionnaires were considered to have consented to participate in the study.Patient participants received a packet of questionnaires that included the JBQ-II, the Japanese version of the Brief Pain Inventory (BPI-J), the six-item Kessler Psychological Distress Scale (K6), and the Subjective Health Scale. Patients completed the questionnaires during a hospital visit and returned them to the researchers. To confirm the test–retest reliability of the JBQ-II, the first 20 patient participants were asked to complete the JBQ-II a second time after a 2-week interval.An anonymous, self-administered questionnaire was distributed to potential health professional participants. This group was asked to complete the JBQ-II as if they were patients with cancer experiencing pain. The health professionals’ questionnaire also covered age, sex, education, length of occupational experience, and length of palliative care experience. Health professional participants completed the questionnaires in their own time and returned them to the researchers by hospital post.The questionnaire used in this survey was created by combining the following tools.The BQ-II is a 27-item instrument that measures patients’ beliefs about cancer pain and use of analgesics . The BQ-Consent to translate the BQ-II into Japanese was obtained from the original author of the BQ-II. The JBQ-II passed through a process of double back translations, was Japanized, and the content verified by the original author. Finally, clinical and research experts examined the content and validity of the JBQ-II. When the BQ-II was developed, the three-item “disease progression” BQ subscale was removed. However, this subscale was returned when the BQ-II was translated into Japanese, making the JBQ-II a 30-item instrument. Disease progression was considered an item culturally relevant in Japan because of Japanese patients’ attitudes toward cancer pain.The BPI is a reliable and valid scale used to assess pain intensity and its effect on daily living; higher scores indicate interference in daily life. This scale is used worldwide –18, inclThe PMI is a scale used worldwide to evaluate pain management , 21. It The K6 has six questions covering a person’s emotional state, and has been validated in Japan , 24. EacThe Subjective Health Scale is a self-assessment of health with four options: “In very good health,” “In fairly good health,” “Not in very good health,” and “Not in good health at all.” The categorization method for this self-assessment of health is frequently used in Japan .Health professionals were asked to answer the JBQ-II subjectively, as if they were a patient with cancer experiencing pain. Information was collected on the appropriateness of the JBQ-II as well as on participant characteristics, length of occupational experience, and length of palliative care experience.Descriptive statistics for each JBQ-II item were calculated to examine ceiling or floor effects and verify that each item reflected the trend of respondents. Reliability was determined by internal consistency using Cronbach’s alpha coefficient, with test–retest reliability assessed using Spearman’s intra-class correlation coefficient (ICC). Construct, discriminant, and criterion-related validity were also assessed.Construct validity was evaluated in two different ways to verify whether it was possible to make theoretical predictions about the concept of a barrier. First, we performed exploratory factor analysis using the principal method and promax rotation, which was the method used for the original scale. The fit of the factor model was evaluated based on the results of the screen test, interpretability, and examination of residuals. Although the BQ-II is a 27-item scale, 30 items (including the three disease progression items) were used for the JBQ-II factor analysis.Next, we used path analysis by structural equation modeling to examine a structural model for pain, barriers, and quality of life (QoL), which was developed based on previous studies Fig.  4, 5, 1, 15, 11.The known group method was conducted to explore discriminant validity . Two groRegarding criterion-related validity, there were no studies available in Japan to assess associations between the barriers score and outcome measures. Therefore, Pearson’s correlation coefficients for the JBQ-II and K6 domains were calculated. The K6 was chosen as an external index because a barrier is a psychological concept. It was proposed that conceptually-related domains would be weakly correlated with each other. In addition, we asked participants to comment on the feasibility and appropriateness of expression of the JBQ-II. Participants were asked whether the JBQ-II content was clear and easy to understand, whether they found it easy to answer the questionnaire, and how long it took to complete the JBQ-II.Questionnaires were distributed to 134 patients, and 123 (91.8%) responded. Valid responses were received from 120 of these respondents (97.5%), after excluding those with missing values or extreme bias. Questionnaires were distributed to 26 health professionals, and 21 (80.8%) responded. Table Health professional participants had a mean age of 33.67 (SD 9.12) years, mean health care experience of 10.81 (SD 9.02) years, and mean palliative care experience of 5.10 (SD 3.18) years. Most of these participants (90.5%) were female.ρ = − 0.19, P < 0.05), which tended to be associated with higher total JBQ-II scores. The JBQ-II total score for patients with previous immunotherapy (n = 21) was 64.7 (SD 19.64), whereas that for patients without previous immunotherapy (n = 99) was 54.10 (SD 19.95), showing a significant difference between the two groups (P = 0.04).The internal consistency for the JBQ-II in the patient sample was 0.90. Total JBQ-II scores ranged from 10 to 98 (possible range 0–150), with a mean of 59.18 (SD 21.22). Analyses were performed to determine whether patients’ JBQ-II scores were related to demographic variables, including age, gender, education, marital status, type of cancer diagnosis, and other significant health problems. The only demographic variable that was correlated with total JBQ-II score was a history of previous immunotherapy , with total scores ranging from 0 to 51. There were no associations between health professionals’ demographic variables  and total JBQ-II scores.Cronbach’s alpha coefficients were 0.90 for the total scale, 0.89 for physiological effects (Factor I), 0.78 for communication (Factor II), 0.86 for harmful effects (Factor III), 0.92 for disease progression (Factor IV), and 0.73 for fatalism (Factor V) (Table ρ) for total JBQ-II scores and each subscale. The coefficient for Factor V was ρ = 0.49, but that of all other factors were ρ > 0.7, indicating that the results can be replicated.Table The construct validity of the JBQ-II was examined by exploratory factor analysis using the principal method and promax rotation Table . Table 5The cumulative contribution rate up to Factor V was 59.33. As the gradient on the scree plot became smaller after Factor V, it was hypothesized that the JBQ-II had a five-factor structure. Factor analysis (specifying five factors) confirmed this structure Table .Table r = 0.20, P < 0.05). Creating two segments for PMI, from − 3 to − 1  and from 0 to 3  and conducting a sub-analysis showed that the total JBQ-II score had weak positive correlations with the K6, indicating a poor emotional state . This demonstrated that patient-related barriers have a weak relationship with emotional state, and indicated the JBQ-II had criterion-related validity.The analysis of the JBQ-II and K6 showed a weak relationship between Factor II (communication) and the K6  reported that the question content was clear and easy to understand, 93 (77.5%) reported that answering the questions was clear, and 85 (70.8%) reported that the questions were clear to understand and answer. The length of time needed to answer the JBQ-II ranged from 2 to 20 min (mean 8.34 ± 4.18), which confirmed the feasibility of the instrument.The overall results showed that the JBQ-II was a reliable and valid instrument for measuring barriers to pain management in Japanese adult patients with cancer. Regarding reproducibility, Factor IV (disease progression) showed a rather low Spearman’s correlation coefficient of 0.49; however, this result might have been affected by patients’ emotional state, symptoms, or condition. The internal consistency of Factor II: Communication and Factor V: Fatalism was slightly lower. Some of the items may have different characteristics than others, but from a content perspective, it makes sense that they belong to the same category. From a holistic perspective of scale, it is a necessary item for observing barriers.In the factor analysis, some BQ-II items were moved to a different factor in the JBQ-II. Other countries’ versions also differ somewhat in terms of the number of questions and how the sub-scales are divided, but the content of the subordinate construct is virtually identical, and the barriers are evaluated stably from the same viewpoint worldwide –13. PatiThe present findings indicated that harmful occurrences or physiological effects might differ depending on the individual’s habits or experiences. It is plausible that the JBQ-II reflects cultural differences regarding awareness of harmful occurrences or physiological effects. In the original questionnaire, subcategories for harmful occurrences included worries about weakening the immune system and concerns about dependence; in Japan, worries viewed as harmful occurrences included weakening the immune system, constipation, and loss of social function or impact on relationships with other people .The path analysis substantiated causal relationships between the JBQ-II → PMI → QoL Fig. , althougTo test the criterion-related validity, we needed to select an external index because there are no indices for the JBQ-II and outcomes. The results showed a correlation between Factor II (communication) and poor emotional state, which is consistent with previous research . StratifThis study demonstrated that the JBQ-II can be applied in daily practice. However, as the present study was conducted in a single institution, similar surveys need to be conducted and verified across other settings to assure the generalizability of the findings. The relationship between patient-related barriers and other factors as well as culturally specific barriers should also be further investigated.There are several limitations to our study. First, this study was conducted in a single institution in Tokyo Japan. Our results may not be applicable other settings. Similar surveys should be conducted in other institutions and regions. A future challenge is to increase the frequency of use and improve the survey’s practical aspects on an ongoing basis. Second, we considered subjective health, emotional state (K6), and daily life (BPI) as potential QoL variables. Therefore, the results might differ if other definitions or a QoL scale were used. Despite these limitations, we conducted multifaceted evaluations of the JBQ-II. Our findings may be useful to assess pain in clinical settings, as well as for epidemiological studies of cancer pain management. It is necessary to provide care that recognizes the presence of barriers to pain management. In addition, from a global perspective, there are many barriers to pain management in Asian and developing countries, which may differ by culture and ethnicity. We believe validated instruments such as the BQ-II need to be available across various languages to integrate global trends.The JBQ-II has construct, criterion-referenced, and discriminant validity, and is a reliable and valid index for assessing barriers to pain management among Japanese adult patients with cancer. Therefore, the JBQ-II may be used routinely to improve pain management in Japan.


G-CSF for stem cell mobilization increases circulating levels of myeloid cells at different stages of maturation. Polymorphonuclear cells (PMNs) are also mobilized in high numbers. It was previously reported that G-CSF primes PMNs toward the release of neutrophils extracellular traps (NETs). Since NETs are often involved in thrombotic events, we hypothesized that high G-CSF blood concentrations could enhance PMN priming toward NET formation in healthy hematopoietic stem cell donors, predisposing them to thrombotic events. However, we found that G-CSF does not prime PMNs toward NETs formation, but increases the serum concentration of cell-free DNA, proteases like neutrophils elastase and myeloperoxidase, and reactive oxygen species. This could possibly create an environment disposed to induce thrombotic events in the presence of additional predisposing factors. Granulocyte colony-stimulating factor (G-CSF) is playing an essential role as a colony-stimulating factor during hematopoiesis. G-CSF stimulates proliferation and differentiation of hematopoietic stem cells (HSC) , differePMNs play a leading role in innate immunity ; they arThe goal of our study was to analyse the occurrence and dynamics of NET formation in healthy stem cell donors at the time of stem cell collection and follow-up.We observed that circulating PMNs mobilized after G-CSF application present markers of immaturity, correlate with high cell-free DNA and increased protease content. This effect was of short duration, with all parameters returning back to baseline 1-month post-G-CSF infusion.https://www.ebmt.org/jacie-standards). G-CSF  was applied at a dose of 10 μg/kg body weight divided into two doses daily  subcutaneously, beginning 5 days before HSC harvest.Allogeneic peripheral stem cell donors were asked to participate in the study. Donor eligibility was assessed according to internal policies and in compliance with the current FACT-JACIE standards .Samples were collected from HSC donors and healthy age-matched controls at various time points; 3–4 weeks prior to G-CSF administration (visit A); at the time of HSC harvesting (visit B); 1 and 3 months post apheresis (visit C and D).Whole blood was collected into EDTA- and silicone-coated tubes (Sarstedt), and analyzed by a Advia (Siemens) for complete blood cell counts. Plasma and serum were collected and processed as described previously . ShortlyFACS: PMNs (5 × 105) were suspended in FACS-buffer  and stained for 30 min at RT using CD66B-FITC (Biolegend), CD34-PE  and CD61-AF647 (Biolegend). Acquisition: Accuri-C6-Plus (Becton Dickinson). PMN  were analyzed using Flow Jo (version 10).Late apoptosis: isolated neutrophils were incubated in 100 μl annexin binding buffer containing phycoerythrin-conjugated Annexin V (Annexin V-PE) and 7-amino-actinomycin D (7-AAD)  for 15 min at room temperature (RT) in the dark. After incubation, 400 μl annexin binding buffer was added, and samples were measured immediately on a BD Accuri™ C6 FACS (BD Biosciences). The Annexin V-PE+/7-AAD+ populations were taken as measurements of late apoptotic cells. Data were analyzed using FlowJo v10 software .NETs quantification: PMN (2.5 × 104) were incubated with 0.2 μM SytoxGreen (Life Technologies), 37°C, 5% CO2, 3 h  following the manufacturing protocol (protocol . NE concprotocol .MPO concentration was measured using the “Hycult Biotech ELISA-Kit” following the manufacturer protocol (protocol .CitH3 concentration was measured using “Citrullinated Histone H3 (Clone 11D3) ELISA-Kit” (Cayman) following the manufacturer protocol.NE activity was measured in serum incubated with the elastase substrate, N-methoxysuccinyl-Ala-Ala-Pro-Val-7-amido-4-methylcoumarin  for 30 min at 37°C, 5% CO2 in the dark. The reaction product was analyzed at 360/455 nm (0/455 nm .PAD4 western blotting: protein extraction using RP1 Buffer (Macherey Nagel), 1% β-Mercaptoethanol (Sigma) and purification using Nucleo Spin (Macherey Nagel). Antibodies: rabbit anti-PAD4 , human anti-HRP  and SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific), Mouse anti-β-Actin (Sigma-Aldrich) as a control. Protein quantification was performed using Image Lab Software (version 5.2.1).ROS measurement: 3.0 × 104 PMNs were incubated with 25 μM DCFDH-DA (Sigma-Aldrich). Fluorescence was recorded at 0, and 30 min using a Biotek Synergy H1 Hybrid plate Reader and expressed as relative fluorescence units (RFU).Statistic: All data is represented as Mean ± SEM. Comparisons between HSC donors visits was performed using ANOVA with Tukey's post-test correction. Data processed using GraphPad Prism version 8.0 (GraphPad Software Inc.).Twenty healthy stem cell donors who were undergoing HSC collection by apheresis after mobilization with G-CSF were included in the study. Demographic and clinical variables of the study population are presented in The formation of PMNs and platelet complexes is an essential parameter indicating the possible initiation of clotting. We observed that the number of platelets  remainedDue to the expression of CD61 on PMNs the aggregation between platelets and PMNs as described in Zarbock et al. , could pA key feature of pro-NETotic PMNs priming is the of citrullination of histone 3 (CitH3) mediated by PAD4. A slightly lower PAD4 expression was observed at visit B compared to visit C and D . SimilarOther factors that could induce injuries in vessels and platelet aggregation are PMN cell-free DNA, proteases MPO and NE, as previously described –21. TherAs recently observed in pregnant women that G-CSF promotes PMN priming and ROS production , we examThis study shows that HSC mobilization with G-CSF increases the concentration of cell-free DNA, of PMN-derived proteases and ROS in serum. This effect is transient and the measured parameters returned to baseline levels in 1-month post-treatment, suggesting the existence of a window of susceptibility to thrombotic events in predisposed subjects.+ cell doses, derived from G-CSF application, lead to better recovery of neutrophils and platelets. With related bone marrow transplant, there is no apparent association of CD34+ cell dose with chronic GvHD, but with unrelated bone marrow, higher CD34+ cell dose does seem to result in improved survival. Functional damage to the host marrow microenvironment through prior therapy and the malignant disease itself cannot be overcome by either increasing stem cell dose or using a graft with different stem cell composition . It was previously shown that increased levels of cell-free DNA in sepsis impaired fibrinolysis by inhibiting plasmin-mediated fibrin degradation . AnotherIt is important to mention that if those concentrations were normalized to the number of PMNs in the blood, the secretion of MPO and NE per cell was the same as in the other time points. This indicates that the total PMN population was not more active or prone to release DNA and proteases. The increased concentration in the serum is the consequence of high numbers of PMNs circulating in the blood and their ability to degranulate. We, therefore, propose that G-CSF creates a transient state of increased risk for hypercoagulability due to the increased concentration of cell-free DNA and proteases in the blood of HSC donors. On the other hand, thrombotic events remain infrequent, suggesting that other factors could contribute to it or prevent it.It was described that ROS could inhibit whole blood aggregation  and we cWe recognize the following limitations of the study: first of all the number of recruited patients/donors is relatively low for a comprehensive study on PMNs behavior, however, since no difference was measured in the NET formation, we are confident that the information we deliver is valuable. Second, not all samples for each time point were included in the presented analysis: for every samples we did run a technical control, if this was resulting in activated PMNs, we did excluded the donor sample from the analysis to exclude a technical error. Third, it would be important to study the interactions between all the immune cells circulating in the blood at time of G-CSF infusion, since high number of PMNs could influence the phenotype and functionality of other immune cells. It would also be interesting to investigate the correlation between neutrophils protease activity due to the increased PMNs presence in the blood and HSC mobilization from the bone marrow niches. Further investigations to better understand the pathophysiology of thromboembolic events in GCS-F mobilized donors to identify a subject at risk are needed.All datasets generated for this study are included in the article.The studies involving human participants were reviewed and approved by Ethical committee of Northern and Central Switzerland (2015-00191). The patients/participants provided their written informed consent to participate in this study.AH, LI, AP, and AB recruited the donor, discussed the manuscript, and organized foundings. MS, SB, GS, LV, SG, SH, and SR processed the samples, analyzed the data wrote, and discussed the manuscript.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.


An absence of any statutory law in Japan regarding donor conception creates uncertainty about the status of donors in relation to the child(ren) born as a result. Laws that provide for certainty regarding the status of the donor are called for, as are laws that address donor anonymity. It would be pragmatic to introduce a prospective system that requires open donation, allowing information to be recorded and released to donor‐conceived people upon request. For past donations, a voluntary register should be established, which would allow those people who are seeking information to register this. Attempts at passing legislation have failed, while academic society guidelines and government documents have been functioning as a soft law. In the meantime, the practice of donor conception using donor sperm and oocytes has proceeded both in country and, in the latter case, using cross‐border reproduction. This has left the status of donors in relation to the children born as a result unclear, and the rights of people born via donor conception to access information about their genetic heritage unsupported. In this paper, the authors outline current situation in Japan regarding donor conception and recommendations are made to improve current and future practices and regulation.2Artificial insemination with donor sperm (AID) has been practiced for many years in Japan to assist heterosexual couples to have a child, with an emphasis on donor anonymity. The total number of births through AID with anonymous sperm donation is not known exactly—some reports varying from more than 10 000 to more than 15 000 individuals. The principle donors have been reported to have been medical students.Keio University Hospital has long been a mainstay in offering anonymous sperm donation for married heterosexual couples since 1949. Since 1995 when Dr Yasunori Yoshimura became a professor at Keio University Hospital (retired in 2014), donor records have been kept rigorously, although still anonymized and never intended for disclosure. Before him, records may have been destroyed.In September 2018, it was reported in the news media that Keio University Hospital had stopped receiving new patients seeking donor sperm. The hospital reported that fewer donors were willing to participate once they were made aware of the spreading global recognition of children's rights to know their biological parents.Private donation in Japan can be provided via organizations or individuals including that in some cases, sperm is delivered by donors to potential parents face to face. In these cases, the donor is only semi‐anonymous. The academic/medical establishment has warned that private sperm donation can be dangerous with respect to legal heritage and infectious diseases.3Demand for oocyte donation has increased over the years, as women are marrying later and age‐related infertility increases. There are again no statutory laws governing oocyte donation in Japan. Government reports maintain that commercial oocyte donation is not permitted, while non‐commercial, anonymous oocyte donation in principle is allowed.Egg donation has been conducted publicly by two organizations. The Japanese Institution for Standardizing Assisted Reproductive Technology (JISART) established in 2003 reported that 77 patients were treated at JISART clinics and 38 children born between 2007 and 2017. Their guidelines allow donated egg from known donor such as relatives. The Oocyte Donation Network (OD‐Network), which was established in 2013,As the domestic pathway for oocyte donation has continued to be extremely limited, some couples have sought overseas oocyte donation programs provided by Japanese agencies. Such arrangements are most often commercial and anonymous. While some travel to the United States, others preferred Thailand due to lower pricing and lax laws. Donor records may have been kept by the clinic or Japanese agent but, as the practice was clandestine, the records are vulnerable unlikely to be kept for many years.When commercialization was prohibited in Thailand in 2015,In Taiwan, remuneration for gamete donation has been legal since the Artificial Reproduction Act was enacted in 2007.4For both AID and oocyte donation, there are no laws in Japan that specifically govern the parent‐child relationships resulting from ART and/or donor conception procedures. However, pursuant to Article 772, para.1 of the Civil Code (Presumption of Child in Wedlock) “a child conceived by a wife during marriage shall be presumed to be a child of her husband,” this would mean that the child born as a result of AID to a married couple would be considered the legal child of the husband. However, note, the presumption is rebuttable, and the status of the donor in such circumstances is not explicitly provided for. The status of the donor in relation to a child born to a single woman or woman in a same‐sex couple who has used donor sperm to conceive that child is unclear.In relation to oocyte donors, the Japanese Civil Code does not address whether the recipient woman, who will also be the birth mother, is the legal mother. However, in a case concerning cross‐border surrogacy and the status of a child born as a result, the Supreme Court of Japan in 2006 held that a woman who had delivered a child (which means surrogate mother) is legal mother of a child. Presumably, this reasoning would also apply to the recipient mother of donated oocytes who gives birth to the child in circumstances that do not involve a surrogacy arrangement. This should be confirmed by legislature to avoid any doubt.It is clear that without specific legislation that clarifies the status of the donor and the recipient parents within the context of donor conception, the rights and/or responsibilities of all involved are uncertain.5Over the last several decades, the regulation of ART, including donor conception, has been drastically changing across the world. In relation to donor anonymity, it appears that a transition to openness and non‐anonymity of donors is growing.all donor‐conceived individuals the opportunity to receive identifying information about past donors regardless of when the donation took place.information about their genetic/biological origin, regardless of when they were born, while providing for a person to file a “contact veto” if they did not want to be contacted.In the Australian state of Victoria, legislation came into force in March 2017, which granted Interestingly, the Swiss laws also allowed for retrospective release of information in 2004, but it is the more recent Victorian laws that have been reported in the news media to have influenced some practices in Japan .At the same time, the situation regarding donor anonymity is changing drastically due to the existence of direct‐consumer DNA testing.6Legislation is called for which must be culturally sensitive, while also recognizing that where donor conception is practiced, there is an increasing likelihood that a person will learn of their donor conception status, and some may seek information about their genetic heritage and relatives. At the same time, it is important to consider that Japan's society is conservative. Much stigma remains around infertility and quite possibly for donors who donated sperm in the past. It may not be appropriate, in the first instance to introduce retrospective legislation that reveals the identity of past donors. Instead, it would be a pragmatic approach to introduce a prospective system that implements an open system of donation that is no longer anonymous. This would seem indicated given the modern social environment for donor conception appears to have moved toward openness since private sperm donation has been increasing recently in Japan.For past donations, it would likely be more socially acceptable to implement a voluntary register, which would allow those people who are seeking information to register their desire to know. It is suggested here that such a register should involve active outreach to donors, should a donor‐conceived person seek information. Note, although it is reported that one‐third of past donor information is missing,Concurrently, opinion surveys among past anonymous donors should be conducted to explore their willingness to disclose identifiable and/or non‐identifiable information. If information release is acceptable, the extent to which they wish to have a relationship with the donor‐conceived individual and what expectations they have could also be explored.Once laws that clarify the status of all parties involved, prospective bans on anonymous donation, and a voluntary register for past donations are established, statistical data and related information can be obtained. Careful monitoring, maintaining a voluntary register, education, and considering the concerns of society and of all parties  indicate an enlightened path forward. This may be effective for removing social stigma and establishing the right to know of donor offspring in a society where donor conception has long been conducted anonymously.Conflict of interest: Yuri Hibino declared that she has received research grants from JSPS (18KK0340) (collaborative study with Dr Sonia Allan). Sonia Allan declared that she is working with Yuri Hibino with research grants from JSPS (18KK0340). Human and Animal Rights: This article does not contain any study with human or animal participants that had been performed by any of the authors.


The objective of the study was to assess the cerebrospinal fluid (CSF) findings in COVID-19 patients.n = 2655) with confirmed COVID-19 between February 15, 2020, and April 15, 2020, in 182 hospitals from a large health system in the USA. The review of data yielded to a total of 79 patients in 20 hospitals who had CSF analysis.This was an observational retrospective cohort from electronic medical records of hospitalized patients  had normal total cell count and normal white blood cell count in CSF. Sixty-three percent (5/8) had elevated total protein. Two patients had normal levels of lactate dehydrogenase (LDH) and 1 patient had significantly elevated (fourfold) neuron-specific enolase (NSE) level in CSF.Unlike bacterial infections, viral infections are less likely to cause remarkable changes in CSF glucose, cell count, or protein. Our observations showed no pleocytosis, but mild increase in protein in the CSF of the COVID-19 patients. The fourfold elevation of NSE may have diagnostic/prognostic value as a biomarker in CSF for COVID-19 patients who have altered mental status. The coronaviruses are RNA viruses that are responsible for zoonotic infections. The strains of coronaviruses that have caused outbreaks in recent history include the systemic acute respiratory syndrome by SARS-CoV and the Middle East respiratory syndrome by MERS-CoV. SARS-CoV was the causal agent of the severe acute respiratory syndrome outbreaks in 2002 and 2003 in China, while MERS-CoV was the responsible agent for the outbreak in 2012 in Saudi Arabia . Seventy-five percent of the patients were 55 years old and above. Sixty-three percent were male. Twenty-four percent of the patients presented neurological symptoms such as encephalopathy, altered mental status, impaired consciousness, dizziness, ataxia, myoclonus, unspecified convulsions, loss of taste or smell, facial weakness nausea, vomiting, aphasia, dysphasia, and lack of coordination. Sixty-six percent stayed in the hospital for more than 3 days and 35% required ventilator support for more than 72 h (Table Twenty of these patients had hypernatremia and 4 (20%) of these had persistent hypernatremia for ≥ 48 h.The CSF was used for testing the presence of bacterial or viral antigens in 73 patients. The primary antigen testing was for Streptococcus pneumonia (74%) followed by adenovirus (17.8%). Ten patients had CSF analysis for proteins, cell count, glucose, and general appearance Table .Table 2Unlike bacterial infections, viral infections are less likely to cause remarkable changes in CSF glucose, cell count, or protein , 21. HowA recent paper from the USA reported increased IgM and cytokine levels in the CSF of three patients with encephalopathy and encephalitis . IncreasAnother interesting observation was that our 6 of the 8 cases had hypernatremia, which was persistent in 4 cases lasting for more than at least 48 h. Our observation with hypernatremia has also been reported recently by other researchers . They obTo our knowledge, this is the largest cohort to report the CSF findings in COVID-19 patients. However, further studies are needed to have a significant analysis to evaluate the association between CSF findings and clinical outcome.


An important aspect of donor management is the optimization of serum sodium levels.To perform a systematic review to determine the effects of donor sodium levels on heart, lung, kidney, and pancreas graft function, recipient mortality, and to identify the optimal donor serum sodium target.We searched MEDLINE, Cochrane, Guideline databases, and trial registries from 1946 to May 2019 for studies investigating the effects of donor serum sodium levels on transplant outcomes in all non-hepatic organs. A two-step independent review process was used to identify relevant articles based on inclusion/exclusion criteria. We describe the results narratively, assess the risk of bias, and apply GRADE methodology to evaluate the certainty in the evidence.We included 18 cohort studies in our final analysis . 3 of 4 studies demonstrated an association between donor serum sodium and successful organ transplantation. 5 studies reported no association with graft function, while 6 studies did. 5 studies reported on recipient survival, 3 of which suggested donor sodium is unlikely to be associated with recipient survival. The included studies had serious risk of bias, and the certainty in evidence was deemed to be very low. In low risk of bias studies, donor sodium dysregulation is unlikely to affect kidney graft function or mortality of heart and kidney recipients, but the certainty in the evidence is very low due to inconsistency and imprecision. Further research is required to refine the serum sodium target range, quantify the dose-response curve, and identify organs most vulnerable to sodium dysregulation. Despite advances in deceased donor management in recent decades, the demand for organs exceeds supply . In lighBoth hypo- and hypernatremia have been linked to adverse effects in transplant recipients. Hyponatremia is common in patients with acute kidney injury (AKI) of pre-renal origin and may be a marker of progressive AKI in donor kidneys . DiseaseWhether sodium dysregulation adversely affects graft outcomes independently or whether it is a surrogate for other processes has not yet been elucidated and generates considerable discussion in the literature . ElevateOur systematic review followed the Cochrane Handbook for Systematic Reviews and Interventions, and reported results according to the PRISMA guideline .Search Question, Population, Eligibility CriteriaThe objective of our review was to determine whether maintenance of sodium in the normal range, compared with hypo- or hypernatremia, improves the organs transplanted per donor, recipient graft function and graft survival, and recipient mortality. For recipient graft function, survival, and recipient mortality, we focused on studies reporting outcomes in heart, lung, kidney, and pancreas recipients. We included studies of pediatric or adult potential organ donors declared deceased by neurological criteria. We defined the normal sodium range as 135–145 mEq/L. Since we anticipated identifying only observational studies, cohorts with mean or median sodium values outside of the normal range were included. We examined the following outcomes: clinical status during the interval between death declaration and organ recovery , organ acceptance/recovery/transplantation, recipient quality of life, graft function, graft survival, and recipient survival, without a pre-specified follow-up duration. We included all prospective and retrospective studies, but excluded scoping reviews, systematic reviews, and meta-analyses. We excluded experimental/animal studies, non-research articles, and conference abstracts. Articles not published in English or French were also excluded.Search StrategyWe performed unrestricted searches in MEDLINE and Cochrane up until May 2019 to identify relevant articles. Trial registry records and clinical practice guidelines were also searched to identify relevant studies. With input from the study investigators, a health information specialist performed the search with appropriate wildcards to consider plurals and variations in spelling. Relevant articles were then reviewed by the health information specialist to confirm the validity of the search strategy and for emerging themes not already accounted for. The search strategy was revised accordingly and the search was repeated. The full search strategy is available in the Appendix. The search included articles published in English and French. Reference lists of relevant articles were manually searched to identify additional relevant articles.Study Selection and Data ExtractionArticles were screened over two stages. In the first stage, two reviewers (LH and PL) independently reviewed the titles and abstracts, and selected relevant articles for full text review. A third reviewer (JB or IB) resolved disagreements. Two reviewers (JB and LH) independently reviewed the full texts of the articles selected in the first stage. Disagreements between reviewers were discussed in conjunction with a third reviewer (IB) in order to reach consensus.Two reviewers (JB and LH) independently extracted data in duplicate through a standardized form. We collected study design, number of patients, donor characteristics, sodium levels, and relevant outcome data. The impact of various donor sodium thresholds on outcomes were summarized primarily by treatment-effect ratios. We contacted the corresponding authors for data points of interest that were not reported in the studies. Disagreements were resolved by discussion first, followed by arbitration by a third party (IB), if required. Evaluating the Risk of Bias and Grading of the EvidenceWe used the Newcastle Ottawa Scale (NOS) to assess the risk of bias across studies . Based oWe evaluated the certainty in evidence using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system . In keepOf the initial 1137 citations identified in the search, 982 unique records were screened. From the screened records, 91 full-text articles were reviewed, and 18 studies met our inclusion criteria . The majOrgans Transplanted Per DonorFour studies reported on donor sodium levels’ association with successful organ transplantation -24. Threet al’s retrospective study on donor management goals (DMGs), achieving a sodium maintenance in the range of 135–160 mEq/L was an independent predictor of four or more organs transplanted per donor   >155 mEq/L) had significantly higher creatinine values in follow-up  . The maximum sodium level was higher in the group with delayed graft function and hypernatremia was identified as an independent risk factor for delayed graft function in their multivariate analysis . Only one study identified hyponatremia as an independent predictor of graft survival in their multivariate analysis over a 162-month follow-up period   mEq/L) and there was no association with delayed kidney graft function, defined as the need for dialysis in the first week post transplantation , 33.et al’s cohort found no difference between donor sodium levels in recipients with and without delayed graft function  [12]. Hypernatremia was not predictive of delayed graft function  or poor kidney function . Delayed graft function was defined as the need for dialysis in the first 7 days post-transplant and poor kidney function was defined as a creatinine clearance less than 50 mL/min/1.73 m2. In Tian, et al’s cohort, donor mean sodium values did not differ between recipients with delayed graft function and those with normal post-transplant function  and did not independently predict acute rejection  [et al’s study of 74 recipients also did not identify donor sodium as an independent risk factor for delayed graft function in a multivariate analysis (OR was not reported) [Three studies reporting on donors with more significant hypernatremia  also found no association on graft function. Baptista, .6–1.03) . Delayedeported) .We identified only one pediatric cohort in the literature, which also had the most extreme donor sodium mean (163.4±10.9 mEq/L). Despite this more extreme range of hypernatremia, there was no significant association between donor sodium levels and delayed graft function in recipients of pediatric kidney grafts (p=0.13) . Recipient MortalityFive studies described the effect of donor sodium on recipient survival for patients receiving heart, kidney, or kidney-pancreas grafts. Three of those studies conclude that sodium dysregulation has no effect on recipient mortality , 33, 34;et al, retrospectively reviewed 4641 orthotopic heart transplants and applied a multivariate analysis to determine predictors of 1-year post-transplantation survival [et al, conclude that donor hyponatremia is an independent predictor of recipient mortality  in their multivariate analysis over a 162-month follow-up period [Of the two studies showing an association between donor sodium and recipient mortality, one evaluated heart donors and the other kidney donors. Hoefer, survival . Recipiep period .et al’s study regarding the effect of sodium levels on the survival of 1800 heart transplant recipients shows no association. Donors were divided into four groups (classified as A through D) based on their sodium levels. The quartiles used to divide donors were: 141, 147, and 154 mEq/L. For each group, 30-day mortality in group A was 9.3%; B, 10.0%; C, 10.0%; and D, 8.8% (p=0.22). Five- and 10-year survival rates in group A were 71.1% and 53.8%; B, 69.3% and 53.9%; C, 72.7% and 61.0%; and D, 71.2% and 62.4%; respectively  [et al, investigated donor variables predictive of 45-day mortality and late 1-year all-cause mortality in heart transplant recipients [et al’s cohort also found no association between donor hypernatremia and kidney recipient survival at 5 years, respectively [Of the four studies showing no association between donor sodium and recipient mortality, two pertained to heart donors, one to kidneys donors, and one to simultaneous kidney-pancreas transplantations. Kaczmarek,  p>0.05) . Chen, ecipients . Donor sectively .  Risk of Bias and GRADE Assessment of the EvidenceAll studies were evaluated for risk of bias using the Newcastle Ottawa Scale. There were seven studies deemed to be high quality with low risk of bias . Only onThe certainty in evidence for organs successfully transplanted was very low due to concerns of inconsistency and imprecision. The certainty in evidence for graft function and survival, as well as recipient mortality, was downgraded to very low due to serious concerns with respect to risk of bias, imprecision, indirectness, and inconsistency. The variability in study design, patient populations, the exposure, and outcomes precluded a meta-analysis. The grading of the evidence is presented in Despite the increasing number of organ transplantations performed in recent years, many patients remain on organ transplant wait-lists, creating a sense of urgency to ensure all potential donors are converted to actual donors by optimizing donor care, 35. NorThere are two perspectives to the deleterious effects of sodium dysregulation in potential organ donors . Sodium Only two studies identified a relationship between donor hypernatremia and kidney graft function for donors with sodium levels >155 mEq/L. Conversely, the mean donor sodium levels in the cohorts of negative studies ranged from 149 to 154.6 mEq/L. The sodium levels in these cohorts may not have been extreme enough to bring about graft dysfunction, raising the question of a dose-response relationship that should be an area of further study. Selecting studies with low risk of bias in kidney recipients suggests donor sodium dysregulation is unlikely to affect kidney graft function. Despite the consistency of the results, the certainty in evidence remains low.et al, found no association between donor sodium levels and recipient mortality, but the division of donor sodium levels into four quartiles may have underestimated the true effect of extreme hypernatremia. Hoefner, et al, found extremes of both hyper- and hyponatremia to be associated with a 25% increased risk of mortality . Donor hemodynamic status was not controlled for, raising concerns for risk of bias in the large effect size they reported. We identified only two studies reporting on heart recipient mortality , 11. Kacet al, but Selck, et al, did not identify donor sodium as a predictor of the number of organs transplanted. Selck, et al, adjusted for confounders such as desmopressin and diuretic administration in their multivariable regression analysis while Malinoski, et al, did not. Desmopressin and diuretic administration were associated with increased organ yield, so undetected collinearity could account for the association of donor sodium identified in the latter.The studies investigating the relationship between donor sodium levels and the success of transplanted organs leave us with some conflicting evidence as well. Achieving a serum sodium target of 135–160 mEq/L predicted successful transplantation of four or more organs per donor in the study by Malinoski, The studies we identified are mixed in their conclusions, and more specifically in their sodium targets, making it difficult to recommend an optimal donor sodium range. Despite this, our review raises a salient point: Perhaps more important than the degree of dysregulation is why the dysregulation is present. The majority of high-quality studies with low risk of bias controlled for donor hemodynamics, volume status, and desmopressin administration, and found no association between dysregulated donor sodium levels and transplant outcomes. Clinical practice guidelines may consider reframing dysregulated donor sodium levels as a symptom of hemodynamic derangement rather than an electrolyte derangement. It is imperative that future studies control for donor volume status, hemodynamics and hormonal therapy when evaluating the clinical effects of donor sodium dysregulation.In the face of inconclusive evidence regarding the effect of donor sodium levels on transplant outcomes, the very low certainty in evidence may still sway clinical practice guidelines to recommend maintaining donor serum sodium concentration in the normal range (135–145 mEq/L) in potential neurologically deceased donors. There could be a potential benefit to normalizing deranged serum sodium levels  and there is no evidence of harm from this practice. Neurologically deceased donors require sodium correction prior to death declaration to eliminate confounders, making this practice recommendation a logical sequela of the current standard of care.Deceased donors with dysregulated donor sodium levels have been considered marginal, a label that risks the discard of a valuable resource . The resStrengths of our review included a sensitive search strategy with no limitation on publication type. Abstract screening, full-text review, data extraction, and risk of bias assessment were done independently by two authors to minimize bias and selective reporting. We applied GRADE to assess certainty in evidence. However, we are restricted in making strong conclusions in the light of the heterogeneous studies and very low certainty in evidence for all of the outcomes. This review is limited by the sampling method used in the study cohorts we included; the majority of these studies recruited patients from registries of transplant recipients and identified relevant donor factors of transplanted organs. This creates a selection bias against potential donors who were never converted to actual donors, whose phenotype is expected to differ when compared to successful donors. In conclusion, the effects of dysregulated donor serum sodium on recipient outcomes are heterogenous across studies. Although based on low certainty in evidence, high quality studies with low risk of bias suggest that dysregulated donor sodium does not independently harm kidney graft function, or increase mortality of heart and kidney recipients. Our review also challenges the notion that donors with dysregulated sodium are “marginal,” underscoring the need for future research to strengthen the certainty in evidence, and inform the optimal sodium target for lung, kidney, heart, and pancreas donors.