The patient underwent anticoagulation and fluid rehydration therapies. No abnormalities were observed in the thoracic MRI scan, but a cervical MRI scan revealed a spontaneous CSF leak. An epidural blood patch with autologous blood was performed, and symptoms completely resolved 3 days after the procedure. This report proposes a diagnostic procedure for detecting rare cases of SIH-induced CVT, thereby preventing future misdiagnoses and delayed treatment. When a patient presenting with CVT in conjunction with intracranial hypotension has no history of trauma or piercing, SIH caused by spontaneous spinal CSF leakage should be considered as a potential cause of secondary low intracranial pressure. For detection of CSF leaks at rare sites, an MRI of the whole spine rather than a localized MRI of the spine needs to be performed to avoid misdiagnosis. An epidural blood patch should be performed as soon as possible as it may shorten the length of hospitalization and improve prognosis.Spontaneous intracranial hypotension (SIH) may lead to cerebral venous thrombosis (CVT). This case report describes the diagnostic and treatment processes used for a patient with CVT caused by SIH due to spontaneous spinal cerebrospinal fluid (CSF) leakage in the high cervical region. Clinical data were collected from a 37-year-old man with an initial symptom of spontaneous posterior cervical pain. The diagnostic and treatment processes of SIH-induced CVT were described. A magnetic resonance imaging (MRI) study showed superior sagittal sinus thrombosis, and a lumbar puncture revealed a low initial CSF pressure of less than 60 mmH Cerebral venous thrombosis (CVT) is a rare neurological disease that may cause life-threatening complications, such as epilepsy, cerebral hemorrhage, and cerebral herniation. The incidence of CVT is approximately five cases per 1,000,000 people per year , a rare cause of CVT occurring in only 2% of SIH cases, is characterized by postural headache and a cerebrospinal fluid (CSF) pressure of less than 60 mmH2O  doctrine2O .Spontaneous spinal CSF leakage occurs when there is a hole or tear in the membrane surrounding the dura. Dura holes or tears can cause a single localized CSF leak or multiple simultaneous leaks, either of which may lead to SIH. The annual incidence of spontaneous spinal CSF leakage is approximately four cases per 100,000 people . SpontanIn this case report, we present a complex case with CVT secondary to SIH caused by a spontaneous spinal CSF leak in the high cervical region.A 37-year-old man experiencing frontal and posterior neck pain while sitting but without any inducement was admitted to the Second Hospital of Hebei Medical University 5 days after symptom onset. The pain severity became aggravated when the patient was in the sitting or standing position but was alleviated in the decubitus position. He was diagnosed with cervical spondylosis in the local hospital, but his headache became progressively worse. One day before the occurrence of left upper and lower limbs weakness and numbness, the patient had a sudden seizure and lost consciousness. There were three seizures in total, each lasting about 1 min. After emergency treatment, including sedation and seizure control, the convulsion was relieved and consciousness was recovered. Subsequently, the patient followed a continuous regimen of valproate sustained-release tablets orally , and no convulsion was observed.The patient had a history of upper respiratory tract infection in the seven days prior to admission, but no history of chronic disease or surgical trauma. He also had no family history of genetic diseases. The patient presented with left hemiplegia. Physical examination revealed a neck stiffness and active tendon reflex in the left. The left Babinski sign is positive.2O, indicating intracranial hypotension. The white blood cell count was 11.70 × 109/L, and the absolute value of the neutrophil was 8.50 × 109/L. The D-dimer was 0.70 μg/mL (↑). The results of a routine CSF examination were normal. Biochemical examination of the CSF showed that the protein content was 2.46 g/L (↑), and the glucose and chlorine levels were normal. The patient was treated with nadroparin calcium (anticoagulant therapy), acyclovir and foscarnet sodium , and cefoperazone sodium/sulbactam sodium (anti-infection therapy) via injection, as well as intravenous rehydration therapy. Symptoms of headache and left limb weakness were slightly alleviated by the treatment but did not completely resolve, and the patient was still unable to walk. Further MRI study of the cervical and thoracic vertebrae showed CSF collection at the C1-C2 level (A head magnetic resonance imaging (MRI) + diffusion weighted imaging (DWI) + magnetic resonance angiography (MRA) study showed enlargement of the pituitary gland, descent of the cerebellar tonsils and brainstem, and crowding of the posterior fossa . An elecC2 level , suggestC2 level . The spiC2 level . The patThe pathogenic mechanisms of spontaneous CSF leaks are still unclear. In some patients with a spontaneous spinal CSF leak, there may be an underlying connective tissue disorder. It is generally believed that there is a structural vulnerability point on the spinal membrane that is more susceptible to holes or tears . Mechani2O. Additionally, he had a positional headache but no history of trauma or piercing, indicating SIH. Further cerebral MRI scan confirmed the diagnosis. Most spontaneous CSF leaks occur at the thoracic level  for the publication of any potentially identifiable images or data included in this article.ML: Writing – original draft. YLi: Writing – review & editing. LT: Writing – review & editing. HL: Investigation, Writing – review & editing. LW: Investigation, Writing – review & editing. YZ: Validation, Writing – review & editing. WF: Data curation, Writing – review & editing. YLiu: Investigation, Writing – review & editing. XL: Resources, Writing – review & editing. JH: Writing – review & editing.


This study aimed to establish the incidence of CSF leakage in children and associated complications after intradural spinal surgery in three tertiary neurosurgical referral centers and to describe the treatment strategies applied.Patients of 18 years or younger who underwent intradural spinal surgery between 2015 and 2021 in three tertiary neurosurgical referral centers were included. Patients who died or were lost to follow-up within six weeks after surgery were excluded. The primary outcome measure was CSF leakage within six weeks after surgery, defined as leakage of CSF through the skin. Secondary outcome measures included the presence of pseudomeningocele (PMC), meningitis, and surgical site infection (SSI).We included a total of 75 procedures, representing 66 individual patients. The median age in this cohort was 5 (IQR = 0-13 years. CSF leakage occurred in 2.7% (2/75) of procedures. It occurred on days 3 and 21 after the index procedure, respectively. One patient was treated with a pressure bandage and an external lumbar drain on day 4 after diagnosis of the leak, and the other was treated with wound revision surgery on day 1 after the leak occurred. In total, 1 patient developed a PMC without a CSF leak which was treated with wound revision surgery. SSI occurred in 10.7%, which included both cases of CSF leak.CSF leakage after intradural spinal surgery in the pediatric population is relatively rare (2.7%). Nevertheless, the clinical consequences with respect to secondary complications such as infection and the necessity for invasive treatment are serious.The online version contains supplementary material available at 10.1007/s00381-022-05797-w. Cerebrospinal fluid leakage is a potentially serious complication after intradural spinal surgery. The complications associated with CSF leakage include wound infection, meningitis, and CSF hypotension. CSF leakage may necessitate invasive treatment, such as surgical wound revision, and prolong hospitalization , 2. In aWatertight closure is thought to be the most important step to prevent postoperative CSF leakage. Surgeons may make use of autologous or synthetic duraplasty material and may choose to use a sealant. Their efficacy in the prevention of CSF leakage, especially in the pediatric population, however, has not been studied.There is variation in the definition of CSF leakage across different studies . Some deThis study aims to establish the risk of CSF leakage and associated complications after intradural spinal surgery in three tertiary neurosurgical referral centers between 2015 and 2021 and to describe the treatment strategies applied. The results will be compared to the existing literature.Evaluation of the up-to-date risk of CSF leakage and associated complications in a multicenter setting may serve as a benchmark and assist in counseling of future patients and parents.This is a multicenter retrospective cohort study of all consecutive pediatric patients in the Wilhelmina Children’s Hospital and Princess Máxima Center for Pediatric Oncology in Utrecht, the Netherlands, and the University Children’s and University Hospital in Zurich, Switzerland. This study was approved by the institutional research boards and the local ethics committee and conducted according to the STrengthening the Reporting of OBservational studies in Epidemiology guidelines .All patients of 18 years or younger who underwent intradural spinal surgery between January 1st, 2015, and June 30th, 2021, with a surgical report available were included. Patients who died within 6 weeks after surgery, were lost to follow-up, or had reoperation within 6 weeks for other reasons than CSF leakage treatment were excluded. Inclusion and exclusion criteria are presented in Online resource Procedures eligible for inclusion were screened based on a prospectively collected database or operation codes in the electronic patient records. Data was collected from the following sources: medical notes, medical imaging, and surgical reports between 6 weeks prior to surgery and 6 weeks after surgery.Surgical characteristics collected were indication of surgery , emergency surgery (yes/no), reoperation , the use of products with the intention to seal the dura (yes/no), duraplasty use (yes/no), attempt to watertight closure (yes/no), suture method used (continuous or standing), and suture material used.The following patient data were collected: age at the time of surgery, steroid use for multiple consecutive days directly before or after surgery, diagnosis of hydrocephalus based on medical imaging or medical notes, perioperative CSF diversion surgery , external lumbar drainage (ELD), ventriculoperitoneal (VP)-shunt, or endoscopic third ventriculostomy (ETV)), perioperative chemotherapy, and perioperative radiotherapy. Patients who used steroids directly before or after surgery, underwent perioperative chemotherapy, or were diagnosed with the auto-immune disease were classified as immunocompromised.Data items related to the clinical course included the total number of admission days, the number of days in the pediatric intensive care unit (PICU), and new neurological deficit after surgery (including worsening of existing deficit). If CSF leakage had occurred, the following additional characteristics were retrieved: the estimated number of extra admission days due to CSF leakage (estimated based on a treatment course for CSF leakage or readmission days), surgical wound revision because of CSF leakage, a CSF diverting procedure as a treatment for CSF leakage, the number of days of CSF diversion, a lumbar puncture to treat CSF leakage, extra suture placement, or pressure bandage because of CSF leakage, puncture of PMC.The primary outcome measure of this study was CSF leakage within 6 weeks after surgery, defined as leakage of CSF through the skin . Secondary outcome measures were the presence of PMC (defined as a subcutaneous collection of CSF), surgical site infection (SSI) , and meningitis  within 6 weeks after surgery.The PubMed and EMBASE database were searched until September 12, 2022, for studies reporting the CSF leakage incidence after spinal surgery in children. Studies including only one specific indication for surgery and case reports were excluded. The following search terms were used:  AND (“pediatric” OR “peadiatric” OR “child*”) AND . Reference lists of included articles were manually searched for additional relevant studies.P-values < 0.05 were considered significant. Because of the high number of potential risk factors for CSF leakage, Bonferroni correction was applied with a baseline significance level of 5%. All analyses were performed in IBM SPSS, version 26.0 .Baseline characteristics and incidence of secondary complications were presented as median with interquartile range (IQR) and compared between patients with CSF leakage and those without, using the Mann–Whitney U test for continuous variables and Fisher’s exact test for categorical variables. Variables with ≥ 5% missing data were not included into statistical analyses. The primary outcome measure is presented as the percentage of the total population. n = 43, 57.3%) (Table n = 39), followed by developmental defects (n = 34) and vascular surgery (n = 2). Seventeen procedures were reoperations. Among the procedures for developmental defects were 11 myelomeningoceles, of which 1 had a preoperative leak (but no postoperative CSF leak).We included 75 procedures, representing 66 individual patients Fig. . The med%) Table . The mosIn total, 10 patients had undergone CSF diversion surgery, of whom 5 preoperatively and 5 postoperatively. In total, 1 patient who underwent tumor resection surgery had undergone preoperative ETV and 4 patients had a VP-shunt, of whom one patient was treated for a tumor and 3 for developmental defects. Another 5 patients received a VP-shunt postoperatively, all developmental defect cases with hydrocephalus.No statistically significant differences were observed between groups for the analyzed variables Table . There wCSF leakage occurred in 2.7% (2/75) of procedures. The first patient was a male newborn who underwent an untethering procedure. CSF leakage occurred on day 21. The patient was readmitted and underwent wound revision surgery 1 day later. The total readmission period was 7 days. The second patient was a 6-year-old boy who underwent a vascular procedure, and the leak occurred on day 3 after the index procedure. A pressure bandage was applied, and on day 4 after diagnosis of the leak, an external lumbar drain (ELD) was placed.The ELD could be removed after 6 days and wound leakage did not recur. Hospital stay was prolonged with an estimated 10 days. The index procedure was the first surgery in both cases, and no dural grafts were used. The patients were not immunocompromised and had no hydrocephalus.p = 0.01). One patient with an SSI (and no CSF leakage) was treated with surgical wound revision. All patients with SSI and CSF leakage received antibiotic treatment. There were no cases of meningitis in this cohort.In total, 3 patients developed a pseudomeningocele or CSF leak (3.8%). There was one case of pseudomeningocele without a CSF fistula, which was treated with wound revision surgery. A total of 8 cases of SSI occurred (10.7%). Patients with a CSF leak had moderate evidence for an increased risk for SSI, where 6 of 73 (8.2%) patients without, and both patients with CSF leak developed an SSI, respectively . This is comparable to the risk of incisional CSF leakage observed in our study (2.7%). The combined CSF leakage and PMC risk in our study is substantially lower: 3.8%. This discrepancy may be the result of the subjective definition of PMC, especially considering the retrospective nature of both studies. Another explanation may be the difference in the distribution of procedures performed between these studies. Kaufman et al. found no CSF leakage in their study reporting on the use of non-penetrating anastomotic clips to close the dura after spinal procedures , 9. CurrTo achieve optimal watertight closure, the first choice is to close the dura primarily using microsurgical techniques. If this is not possible, the use of autologous duraplasty material is the second choice. The third choice would be to make use of an allogenic duraplasty. The low leakage rate observed in this spinal cohort is in line with the observation that for the largest group in this cohort, tumor resection cases, we generally find an intact dura to be opened surgically during the procedure, which we are able to close primarily, without the need for duraplasty. In the vast majority of cases, this involves laminotomy with the replacement of the lamina. For the other large group in our cohort, developmental disorders, we more often see a defect in the dura, for which a duraplasty has to be used. We advise to leave any subcutaneous lipoma in place for proper closure of the skin tissue to reduce the relatively higher leakage risk in these cases.The CSF leakage risk found in this spinal cohort is lower than that found in the cohort of intradural cranial surgery procedures 7.5%) in the same centers for this time period . This al% in the The percentage of surgical site infections in our series is relatively high which may partially be explained by our lenient definition of SSI (which does not require a positive wound culture). Previous studies report SSI risks between 0.3 and 12.7% , 14–17. Risk factors identified for CSF leakage in the study by Liu et al. after intradural spinal surgery in the pediatric population are previous spinal surgery, the use of a dural graft, older age, the use of non-locked continuous suturing, and the procedure performed . Cord unCSF leakage was treated with invasive treatment  in both cases in our study. Balasubramaniam et al. also report invasive treatment with lumbar drainage, placed a few millimeters from the incision, preferably one or two levels above, in all procedures and reoperation in 23% . Liu et N = 2), which limits the validity of statistical analyses performed. Furthermore, this retrospective study is vulnerable to reporting bias as it depends on existing clinical records. The variables with missing data for suture type, suture technique used, and attempt of watertight closure reflect this bias. These variables could therefore not be incorporated into our statistical analyses. Furthermore, we did not collect data on bedrest prescription and level of the surgery. Yet, these factors may actually be of influence on the risk of CSF leakage and should thus be further evaluated in future studies.The most important limitation of this study is the small sample size and the low number of CSF leaks . Nevertheless, the clinical consequences with respect to complications such as infection, necessity for invasive treatment, and prolonged hospitalization are serious. Future studies into the development of preventative strategies for spinal surgery in the pediatric population are warranted.Supplementary file1 (DOCX 12 kb)Below is the link to the electronic supplementary material.


Neurofilament light chain protein (NFL), a marker of neuronal axonal degeneration, is increased in cerebrospinal fluid (CSF) of patients with idiopathic normal pressure hydrocephalus (iNPH). Assays for analysis of NFL in plasma are now widely available but plasma NFL has not been reported in iNPH patients. Our aim was to examine plasma NFL in iNPH patients and to evaluate the correlation between plasma and CSF levels, and whether NFL levels are associated with clinical symptoms and outcome after shunt surgery.Fifty iNPH patients with median age 73 who had their symptoms assessed with the iNPH scale and plasma and CSF NFL sampled pre- and median 9 months post-operatively. CSF plasma was compared with 50 healthy controls (HC) matched for age and gender. Concentrations of NFL were determined in plasma using an in-house Simoa method and in CSF using a commercially available ELISA method.Plasma NFL was elevated in patients with iNPH compared to HC (iNPH: 45 (30–64) pg/mL; HC: 33 (26–50) , p = 0.029). Plasma and CSF NFL concentrations correlated in iNPH patients both pre- and postoperatively . We found only weak correlations between plasma or CSF NFL and clinical symptoms and no associations with outcome. A postoperative NFL increase was seen in CSF but not in plasma.Plasma NFL is increased in iNPH patients and concentrations correlate with CSF NFL implying that plasma NFL can be used to assess evidence of axonal degeneration in iNPH. This finding opens a window for plasma samples to be used in future studies of other biomarkers in iNPH. NFL is probably not a very useful marker of symptomatology or for prediction of outcome in iNPH. Idiopathic normal pressure hydrocephalus (iNPH) is a treatable neurological disease in the elderly, characterized by gait and balance disturbance, cognitive decline and urinary incontinence in combination with ventriculomegaly . The etiCSF) is a biomarker reflecting neuronal death and axonal degeneration [CSF has proven rather unspecific for neurological disorders [CSF is elevated in patients with iNPH in comparison to neurologically healthy individuals [Cerebrospinal fluid (CSF) neurofilament light chain protein , allowing monitoring of disease related axonal degeneration without a lumbar puncture [plasma has been tested successfully in other neurological disorders, such as Alzheimer’s disease (AD) [plasma in patients with iNPH.It is now possible to determine NFL in plasma  , parkinsase (AD) , 21 and ase (AD)  and coulase (AD) . As the plasma is elevated in patients with iNPH in comparison to healthy individuals; (2) to explore the association between NFLplasma and NFLCSF in iNPH patients pre- and postoperatively; (3) to explore the associations between NFL concentrations in plasma and CSF and severity of symptomatology and outcome after shunt surgery in iNPH.The aim of this study was (1) to explore if NFLFifty patients (34 men and 16 women) aged 73; 63–78 years ) consecutively diagnosed were treated with shunt insertion for iNPH according to the international guidelines  at the HSymptoms and signs were assessed on the iNPH scale  comprisiAll patients received a Medtronic Strata ventriculoperitoneal shunt with an adjustable valve. All patients were operated on by frontal approach. All shunts were set to 1.5 opening pressure at time for the insertion. No adverse events were recorded. All shunts were checked for patency and were working at the time of follow-up.plasma [Fifty healthy individuals from the H70-study population based sample reported elsewhere ) were included as controls for NFLplasma –27. All Lumbar CSF was obtained from the iNPH patients at the pre-op clinical work-up and at the post-op examination after median 9 months (Q1–Q3 8–11). Lumbar puncture (LP) was performed in the L3/L4 or L4/L5 interspace with the patient in a lateral recumbent position. The CSF opening pressure was measured. The CSF was collected in polypropylene tubes, centrifuged, aliquoted, and analyzed according to standardized procedures.Blood samples were collected in EDTA tubes from the iNPH patients at the time for LP both pre- and postoperatively and for controls in connection with the clinical study evaluation . Plasma CSF was measured by the NF-Light ELISA as described by the manufacturer .The concentration of NFLplasma was measured using the in-house Simoa NFL assay which has been described in detail elsewhere [d-galactopyranoside (Quanterix) was added and the immunocomplex was applied to a multiwell array designed to enable imaging of every single bead. The average number of enzymes per bead (AEB) of samples was interpolated onto the calibrator curve constructed by AEB measurements on bovine NFL (UmanDiagnostics) serially diluted in assay diluent. The average inter-assay CV was 7.9%CV for QC1 and 13.4%CV for QC2. The limit of detection (LOD), determined as the mean blank signal + 3 SD for the Simoa NFL assay was 0.3 pg/mL, and the lower limit of quantification (LLOQ) determined as the mean blank signal + 10 SD was 2.7 pg/mL when compensated for a four-fold sample dilution.The concentration of NFLlsewhere . Brieflys). The level of significance chosen was 0.05, if not otherwise stated. No correction for the mass-significance effect was made. Statistical analyses were performed using IBM® SPSS® Statistics for Windows version 24.Non-parametric methods were used for all analyses due to markedly skewed distributions of concentrations in CSF and plasma and because of the ordinal level of measurement used in some of the domains of the iNPH scale. The Chi-square test was used to compare frequencies across groups. When analysing the relation between paired variables, the Related-Samples Wilcoxon Signed Rank Test was used. For unpaired variables, Mann–Whitney U-test was performed. For associations between two independent variables, the Spearman Rank Order Correlation test was used (rThirty-six 72%) patients improved after shunt surgery. The total iNPH scale score increased from 51; 37–72  to 70; 53–83 (p ≤ 0.001) and increased scores were seen in all domains (Table % patientplasma in iNPH patients was 45 (30–64) pg/mL  compared to 33 (26–50) in controls (p = 0.029)  ng/L in iNPH-patients. Seventeen of the 50 patients (34%) had elevated NFLCSF concentrations compared to the laboratory reference value < 1850 ng/L.Preoperatively, NFLmedian; Q–Q3 compaplasma remained unchanged (43 (28–55) pg/mL, p = 0.54) whereas NFLCSF increased (1955 (1500–2083), p < 0.001). This increase was seen in both patients with normal, or increased, NFLCSF preoperatively.Postoperatively, NFLplasma correlated with NFLCSF both pre- and postoperatively (iNPH patients); rs = 0.629 (p =  < 0.001) and rs = 0.722 (p =  < 0.0001) respectively  as well as on the domains of gait , balance  and neuropsychology . A higher NFLplasma correlated weakly with a lower score on the total iNPH scale  as well as on the neuropsychology domain . Postoperatively, no significant correlations were seen, except between NFLCSF and the balance domain . Neither did the clinical improvement correlate to concentration of NFL in plasma or in CSF, pre- or postoperatively. Patients who improved after shunt surgery did not differ from those who did not improve in baseline concentrations of NFLCSF or NFLplasma (p = 0.12 and 0.72 respectively).Preoperatively, a higher NFLCSF or NFL plasma was not correlated with age in iNPH patients , while there was a weak correlation between NFLplasma and age in controls . There was no correlation between NFLCSF or NFLplasma and disease duration (iNPH patients) .NFLWe report, for the first time, an elevation of plasma NFL in patients with iNPH in comparison with healthy individuals matched for age and gender and that CSF NFL concentrations are correlated with plasma concentrations both pre- and postoperatively. We found only weak correlations between plasma or CSF NFL and measures of clinical symptoms. A postoperative increase of NFL was seen in CSF but not in plasma.The elevated concentrations of NFL in plasma in comparison with healthy controls reported here corroborate earlier studies using CSF samples –16. NeurEven if most studies report CSF NFL to be elevated in iNPH in comparison to healthy controls, the elevation seen in the clinical setting is normally mild-moderate and according to our experience not necessarily above laboratory reference value. In our material, 34% of the patients had concentrations above this reference value which is in accordance with this notion. In contrast, markedly elevated levels of NFL are often seen in some of the clinical mimics of iNPH, such as atypical parkinsonian disorders, Alzheimer’s disease and frontotemporal dementia , 30. ThuPlasma NFL concentrations were significantly correlated with CSF NFL concentrations both pre- and postoperatively with a strength of associations similar to that in other neurological disorders  supportiCSF correlated with worse gait, balance and cognitive performance with stronger correlations for some of the clinical measures possibly related to the inclusion of secondary cases [We found only weak associations between NFL in CSF and plasma and clinical symptoms and no association to outcome after shunt surgery, age or disease duration. A previous study including patients with idiopathic and secondary NPH found that increased NFLry cases . Recentlry cases . These fry cases . Howeverplasma and age. These findings are consistent with other studies, where there seems to be a clearer age-related increase in NFL in healthy individuals, whereas there are more divergent results in cohorts with neurological disease [We found no association between NFL and age in patients with iNPH, in controls however, there was a weak correlation between NFL disease . In the CSF increased postoperatively while NFLplasma did not. Studies describing postoperative changes of NFLCSF have been contradictory and both increases [In this study, NFLncreases , 14 decrncreases  and unchncreases  in lumbancreases  and in ancreases . The posWe consider the sample of iNPH patients representative and comparable to earlier studies examining the concentration of NFL in CSF in patients with iNPH in comparison to controls. Patients were diagnosed according to diagnostic guidelines and well characterized regarding clinical symptoms and outcome. Controls were recruited from a population-based sample and matched for age and gender. Limitations include the lack of CSF in controls which limits the possibility to compare CSF concentrations of NFL between patients and healthy individuals, but this has been done in numerous previous studies , 38. FurCSF was determined using a standardized ELISA and routine samples were used. Determining the concentration of NFLCSF on the Simoa might increase the correlation [CSF is determined by ELISA and NFLplasma by Simoa and makes it easier to transfer results to everyday routine. We used standard CSF samples in combination with stored plasma samples. Even though this could affect the results, NFL has shown high stability under different pre-analytical conditions, such as contamination, repeated freeze–thaw, delayed processing or long-term storing [NFLrelation . On the  storing .Plasma NFL is increased in iNPH patients compared with healthy controls and concentrations correlate with CSF NFL, implying that plasma NFL can be used to assess evidence of axonal degeneration in iNPH. This finding supports the view that plasma NFL concentrations reflect CSF concentrations, which opens a window for plasma samples to be used in future studies of other biomarkers in iNPH. NFL is however probably not a clinically useful marker for diagnosis or for prediction of outcome in iNPH.


Odorrana tormota exhibit a diverse range of calls that are rich in NLP components. Previous field playbacks have shown that the female calls can elicit responses from male frogs. Therefore, we conducted a phonotaxis experiment to investigate the differential effects of different NLP calls by female O. tormota on the vocal behavior of male frogs. The results revealed that calls with subharmonics elicited a greater number of short calls and answering calls from male frogs compared to calls with frequency jumps. However, calls with frequency jumps triggered more staccato calls from males than calls with subharmonics. Additionally, during the phonotaxis experiments, we recorded the initial vocalizations of males in response to playbacks of female calls. The majority of males first produced short calls. Under calls with frequency jumps, most of male frogs approaching within 10 cm of the loudspeaker produced staccato calls instead of “meow” calls or short calls. While under calls with subharmonics, most male frogs preferred to produced short calls. Our findings demonstrate that frequency jumps and subharmonic components in the calls of female O. tormota have different effects on male vocal behaviors. The current study lays a foundation for a further understanding of the function of anuran NLP components.Studies have demonstrated that the sounds of animals from many taxa with nonlinear phenomena (NLP)—caused by nonlinear characteristics of vocal organ dynamics that lead to nonlinear vocal phenomena—can influence the behavior of receivers. However, the specific functions of different NLP components have received less attention. In most frog species, females produce few or no vocalizations; in contrast, female The online version contains supplementary material available at 10.1186/s12983-023-00517-9. Currentics, Bp) –9 and evics, Bp) . Compareics, Bp) , 12. Manics, Bp) , making ics, Bp) , 15, encics, Bp) ), attracics, Bp) , 15, incics, Bp) , 17, andics, Bp) . HoweverOdorrana tormota), a nocturnal species that inhabits vegetation along noisy streams, produce courtship calls with ultrasonic and NLP components [O. tormota increase sexual attractiveness [Researchers have found that male and female concave-eared torrent frogs  accounted for more than 50% of all calls containing NLP components. The remaining calls containing NLP had at least two types of NLP components. Therefore, the purpose of the present study was to investigate whether frequency jumps and subharmonics in the calls of female frogs differentially affect the vocal behaviors of male frogs.A field study was performed in the mountain range of Huangshan  in the village of Fuxi along Fu Creek , in March and April of 2021, 2022 and 2023. During the breeding period, the male concave-eared torrent frogs usually appeared at approximately 16:00 and called from rocks or vegetation on the banks of the stream, and at 22:00, the calling behavior of male frogs has mostly ceased. During the study period (18:00–23:00), the nightly ambient temperature at the study site fluctuated widely, but the temperature and humidity at which males were observed in the field ranged from 15.0 to 20.0 °C and from 85 to 95%, respectively.All female calls were recorded using a sound recorder  and an AKG microphone  with the microphone placed approximately 5–10 cm above the female frog’s head. Recordings were carried out during the peak activity period (mainly between 19:00 and 23:00) in the breeding season (March and April) under similar ambient conditions .A total of 120 male frogs were captured in the field and placed in individual plastic terraria for the playback experiment. We used acoustic foam (thickness: 180 mm) to construct a platform (L × W × H: 170 × 120 × 150 cm) in a dark and quiet room  and the last call  to determine whether the vocal behaviors of male frogs were influenced by the distance between males and females in the field. Different call stimuli can result in varying phonotaxis durations; hence, we conducted a statistical analysis of the latency of male frogs to approach within 10 cm of the loudspeakers. Additionally, we documented the frequency of each call type emitted by individual male frogs during the phonotaxis experiment to analyze the impact of various NLP components on vocalization behavior. In summary, we recorded 7 variables for the phonotaxis experiments: (1) the number of short calls produced (C-Sc), 2) the number of “meow” calls produced (C-Mw), (3) the number of staccato calls produced (C-St), and (4) the number of answering calls produced . We used likelihood ratio tests to calculate the significance of each fixed term. If there was a significant impact of NLP on vocal behavior, we employed Wilcoxon tests to further explore whether there were differences in C-Sc, C-Mw, C-St, and C-Ac produced in response to different NLP components. Finally, we used a chi-square test to examine which call types male frogs preferred to produce as the first calls and last calls, as well as to identify potential differences between the first calls and last calls.We used generalized linear mixed models (GLMMs) for primary analyses; these included the NLP component as a fixed term and the call stimulus as a random effect. Due to variation in the latency of male frogs to approach within a 10 cm of the speaker, we include the log-transformed T-Sp as a fixed factor. We employed the “P = 0.29; P < 0.001; P < 0.001; P < 0.001). The male frogs exhibited significant differences in C-Sc, C-St, and C-Ac in response to vocal stimuli with different NLP components, whereas C-Mw was not affected   are widely reported in animal vocalizations, often generating harsh or rough sounds . Severallls Fig. .Anomaloglossus beebei) [O. tormota are affected by their distance from a female.In the phonotaxis experiment, no male frogs produced long calls in response to female calls with frequency jumps and subharmonics. This is attributed to the use of long calls to advertise their sexual readiness and defend their territories , rather  beebei) , whose c beebei)  and thenO. tormota [This study showed that a sudden frequency change  in a female call elicited a greater number of staccato calls from males. Both field and indoor amplexus experiments revealed that male frogs typically emitted staccato calls before engaging in amplexus. Combining these findings with the results of this experiment, we propose that female frog calls containing Fj components may elicit a stronger amplexus response in males  emit sounds with relatively high levels of subharmonics when they reject male mating attempts. Surprisingly, although females signal their rejection with these calls, subharmonics attract male attention, leading males to continue to vocalize [O. tormota may elicit further vocalizations from male frogs, thus enabling female frogs to assess the suitability of potential males.Signalers can use NLP components in their calls to convey their state, as well as to influence the behavior of conspecifics and/or heterospecifics , 34–38. vocalize . The prevocalize , we specO. tormota that contain frequency jumps and subharmonics elicit different vocal behavior from male frogs; the call types that males produced during the phonotaxis experiment were not emitted at random. Males preferentially produced short calls, meows, and staccato calls but not long calls. Future studies should examine the effect of NLP components in male calls on the vocal behaviors of female frogs and investigate how different NLP components affect competitive vocal behaviors among male frogs. These findings may help us to better understand the evolution of calls containing NLP components, and the benefits of such calls for the reproductive behavior of anurans.This study supported our hypothesis that calls of female Additional file 1. Supplementary Materials.Additional file 2. Supplementary video S1.Additional file 3. Supplementary video S2.


Single-molecule FRET (smFRET) is a powerful imaging platform capable of revealing dynamic changes in the conformation and proximity of biological molecules. The expansion of smFRET imaging into living cells creates both numerous new research opportunities and new challenges. Automating dataset curation processes is critical to providing consistent, repeatable analysis in an efficient manner, freeing experimentalists to advance the technical boundaries and throughput of what is possible in imaging living cells. Here, we devise an automated solution to the problem of multiple particles entering a region of interest, an otherwise labor-intensive and subjective process that had been performed manually in our previous work. The resolution of these two issues increases the quantity of FRET data and improves the accuracy with which FRET distributions are generated, increasing knowledge about the biological functions of the molecules under study. Our automated approach is straightforward, interpretable, and requires only localization and intensity values for donor and acceptor channel signals, which we compute through our previously published smCellFRET pipeline. The development of our automated approach is informed by the insights of expert experimentalists with extensive experience inspecting smFRET trajectories (displacement and intensity traces) from live cells. We test our automated approach against our recently published research on the metabotropic glutamate receptor 2 (mGluR2) and reveal substantial similarities, as well as potential shortcomings in the manual curation process that are addressable using the algorithms we developed here. Over the years, researchers have developed various analysis methods for the automated processing of single-molecule fluorescence resonance energy transfer (smFRET) data to generate insights into the structure and function of mostly purified and reconstituted biomolecules in surface-immobilized preparations . At the Using technical advances we developed for smFRET imaging, including the smCellFRET software, we recently successfully carried out smFRET experiments in living Chinese Hamster Ovary (CHO) cells in order to study G protein-coupled receptor (GPCR) dimerization, in addition to structural changes within metabotropic glutamate receptor 2 (mGluR2) . With thsmCellFRET is an open-source software package for use in the MATLAB environment that processes smFRET data of labeled donor and acceptor molecules diffusing in the cell plasma membrane. Data processing in smCellFRET utilizes tracking and motion classification data generated with the SPT software u-track and the DC-MSS analysis method . We usedIn our early experiments, once we had generated smFRET trajectories, we recognized that multiple particles would occasionally cross into a given donor or acceptor’s path. During such crossing-over events, fluctuations in the donor and acceptor channel ROIs influence the calculation of the FRET value and lead to distortions in the FRET value distributions generated from trajectories where such crossings occurred. To guarantee that crossing-over events were not contributing to the FRET value distributions, a trained specialist would manually inspect both the fluorescence emission in the donor channel and sensitized acceptor emission in the acceptor channel, for every smFRET trajectory used to generate smFRET histograms. This process required watching a video of every smFRET trajectory over its entire trajectory, and we quickly realized that excising individual frames would be unfeasible for hundreds of trajectories per cell, each several hundred frames long. As a compromise, the experimentalist would inspect the video frames of each donor-acceptor trajectory, checking for any crossing-over events, and only preserve those trajectories for further analysis that were entirely free of crossing-over. Given that an experimenter can make a determination at a rate of one frame per second, 8 h is a conservative estimate for the time it would take to hand-curate all of the trajectories from only a single cell. In our previously published dataset, 69% of all traces were rejected, representing 77% of all FRET events. Importantly, much of the excluded data consists of trajectories where interference by other particles happens only in a small subset of frames during that trajectory’s lifetime.As noted, in our previous work, because of the complexity associated with manually removing these crossings, the entire trajectory would be removed from inclusion in the computation of the FRET distribution, as frame-by-frame analysis and labeling for crossing-over would have been too time-consuming. Background autofluorescence in the cell and heterogeneities across the cell surface made the observation and discrimination of crossing-over events by a human both time-consuming and subjective, leading to potential biases and reduced reproducibility. To overcome these obstacles, we have now developed the necessary analysis for automating the removal of frames with crossing-over events. This allows us to generate an accurate FRET distribution from the data. We additionally provide an intensity-thresholding operation that selects trajectories based on a template-matching approach, using a small subset of trajectories where acceptor photobleaching coincides with a simultaneous rise in donor intensity. The automation we have developed here provides two basic enhancements:  at the fTo develop methods for the automation of crossing-over event removal, we sought to replicate the performance of a manually-curated analysis approach on a dataset obtained using total internal reflection fluorescence microscopy (TIRF) imaging of the SNAPf-tagged membrane-embedded protein, mGluR2, controllably expressed at low levels . The proFor the dataset we used in this study to automate the removal of crossing-over frames, we studied mGluR2 dimers with an acceptor and donor fluorophore on each protomer . When diDue to the nature of stochastically labeled particles, signals in the donor channel can come from receptor dimers labeled with one donor and one acceptor, which represents the population of receptors with smFRET signal, but also from dimers containing two donors or only one donor and no acceptor. Thus, there are far more signals in the donor channel compared to the acceptor channel, which only contains sensitized acceptor particles in an smFRET experiment, leading to relatively uncomplicated tracking in the acceptor channel. Because of the relatively high particle density in the donor channel, there is a potential for overestimating the intensity within the ROI if multiple signals contaminate the donor ROI. Whenever another molecule or aberrant fluorescent signal appears in the proximity of the donor ROI, the computation of the total photons emitted from the original acceptor molecule and donor molecule is compromised. Thus, the accurate calculation of the intensity of the donor and acceptor requires knowledge of every fluorescent signal within a region near the ROI. We calculate the FRET efficiency value, E(t), at each frame in time based on the intensity of the donor and acceptor, with the appropriate corrections α, δ, and γ, for donor-to-acceptor bleed-through, acceptor direct excitation at our imaging wavelength, and the relative detection efficiencies and quantum yields of the fluorophores, respectively :Et=IaccIn a manual-curation approach, one must verify the integrity of the photon calculation for a given acceptor fluorophore and donor fluorophore by scanning every frame of an smFRET trajectory to determine whether the search area around the ROI has been breached by another molecule or signal . MultiplDtrajectory→acceptor and Dtrajectory→donor which contain, for each frame at all times t when a donor-acceptor trajectory is tracked, the distance between the acceptor in the trajectory and the entire set of signals detected in either the acceptor channel or donor channel . To accomplish this, we first compute two time-varying trajectory-wise distance matrices nnel Eq. . In our D are computed, a density can be computed at each timepoint, ρi(t) for the ith donor-acceptor trajectory, as the count of all signals in either the acceptor or donor channel located at a distance less than dmin:Once the trajectory-wise distance matrices total number of located particles  that may cause interference at a given timepoint t, in the acceptor or donor channel, respectively. The density function is a sum at each timepoint over all of that timepoint’s potential interfering particles, and the identification function (script 1 in min). We use the notation pair→acceptor and pair→donor to describe the distance between the acceptor/donor pair under examination and any other particles in the ROI of either the acceptor or donor, respectively. Note that the density must return 1 at a timepoint where the original donor and acceptor of the donor-acceptor trajectory have been successfully located by u-track. At some timepoints, when the smFRET efficiency is very high and approaches 100%, the donor may be nearly completely quenched with little to no detectable fluorescence, leading to no donor track, and ρdonor would be zero for the donor-acceptor at those timepoints. With regard to the choice of dmin, to reduce the possibility of introducing photons from another signal’s Airy disk, we chose 6 pixels for our value of dmin, as the ROI we used to calculate intensity around the acceptor and donor is 5 pixels x 5 pixels. In the ideal situation where no crossing-over is ever present in the data, there would be N functions for ρ(t), which would be equal to 1 for all timepoints, and the number of acceptor signals N would be equal to the number of donor signals M. In our data, M >> N and ρ(t) would exceed 1 at many timepoints. For the ith donor-acceptor trajectory, we preserve the subset of FRET values where for a given timepoint, both ⍴iacceptor(t) and ⍴idonor(t) remained less than or equal to 1  the high FRET, medium FRET, and low FRET regions. In our analysis, we accepted for analysis those donor-acceptor trajectories with acceptor and donor intensities falling within one of the three regions delimited by our inferred Iolutions , we usedolutions . Thus, wolutions . Indeed,olutions . The remN = 6 cells for each of the three conditions   . In computamate) . Notablyutamate) . To ensuWe also applied our new method to a negative control dataset acquired in the same experiment. For the Low Density Lipoprotein transmembrane domain (LDL-TM), a known monomer , our algIn our previous work tracking smFRET in live cells, we optimized smFRET particle tracking by expressing low levels of labeled molecules, tracking sensitized acceptor molecules and using these locations to create ROIs for the acceptor channel, as well as for the donor channel via a mapping function, and tuning u-track to provide highly reliable tracking at subpixel resolution . NeverthOur intensity thresholding approach here does not rely on visual inspection of images or intensity histograms over the dataset but rather a template-matching approach. We use smFRET donor-acceptor trajectories with a known signature of FRET, anti-correlated bleaching, to justify the selection of a lower boundary on the acceptor and the upper boundary on the donor. It is clear that this logic will apply best in experimental systems where a large amount of data is collected for a given fluorophore, where one donor is expected to meet one acceptor, and where at least some acceptor photobleaching is observed. In some future smFRET experiments, photobleaching may not be observed or expected, in which case another basis for discriminating reliable trajectories would need to be used. For example, a trajectory may reveal a high level of anti-correlation, increasing our confidence that the trajectory is generated by a donor-acceptor pair undergoing smFRET. Such anti-correlated trajectories could be collected to create templates, as we have done here, for estimating the range of intensity values for single acceptor and donor molecules. Alternatively, given a large enough dataset, a spectral clustering approach on the intensity space values could be attempted for sequestering the cloud of template trajectories from background noise. In either case, it is crucial to examine the underlying assumptions of any clustering approach and ensure their validity for the system under investigation.Our automated approach remedies the manual trajectory selection process’ bias away from high FRET values, introduced by the complete exclusion of donor-acceptor trajectories with any crossing-over frames required by the hand-selection process. By mapping our data in intensity space onto FRET space, we observe that trajectories with high acceptor fluorescence, and therefore higher FRET, invariably persisted for more frames in our experimental pipeline, because we tracked sensitized acceptors and only infer donor locations. The higher intensity of an acceptor leads to tracking over a longer time frame, leading to more opportunities for a manual observer to perceive a crossing-over event. This slight bias toward shorter trajectories and therefore away from high acceptor particles diminished the observation of the high FRET state for mGluR2. The finding of an enhanced proportion of high FRET values is reflected in our previous publication , where wUltimately, the enhancements we pursued here led to a nearly two-fold increase in the total data spared after automated curation. While it is possible that by reintroducing donor-acceptor trajectories and smFRET traces that had been rejected by a specialist, we have injected some other type of error. For instance, the minority fraction of trajectories that were rejected by the specialist but contained no detectable crossing-over events may reflect a lack of detection by u-track based on the settings we used. These crossing-over events may have been due to particles that were too dim or not well-fit to a Gaussian and therefore were not tracked by the software. Were it important to consider these low-intensity interferences, one could turn to intermediate data structures within the u-track pipeline, which contain all the local maxima present in an image even before fitting with a Gaussian kernel and could be the basis for a more conservative detector of crossing-overs. Here, we chose to only focus on those tracks which u-track accepted as sufficiently Gaussian and having an adequate signal-to-noise ratio. A track originating from a spot that does not meet these basic requirements would not have sufficient intensity to materially alter FRET, unless the donor and acceptor both had low intensities themselves, a situation that we mitigate against using our thresholding step. For the tracks in the donor and acceptor channels that are identified as potentially interfering with our smFRET donor-acceptor trajectories, our automated approach outputs all the track identities and intensity information. This could enable future studies in which interactions between smFRET donor-acceptor trajectories are studied, or the density of donor-acceptor trajectories in defined membrane areas are related to the FRET values computed. Indeed, our crossing-over detection algorithm can easily be used as a method for obtaining the local density of smFRET donor-acceptor trajectories along an individual trajectory’s trajectory.We showed that the results of our analysis could be replicated in a different dataset collected at a different time from a different set of cells. Namely, the presence of a high-FRET state we detected through automated analysis of our original, published work can be recaptured in this distinct dataset, at remarkably similar proportions. This bolsters the claim that the glutamate receptor, in living cells, visits at least two distinct conformations even in a condition with no ligand added. The persistence of this state even in increasing levels of ligand provides an interesting insight into the nature of this conformation.Through our intensity space representations, we sought to develop an intuitive approach for scrutinizing donor-acceptor trajectories and viewing results of selection routines imposed on them. This representation of each donor-acceptor trajectory in intensity space allows us to infer reasonable values for thresholds from mapping template particles, those which had the signature FRET anti-correlated bleaching behavior, onto the overall distribution of particles. Looking within intensity space also provides hints as to where hand-curation may have introduced biases. Moreover, it could help tune tracking, as the donor-acceptor trajectory in the intensity space could be color-coded by its lifetime, revealing potential variability due to particle intensity. Further implementations of this view could be used to examine the diffusion states of molecules spanning intensity space, which would allow probing of the relationship between FRET and diffusion. In another embodiment, points in the intensity space could be color-coded based on their proximity to a biologically relevant landmark in the cell. These relationships could motivate various discriminative classifiers that would give experimentalists novel insights into decoding the relationships between FRET and the cellular milieu in which molecules diffuse.Ultimately, we believe the optimal approach to scrutinizing smFRET values will integrate a small amount of manual inspection by a trained specialist, especially for populations of donor-acceptor trajectories that are dissimilar from their neighbors in terms of track length, mobility, signal-to-noise, or other features accessible through upstream tracking software such as u-track. The insights of such a specialist can be used to guide and coordinate the automated approaches that can be developed by a data analyst working in conjunction with the experimental team. As imaging experiments become increasingly ambitious and more particle features are used to analyze tracking accuracy and biological variables of interest , the amo


Wearable surface electromyography (sEMG) signal-acquisition devices have considerable potential for medical applications. Signals obtained from sEMG armbands can be used to identify a person’s intentions using machine learning. However, the performance and recognition capabilities of commercially available sEMG armbands are generally limited. This paper presents the design of a wireless high-performance sEMG armband (hereinafter referred to as the α Armband), which has 16 channels and a 16-bit analog-to-digital converter and can reach 2000 samples per second per channel (adjustable) with a bandwidth of 0.1–20 kHz (adjustable). The α Armband can configure parameters and interact with sEMG data through low-power Bluetooth. We collected sEMG data from the forearms of 30 subjects using the α Armband and extracted three different image samples from the time–frequency domain for training and testing convolutional neural networks. The average recognition accuracy for 10 hand gestures was as high as 98.6%, indicating that the α Armband is highly practical and robust, with excellent development potential. Surface electromyography (sEMG) signals reflect the comprehensive effects of surface muscle and nerve trunk electrical activity on the skin. sEMG data can be acquired noninvasively through simple operations . DiffereSince the introduction of the Myo Gesture Control Armband by Thalmic Labs, the market for sEMG data-acquisition systems has grown rapidly. However, the Myo Armband is limited by its eight channels, 8-bit analog-to-digital converter (ADC) resolution, 200 sps sampling rate, and bandwidth of 5–100 Hz. The Biomedical Microsystems Laboratory at Laval University designed a 3DC Armband, which had the following improved device parameters: a 10-bit ADC resolution, 1000 sps sampling rate, and bandwidth of 20–500 Hz. Under the same experimental conditions, the 3DC Armband significantly outperformed the Myo Armband [Phinyomark et al. compared sEMG recognition results obtained with sampling rates of 200 and 1000 sps and found that the 200-sps sampling rate significantly reduced the amount of discriminatory information used for sEMG control, and the difference in frequency information features significantly affected the classification results. The accuracy of recognizing multiple hand and finger movements was significantly increased by high−frequency components, particularly for individuals who had undergone radial artery amputation, with differences exceeding 10% . In addiIn summary, the performance of wearable sEMG data-acquisition devices needs to be improved. In this study, we designed a 16-channel sEMG Armband with wireless communication, a 16-bit ADC resolution, an adjustable bandwidth (0.1–20 kHz), and an adjustable sampling rate with a maximum value of 2000 sps/channel. According to the Nyquist–Shannon sampling theorem, this device can effectively acquire sEMG signals up to 1000 Hz. The high sampling rate allows the accurate identification and classification of sEMG signals through close approximation of the original waveform [The surface electrodes at the front-end of the sEMG acquisition system convert the ionic currents generated by human electrochemical activity into electronic currents, which are detectable by the electronic detection system . Wet eleRHD2216  is a low-power physiological signal-acquisition chip with dimensions of only 8 mm × 8 mm. It features 2.4 µVrms input reference noise, a 16-bit ADC, and 16 differential channels. The amplifier bandwidth can be changed by configuring the registers, and the amplifier offset can be eliminated by an internal digital high-pass filter. The 16-channel differential inputs connect to the differential electrodes and the surface EMG signals are collected in parallel and sent to the microcontroller unit (MCU) in real time through the serial peripheral interface.STM32F765VIH6 is a high-performance controller in the ARM Cortex-M7 series from STMicroelectronics . It has a digital signal processor (DSP), a floating-point unit (FPU), and an adaptive real-time (ART) accelerator, with a maximum clock frequency of 216 MHz and high-speed data-processing capabilities. In addition, it uses thin fine pitch ball grid array (TFBGA) packaging, with dimensions of only 8 mm × 8 mm. Because the surface of the EMG signal-acquisition system needs to contact the body surface, the system volume and weight are critical parameters. The controller is ideal for sEMG signal acquisition owing to its excellent data-processing capability and small size. The MCU continuously monitors the battery status in the idle mode and provides feedback to the user through indicator lights and motors. The MCU receives instructions from the base station through interrupts and configures the upper and lower cutoff frequencies , the sEMG sampling rate (up to 2000 sps/channel), and the inertial measurement unit (IMU) sampling rate (up to 100 Hz), according to the instructions. The parameter configurations are stored in flash memory and are read when the device restarts to perform configuration operations. The MCU reads the values of the corresponding registers to determine whether the configuration was successful and provides feedback on the configuration results to the base station. After receiving the acquisition instructions, the MCU collects sEMG and IMU signals at the configured sampling frequency through direct memory access (DMA), until a stop instruction is received or the device’s power is insufficient. The device supports both wired and wireless transmission channels, with low-power Bluetooth for wireless communication and a universal synchronous/asynchronous receiver/transmitter (USART) for wired communication. The MCU determines the source of the instructions and replies with data on the corresponding channel.The IMU uses ICM-20948 , which has a built-in three-axis gyroscope, three-axis accelerometer, three-axis compass, and digital motion processor (DMP). It is encapsulated in a 3 mm × 3 mm × 1 mm (24-pin quad flat no-led (QFN)) package. The system supports multiple 200-mAh lithium batteries in parallel or a Type-C power supply. BQ24075 is responsible for charging and discharging management, and the LP5912 series low-dropout regulator (LDO) chip provides 1.8- and 3.3 V voltages. BQ27220 is responsible for monitoring the battery level and other parameters. The low-power Bluetooth module HJ-185IMH with dimensions of 5.5 mm × 5 mm × 1.3 mm is used as the wireless transceiver. It can support a maximum speed of 1 Mbps. The sEMG and IMU data are received through a USB dongle (HJ-380IMH).The α Armband is shown in The authors designed a user-friendly software for the α Armband through MATLAB R2022a which facilitates data interaction with the hardware through the manipulation of the USB dongle. The software displays real-time sEMG waveforms of 16 channels and has functions for configuring hardware parameters and saving data .To prevent the loss of high-frequency components of sEMG data, a sampling rate of 2000 sps was used in this experiment. The upper computer software received sEMG data in real time through Bluetooth and saved the received data. Finally, the data were analyzed and processed using MATLAB.During the experiment, the α Armband was worn on the right forearm of the subject, who had rested adequately to ensure that muscle fatigue would not introduce interference. Additionally, the electrode placement area was cleaned and disinfected before the experiment. The participants were asked to perform 10 common hand gestures: Power Grip, OK Hand (in China), Thumb Up, Thumb Down, Scissorhands, Palm Up, Palm Down, Palm Outward, Palm to the left, and Palm to the right .The experiment involved 30 participants, including 20 males and 10 females between the ages of 20 and 50 years. Among them, 24 were right-handed, and 6 were left-handed. Each participant was asked to maintain a gesture for 10 s, followed by a 3 s rest period, and then to repeated the same gesture 10 times. This process was repeated for all 10 gestures. The experiments lasted for a week, with one experiment conducted per day. The data used to the train machine-learning were the average values for the 10 repetitions of each gesture. All participants agreed to allow their collected sEMG data to be used free of charge for academic research purposes.sEMG signals are often contaminated by noise due to interference from the surrounding environment and the circuitry of the acquisition device, which can sometimes overpower the sEMG signal. To obtain the true sEMG signal, noise reduction measures must be taken ,11,12.On the hardware side, the cutoff frequency of the built-in high-pass filter of RHD2216 was configured to 2 Hz. The sampling frequency in the experiment was 2000 Hz. To comply with the Nyquist–Shannon sampling theorem, the cutoff frequency of the low-pass filter was configured to 1000 Hz for constructing a bandpass filter that screened out noise outside of the frequency band. The built-in custom digital module of RHD2216 was used to implement a single-pole high-pass filter on each sampling amplifier channel, with the aim of eliminating the residual direct-current (DC) offset voltage associated with the analog amplifier.Electric-field interference can affect sEMG detectors through capacitive coupling. In a 50-Hz alternating-current (AC) power environment , 50 Hz AC power can easily interfere with sEMG acquisition equipment. Poor grounding and many other factors can induce powerline frequency interference into the equipment . The colHigh-accuracy gesture recognition is a key goal pursued by researchers. Thus, feature extraction and classification are among the most critical stages in sEMG control systems. Classification techniques based on sEMG have been extensively studied , and sEMThe manual extraction of sEMG signal features often leads to significant errors. Deep learning can automatically extract features at different levels from a large number of input samples, avoiding the complex and cumbersome process of manual feature extraction and selection . In receIn our experiment, two comparative methods were designed on the basis of the CNN architecture. The data of 8 sEMG channels of the α Armband were used as one group (distributed evenly across 8 channels) and the data of 16 sEMG channels were used as the other group to examine the effect of the number of channels on the gesture recognition accuracy. The collected sEMG data were divided into three types according to the time–frequency domain: time-domain image inputs, multi-channel spectral image inputs, and feature-enhanced multi-channel spectral image inputs.For real-time wearable sEMG devices, the total response time of myoelectric control should be limited to 300 ms ,26,27. AAs shown in In this experiment, a sliding window of the Hann function was used to perform the short-time Fourier transform (STFT) on sEMG signals for acquiring the spectral features of the signals. A 100 ms window with a 50 ms increment was used to segment a 250 ms sEMG signal, resulting in a 4 × 129 output matrix. As shown in The CNN architecture for the multi-channel spectral images was similar to that of the time-domain images. Some parameters were adjusted according to the input images. As shown in To enhance the feature representation of the signal, Duan Na et al. used the Myo Armband to extract the maximum value of the sEMG signal from each channel as an auxiliary channel signal. They merged the spectrograms of the eight original channels and one auxiliary channel into a single image as the input of the CNN. The test results indicated that after multiple iterations, the accuracy of the CNN reached 94.06% . In thisIn According to Considering the input model, for the eight-channel experiments, the average accuracies of the time-domain image model and spectral image model were 88.6% and 90.3%, respectively. However, the average accuracy of the feature-enhanced spectral image model was 95.4%, which was 6.8% and 5.1% higher than those of the previous two models, respectively. For the 16-channel experiments, the average accuracies of the time-domain image model and spectral image model were 93.9% and 95.9%, respectively. However, the feature-enhanced spectral image model achieved an average accuracy of 98.6%, which was 4.7% and 2.7% higher than those of the previous two models, respectively. The lowest recognition accuracy of the 16-channel feature-enhanced spectral image model was 96.7%, and the highest recognition accuracy was 99.7%, which was almost perfect. The results indicate that the spectral images carry more prominent features than time-domain images and that extracting the maximum value as an auxiliary channel from the α Armband’s sEMG channels is feasible and helps the CNN extract important features more accurately.Regarding the number of channels, in Ulysse Côté-Allard et al.’s comparative study, the 3DC Armband  outperformed the Myo Armband  in all gesture recognition experiments. The average accuracies of the 3DC Armband and Myo Armband were 89.47% and 86.41%, respectively ,30.In our future work, we will persist in improving the α armband, such by enabling it to detect deep muscles, because deep muscles and surface muscles work together for motion control ,32. Howe(1)Feature-enhanced spectral images enhanced the CNN’s gesture recognition accuracy by at least 3% compared with conventional spectral images;(2)The 16-channel sampling increased the CNN’s gesture recognition accuracy by at least 3.2%.The α Armband designed in this study has more data-acquisition channels, a higher sampling rate, and a larger bandwidth than traditional sEMG armbands. These parameters can be easily configured through Bluetooth, making the α Armband highly adaptable to various working conditions. Our experiment involving the α Armband confirmed its high reliability; it achieved an average recognition accuracy of 98.6% under the feature-enhanced spectral image model. Additionally, our experiment indicated the following:In future research, the authors will test a wider variety of gestures, finger movements, and limb movements and develop more feature extraction and classifier models, facilitating the application of the α Armband in a wider range of medical working conditions.


Molecular analyses did not identify common mutations or chromosomal abnormalities seen in clear cell renal cell carcinoma or ELOC-mutated renal cell carcinoma. This case highlights that rare renal cell tumors remain difficult to classify and the distinction between CCPRCT and CCPRCT-like tumors remains to be better defined.Clear cell papillary renal cell tumor (CCPRCT) is a distinct clinical entity with characteristic pathological features and non-aggressive clinical behavior. Diagnostically challenging cases present when there are immunomorphological findings of CCPRCT associated with heterogeneous morphologies, aggressive histological features, and advanced pathological stages—so-called CCPRCT-like tumors. In this report, we describe a heterogeneous, multifocal renal tumor with immunomorphological characteristics of CCPRCT but with associated aggressive features such as sarcomatoid and necrotic areas, perirenal and sinus fat involvement, and most notably, lymph node metastasis composed entirely of classic CCPRCT morphology and immunophenotype. Immunohistochemical and fluorescence  An 83-year-old female was incidentally found to have a renal mass and retroperitoneal adenopathy on imaging. Her medical history was significant for a transurethral resection of a noninvasive low-grade papillary urothelial carcinoma. Computed tomography (CT) scan revealed a predominantly solid, 3.2 cm mass with cystic areas in the left kidney with a predominantly cystic, 4.0 cm left periaortic mass. Renal biopsy identified an unclassifiable renal cell carcinoma (RCC) with sarcomatoid differentiation. Lymph node biopsy identified minute fragments of tubulopapillary structures suggestive of metastatic RCC. Preoperative bone scan and CT thorax did not show bone or pulmonary metastases. The patient was scheduled for a left open radical nephrectomy and lymph node dissection.in situ hybridization (FISH) with dual color break apart probe for TFE3 did not identify TFE3 gene rearrangements, thus eliminating TFE3-rearranged RCC. Given the morphological and immunohistochemical findings, tumor 1 was diagnosed as RCC with clear cell papillary and sarcomatoid features (see Macroscopic examination of the left nephrectomy specimen identified two tumors: the first (tumor 1) measured 3.0 × 2.7 × 2.5 cm and the second (tumor 2) measured 3.7 × 2.4 × 2.0 cm. Tumor 1 showed histological features typical of clear cell papillary renal cell tumor (CCPRCT), merging with areas displaying more compact cellular growth as well as spindle cell sarcomatoid differentiation. Immunohistochemistry revealed the following staining pattern in the clear cell papillary component: diffuse strong staining for PAX8, keratin AE1/AE3, keratin 8/18, keratin 7, GATA3, carbonic anhydrase 9 (CA9) in a “cup-like” fashion, apical staining for CD10, and negative staining for AMACR. The sarcomatoid component was diffusely positive for CA9, CD10, and GATA3 and negative for AMACR and keratin 7. Fumarate hydratase (FH) and succinate dehydrogenase (SDH) were both retained, thereby ruling out FH-deficient and SDH-deficient RCCs. TFE3, cathepsin-K, MART-1, and HMB45 were also performed to rule out microphthalmia transcription factor family translocation RCCs and were negative. Confirmatory fluorescence ures see  and 2. Tures see  and immuELOC (formerly TCEB1)-mutated RCC. In order to do so, we interrogated the DNA sequences of different tumor components , using an RCC-focused next-generation sequencing (NGS) gene panel followed by whole exome sequencing on an iSeq 100 and NovaSeq, respectively (paired-end 150). Bioinformatic analysis to detect germline and somatic variants was conducted as previously described. The analysis did not detect any mutations in the most commonly mutated genes in RCC, including VHL, ELOC, PBRM1, SETD2, BAP1, MET, ATM, ARID1A, ATP9B, CDKN2A, CDKN2B, COL11A1, CSMD3, DMD, KDM5C, KDM6A, MTOR, NF2, NFE2L3, PTK7, PTEN, PIK3CA, TP53, TRRAP, TSC1, and TSC2 in any of the tumor components. Furthermore, copy number variation analysis did not detect loss of the VHL gene or whole chromosome losses in any of the areas examined.Due to the highly unusual finding of a CCPRCT with sarcomatoid features and advanced pathological stage, further studies were needed to exclude other renal tumors that could show significant overlapping features with CCPRCT, such as clear cell RCC (CCRCC) and Five months post resection, the patient was found to have a solitary lytic lesion in the vertebral body of L2 suspicious for metastasis that measured 1.4 cm on bone scan and remained stable on follow-up imaging. No other evidence of metastasis was identified on CT imaging of the chest, abdomen, or pelvis at the time of this publication. The tumor was renamed as a reflection of its almost invariably low-pathological stage and indolent behavior. Although initially described in the setting of end-stage renal disease, it is currently known to be a common tumor affecting normal and abnormal kidneys in a sporadic setting.6 Macroscopically, tumors tend to be solid or cystic, usually tan-white and fibrous. Histologically, CCPRCT shows tubular and papillary structures, as well as frequent cystic components.8 Cells characteristically have clear cytoplasm with nuclei aligned away from the basement membrane resembling secretory endometrium.9 CCPRCT classically tends to be WHO/International Society of Urological Pathology (WHO/ISUP) grades 1 or 2, with only a minority of reported tumors that have grade 3 nuclei.CCPRCT, previously known as clear cell papillary RCC, was first recognized in 2006 and included in the 2016 World Health Organization (WHO) as a novel subtype.7 In the areas with CCPRCT morphology, our tumor showed an immunohistochemical profile most consistent with CCPRCT. Although CD10 staining was present, it showed membranous staining in only the apical aspect of cells. Interestingly, minor labeling for CD10 has been described to have focal positivity in 24% of tumors, and when present is frequently luminal with accentuation in cells lining cystic foci as seen in our patient's tumor. In the areas with more solid nested and sarcomatoid morphology, the tumor stained similarly although showed loss of keratin 7.Immunohistochemically, CCPRCT shows cup-like staining pattern with CA9, and is typically diffusely positive for keratin 7. GATA3 can be positive while AMACR and CD10 are either negative or focally positive.TFE3-rearranged RCC, or ELOC (formerly TCEB1)-mutated RCC.1015 In recent years, a range of renal tumors displaying CCPRCT-like areas have been increasingly recognized and documented. Therefore, with current evidence suggesting that CCPRCTs are almost invariably indolent, the standard practice has been to use strict morphological and immunohistochemical criteria  when making a diagnosis of CCPRCT, as the current terminology implies those tumors are not even malignant. When faced with a CCPRCT-like tumor showing either an unusual morphology , unusual immunophenotype (such as a box-like staining pattern for CA9), unusual tumor characteristics , one has to assume that such tumors represent entities other than CCPRCT, with CCRCC being the most common tumor type reported to mimic CCPRCT. From a practical standpoint, if such further molecular characterization is unavailable, CCPRCT-like tumors with unusual or aggressive features likely need to be regarded and treated as an RCC.Molecularly, CCPRCT does not appear to have a distinct profile and is better characterized by a lack of the characteristic genetic alterations identified in other subtypes such as CCRCC, papillary RCC, VHL, PBRM1, SETD2, BAP1, and MET, among others as well as copy number variation analysis may be helpful to help exclude both CCRCC and papillary RCC. Translocation RCCs are also well-known mimickers of many RCC subtypes, including CCRCC and CCPRCT.1719 Skala et al specifically observed that TFE3-rearranged RCCs may have focal or diffuse subnuclear clearing and linear array of mid-to-apical nuclei on morphology akin to CCPRCT. Therefore, FISH testing for TFE3 in addition to immunohistochemical studies for TFE3 may be considered. ELOC (formerly TCEB1)-mutated RCC are similarly known to have overlapping morphology with CCRCCs and CCPRCTs. ELOC mutation testing may also be considered in an NGS panel for CCPRCT-like tumors depending on available resources.If access is available, testing for molecular alterations seen in CCRCC, papillary RCC, or molecularly defined RCCs may be considered for CCPRCT-like tumors with high-grade morphology and advanced stage. An NGS panel for genes such as  our patient's tumor was unique for several reasons. First, the presence of typical CCPRCT areas, associated with the characteristic immunoprofile in both the primary and the metastatic tumors, is striking. Extraordinarily, the entirety of the metastatic deposit was CCPRCT-like. Second, the CCPRCT-like areas were notably intertwined with those with sarcomatoid morphology. Finally, we did not identify molecular evidence that would exclude a diagnosis of CCPRTC using an extensive molecular panel. More specifically, the tumor did not show any of the most commonly CCRCC mutations , mutations of sarcomatoid transformation in CCRCC , nor chromosome 3p loss in any of the components .2225 As TSC1, TSC2, MTOR, or ELOC (TCEB1) mutations were also not identified, diagnoses such as RCC with fibromyomatous or leiomyomatous stroma and ELOC-mutated RCC similarly are not supported.26 The immunoprofile was also not in favor of a diagnosis of FH-deficient or SDH-deficient RCCs. Finally, immunohistochemical studies  and confirmatory FISH analysis for TFE3 did not support a TFE3-rearranged RCC.Although case reports of CCPRCT-like metastatic tumors with negative genetics have been reported,The present tumor illustrates that even when using strict criteria and with extensive molecular characterization, rare cases remain perplexing and difficult to classify. Whether this tumor represents an extremely rare report of CCPRCT with sarcomatoid differentiation and metastasis, or an unclassified RCC closely mimicking CCPRCT, cannot be entirely determined. However, it clearly highlights that the entity CCPRCT versus CCPRCT-like tumors is still an evolving concept that is yet to be fully defined. Future large studies with long-term follow-up, and similar case reports with molecular characterization will be key in determining the extent to which the diagnosis of CCPRCT can be confidently made solely on morphological and immunohistochemical grounds. Ultimately, genome-scale molecular studies on similar unusual tumors would help to identify driver molecular alterations that would eventually help to refine the diagnosis or describe new unreported entities.


This retrospective review examines the utility of phase-contrast magnetic resonance imaging (PC-MRI) to calculate flow through the aqueduct between the third and fourth ventricles to estimate cerebrospinal fluid (CSF) production. Imaging software quantified the CSF flow rate across the aqueduct of four females and two males at a single center, and the mean of these results was compared to the established mean CSF production calculated by invasive techniques. There was no significant difference between the means, contributing to the body of literature suggesting the utility of PC-MRI in estimating CSF production rates. Cerebrospinal fluid (CSF) is critical to maintaining the central nervous system (CNS). According to Cushing’s "third circulation" description, CSF is produced mainly by the choroid plexus in the ventricles and exits by the foramen of Luschka and Magendie to fill the subarachnoid space and disperse to the rest of the CNS . In addiPopulation and study designPatients were selected for the study from the outpatient elective MRI schedule. The selected patients presented with indications for elective routine brain MRI scans. The patients were notified by phone prior to the day of their scheduled MRI and asked if they would be willing to participate in the study. Upon arrival for the MRI scan, the patients consented to an additional phase-contrast series and thin-cut high-resolution axial sections along the length of the cerebral aqueduct. The study was approved by the Augusta Institutional Review Board (IRB), Augusta, Georgia. Enrollment took place between November 2019 and January 2020. Inclusion criteria were patients scheduled for elective MRI scans of the brain between November 2019 and January 2020. Exclusion criteria included a history of hydrocephalus or pathology requiring shunt placement and contraindications to MRI scanning, including claustrophobia, anxiety, or the presence of metal implants or devices in the body. Eight patients consented and were scanned, but two patients were excluded from the data due to corrupted images. The scans of four females and two males were evaluated, and the average age was 25.7 years. Two patients received scans to trend lesional pathology; three patients were evaluated for seizures; and one patient presented with nocturnal vomiting. Patients did not undergo invasive testing.Imaging technique and data calculationFor each patient, the phase-contrast time sequence was recorded across a single axial MRI slice. Imaging software Phillips IntelliSpace portal Q-flow analysis quantifies the flow rate across the cross-section of the cerebral aqueduct, identified by a user-defined polygonal outline. The area of interest along the aqueduct was chosen by a board-certified neuroradiologist with expertise in phase-contrast MRI interpretation. The peak velocity was extrapolated from the sequence and used as the velocity factor in the flow rate equation. The radius of the aqueduct was measured from the separate, high-resolution axial MRI cut at the same level to find the area of the circle formed by the two-dimensional cut. The flow rate of CSF (cm/sec) was multiplied by the area of the circle (cm2) to calculate volumetric CSF flow (cm3/min) and estimate production.Statistical analysisA two-tailed, one-sample t-test was used to compare the mean calculated CSF production rate of these data (n=6) with the mean CSF production rate calculated via invasive methods by Rubin et al. . The levEight patients were enrolled in the study, but two were excluded from the data collection due to corrupted data obtained in the imaging process. Each study patient was scanned on the same Philips 3 Tesla (3T) magnetic resonance imaging scanner. The brain MRI scans of the four female and two male study participants were individually evaluated by a board-certified neuroradiologist with expertise in phase-contrast MRI. The average age of the participants enrolled in the study was 25.7 years. The ages ranged from six months old to 76 years old. One study participant had a known meningioma along the convexity and did not have a connection to the ventricular system. Another participant had been diagnosed with an incidental arachnoid cyst that was being followed for interval changes. The arachnoid cyst was located in the middle cranial fossa and had no communication with the ventricular system. A third study participant was evaluated with an MRI for nocturnal vomiting, and the MRI did not reveal any abnormal findings. Three study participants were evaluated with an MRI for new-onset seizure activity. No abnormal intracranial pathology was identified in any of these three patients. The mean radius, in centimeters, of the cerebral aqueduct was 0.097, ranging from 0.075 cm to 0.120 cm. The mean area of the aqueduct, calculated by using the equation for the area of a circle, A = pi r2, was 0.030 cm2. The mean area of the aqueduct ranged from 0.018 cm2 to 0.045 cm2. The mean calculated peak velocity was 10.0662 cm/s. The calculated velocities ranged from 8.220 cm/s to 16.540 cm/s  production in rabbits in 1963 compared relative cell volumes between the choroid plexus venous blood and aortic arterial blood . The metPC-MRI sequences, first described in 1986, use a combination of phase contrast injection and MRI to generate a distinction between stationary and mobile hydrogen nuclei and quantify the direction and velocity of blood flow . It was The average CSF flow rate across the aqueduct in this single-center study was not significantly different from the average flow rate from the lumbar perfusion series, contributing to the growing body of literature suggesting that PC-MRI could serve as an accurate noninvasive method to measure CSF production for these patients.There are disadvantages inherent in using still images to measure a dynamic system. PC-MRI does not account for CSF produced in the fourth ventricle or from extra-choroidal sources. Given its brief window of capture, PC-MRI is also limited in establishing an average flow rate over an extended period of time, which can reduce its accuracy in cases of dynamic flow and production rate changes due to physiologic processes or anatomical features . Net CSFLimitationsThis study is limited by its sample size of six patients. The flow rate obtained from the aqueduct is also the only data point obtained for each patient in a single sitting. Therefore, it is not known if these estimates would be consistent if compared to additional scans or if the production rates were calculated using other methods.A reliable, non-invasive measurement of CSF production could be a valuable tool in determining the underlying causes of many pathological conditions within the central nervous system. Invasive methods performed over fifty years ago quantified an average CSF production rate in humans that is generally extrapolated to patients, despite being measured in patients with mass lesions. Recent studies of CSF composition and flow dynamics, combined with advances in imaging, have cultivated an interest in quantifying CSF dynamics in the individual patient. This single-center study suggests that phase-contrast magnetic resonance imaging, although somewhat limited by temporal, spatial, and physiologic factors, shows promise as a safe, non-invasive tool to measure CSF flow through the aqueduct and approximate CSF production.