Suffering an acute spinal cord injury (SCI) can result in catastrophic physical and emotional loss. Efforts to translate novel therapies in acute clinical trials are impeded by the SCI community’s singular dependence upon functional outcome measures. Therefore, a compelling rationale exists to establish neurochemical biomarkers for the objective classification of injury severity. In this study, CSF and serum samples were obtained at 3 time points  from 30 acute SCI patients . A differential chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) with a universal metabolome standard (UMS) was applied to the metabolomic profiling of these samples. This method provided enhanced detection of the amine- and phenol-containing submetabolome. Metabolic pathway analysis revealed dysregulations in arginine-proline metabolism following SCI. Six CSF metabolites were identified as potential biomarkers of baseline injury severity, and good classification performance (AUC > 0.869) was achieved by using combinations of these metabolites in pair-wise comparisons of AIS A, B and C patients. Using the UMS strategy, the current data set can be expanded to a larger cohort for biomarker validation, as well as discovering biomarkers for predicting neurologic outcome. Acute spinal cord injury (SCI) often causes paralysis and severe disability, for which there are few treatments that even marginally improve neurologic outcome. In Canada, the incidence of acute traumatic SCI is over 1,500 per yearEfforts to validate potential interventions in the acute SCI setting have been hampered by the reliance upon clinical assessments to quantify neurologic impairment after SCI and to stratify the severity of injury. These assessments are imprecise predictors of neurologic recovery and are often impossible to even obtain in the acute setting due to intoxication/sedation or concomitant injurieshuman SCI has not been previously reported.Additionally, the scientific development of novel therapies for acute SCI depends upon an understanding of the complex pathophysiologic mechanisms that are triggered after acute SCI. Traumatic injury to the spinal cord induces multiple disturbances in the human metabolic network, including oxidative stress, glycolysis, and amino acid and lipid metabolism4568910In this study, we describe a parallel metabolomic profiling of cerebrospinal fluid (CSF) and serum from acute SCI patients using a newly developed chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) platform with a universal metabolome standard (UMS)http://asia-spinalinjury.org/wp-content/uploads/2016/02/International_Stds_Diagram_Worksheet.pdf and this form is also available from the corresponding authors upon request. The baseline American Spinal Injury Association (ASIA) Impairment Scale (AIS) grade was A for 10 patients, B for 12, and C for 8. In general terms, AIS A denotes those with complete motor and sensory paralysis (the most severe neurologic impairment), AIS B denotes those with complete motor paralysis but some preserved sensation, and AIS C is assigned when there is some preserved motor and sensory function. A lumbar intrathecal catheter was inserted at the time of surgery and maintained for 3–5 days post-injury for the acquisition of serial CSF samples. CSF and serum samples were obtained at 3 time points: approximately 24, 48, and 72 hours post-injury. The CSF and blood samples were drawn within a few minutes of one another and were processed, centrifuged, and frozen at the bedside. The patients were examined clinically at 6 months post-injury to determine AIS grade and motor score recovery.We prospectively enrolled 30 acute traumatic SCI patients who had suffered a cervical or thoracolumbar injury (C3-L1) in whom a valid baseline neurologic examination could be performed in accordance with the International Standards for Neurologic Classification of SCI (ISNCSCI). This clinical examination was done typically within 24 hours of injury. The ISNCSCI examination and its scoring conventions are available from 12C-labeled individual CSF or serum sample was combined with an equal mole amount of the corresponding 13C-labeled pooled sample which served as the UMS. The relative concentration of each metabolite in an individual sample to that of the corresponding metabolite in UMS was measured using the intensity ratio of the 12C/13C peak pair. Since the same UMS was spiked into all the comparative samples, the peak ratio values of a given metabolite in individual samples reflected their concentration differences in these samples. The use of 13C-labeled UMS as internal standards enables more accurate quantification of the 12C-labeled metabolites. In addition, any future samples could be 12C-labeled and then compared to the 13C-labeled UMS, thereby allowing expansion of the current dataset to a larger cohort. Dansylation LC-MS has the advantage of improved chromatographic separation and enhanced electrospray ionization (ESI) response, resulting in 10- to 1000-fold increase in detection sensitivity12C/13C peak pairs from CSF and serum, respectively. By matching accurate mass and retention time with the dansyl standard library1 (16–32 hours), t2 (40–56 hours) and t3 (64–80 hours). 2 and Q2 the slopes are positive and the permutation data are lower than the original point, suggesting that the PLS-DA models are valid. A clear separation is observed between samples collected at t1 and t3, while the t2 samples fall in between t1 and t3 with some overlap. This pattern suggests a gradual change in the metabolic profile from t1 to t3.Principle component analysis (PCA) was first applied to provide an overview of the metabolomic dataset. 0), we found that the temporal changes follow two major patterns. The first pattern is a gradual increase or decrease of the metabolite level over time (see 1 samples), followed by a gradual restoring of the metabolite level back to the non-injured state , 376 serum and 73 CSF metabolites that exhibit statistically significant temporal changes were chosen. Among these differentiating metabolites, 20 of them were commonly detected in serum and CSF. The lower number of significant metabolites in CSF is not unexpected as the breadth of the metabolome in CSF is much smaller compared to serum. Among the 376 serum metabolites, 142 (38%) of them follow pattern 1 and 227 (60%) conform to pattern 2. For the 73 CSF metabolites, pattern 1 accounts for 13 (18%) and pattern 2 explains 49 (67%) of the temporal changes. This observation suggests that most of the metabolic changes occur at an early stage of the injury . Metabolic changes in the first 16 to 32 h after injury are of great interest because they are responses that represent targets for acute pharmacologic/biologic interventions and are also potentially useful for objectively assessing injury severity at an early stage. Therefore, we focused our analysis on the t1 samples for the following metabolomics analysis.With the non-injured controls serving as the reference point  in at least one of the three pairwise comparisons was illustrated by the Venn diagram  with the non-injured controls (N). As the total number of samples was relatively small, we only performed univariate analyses to identify metabolites that were significantly altered. The number of metabolites that met the selection criteria  value obtained from pathway enrichment analysis. p)  and high pathway impact values. As shown in For the 24 CSF and 13 serum metabolites whose levels were significantly different in SCI patients as compared to non-injured controls, a metabolic pathway analysis (MetPA) was performed to evaluate their associated pathways. post-hoc test to select metabolites that had significantly difference within-group and between-group means (p < 0.05). It was observed from the PCA score plots  with Tukey’s re plots  and B thWe then examined whether the selected metabolites or combinations thereof could be used to classify AIS A, B and C levels. We first evaluated the receiver-operating characteristic (ROC) curves for each pairwise comparison . 1 was missing). Then, the non-AIS A samples identified from step 1 were subjected to a second logistic analysis that predicted AIS C vs. non-AIS C. The samples that were predicted as non-AIS C in the second step were assigned AIS B. A 3 × 3 confusion matrix was then built based on the predicted results. The results are shown in In addition to ROC analysis, we also applied logistic regression to create prediction models in combination with leave-one-out cross validation to evaluate prediction accuracy. The prediction model was built in two steps. Firstly, a logistic regression model was applied for predicting AIS A vs. non-AIS A on 29 samples  could build prediction models with improved classification accuracy. All serum and CSF metabolites with ANOVA tes only , using stes only , and usites only . The ovetes only , the useRecently, it has been shown that metabolomic screening of rat plasma samples can be used to establish an injury severity evaluation model based on the identified metabolomic fingerprints1618While CSF and serum are both promising biofluids for discovery of potential SCI biomarkers, the metabolome analysis of CSF is challenged by the relatively low metabolite concentrations. In this study, we applied a dansylation labeling technique for metabolomic profiling of CSF and serum samples, which provides a signal enhancement of 10- to 1000-fold on its targeted submetabolome. As shown in 17A comparison between non-injured controls and the three injury groups revealed a number of metabolites that show significant alterations induced by SCI. In particular, it was found that uridine was down-regulated considerably in both CSF and serum. Uridine is an important metabolic precursor of phosphotidylcholine, a major class of the cell membrane phospholipids that plays a key role in cell growth and repairSubsequent pathway analysis of these differentially expressed metabolites indicated a perturbation in the arginine and proline metabolism. Among the 11 metabolites detected in this study, 6  and 3  of them were found to be significantly altered in at least one injury group in CSF and serum. Analysis of the CSF arginine and proline pathway revealed a significant decrease in arginine and increases in a few downstream metabolites towards the synthesis of homocarnosine. Arginine is the precursor of the biologically active molecule, nitric oxide (NO), through a reaction catalyzed by the nitric oxide synthase (NOS) family. NO is known to play a key role in the pathogenesis of acute CNS injury2728The metabolic changes associated with serum arginine and proline metabolism are quite different from that of CSF. For example, in contrast to CSF, we found that arginine levels in the serum did not change significantly. In addition, the brain-specific metabolite homocarnosine could not be detected in serum. On the other hand, metabolites that exhibited significant alterations in serum, such as 4-hydroxyproline and sarcosine, did not show any difference between non-injured and injured CSF samples. This observation highlights the importance of performing parallel metabolomic profiling of serum and CSF at the discovery stage. While there were many common metabolites in serum and CSF, the metabolic responses to SCI were found to be very different. The immediate proximity of the CSF to the injured spinal cord makes it the most direct reflection of injury responses within the cord; accessing CSF, however, is obviously far more challenging than obtaining blood samples for biomarker studies. A parallel analyses such as ours enables one to compare the responses in both biofluids and determine how well processes within the CSF are manifested in the serum.From the clinical perspective, it would be of great value to establish specific metabolites that can accurately classify injury severity, especially in situations where the functional measures are difficult to assess and are poor predictors of outcome . In this study, we identified six CSF metabolites and one serum metabolite that show promise as markers for evaluation of SCI injury severity. This targeted metabolic profiling result promotes the use of CSF for severity analysis because of the greater number of differentially expressed metabolites within CSF. For these six CSF metabolites, the metabolite levels were the highest in the AIS A group, and followed the trend A > B > C. This observation is consistent with the notion that the AIS A group represents the greatest injury severity  and the severity level lowers from B  to C . Therefore, we would expect the greatest metabolic disturbance in AIS A. By using a combination of these metabolites, we have shown that good classification performance can be achieved in pair-wise comparisons . In addition, some of the metabolic changes can be rationalized by considering their biological functions.Firstly, citrulline is produced from arginine, and is the by-product of the reaction catalyzed by NOS that releases NO. As described earlier, activation of the NOS family following SCI would promote generation of NO as well as the by-product citrulline. On the other hand, the NO and citrulline levels are also regulated by activation of arginase, an enzyme that catalyzes a competing reaction and consumes arginine. While a decrease in arginine has been consistently detected in all three injury groups, the production of citrulline was found to be severity-dependent. For patients associated with “complete” paralysis (AIS A), the elevation in citrulline level is more pronounced compared to AIS B and C. On the other hand, patients in the AIS C category did not show significant change in citrulline level compared to the non-injured controls. This observation suggests that regulation of these two enzyme families (NOS and arginase) is related to the injury severity.2+ 32We have also identified glycerol and lactate to be differentially expressed among three injury groups. Glycerol is the end-product of phospholipid degradation. Activation of phospholipases A and C is known to occur after traumatic brain or spinal cord injury, which is thought to be associated with an increase in intracellular free CaAnother metabolic change that is worth noting is the elevation of two acetylated polyamines, N-acetylputrescine and N1, N12-diacetylspermine, following acute SCI. In particular, N1, N12-diacetylspermine is not detected in the non-injured samples, but is highly elevated in the AIS A group. Acetylation of polyamines is catalyzed by spermidine/spermine N1–acetyltransferase (SSAT) as part of the polyamine catabolism process. Although the relationship between polyamine acetylation and spinal cord injury has not been extensively reported, a recent study demonstrated an enhanced polyamine catabolism, including acetylation, after traumatic brain injury13C-UMS will be used to analyze new batches of samples, the peak ratio values obtained from these individual samples will be fully compatible to those shown in this studyIn summary, six identified CSF metabolites have emerged as potential biomarkers for assessment of injury severity, and good classification performance has been achieved by using a combination of these metabolites in pair-wise comparisons of AIS A, B and C patients. In addition, we have shown that using significantly changed metabolites that have not been positively identified at this stage can further improve the prediction accuracy. For future work, more samples will be used to validate these potential biomarkers as well as examine the possibility of finding biomarkers that predict neurologic recovery or that could be used as surrogate outcome measures. In this regard, the use of UMS allows us to readily expand the current dataset by analyzing new samples. Because the same or comparable 1, 16–32 hours), 48 hours  and 72 hours post-injury .Patients with acute traumatic SCI were enrolled in a prospective observational trial to obtain CSF, entitled CAMPER (the Canadian Multicenter CSF Pressure and Biomarker Study). Participating sites included Vancouver (British Columbia), Montreal (Quebec), Halifax (Nova Scotia) and London (Ontario). The CAMPER trial is registered with ClinicalTrials.gov  and is conducted in accordance to the Declaration of Helsinki ethical principles for medical research involving human subjects. The CAMPER clinical trial protocol and experimental procedures  received ethics approval from each participating center’s Institutional Review Board (IRB): Vancouver site from University of British Columbia Clinical Research Ethics Board, London site from Western University Health Sciences Research Ethics Board, Montreal site from Hôpital du Sacré-Coeur de Montréal Ethics Committee of Research and Halifax site from Capital Health Research Ethics Board. Additional sites including San Francisco and Stanford have been added to enroll more patients for future research; no samples from these new sites were used in this current study. Ethics approval was received from the University of California San Francisco, Human Research Protection Program Committee on Human Research and Stanford University Institutional Review Board. Each SCI patient provided his/her informed consent for the study. Intrathecal catheters were inserted pre-operatively into 30 acute SCI patients with baseline ASIA Impairment Scale (AIS) grades of A (n = 10), B (n = 12) and C (n = 8). These catheters were left in place for 5 days, during which CSF/serum samples were drawn approximately every 8 hrs. The CSF and serum samples were centrifuged at the bedside and the supernatant was immediately frozen on dry ice. The collected samples were shipped on dry ice and stored at −80 °C until further use. For each patient, three CSF and serum samples drawn at different time points were used for this study. These samples were grouped into three approximate daily stages: 24 hours (t12C-dansyl chloride solution (18 mg/mL) (for light labeling) or 13C-dansyl chloride solution (18 mg/mL) (for heavy labeling). The reaction was allowed to proceed for 1 hr at 40 °C. After 1 hr, NaOH  was added to the reaction mixture to quench the excess dansyl chloride. The solution was then incubated at 40 °C for another 10 min. Finally, formic acid in 50/50 ACN/H2O  was added to consume excess NaOH and to make the solution acidic.Metabolites were extracted from serum and CSF samples via methanol protein precipitation. Three volumes of ice-cold methanol were added into 25 μL of serum/CSF, vortexed and incubated on ice for 15 min. This was followed by centrifugation at 20 817 g for 15 min. The supernatants (75 μL for serum and 90 μL for CSF) were dried using a SpeedVac and resuspended in 25 μL of water. For the labeling step using the dansyl chloride agents available at MCID.chem.ualberta.ca, the extracted solution was mixed with 12.5 μL 250 mM sodium carbonate/sodium bicarbonate buffer and 12.5 μL ACN. The solutions were vortexed, spun down and mixed with 25 μL freshly prepared 12C-dansyl chloride as three experimental replicates, while the remaining liquids were mixed together to generate a pooled sample which was subsequently labeled by 13C-dansyl chloride. The same procedure was performed for serum samples. In all other studies, aliquots of all individual CSF/serum samples were mixed together to generate a pooled CSF/serum sample. For comparison between the serum and CSF metabolome, equal aliquots of pooled CSF and serum were combined and labeled with 13C-dansyl chloride, while the pooled CSF and pooled serum samples were labeled with 12C-dansyl chloride. For metabolomic profiling of the entire sample set, all individual samples were labeled with 12C-dansyl chloride, and the pooled CSF and pooled serum samples were labeled with 13C-dansyl chloride. Prior to LC-MS analysis, the 12C-labeled samples were combined with an equal amount of the corresponding 13C-labeled reference. Quality control (QC) samples were prepared by 1:1 volume mix of a 12C-labeled and a 13C-labeled pooled sample.In the analysis of analytical variability, a 90 μL aliquot was taken from one CSF sample of each patient group . Three of the 25 μL portions from each sample were labeled with The labeled metabolites were analyzed using a Bruker Maxis Impact QTOF mass spectrometer  linked to an Agilent 1100 series binary HPLC system . The samples were injected onto an Agilent reversed phase Eclipse Plus C18 column  for separation. Solvent A was 0.1% (v/v) formic acid in 5% (v/v) acetonitrile, and solvent B was 0.1% (v/v) formic acid in acetonitrile. The chromatographic conditions were: t = 0 min, 20% B; t = 3.5 min, 35% B; t = 18 min, 65% B; t = 24 min, 99% B; t = 31 min, 99% B, t = 32 min, 20% B. The flow rate was 180 μL/min. All MS spectra were obtained in the positive ion mode with a scan range of 150 to 1000 m/z. The capillary voltage was 4500 V and nebulizer pressure was 1.8 bar. The dry gas flow was set to 8 l/min and the dry gas temperature was set to 230 °C. Quality control samples were analyzed between every 20 sample runs to monitor instrument performance. For each LC-MS run, masses were calibrated to the spectrum that contained the dansyl-amine peaks at m/z 242.57160 (two tags two charges), m/z 484.13592 (two tags one charge) and m/z 971.27799 (dimer) using the Data Analysis software and the calibration result was applied to all the other spectra in the same LC-MS run. The resulting MS data were processed using a peak-pair picking software, IsoMSwww.metaboanalyst.ca)p < 0.05. For comparisons between two groups, the fold change and p-value by Student’s t test were calculated using Microsoft Excel. The multiple-testing-corrected p-value  was calculated using R and BioConductor (www.bioconductor.org)p < 0.05 from the analysis of variance were ranked using the greedy stepwise approach. The prediction model was established in two steps, and leave-one-out cross validation (LOOCV) was utilized to establish the prediction accuracy. Firstly, logistic regression was applied for predicting AIS A vs. non-AIS A on all samples . Then, the predicted non-AIS A underwent a second logistic regression LOOCV for predicting AIS C vs. non-AIS C. In this step, all non-AIS Cs would be assigned as AIS B. In each step, the optimal number of top ranked metabolites for building prediction models was determined based on the highest prediction accuracy, with a restriction of 6 metabolites at maximum. The final logistic regression equation has been calculated using all of the samples involved in that step. A 3 × 3 confusion matrix table was constructed, and the overall prediction accuracy was calculated as the percentage of samples that have been correctly assigned as A, B or C .Only those peak-pair features shared by more than 50% of the samples were retained for statistical analysis. Multivariate statistical analysis including principle component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) was carried out using SIMCA-P+ 12.0 . PLS-DA validation was performed using 20-permutation test built in the SIMCA-P+ 12.0 program. The threshold for selection of significant features in PLS-DA was VIP > 1.5. Analysis of variance (ANOVA) and Tukey’s test were performed by Metaboanalyst .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Infections and inflammatory processes have been associated with the development of schizophrenia and affective disorders; however, no study has yet synthesized all the available data on cerebrospinal fluid (CSF) immune-alterations. We conducted the first systematic review of the immunological findings from CSF studies among patients with schizophrenia or affective disorders.All studies investigating CSF inflammatory markers in persons with schizophrenia or affective disorders published prior to March 23, 2017 were identified searching PubMed, CENTRAL, EMBASE, Psychinfo, and LILACS. The literature search, data extraction and assessment of risk of bias were performed by two independent reviewers. Meta-analyses with standardized mean difference (SMD) including 95% confidence intervals (CI) were performed on studies including healthy controls.We identified 112 CSF studies published between 1942–2016, of which 32 were included in meta-analyses; however, only few studies investigated identical biomarkers. The CSF/serum albumin ratio was increased in both schizophrenia  and affective disorders , compared to healthy controls. Total CSF protein was elevated in both schizophrenia  and affective disorders . The IgG ratio was increased in schizophrenia , whereas the IgG Albumin ratio was decreased . Interleukin-8 (IL-8)  and IL-6 levels  were increased among individuals with schizophrenia but not significantly increased in affective disorders. None of the remaining inflammatory markers were significantly different compared to healthy controls in the meta-analyses. However, in the studies which did not include healthy controls, CSF abnormalities were more common, and CSF dependent re-diagnosis occurred in 3.2–6% in the two studies investigating this.Our findings suggests that schizophrenia spectrum and affective disorders are associated with CSF abnormalities including signs of blood-brain barrier impairment and inflammation, supporting a role of the immune system in mental disorders. However, only few studies investigated the same parameters with healthy controls and high quality longitudinal CSF studies are lacking, including impact of psychotropic medications and potential benefits of anti-inflammatory treatment in subgroups with abnormal CSF inflammatory markers


By processing the data obtained with this CSF MRI sequence, brain parenchymal volume (BPV) and intracranial volume (ICV) can be automatically obtained. The aim of this study was to assess the precision of the BPV and ICV measurements of the CSF MRI sequence and to validate the CSF MRI sequence by comparison with 3D T1-based brain segmentation methods.In previous work we have developed a fast sequence that focusses on cerebrospinal fluid (CSF) based on the long T1-weighted sequence (6:47min). Data of the HR 3D-T1-weighted images were downsampled to obtain LR T1-weighted images (reconstructed imaging time: 1:59 min). Data of the CSF MRI sequences was automatically segmented using in-house software. The 3D T1-weighted images were segmented using FSL (5.0), SPM12 and FreeSurfer (5.3.0).Ten healthy volunteers  were scanned (3T MRI) twice with repositioning in between. The scan protocol consisted of a low resolution (LR) CSF sequence (0:57min), a high resolution (HR) CSF sequence (3:21min) and a 3D T2 .67–.97). The results from the downsampled data and the high-resolution data were similar.The mean absolute differences for BPV and ICV between the first and second scan for CSF LR (BPV/ICV: 12±9/7±4cc) and CSF HR (5±5/4±2cc) were comparable to FSL HR (9±11/19±23cc), FSL LR , FreeSurfer HR (5±3/14±8cc), FreeSurfer LR , and SPM HR (5±3/4±7cc), and SPM LR . The correlation between the measured volumes of the CSF sequences and that measured by FSL, FreeSurfer and SPM HR and LR was very good (all Pearson’s correlation coefficients >0.83, R1-based automated segmentations methods for the segmentation of BPV and ICV. However, the short imaging time of the fast CSF MRI sequence is superior to the 3D T1 sequence on which segmentation with established methods is performed.Both CSF MRI sequences have a precision comparable to, and a very good correlation with established 3D T In addition, the long scan time makes it susceptible to motion artefacts. The 3D T1-weighted sequence is aimed at T1 contrast, which gives good contrast between gray matter and white matter while CSF appears dark on the images. Recently, we discovered that data of a fast (<1-3min) cerebrospinal fluid (CSF) MRI sequence [1-weighted sequence and this facilitates a more widespread use of brain volume measurements [1 and T2 of CSF. The CSF MRI sequence relies on the longer transverse (T2) and longitudinal (T1) relaxation rate of the CSF compared to surrounding tissues and allows segmentation of the CSF and brain parenchymal volume (BPV). The obtained images with the CSF MRI sequence show no contrast between gray matter and white matter, and a good contrast between CSF and brain parenchyma. In a previous study, we showed a good correlation between the CSF MRI sequence and qualitative brain atrophy scores, and a good correlation with brain parenchymal volume (BPV) measured by FMRIB Software Library (FSL) in a simple feasibility study [1-weighted methods . The aim of this study was to assess the precision of the BPV and intracranial volume (ICV) measurements of the CSF MRI sequence and to validate the CSF MRI sequence by comparison with 3D T1-based brain segmentation methods.Brain volume measurements on magnetic resonance (MR) images is often used as an etiological and prognostic biomarker , 2. Alzhsequence  can be pFor this study ten healthy volunteers  were recruited with a median age of 28 years (range 24–41). Participants were recruited between 2-9-2015 and 22-9-2015, and all participants completed the scan protocol. Recruitment was performed on the university campus. Subjects who demonstrated interest in participating in MR research were approached and screened for MR eligibility. Of the 10 subjects who were approached, all subjects were MRI eligible. This study was approved by the Medical Ethical Committee of the University Medical Center Utrecht under protocol number NL39070.041.11. All participants provided written informed consent.1-weighted sequence. To assess the precision, the scan protocol was performed twice with repositioning of the subjects in between both scans (scan 1 and scan 2). The data of the 3D T1-weighted sequence was also downsampled to the voxel size of the HR CSF MRI sequence.MR imaging was performed on a 3 Tesla Philips Achieva System  with a quadrature body coil as transmitter and an 8-channel head coil as a signal receiver. The MRI scan protocol consisted of the following sequences: a low resolution (LR) and a high resolution (HR) CSF MRI sequence, and a high resolution 3D T2-preparation, and a readout part (multi-slice echo planar imaging (EPI) readout) [2-preparation part, a Malcolm Levitt’s CPD (MLEV) T2-preparation scheme was used with a τCPMG of 70 ms and 0, 4, 8 and 16 refocusing pulses resulting in effective TEs (eTEs) of 0, 280, 560, and 1120 ms  . For the 1120 ms . To compData of the CSF MRI sequences was fully automatically processed using IDL 6.1 for Windows .2 of the CSF was performed using the following formula:a) is the signal recovery at time Ta, the constant A is the recovered magnetization at time Ts after the non-selective saturation pulse, T1,CSF and T2,CSF are the longitudinal and transverse relaxation of the CSF. Second, the M0 of the voxels were obtained by extrapolating the T2 of the CSF to the intersect at eTE = 0 ms. From this, the actual volume fraction of CSF (CSFvf) in a voxel is obtained by relating the M0 of a voxel to the M0 of reference voxels. Only last 3 echo times are used to extrapolate the M0 of CSF in order to exclude parenchymal tissue. The reference voxels free of partial volume are automatically selected based on a histogram analysis of signal intensities. The CSF volume was based on the CSF M0 map which was smoothed with a 7mm Gaussian filter. The signal value at 99.5% of the nonzero histogram was used to determine the reference voxels filled with CSF. The upper 0.5% of values were discarded to avoid artificially high reference values caused by noise. Third, total CSF volume (Vcsf) was obtained by multiplying the volume fraction by the voxel size (mm3).First, signal decay curves were obtained for each voxel from which a voxelwise estimation of the TFourth, based on the first acquired echo (eTE = 0) of the CSF MRI sequence which includes both parenchymal and CSF signal, the intracranial volume (ICV) was measured. A conservative brain extraction (BET) mask from FSL  was applThe postprocessing time was 1 minute for the low resolution CSF MRI sequence (CSF LR), and it was 25 minutes for the high resolution CSF MRI sequence (CSF HR).1-weighted MRI sequence were; matrix size 256x256, FOV 232x256x192, voxel size 1.0x1.0x1.0 mm3, TR = 8187ms, TE = 4.5ms, scan time 6:47 min. The data were downsampled to LR 3D-T1-weighted images with a resolution similar to the CSF HR MRI sequence through registration to the CSF HR images: voxel size 1.0x1.0x3.5 mm3, scan time 1:59 min . The 3D T1-weighted images were used for brain segmentation by FMRIB Software Library 5.0 (FSL), FreeSurfer (5.3.0) and Statistical Parametric Mapping 12 (SPM).MR imaging parameters of the HR 3D-TFSL is software created by the Analysis Group, FMBRIB, Oxford, UK. In FSL, Structural Image Evaluation using Normalization of Atrophy Cross-sectional (SIENAX) was used to obtain brain volumes normalized for skull size , 9. The—FreeSurfer is open source software developed by the Athinoula A. Martinos Center for Biomedical Imaging, Harvard-MIT, Boston. The volume-based stream consists of five steps, including registration to standard space and skull stripping . In Freehttp://www.fil.ion.ucl.ac.uk/spm). This function uses the Unified Segmentation algorithm, which combines image registration, tissue classification and bias correction [SPM12 is developed by the Wellcome Department of Cognitive Neurology, Institute of Neurology, Queen Square, London [rrection . As suchStatistical analysis was carried out with R .1-based methods . The mean difference between the results of scan 1 and scan 2 reflects whether a systematic bias exists in the test-retest measurements. The mean absolute difference reflects the total test-retest volume error between the results of scan 1 and 2. Wilcoxon signed rank tests were used to evaluate whether there was a significant difference between the test-retest measurements. Bland-Altman plots were used to visualize these differences [1-based methods.For both BPV and ICV the precision was calculated by the mean (absolute) differences between the measurements of scan 1 and scan 2 for the CSF sequences and the 3D Tferences . Precisi1-based methods  were evaluated for significant differences (Wilcoxon signed rank). To test for correlation between the CSF sequences and the 3D T1-based methods, Pearson’s correlation coefficients were calculated. Bland-Altman plots comparing the volumes obtained with the CSF sequence with those of the 3D T1-based methods were used to visualize differences. The results from the first scan were used for the Bland-Altman plots. The degree of variance was calculated (R2) with 90% confidence intervals (CI).The absolute volumes of BPV and ICV between the CSF sequences and the 3D TAll segmentations of all methods were considered of good quality , except for CSF LR versus FSL HR , CSF LR/SPM LR (11 cc), and CSF LR/FSL HR (24 cc), CSF HR/FSL LR (39 cc) and CSF LR/SPM HR (39 cc), (1-based methods. The limits of agreement were the most narrow for CSF LR/FreeSurfer HR (49–133 cc), CSF LR/FreeSurfer LR (35–130), CSF HR/FreeSurfer HR (79–185 cc), and CSF LR/FSL LR (-61–56) , . The lim(-61–56) . The deg1-based brain segmentation methods , but this was only significantly different for the CSF sequences versus FSL (p<0.05). The CSF sequences measured a lower ICV than FSL LR and FreeSurfer LR (p<0.05). There was a good to very good correlation for ICV measurements between the CSF sequences and the 3D T1-based methods , except for FreeSurfer  and obtained volumes as conventional 3D T1-weighted images between the tabula interna of the skull and the dura mater on one side, and the CSF on the other side [1-weighted images can be troublesome and several segmentation methods show difficulties in calculating the ICV [The precision and performance of commonly used MRI-based segmentation methods varies across different methods and also varies dependent on the application –17. The her side . As a re the ICV –20. Espe the ICV , 16. In 1 based methods, because it facilitates an easy addition of this sequence to a scan protocol. The 3D T1-weighted sequence used in our study had a scan time of nearly 7 minutes and this scan could thus be avoided when a high-resolution T1- weighted scan is not necessary to answer any of the other clinical or research questions. Nowadays, shorter 3D T1-weighted sequences that still provide sufficient image quality for most radiology exams are available, and we simulated the precision of such a sequence by downsampling the data of the high resolution T1-weighted sequence. The results of this analysis demonstrated that global measures from automated segmentation methods—BPV and ICV—are not sensitive to a somewhat lower scan quality. The precision of our LR CSF sequence was similar to the precision of both the HR and LR T1-weighted sequence, but then with a scan time half of the scan time of the LR T1-weighted sequence. Another strength of our CSF sequences is the good contrast between the skull and CSF, which is often limited in 3D T1-weighted images. This leads to an very good precision of our CSF sequences for ICV. A limitation of our CSF sequences is that there is almost no contrast between the white and gray matter. Consequently, both gray and white matter atrophy and cortical thickness cannot be distinguished. Another limitation of the CSF LR and HR sequences could be that the obtained volumes are not similar to the volumes obtain with SPM, FreeSurfer or FSL. However, this also holds for comparisons across these 3D T1-based segmentation methods [The short imaging time of 57 seconds is a major advantage of the CSF LR sequence over 3D T methods , 16. Mor2, of CSF may be related to oxygen partial pressure [The purpose of this paper was to cross-validate the CSF MRI sequences for volume estimates. Future work could focus on further image processing of the CSF images such that regional brain atrophy can be determined. This would allow detection of different patterns of brain atrophy. This is a useful addition, as several diseases are marked by a characteristic pattern of brain atrophy, for example frontotemporal dementia . The pospressure , 22.1-based automated segmentations methods for segmentation of BPV and ICV. However, the short imaging time of the fast CSF MRI sequence is superior to the 3D T1-weighted sequence on which segmentation with established methods is performed.In conclusion, both CSF MRI sequences have a precision comparable to, and a very good correlation with established 3D TS1 Dataset(XLSX)Click here for additional data file.S1 FigCSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan.(PNG)Click here for additional data file.S2 FigCSF LR, CSF HR (top row), FSL LR, FreeSurfer LR, and SPM LR (bottom row). The dashed lines represent the limits of agreement (mean difference ±1.96*SD of the differences). LR = low resolution, downsampled scan. CSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan; FS = FreeSurfer.(PNG)Click here for additional data file.S3 FigThe dashed lines represent the limits of agreement (mean difference ±1.96*SD of the differences). CSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan; FS = FreeSurfer.(PNG)Click here for additional data file.S4 FigCSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan.(PNG)Click here for additional data file.S5 FigCSF LR, CSF HR (top row), FSL LR, FreeSurfer LR, and SPM LR (bottom row). The dashed lines represent the limits of agreement (mean difference ±1.96*SD of the differences). LR = low resolution, downsampled scan. CSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan; FS = FreeSurfer.(PNG)Click here for additional data file.S6 FigThe dashed lines represent the limits of agreement (mean difference ±1.96*SD of the differences). CSF LR = CSF low resolution MRI scan; CSF HR = CSF high resolution MRI scan; FS = FreeSurfer.(PNG)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


The TlBi(Sx)2 system is interesting because it shows a topological phase transition, where a topologically nontrivial phase changes to a trivial phase without changing the crystal structure qualitatively. In addition, Dirac semimetals whose bulk band structure shows a Dirac-like dispersion are considered to exist near the topological phase transition. The technique shown here is also generally applicable for other chalcogenide topological insulators, and will be useful for studying topological insulators and related materials.In this article, practical methods for synthesizing Tl-based ternary III-V-VI This value is smaller than n obtained from the Hall coefficient, but it can be understood if the FS is elongated along the S. The negative sign of S is consistent with n-type doping in the present system.In this section, let me discuss the magneto transport of the best sample of TlBiSeS result . If a sp4.4.x = 0.1 and 0.4 samples show a significant increase up to 500 K, whereas the increase is modest in x = 0.6 and 1.0 samples. In all these samples, the charge carriers are electrons, and thus the chemical potential is located in the conduction band. The observed behavior suggests that the responsible bands for the Se-rich composition and the other are different from each other, and thus it is suggested that band inversion occurs when x changes from 0.4 to 0.6 in the present system.From the transport properties at high temperatures, one may extract the energy gap if an activation behavior is observed . In the 5.x)2. The growth condition of TlBiSe2 is optimized to obtain higher mobility using transport properties. XRD analysis confirmed that the change in the crystallographic structure is gradual in TlBi(Sx)2, and this system is useful for studying the topological phase transition and related Dirac semimetals.This article provides detailed information on the synthesis and characterization of TlBi(S


A 46-year-old woman with a history of dasatinib-resistant chronic myeloid leukaemia, clonal evolution and monosomy 7 underwent reduced intensity conditioned in vivo T-cell-depleted allogeneic haematopoietic stem cell transplantation (HSCT) from a matched unrelated donor. Following the transplantation, she developed recurrent cutaneous graft versus host disease (GvHD), which required treatment with systemic immunosuppression and electrocorporeal photophoresis. Concurrently, she developed a lichenoid rash with granulomatous features suggestive of cutaneous sarcoidosis. Additional treatment with hydroxychloroquine was initially successful, but 2 months later, she developed erythroderma with palpable lymphadenopathy. Repeated histological analysis established a diagnosis of folliculotropic mycosis fungoides stage IVA2, and the malignant clone was confirmed to be of donor origin. A positive response to brentuximab has been shown. This is the first reported case of primary mycosis fungoides after matched unrelated donor HSCT, and in a patient still undergoing treatment for GvHD. Allogeneic haematopoietic stem cell transplantation (HSCT) was the standard of care for patients for chronic myeloid leukaemia (CML) until the advent of tyrosine kinase inhibition. Since then, HSCT has been reserved for those with resistance to tyrosine kinase inhibition or advanced disease.2 for 5 days and melphalan 140 mg/m2 for 1 day) in vivo T-cell-depleted  unrelated donor allogeneic haematopoietic stem cell transplant (HSCT) in August 2010. Her donor was a man and matched for the human leucocyte antigens (HLA) A, B, C, DRB and DRQ. Graft versus host disease (GvHD) prophylaxis with ciclosporin was initiated 2 days pre-transplant.A 46-year-old woman with a history of CML diagnosed in the first chronic phase demonstrated dasatinib resistance associated with clonal evolution and monosomy 7. Consequently, she underwent a reduced intensity conditioned  imaging revealed lymphadenopathy above and below the diaphragm. The largest lymph nodes measured up to 3.2×1.7 cm were located in the pre-pectoral and axillary regions, and were the most avid  max 5.8). Histological examination of the skin at this point revealed a loss of features characteristic for GvHD or granulomatous infiltrate, but displayed an atypical T-lymphocytic infiltrate C, D withClonality studies confirmed the presence of an identical clonal TCRβ rearrangement in skin and lymph node biopsies, thus confirming a diagnosis of cutaneous T-cell lymphoma (CTCL) A, B, sta10Microsatellite and XY-FISH studies confirmed that the malignant infiltrate was of donor origin.Please see the ‘Outcome and follow-up’ section.The patient remained erythematous (mSWAT score of 200) and failed to respond to 4 cycles of gemcitabine monotherapy and taper of immunosuppression. Further FDG-PET imaging revealed an increase in metabolic activity of affected nodes (axillary lymph node SUV max 6.9), along with new splenomegaly (24 cm) exhibiting increased diffuse activity. While still receiving ECP for GvHD, she initiated brentuximab (1.8 mg/kg dose) with the aim of disease control. After 3 cycles, she experienced significant diarrhoea and grade 1 peripheral neuropathy, and the dose of brentuximab was reduced to 1.2 mg/kg. She has subsequently received a further four cycles with no further detrimental effects, and has experienced a significant reduction in lymphadenopathy and B symptoms. Her quality of life has improved, and her Eastern Cooperative Oncology Group (ECOG) has dropped from 3 to 1. However, despite these improvements, she remains pruritic with an mSWAT score of 100 D.We report the first case of erythrodermic mycosis fungoides following allogeneic HSCT to derive from a matched unrelated donor. ‘Transmission’ of donor-derived T-cell lymphoma and leukaemia via HSCT has been reported previously.9Here, information related to the anonymous unrelated donor was understandably limited, but the relevant donor registry was informed of the potential risk of malignancy for the donor.It is known that CTCL may present indolently,Survivorship following haematopoietic stem cell transplantation (HSCT) is of increasing importance as the safety of the procedures improves. Secondary malignancy is a devastating complication that contributes to the morbidity and mortality after HSCT.We present the first case of folliculotropic mycosis fungoides (stage IVA2) following HSCT to derive from an unrelated donor. A positive response to brentuximab is reported.We describe an evolving pathology with a multifactorial aetiology including intractable cutaneous graft versus host disease and long-term profound immunosuppression.Diagnosis required a high index of clinical suspicion and a cohesive multidisciplinary approach. This case highlights the vigilance required while monitoring patients following HSCT, and others on long-term immunosuppression for alternative diagnoses.


For the foreseeable future, more individuals will need a kidney than there are kidneys available for transplant. This is not a new issue, and it is one that will not likely be solved anytime soon. While recent initiatives have focused on efficiently allocating kidneys in order to maximize supply, a shortage will remain. Currently, organs are made available for transplant through three different processes: donation after brain death declaration (BD), donation after circulatory death (DCD), and living donation . The objective of this article is to discuss the possibility of a fourth option in imminent death single kidney donation (IDSKD) and its potential effects on the future of donation and transplantation. During our study, IDSKD had the potential to increase the number of kidneys transplanted in our service area by approximately 5%. The purpose of this study was to identify an alternative group of patients who were unable to become an organ donor after brain death declaration or donation after circulatory death. The primary reason these patients were unable to donate is due to intact neurological responses, which typically would prevent the patient from cardiac arresting within a given timeframe. All patients who were focused on during the study were referred to the Louisiana Organ Procurement Agency (LOPA) due to being hospital-bound, ventilator dependent, and their families had made the decision to terminally extubate. Imminent death single kidney donation (IDSKD) would allow a patient population who currently are deemed unsuitable for organ donation to become a donor. Once the patient’s family has decided to terminally extubate, the patient would be evaluated for donation after circulatory death (DCD). If the organ procurement organization (OPO) determines that DCD is not an option, then the patient could be evaluated for IDSKD. This process allows a family to authorize the removal of a single kidney from the potential organ donor. If the family approves of this option, the OPO and hospital would complete all legally required paperwork and required testing, per the United Network for Organ Sharing (UNOS) policy. Once a recipient is identified, the donor would be brought to the operating room, and a nephrectomy would be performed by the recipient’s transplant surgeon. Once the surgery is complete, the donor would then be brought back to the ICU, or designated area. The family’s decision to terminally extubate the patient could then be carried out by the appropriate hospital staff.While this process is not an original thought, addressing the issue of an eligible patient population is. To date, no known long-term study has been completed to assess the potential for this type of organ donation. This study focused on data collected from 2014-2015. The original use of the data used in this study was not intended for this project. This study is a byproduct of research currently being completed by the Louisiana Organ Procurement Agency (LOPA) attempting to accurately predict the likelihood a patient would cardiac arrest in a timely manner in order to facilitate DCD. In order for donation after circulatory death to take place, the potential donor must cardiac arrest within two hours. Outside of the allotted time, the potential donor is no longer eligible for organ donation. The original research evaluated numerous patient demographics including, but not limited to, age, body mass index (BMI), cause of death, intact neurological responses, and various lab values. Patients would be evaluated once the LOPA was informed of a family’s decision to terminally extubate a patient who was ventilator dependent and hospital-bound. The LOPA clinical staff were responsible for the initial data collection and evaluation.Under the advisement of the LOPA’s medical director, additional metrics would be necessary to properly assess the potential donor pool for IDSKD, primarily a kidney donor profile index (KDPI), history of diabetes, and the patient’s age. A patient’s  KDPI is determined using the KDPI calculator provided by the Organ Procurement and Transplantation Network (OPTN). Variables used in the calculation are as follows: age, height, weight, ethnicity, history of hypertension, history of diabetes, cause of death, serum creatinine, as well as Hepatitis C status. Once all information is entered, the calculator provides a percentage. According to OPTN, “lower KDPI score is associated with longer estimated function, while higher KDPI scores are associated with shorter estimated function. For example, a kidney with a KDPI of 20% is expected to have shorter longevity than 20% of recovered kidneys ” . Figure Since IDSKD is neither a deceased donor nor is it a traditional living donor, a metric, such as a KDPI, was needed for evaluation. Given that it provides an estimation of graft function, it was determined to be a pertinent metric for evaluating the donor population for IDSKD. Traditionally, a potential donor’s KDPI is only calculated in a deceased donation scenario, not for living donors. In imminent death single kidney donation, the patient does not fit the definition for either type of donation, making the use of KDPI unique for the current study.Calculation of the potential donor’s KDPI was completed by the LOPA’s Data Manager. All necessary data needed for completion of the KDPI calculation were obtained after a potential donor was referred to the LOPA. Once data collection was completed, it then needed to be filtered according to the LOPA’s medical director’s criteria. The following criteria were used as filters: patients over the age of 60 were removed, those patients that had become a donor, those declared brain dead, history of diabetes, a KDPI above 85%, or if the family authorized for donation but the process was stopped due positive serological results, poor organ function, or inability to locate a potential recipient.Twenty-five potential imminent death single kidney donors were identified. These patients' ages ranged from 14 to 59 years of age, with a median age of 45 and mean of 44. Calculated KDPI’s ranged from 15% to 84%, with a median of 59% and a mean of 56%.Table During the data collection time period, the LOPA transplanted 483 kidneys. The additional 25 possible IDSKD would have accounted for an increase of 5.17% kidneys transplanted. During this same collection period, 23,820 kidneys were transplanted nationally. A 5% increase nationally could have yielded an estimated 1,191 live-saving transplantable kidneys.The patient population on whom this study was focused were referred to the LOPA during their most current hospital admittance. Average times from admittance to extubation ranged from 10 hours to 690 hours. The average time from admittance to extubation was 140.8 hours, or 5.8 days. There was no correlation between the length of time to extubation and an increase in a patient’s KDPI. Extubation time for case #14506202425 was not readily available, therefore it was not included in the average calculation.At this time, the LOPA is formulating an implementation strategy for IDSKD. The most likely scenario for implementation will require a trial period at a local transplant facility. The LOPA would initially evaluate a patient's suitability for donation after circulatory death. If the patient does not meet the criteria, they would then be evaluated for IDSKD. By trialing IDSKD in a local transplant facility, it would allow greater control of the possible variables involved until the process is perfected. Educating and preparing the hospital staff would be considered paramount during this trial period.As preparation continues, the OPO industry is faced with a primary issue. The United Network for Organ Sharing (UNOS) currently does not recognize imminent death single kidney donation. Therefore, there are no policies in place governing recovery and allocation of recovered kidneys through this process. Additional communication and process formalization with UNOS will be required to move forward in the future.IDSKD will undoubtedly bring in ethical questions. An anticipated concern is hastening the death of the donor due to the removal of a kidney. According to the National Kidney Foundation, “Living donation does not change life expectancy, and does not appear to increase the risk of kidney failure. In general, most people with a single normal kidney have few or no problem...” . While IWe realize limitations may exist by using the Kidney Donor Profile Index. Primarily, the fact that it does not calculate how well a kidney will actually function once transplanted or just how well it might outperform other kidneys transplanted based on historical data. Fortunately, our goal was not to determine the functionality of the kidneys once transplanted. The goal was to identify a group of patients who, based on criteria provided by our medical director, would possibly be considered suitable for donation. Using the KDPI allowed us to take into account many different patient demographics and provided a metric on which we would be able to filter results. Ultimately, each transplant facility would be responsible for determining their own criteria for acceptance of a kidney based on the information provided to them.Another task that will need to be addressed lies in how OPOs will be judged by governing agencies, primarily the federal government. Currently, OPOs are judged based on metrics that rate how productive and efficiently they are able to maximize organs from a potential donor. IDSKD would have a direct and negative impact on their efficiency rating.While it is clearly evident that imminent death single kidney donation will not completely address the entire shortage of kidneys the nation is currently facing, it does have the potential to provide more life-saving transplantable kidneys to patients in need. Ultimately, our responsibility as the organ procurement and transplant community is to ensure that those in need of a transplant are provided with said opportunity. We have a responsibility to be innovative in pursuing all legal and ethical options to ensure that no person dies while waiting for a transplant.


CSF2RA-mutated, elderly-onset hereditary (h) PAP.Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by surfactant accumulation, and is caused by disruption of granulocyte/macrophage colony-stimulating factor (GM-CSF) signaling. Abnormalities in CSF2 receptor alpha (CSF2RA) were reported to cause pediatric hereditary PAP. We report here the first case of CSF2RA. High-resolution analysis of single nucleotide polymorphism array confirmed a 22.8-Mb loss of heterozygosity region in Xp22.33p22.11, encompassing the CSF2RA gene. She was successfully treated with whole lung lavage (WLL), which reduced the serum levels of interleukin (IL)-2, IL-5, and IL-17, although IL-3 and M-CSF levels remained high.The patient developed dyspnea on exertion, and was diagnosed with PAP at the age of 77 years, based on findings from chest computed tomography scan and bronchoalveolar lavage. She tested negative for GM-CSF autoantibodies, with no underlying disease. Her serum GM-CSF level was elevated (91.3 pg/mL), indicating GM-CSF signaling impairment and genetic defects in the GM-CSF receptor. GM-CSF-stimulated phosphorylation in signal transducer and activator of transcription 5 (STAT5) was not observed, and GM-CSF-Rα expression was defective in her blood cells. Genetic screening revealed a homozygous, single-base C > T mutation at nt 508—a nonsense mutation that yields a stop codon (Q170X)—in exon 7 of CSF2RA mutation, which caused defective GM-CSF-Rα expression and impaired signaling. The analyses of serum cytokine levels during WLL suggested that GM-CSF signaling might be compensated by other signaling pathways, leading to elderly-onset PAP.This is the first known report of elderly-onset hPAP associated with a The online version of this article (doi:10.1186/s12890-017-0382-x) contains supplementary material, which is available to authorized users. The lungs reduce alveolar surface tension by producing pulmonary surfactants, which are essential for maintaining smooth breathing. The levels of pulmonary surfactants are balanced between their production by alveolar epithelial cells and clearance by alveolar macrophages. Granulocyte/macrophage colony-stimulating factor (GM-CSF), a 23-kDa monomeric glycoprotein, regulates various functions of the alveolar macrophages, including surfactant catabolism, and plays an important role in pulmonary surfactant homeostasis.Pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by the accumulation of surfactant proteins, which causes progressive respiratory insufficiency –3. ThreeThe GM-CSF signal is conveyed through the GM-CSF receptor on cell surfaces, via signal transducer and activator of transcription 5 (STAT5). The GM-CSF receptor is a dodecameric complex consisting of a ligand-specific alpha  and a beta  subunit, which is shared with the interleukin (IL)-3 and IL-5 receptors , 10.CSF2RA) cause hereditary (h) PAP, presenting as insidious, progressive dyspnea in children; and that increased serum GM-CSF is useful to identify such individuals [Recently, it has been reported that abnormalities in the GM-CSF-Rα-encoding alleles .The concentration of GM-CSF autoantibodies in the bronchoalveolar lavage fluid (BALF) or serum were measured by sandwich enzyme-linked immunosorbent assay (ELISA), performed as described previously , 17, wit5 cells/well. The cells were incubated with GM-CSF (0–1000 ng/mL) for 15 min. Lysed protein extracts were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and assessed by standard western blotting procedures, as previously described [Peripheral blood mononuclear cells (PBMCs) were cultured on 24-well culture plates at 5 × 103/PBS solution and incubated with Alexa Fluor 647-labeled anti-pSTAT5 (BD Bioscience). Cells with phosphorylated STAT5 were detected by flow cytometry .Phosphorylated STAT5 detection by flow cytometry was described previously . HeparinCSF2RA-specific primers , and was reverse transcribed with random hexamer primers and the SuperScript III First-Stand Synthesis System for RT-PCR . The cDNAs were subjected to semiquantitative RT-PCR analysis with PrimerSTAR GXL DNA polymerase  with CSF2RA gene, which were purified using the QIAquick DNA extraction kit (QIAGEN) and subjected to nucleotide sequencing using the BigDye Terminator v3.1 cycle sequencing kit  and CSF2RA-specific primers (Supplementary Table). The resulting sequences were compared with published sequences for CSF2RA (GenBank/EMBL/DDBJ under accession number NM_006140).The PCR was performed to generate products spanning exons and flanking sequences of the aCGH analysis was performed using the CytoScan HD array kit according to the manufacturer’s protocol . Genetic counseling was performed for the patient before and after genetic analyses.IL-2, IL-3, IL-4, IL-5, IL-17, and M-CSF were analyzed using ELISA kits  according to the manufacturer’s protocol.A previously healthy, 77-year-old, non-smoking woman, with normal chest radiograph at a medical check-up three years earlier, was referred to our hospital for dyspnea on exertion, which occurred one month earlier. She was a homemaker with no remarkable family history.2) of 47 mmHg just before WLL. BALF presented a milky appearance with lymphocytosis , foamy macrophages, and amorphous materials  scan indicated ground glass opacity in both lower lungs, of which crazy-paving appearance was confirmed upon high-resolution CT scan Fig. . Laboratals Fig. .Fig. 1aThe patient was diagnosed with PAP, based on typical findings from chest CT scan and bronchoalveolar lavage. She had no underlying disease. GM-CSF autoantibodies were not detected in either the serum or the BALF. On the other hand, a high level of serum GM-CSF was detected (91.3 pg/mL), indicating disrupted GM-CSF signaling and genetic defects in the GM-CSF receptor. We decided to screen for GM-CSF signaling abnormalities because GM-CSF concentration was extremely high in her BALF.To investigate signaling activated by GM-CSF in the PBMCs, we first probed the presence of phosphorylated STAT5 upon stimulating the PBMCs with increasing concentrations of GM-CSF (0–1000 ng/mL). Phosphorylated STAT5 was observed in the control but not in the patient PBMCs, indicating defective GM-CSF signaling in the latter Fig. . To confNext, we investigated the expression of the α and β chains of the GM-CSF receptor on the PBMCs, by western blotting. The expression of GM-CSF-Rβ and IL-3Rα on patient monocytes was comparable with that on the controls; however, expression of GM-CSF-Rα was not detected in the former Fig. . The resCSF2RB, reverse-transcribed from the mRNA of the patient PBMCs, harbored no mutations or deletions (data not shown). PCR amplicons for each exon of CSF2RA from the patient PBMCs were screened for their nucleotide sequences, revealing a homozygous single-base mutation C > T at nt 508 in exon 7  in Xp22.33p22.11, encompassing the 2 by 26 mmHg (Fig. To investigate the functional background underlying the late onset of the symptoms in the present case, we compared the changes in serum levels of IL-2, IL-3, IL-4, IL-5, IL-17, and M-CSF before and after WLL that improved PaOCSF2RA mutation. The patient had a homozygous mutation that changed a codon for glutamine 170 to a stop codon, disrupting normal expression of GM-CSF-Rα and proper signaling of GM-CSF. We previously reported a case of adult-onset hPAP that was due to homozygous nonsense mutation in the CSF2RB gene [CSF2RA gene, in which the first child case for hPAP also had a mutation [The present study reports the first case of elderly-onset hPAP with 2RB gene . The premutation .Table 1CTo investigate the background of the late-onset hPAP in this case, we studied her serum profile for various cytokines. The patient was treated with WLL, which greatly improved oxygenation indices and reduced the serum levels of various cytokines including IL-2, IL-5, and IL-17, indicating relieved inflammatory activation of Th1 and Th17 lymphocytes. In contrast, the serum levels of IL-3 and M-CSF remained high after WLL, indicating that they might be needed even after the treatment procedure. The high activities of IL-3 and M-CSF in the patient might be associated with the clinical course of her late-onset hPAP.It is notable that GM-CSF, IL-3, IL-4, and IL-5 are coded by neighbor genes located at 5q31.1, and affect the functions of monocytes and macrophages. The serum levels of IL-4, IL-5, and GM-CSF decreased after WLL, while IL-3 levels remained high. Receptors of IL-3 and IL-5 share beta chains with that of GM-CSF. Disruption of the alpha chain of the GM-CSF receptor might affect stoichiometry of the common beta chains, presumably causing substantial alterations in IL-3 and IL-5 signaling. Interestingly, WLL differentially affected the levels of these cytokines in the serum of the patient in this study.Similar to previous pediatric hPAP cases, the present case also demonstrated a high level of serum GM-CSF and a good response to WLL Table . The incCSF2RA, which caused defective GM-CSF-Rα expression and impaired signaling. These results suggest that GM-CSF signaling is compensated by other signaling pathways, leading to elderly-onset or non-symptomatic PAP.This is the first known report of elderly-onset hPAP associated with a genetic defect in


Objective: Examine relationships among neurodegenerative biomarkers and PD motor and nonmotor symptoms.Background: CSF alpha‐synuclein is decreased in PD versus healthy controls, but whether plasma and saliva alpha‐synuclein differentiate these groups is controversial. Correlations of alpha‐synuclein among biofluids  or biomarkers  are not fully understood. The relationships of these biomarkers with PD clinical features remain unclear.Methods: BioFIND, a cross‐sectional, observational study, examines clinical and biomarker characteristics in moderate‐advanced PD and matched healthy controls. We compared alpha‐synuclein concentrations across diagnosis, biofluids, and CSF biomarkers. Correlations of CSF biomarkers and MDS‐UPDRS, motor phenotype, MoCA, and rapid eye movement sleep behavior disorder questionnaire scores in PD were examined.Results: CSF alpha‐synuclein was lower in PD versus controls (P = .01), controlling for age, gender, and education. Plasma and saliva alpha‐synuclein did not differ between PD and controls, and alpha‐synuclein did not significantly correlate among biofluids. CSF beta‐amyloid1‐42 was lower in PD versus controls (P < .01), and correlated weakly with MoCA recall scores . CSF alpha‐synuclein was lower in the postural instability/gait difficulty phenotype than other motor phenotypes (P < .01). No CSF biomarkers predicted or correlated with total motor or rapid eye movement sleep behavior disorder scores. CSF alpha‐synuclein correlated with beta‐amyloid1‐42, total‐tau, and phosphorylated‐tau .Conclusion: Lower CSF alpha‐synuclein is associated with diagnosis and motor phenotype in moderate‐advanced PD. Plasma and saliva alpha‐synuclein neither correlate with CSF alpha‐synuclein, nor distinguish PD from controls. CSF beta‐amyloid1‐42 remains a potential biomarker for cognitive impairment in PD. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. Biomarkers have the potential to play a role in establishing PD diagnosis, understanding disease progression or disease‐related features, and monitoring therapeutic effects. In PD, candidate biofluids include cerebrospinal fluid (CSF), plasma/serum, saliva, and urine, among others.Several studies have examined CSF markers of neurodegeneration in PD , but most studies focus on either de novo PD patients , or specifically on cognitive measures in PD cohorts.1‐42, t‐tau, p‐tau) in PD and HC in the BioFIND cohort and (b) in PD participants, the relationships among CSF, plasma, and saliva α‐syn levels; (2) determine differences between PD and HC in CSF α‐syn, Aβ1‐42, tau, and p‐tau and in plasma and saliva levels of α‐syn; and (3) investigate the associations of CSF markers with specific motor and nonmotor features of PD.BioFIND, a cross‐sectional, observational study of moderate‐advanced PD patients and matched HCs evaluated with standardized clinical and biospecimen acquisition protocols, provides a unique resource for examining biomarkers in a well‐characterized, “typical” (including good response to levodopa) PD population.BioFIND includes moderate‐advanced PD participants and HCs enrolled at 8 sites in the United States.Clinical data and biospecimen collection occurred on 2 visits . For PD patients, V1 was performed in the on state (1‐3 hours after the last PD medication dose) and V2 was performed in the practically defined off state (early morning before PD medications and approximately 12 hours after the last dose the night before). V1 included collection of blood for DNA and plasma and assessments including the International Parkinson and Movement Disorder Society–Sponsored revision of the UPDRS (MDS‐UPDRS) parts I (nonmotor experiences of daily living), II (motor experiences of daily living), III (motor examination), and IV (motor complications) for PD patients and part III only for HCs.For PD patients, motor phenotype was classified as tremor dominant (TD), postural instability/gait difficulty (PIGD), and indeterminate,The study was approved by the institutional review boards for the University of Rochester Clinical Trials Coordination Center and study sites. Written, informed consent was obtained from the study participants.Concentrations of α‐syn in plasma, CSF, and saliva samples were analyzed using ELISA assays . The concentration of α‐syn in each sample was determined by interpolation of values against the standard curve established by the reference standards (range 1500 pg/ml‐6.1 pg/ml) using a 4‐parameter regression. Recombinant α‐syn from rPeptide was used as the standard in this assay. Each sample was analyzed in duplicate at appropriate dilutions . Concentrations of hemoglobin in CSF, plasma, and saliva samples were analyzed using ELISA assays . Reference standards used in the assay ranged from 7.5 ng/ml to 125 ng/ml. The concentration of hemoglobin in each sample was determined by interpolation of values against the standard curve established by the reference standards using a 4‐parameter regression. The assay has been validated in multiple matrices including in CSF, plasma, and saliva . Interassay precision and spike/recovery data for saliva are depicted .1‐42, t‐tau, and p‐tau in CSF was performed using a highly standardized micro‐bead based research‐use‐only immunoassay .1‐42 and 6 t‐tau results had microbead counts <50 and 1 p‐tau181 result had duplicate precision (%CV) > 25%; therefore, these 9 results were invalidated.Simultaneous analysis of Aβt tests. Gender and dichotomous variables  were compared between groups using chi‐square tests. Between‐subjects tests were used to examine MoCA total and subdomain scores controlling for age and gender and CSF biomarker levels in motor phenotypes controlling for age, gender, and duration of disease. Pearson and Spearman rho correlations were used to examine the relationships among biofluids, CSF biomarkers, and for CSF with motor and nonmotor variables, as appropriate and controlling for age, gender, and education. Regression models controlling for age, gender, and education were used to examine CSF biomarker predictors of PD diagnosis.The analyses were conducted in IBM SPSS Statistics version 23 . Demographic data, disease characteristics, and biomarker concentrations were compared between PD and HC groups using P < .01), with mean age of disease onset 59.9 years, disease duration 8.34 years, MDS‐UPDRS part III total motor score (off) 39.13, and a median Hoehn and Yahr stage of 2. PD motor phenotypes included 51.3% TD, 36.5% PIGD, and 12.2% indeterminate. MoCA total scores were significantly lower in PD patients when compared with HCs (P < .001) as expected because HCs with MoCA scores ≤ 26 were excluded. Of the PD participants, 45.2% had RBD scores > 5, compared to 6.8% of HCs (P < .001).Clinical characteristics of BioFIND participants who provided at least one of the biomarker specimens are presented in Table r = 0.81) and modestly with p‐tau (r = 0.43) and Aβ1‐42 , α‐syn , and t‐tau . Among PD participants only, correlations between CSF α‐syn concentrations and Aβ1‐42, t‐tau, and p‐tau remained consistent with those found in the complete BioFIND cohort.In the complete BioFIND cohort (PD and HC), CSF α‐syn concentrations were most strongly correlated with t‐tau r = 0.8 and moder = 0.41, P = .04), but not in plasma  or CSF  in the BioFIND cohort. Saliva samples with hemoglobin concentrations > 1200 ng/ml were excluded to avoid potential impact of contaminant hemoglobin on α‐syn levels.Hemoglobin and α‐syn levels were modestly correlated in saliva , CSF and saliva , or plasma and saliva . Likewise, among PD patients, there were no significant correlations between α‐syn concentrations in CSF and plasma , CSF and saliva , or plasma and saliva .Comparing α‐syn across the different biofluids in the BioFIND cohort, we did not find significant correlations for α‐syn concentrations between CSF and plasma (P = .01) levels were lower in PD patients when compared with HCs (Table P = .64) and saliva (P = .23), however, did not differ between PD and HCs. CSF Aβ1‐42 (P < .01) and p‐tau (P = .05) levels were lower in PD patients when compared with HCs, but t‐tau (P = .08) levels did not differ between PD and HCs , with lower concentrations in participants with PIGD phenotypes (1215.40 [491.56] pg/ml) than indeterminate (1682.99 [806.14] pg/ml) and TD phenotypes (1594.85 [702.45] pg/ml). Post hoc comparisons revealed differences between PIGD and TD . The significant difference between motor subtypes was noted when covariates of age, gender, and disease duration were considered. CSF Aβ1‐42, t‐tau, and p‐tau did not differ significantly among the PD motor phenotypes. None of the CSF biomarkers correlated with factors of the MDS‐UPDRS parts I to IV.None of the CSF biomarkers correlated with, or predicted, MDS‐UPDRS total motor scores. However, CSF α‐syn levels differed among motor phenotypes  and attention subscores . There were no significant correlations between any of the CSF biomarkers and dichotomized RBD scores in the PD patients.The only CSF biomarker associated with cognition was Aβ1‐42, t‐tau, and p‐tau) in a large sample of “typical,” moderate‐advanced PD and matched healthy control participants. CSF α‐syn strongly correlated with t‐tau and modestly, but significantly, with Aβ1‐42 and p‐tau. α‐syn levels in different biofluids such as CSF, plasma, and saliva did not significantly correlate with each other. Overall, CSF α‐syn, Aβ1‐42, and p‐tau concentrations were lower in PD when compared with healthy control participants, whereas t‐tau levels did not significantly differ between groups. Furthermore, the concentration of CSF α‐syn, but not the other CSF neurodegenerative markers, was significantly lower in those PD patients with PIGD motor phenotypes than in TD and indeterminate phenotypes. Finally, CSF Aβ1‐42 levels significantly correlated with aspects of cognition on the MoCA examination.This cross‐sectional, observational study examined the levels of α‐syn across multiple biofluid matrices  and multiple CSF neurodegenerative biomarkers  of CSF and total plasma α‐syn,Our findings provide insights into the α‐syn levels across multiple biofluid matrices. Peripheral manifestation of Lewy type pathology has been explored as a diagnostic marker. One study noted a weak correlation .1‐42, and tau may interact on cellular and pathophysiological levels in PD. Interactions of α‐syn and tau proteins have been described in animal models and postmortem brain studies, including colocalization of tau‐positive tangles and α‐syn‐positive Lewy bodies,1‐42 may have a synergistic relationship.Consistent with published literature including a study of 63 de novo PD and 32 healthy control participants in PPMI, CSF α‐syn levels in PD strongly correlated with t‐tau, and to a lesser degree, p‐tau.P = .05). Aβ1‐42 also differentiated PD patients when compared with healthy controls. We recognize, however, that many studies represent healthy controls as the ceiling of health, such as our enrollment criteria of controls with MoCA scores ≥ 26; these groupings may introduce potential bias by detecting greater differences between disease and healthy states. However, it is interesting to note that CSF Aβ1‐42 may relate to certain cognitive domains such as remote memory; this finding supports CSF Aβ1‐42 as an important potential biomarker for cognitive impairment in PD.In agreement with previous reports, CSF α‐syn levels were lower in PD than in HCs and, in classification and regression models, distinguished these groups from each other.It is notable that CSF α‐syn levels did not correlate with several disease‐related characteristics, including MDS‐UPDRS total motor scores (part III), motor complications (part IV), and motor experiences of daily living (part II). Furthermore, CSF α‐syn did not correlate with nonmotor features including RBD, cognition as measured by the MoCA, or nonmotor experiences of daily living (MDS‐UPDRS part I). The lack of a relationship between RBD and CSF α‐syn in our cohort differs from a study in which oligomeric α‐syn levels were elevated in the CSF and serum in PD patients with RBD when compared with PD patients without RBD and controls. The different methodologies used and the more advanced PD participants included in our study may contribute to different study findings.CSF α‐syn levels differed among the PD motor phenotypes, with lower levels in the PIGD subtype. Although lower CSF α‐syn levels may not predict motor scores, they may be useful in distinguishing motor phenotypes and for prognosticiation because PIGD deficits may portend a worsened motor and cognitive course of PD. The relationship of CSF α‐syn, but not other CSF markers, to PIGD motor phenotype in PD also occurs in early PD as described in the PPMI cohort.The strengths of our study include well‐characterized and “typical” PD patients, rigorously collected and detailed clinical and biospecimen data, and simultaneous measurement of multiple CSF neurodegenerative markers and of α‐syn across multiple biofluid sources. We probed relationships of CSF biomarkers with motor and nonmotor variables, such as RBD and MDS‐UPDRS factors, which have received limited attention to date. We note several study limitations, including its cross‐sectional design and lack of clinico‐pathological correlations, which preclude our ability to determine relationships between biomarkers and disease progression or neuropathology. RBD assessment was based on self‐report without confirmation by polysomnography, and cognition was measured with a screening questionnaire rather than a full cognitive battery. Despite this, our findings provide direction for design of more in‐depth future studies. Lastly, although our CSF and plasma sample sizes are large, the number of salivary specimens was small because this assessment was added after the study had begun, and thus larger studies are needed.The BioFIND cohort offers a unique opportunity to compare α‐syn across different biofluids. In our cohort, α‐syn levels in different biofluids such as CSF, plasma, and saliva did not significantly correlate with each other. In addition, the finding that CSF α‐syn levels are associated with PIGD motor phenotype suggests that this marker may also be useful for providing prognostic information for PD patients.1) Research project: A. Conception, B. Organization, C. Execution; 2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique; 3) Manuscript: A. Writing of the first draft, B. Review and Critique.J.G.G.: 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3BH.A.: 2A, 2B, 2CA.A.: 1A, 1B, 1C, 3A, 3BA.N.: 1A, 1B, 1C, 2A, 2B, 2C, 3BR.N.A.: 1A, 1B, 1C, 2A, 2B, 2C, 3A, 3BL.M.S.: 1A, 1B, 1C, 3BP.T.: 1A, 1B, 1C, 3BT.X.: 1A, 1B, 1C, 3BP.T.: 1A, 1B, 1C, 3BC.H.: 1A, 1B, 1C, 3BP.H.: 1A, 1B, 1C, 3BS.F.: 1A, 1B, 1C, 3BM.H.S.H.: 1A, 1B, 1C, 3BM.F.: 1A, 1B, 1CV.A.: 1A, 1B, 1CA.N.R.: 1A, 1B, 1CM.S.: 1A, 1B, 1CC.S.F.: 1A, 1B, 1CK.G.: 1A, 1B, 1CU.J.K.: 1A, 1B, 1C, 2A, 2B, 2C, 3B1‐42, t‐tau and p‐tau in this study; consulting and advisory board membership with honoraria: Eli Lilly, Roche; grants and research: The Michael J. Fox Foundation, NIH(NIA), provide QC oversight for AD CSF immunoassays in the past for AlzBio3 immunoassay (Fujirebio) and currently for the Roche Elecsys fully automated CSF AD immunoassays; Employment: University of Pennsylvania. P.T. is an employee of BioLegend, the entity responsible for the manufacture and commercialization of the alpha‐synuclein ELISA (Cat. 844101) used in this study. BioLegend received funding from the Michael J. Fox Foundation to complete the analysis of alpha‐synuclein and hemoglobin in samples collected from the study cohort. P.T. reports the following: grants and research: BioLegend received funding from the Michael J. Fox Foundation to complete the analysis of alpha‐synuclein and hemoglobin in samples collected from the study cohort; employment: BioLegend. R.N.A. reports the following: grants and research: Parkinson's Disease Foundation, the National Institutes of Health (K02NS080915), and The Michael J Fox Foundation; agency honoraria: Genzyme/Sanofi, Denali, and Prophase; employment: Columbia University. T.X. reports the following: grants and research: The Michael J Fox Foundation, Bristol‐Myers Squibb; agency honoraria: CVS/Caremark; employment: University of Chicago Medicine. P.T. reports the following: grants and research: NIH, Bristol‐Myers Squibb, Biogen, Northwestern University, The Michael J. Fox Foundation, Biotie, Kyowa, and University of Minnesota; employment: University of Minnesota and University of Minnesota Physicians. C.H. the following: consulting and advisory board membership with honoraria: ACADIA ‐ ad hoc advisory board participation; US WorldMeds: ad hoc advisory board participation; Gerson Lehman Group: consultation; grants and research: NIH/NINDS; NIH/NOIT; New York State Department of Health /Empire State Stem Cell Board; employment: Weill Cornell Medical College. P.H. reports the following: grants and research: The Michael J. Fox Foundation; employment: Oregon Health Science University. S.F. reports the following: grants and research: Teva Pharmaceuticals; employment: Beth Israel Deaconess Medical Center. M.H.S.H. reports the following: consulting and advisory board membership with honoraria: Delsys Inc, Acorda; grants and research: MJFF, NIH, Acorda, Auspex; employment: Boston University. M.F., V.A., and A.N.R. report the following: employment: The Michael J. Fox Foundation. M.S., C.S.F., and K.G. report the following: employment: National Institute of Neurological Disorders and Stroke. U.J.K. reports the following: consulting and advisory board membership with honoraria: PF SAB, APDA SAB; grants and research: NIH R01 NS101982, R03 NS096494, U01 NS100600, DoD/CDMRP Discovery Award (PR161817), The Michael J. Fox Foundation, and Parkinson's Foundation; agency honoraria: NIH study section, PF, The Michael J. Fox Foundation grant review; employment: Columbia University.J.G.G. reports the following: consulting and advisory board membership with honoraria: Acadia, Aptinyx, Biogen; grants and research: Acadia, Biotie (site‐PI), Consolidated Anti‐Aging, National Institutes of Health, National Parkinson Foundation, The Michael J. Fox Foundation, Rush University, Agency Honoraria: American Academy of Neurology, International Parkinson and Movement Disorder Society, MedEdicus; employment: Rush University. H.A. reports the following: consulting and advisory board membership with honoraria: The Michael J. Fox Foundation; Employment: Columbia University. A.A. reports the following: consulting and advisory board membership with honoraria: advisory board for Jazz Pharmaceuticals; grants and research: NIH NINDS, NIA, Parkinson's disease Foundation, Rehabilitation Research Resource to Enhance Clinical Trials, The Michael J. Fox Foundation, AbbVie, Jazz Pharmaceuticals, Axovant Sciences, Ltd.; Employment: University of Alabama at Birmingham. A.N. reports Employment: The Michael J. Fox Foundation. L.M.S. reports funding from the Michael J. Fox Foundation to complete the analysis of CSF AβAdditional Supporting Information may be found in the online version of this article at the publisher's websiteSupporting Information 1Click here for additional data file.


To determine the rate of donor-site morbidity after osteochondral autologous transplantation (OATS) for capitellar osteochondritis dissecans.A literature search was performed in PubMed/MEDLINE, Embase, and Cochrane Library to identify studies up to November 6, 2016. Criteria for inclusion were OATS for capitellar osteochondritis dissecans, reported outcomes related to donor sites, ≥10 patients, ≥1 year follow-up, and written in English. Donor-site morbidity was defined as persistent symptoms (≥1 year) or cases that required subsequent intervention. Patient and harvest characteristics were described, as well as the rate of donor-site morbidity. A random effects model was used to calculate and compare weighted group proportions.Eleven studies including 190 patients were included. In eight studies, grafts were harvested from the femoral condyle, in three studies, from either the 5th or 6th costal-osteochondral junction. The average number of grafts was 2 (1–5); graft diameter ranged from 2.6 to 11 mm. In the knee-to-elbow group, donor-site morbidity was reported in 10 of 128 patients (7.8%), knee pain during activity (7.0%) and locking sensations (0.8%). In the rib-to-elbow group, one of 62 cases (1.6%) was complicated, a pneumothorax. The proportion in the knee-to-elbow group was 0.04 (95% CI 0.0–0.15), and the proportion in the rib-to-elbow group was 0.01 (95% CI 0.00–0.06). There were no significant differences between both harvest techniques (n.s.).Donor-site morbidity after OATS for capitellar osteochondritis dissecans was reported in a considerable group of patients.Level IV, systematic review of level IV studies.The online version of this article (doi:10.1007/s00167-017-4516-8) contains supplementary material, which is available to authorized users. Osteochondritis dissecans (OCD) of the capitellum is a disorder of the subchondral bone and articular cartilage , 23, 32.OATS has become a popular treatment option for large, unstable lesions (diameter > 10 mm) with lateral wall involvement, as well as also for athletes without an acceptable outcome after less invasive techniques , 22, 24.The purpose of this study was to determine the rate of donor-site morbidity following osteochondral autologous transplantation for capitellar osteochondritis dissecans. The hypothesis op the study was that there would be no difference in the proportion of donor-site morbidity between graft harvesting from the femoral condyle or costal-osteochondral junction.The findings of this systematic review were reported according to the Preferred Reporting Items for Systematic Review and Meta-Analyses (PRISMA) guidelines .Studies that met the following inclusion criteria were included: (1) osteochondral autologous transplantation for capitellar OCD, (2) reported outcomes related to the donor site, (3) minimum inclusion of 10 patients, (4) minimum follow-up of one year, and (5) written in English. Case reports, reviews, and cadaveric studies were excluded. Donor-site morbidity was defined as the presence of persistent symptoms (≥1 year) after graft harvesting, as well as the need for subsequent intervention to treat complications related to the donor site.A literature search was performed in the following databases up to November 6, 2016: PubMed/MEDLINE, Embase, and the Cochrane Library. The PubMed/MEDLINE search strategy was adjusted into similar search strategies for other databases (Online Appendix 1). Reference lists of retrieved studies were manually searched for additional studies potentially meeting the inclusion criteria.Study selection was independently performed by two authors. First, title and abstract were screened to identify potentially relevant papers (R.B. and P.O.). These results were verified by two senior authors (L.O. and M.v.d.B.). Subsequently, manuscripts were retrieved when title or abstract revealed insufficient information to determine the appropriateness for inclusion. Disagreement was resolved by discussion or with arbitration when necessary by the senior authors (L.O. and M.v.d.B.) when differences remained.The main outcome of interest was the presence or absence of donor-site morbidity (persistent symptoms or need for subsequent intervention) after capitellar OATS. The following data were extracted from each study: author names, year of publication, patient demographics, follow-up time, harvest method, graft characteristics, and the use of fillers. The following outcomes related to the donor site were extracted: symptoms , physical examination , complications , patient reported outcome scores, imaging evaluation , and anatomic and histological outcomes. Data extraction was independently performed by two authors (R.B. and P.O.) and verified by two senior authors (L.O. and M.v.d.B.). Articles were not blinded for author, affiliation, and source.Two authors (R.B. and P.O.) independently judged the methodological quality of included studies using the checklist for quality appraisal of case series studies that was developed at the Institute of Health Economics (IHE) , 20. EacCategorical data were displayed as absolute numbers with frequencies; continuous data were displayed as means with sample range.p value <0.05 was considered significant. Statistical analyses were performed with the use of Stata 13.0 .The main outcomes of interest were the rate of donor-site morbidity within the knee-to-elbow group and rib-to-elbow group. Proportions of donor-site morbidity were calculated for each study using the Freeman-Tukey double arcsine transformation. Subsequently, a random effects model, to account for heterogeneity across studies, was used to calculate weighted group proportions for each harvest technique, and to compare proportions between the two techniques , 34. A pA detailed summary of patient and harvest characteristics for each study is given in Table The knee served as the donor site in 128 patients across eight studies (Table A detailed summary of donor-site effects per study is given in Table p > 0.05).The proportion of donor-site morbidity in the knee-to-elbow group was 0.04 (95% CI 0.0–0.15), and the proportion in the rib-to-elbow group was 0.01 (95% CI 0.00–0.06) Fig. . There wThe results of methodological quality assessment of individual studies using the IHE scale are presented in Table The most important finding of the present study is that donor-site morbidity after osteochondral autologous transplantation for capitellar osteochondritis dissecans occurred in 7.8% within the knee-to-elbow group and 1.6% within the rib-to-elbow group. In the knee-to-elbow group, knee pain during daily activities (7.0%) and locking sensations (0.8%) were reported; in the rib-to-elbow group, one case was complicated by a pneumothorax. There was no significant difference in proportion of donor-site morbidity between the two harvest techniques. The findings of this systematic review emphasize the importance of associated donor-site morbidity following graft harvesting in the treatment of capitellar OCD.The rate of donor-site morbidity in our study is comparable to the morbidity rate after knee-to-knee OATS (6%), as reported by Andrade et al. . InteresIn the present study, the rate of donor-site morbidity ranged from 0 to 57% in studies in whom grafts were harvested from the knee. Weigelt et al. reported morbidity in eight of 14 patients (57%): occasional pain during heavy lifting in seven patients and intermittent locking sensations in one patient . The advThree studies that were included in this review harvested grafts from either the 5th or 6th costal-osteochondral junction to repair capitellar OCD , 28, 30.Using the IHE scale to evaluate the methodological quality of included studies , 20, theFuture studies should comprehensively evaluate effects related to the donor site. Patient’s symptoms and physical examination should be reported for each patient. Additionally, the use of a patient reported outcome measure to assess knee function should be a routine part of clinical evaluation, both preoperatively and postoperatively. Radiographs should be performed to evaluate potential progression to osteoarthritis at 1 year and may be performed in cases in which donor fillers were used. MRI evaluation should, because of cost-effectiveness reasons, only be ordered in case of persistent symptoms. Besides the evaluation of donor-site effects, harvest characteristics should be reported in great detail, such as donor location, number of grafts, graft diameter, and depth. Also, alternative harvest methods should be investigated to have no longer the need to violate the integrity of an asymptomatic knee or rib in an adolescent athlete.The findings of the present investigation demonstrate a considerable risk for donor-site morbidity following capitellar OATS. Although good-to-excellent results related to the elbow have been reported after capitellar OATS, surgeons should be aware of the risk for donor-site morbidity and patients should be counseled about this issue. Knowing the overall risk for donor-site morbidity is also relevant in surgical decision making. Taking this into consideration, surgeons could consider other resurfacing techniques such as allografting or autologous chondrocyte transplantation.Osteochondral autologous transplantation in the treatment of capitellar osteochondritis dissecans may lead to donor-site morbidity in a considerable group of patients, either after harvesting from the femoral condyle (7.8%) or costal-osteochondral junction (1.6%).Below is the link to the electronic supplementary material.Online Appendix 1 PubMed/MEDLINE Search Strategy (TIF 28 KB)