Early clinical successes are driving enthusiasm for fecal microbiota transplantation (FMT), the transfer of healthy gut bacteria through whole stool, as emerging research is linking the microbiome to many different diseases. However, preliminary trials have yielded mixed results and suggest that heterogeneity in donor stool may play a role in patient response. Thus, clinical trials may fail because an ineffective donor was chosen rather than because FMT is not appropriate for the indication. Here, we describe a conceptual framework to guide rational donor selection to increase the likelihood that FMT clinical trials will succeed. We argue that the mechanism by which the microbiome is hypothesized to be associated with a given indication should inform how healthy donors are selected for FMT trials, categorizing these mechanisms into four disease models and presenting associated donor selection strategies. We next walk through examples based on previously published FMT trials and ongoing investigations to illustrate how donor selection might occur in practice. Finally, we show that typical FMT trials are not powered to discover individual taxa mediating patient responses, suggesting that clinicians should develop targeted hypotheses for retrospective analyses and design their clinical trials accordingly. Moving forward, developing and applying novel clinical trial design methodologies like rational donor selection will be necessary to ensure that FMT successfully translates into clinical impact. C. difficile infection (CDI) across multiple randomized, placebo-controlled trials [Fecal microbiota transplantation (FMT) is the transfer of gut bacteria through whole stool from a healthy donor to a recipient. FMT has demonstrated high cure rates in recurrent d trials  and has d trials –4. Beyond trials –10.Despite these early successes, the underlying mechanism of FMT across all disease indications, including CDI, remains unclear. However, it is generally considered that FMT restores gut microbial community perturbations from a dysbiotic state to a healthy stable state with engraftment of donor strains, or perhaps through other donor-dependent features such as the abundance of non-bacterial components or donor clinical features ,12. HoweUnlike FMT trials in CDI, where selecting donors based on specific clinical or microbiome profiles does not seem to affect clinical response rates, donor selection is likely to be crucial to trial outcomes in diseases with more complex host-microbiome interplay or distinct disease-associated perturbations . Most noTo date, the typical approach for donor selection in FMT trials is to use a single healthy donor or to randomly select multiple donors from a set of screened potential donors ,18,19. HUnlike traditional clinical trials which test well-defined small molecules, FMT trials test the donor microbiome, which is variable . FortunaIn this paper, we present a framework to guide donor selection for FMT trials. The mechanism by which the microbiome is hypothesized to be associated with a given indication should inform how donors are selected for FMT trials, and we describe different disease models which may underlie microbiome-mediated conditions . We descFMT trials are pursued when research or clinical experiences suggest that the microbiome may be causing or exacerbating a disease. Here, we propose four different models which may underlie microbiome-mediated etiologies and their corresponding rational donor selection strategies . UltimatMost of the donor selection strategies described below can be modified to incorporate matching between patients and donors. More specifically, donors can be tailored to individual patients to specifically make up for the unique taxonomic or functional deficiencies in that patient’s microbiome . With thIn all cases, ensuring patient safety through carefully screening donors and following all regulations relating to pursuing an FMT trial is of paramount importance and supersedes any other considerations regarding donor selection. While FMT has shown promise and is defined in some clinical guidelines as standard of care for multiply recurrent CDI –4, it isClostridium difficile infection (rCDI), a disturbed microbial community is unable to prevent colonization by or overgrowth of the pathogen, leading to recurrent overgrowth of C. diff and clinical symptoms [An acutely dysbiotic gut microbial community is broadly dysfunctional and can no longer maintain the health of the host. For example, in the case of recurrent symptoms . Acute dsymptoms  In this symptoms .In this model, the host simply needs to return to a “healthy” microbiome and thus choosing any healthy FMT donor should be sufficient to induce clinical improvements. Because there is no specific disease-associated microbial community and deviation from health is instead the more important factor, simply replenishing the microbiome with a healthy configuration should be sufficient. Indeed, FMT trials have demonstrated that rCDI can be effectively treated by almost any choice of healthy donor . In thisAbsence of beneficial taxaR. obeum, restricted infection by V. cholerae through quorum-sensing-mediated mechanisms [Lactobacillus was sufficient to ameliorate salt-induced hypertension in mice, and follow-up studies indicate that similar mechanisms may be involved in salt-sensitive high blood pressure in humans as well [In other cases, perhaps a disease is being caused or exacerbated by the lack of certain specific microbes, and replenishing these few taxa would be sufficient to restore the host to health . For exachanisms . Surpris as well .In these cases, the donor selection strategy should focus on maximizing the probability of engraftment of the beneficial taxa. In cases where the unique taxa are not specifically known or are rare members of the human microbiota, many healthy donors could be pooled together or a donor with a high alpha diversity could be selected in order to maximize the probability that the transplanted sample contains the necessary taxa . HoweverPresence of harmful taxaFusobacterium has been found to be more abundant in colorectal cancer patients, specifically enriched in the tumors themselves [Fusobacterium, inflammatory transcriptional signatures, and tumor growth in mouse and human models of colorectal cancer, pointing to a causal role for Fusobacterium in colorectal cancer progression [Rather than being characterized by the absence of individual bacteria, perhaps a disease is instead mediated by the presence or overabundance of specific microbes, and removing these bacteria in a targeted fashion could lead to improvements in disease progression. For example, emselves . Multiplgression , 30. Recgression .Removing and replacing these bacteria should be the goal of FMT in cases where this disease model applies. This can be achieved by first removing the harmful bacteria (e.g. via antibiotic treatment) with follow-up FMT to re-establish a healthy community that prevents their re-colonization. In all cases, donors should be screened to exclude any samples which contain the harmful bacteria. Donor samples can then be selected based on the abundance of bacteria which are known to out-compete the harmful taxa. Competitors can be identified by searching the microbiology literature to identify bacteria which live in the same niche or which have been experimentally shown to directly out-compete the undesirable taxa, or they can perform these competition assays themselves. If resources to perform competition assays are not available and the literature is sparse, researchers can also mine existing microbiome data to find bacteria which consistently anti-correlate with the harmful taxa, and choose donor samples with a high abundance of these putative competitors .Patient matchingTaxa-based donor selection strategies are particularly amenable to patient-matching, when both patient and donor microbiome data are available prior to the start of a trial . For exaCase study: Inflammatory bowel diseaseAn example where the “missing taxa” model may be applicable is in inflammatory bowel disease (IBD). Butyrate has long been associated with IBD , 33, andGiven the availability of microbiome data from completed FMT studies, a clinician leading an IBD FMT trial may elect to perform rational donor selection based on the “absence of beneficial taxa” disease model. To illustrate this process, we re-analyzed microbiome data from three completed IBD FMT trials which provided publicly available sequencing data for patient and donor samples –38. We sDespite their accessibility, taxonomy-based approaches are limited in their ability to identify functional or strain-level associations. For example, in this 16S-based analysis, we were unable to identify any of the Eubacteria taxa which are major contributors to SCFA production in the human gut , 40. MorSome microbiome-associated diseases may not be addressable by replenishing the patient with a generically healthy community or by targeting individual taxa, and may instead be mediated by the microbiome through a community-level function. Here, there may not be a consistent disease-associated microbiome across patients in terms of taxonomic composition, but patients may be characterized by having microbiomes which are similarly missing or enriched in some core functionality. This model may also apply to conditions where there are consistent disease- or health-associated taxa, but in which their collective functioning is the more important mediator of disease. The IBD case study described above may reflect this situation: although depletion of butyrate producers is strongly associated with IBD throughout the literature, a successful donor selection strategy may need to consider butyrate production directly rather than through the proxy of taxonomy , 34, 35.Missing community-level functionIn the case where a community-level function is missing from patients’ microbiomes, the goal of FMT should be to replace the deficient community with a beneficially functional microbiome. Here, it is important that a single donor with an intact microbial community is used, rather than a mixture of donors which may not yield the desirable community composition at steady-state after FMT. To choose a donor, molecules which can serve as proxies for the metabolic output of the microbial community can be measured directly in donor stool, and donors can be selected based on the abundance of these molecules.Lachnospiraceae and Ruminococcaceae, taxa which were were previously found to be depleted in HE patients based on cross-sectional microbiome data. The clinical trial was a success, but it remains unclear from this trial whether the donor's strains engrafted in the patients post-FMT and whether this played any role in the successful FMT responses. The exact mechanisms of action of these strains remain unknown, though both bacterial families are known short chain fatty acid producers (in particular butyrate) [Like IBD, hepatic encephalopathy (HE) is an example where community functionality is likely more relevant to FMT outcome than specific taxa. A previous trial in HE  rationalutyrate) . Recent utyrate) ). As in ex vivo assays, in which each donor sample is homogenized and provided with the substrates (e.g. fiber) needed to produce the desirable output (e.g. short-chain fatty acids like butyrate). In this way, the donor community function can be measured directly [While measuring metabolites in stool as a proxy for community production will likely be an improvement over taxonomy-based approaches in most cases, these measurements are also complicated by potential host effects. For example, different hosts may absorb these molecules at different rates, and so measuring them in stool may not be an accurate reflection of each donor community’s productive potential. Additionally, community function may depend on non-biologically relevant factors like the donor’s diet and time that they provided their sample. As an example, bile acid production spikes after meals , so the directly , 45.Overactive functionA disease may also be mediated by an overactive microbiome doing something harmful to the host. For example, TMAO produced by the microbiota contributes to atherosclerosis , 47. HerDiseases with more complex etiologies may not have a direct taxonomic or functional association with the microbiome but instead be related through some intermediate host phenotype which needs to be improved or corrected. For example, severe acute malnutrition has been associated with a gut microbiota which is not fully mature, with mouse experiments suggesting that this association may be causal , 49. OthFor these more complex cases, models can be trained from existing datasets to learn the community signatures linked to the disease-associated phenotype. In some cases, it may be possible to develop computational models which directly predict the phenotype of interest. For example, Stein et al. developed a model to predict the induction of regulatory T-cells by microbial communities . In otheIn some conditions, there may not be enough understanding of the underlying microbiome-based etiology to inform donor selection in an FMT trial. It may also be the case that there are no existing datasets on which to train models, existing datasets are not sufficiently powered to distinguish the different potential underlying models, or logistical considerations constrain a clinician’s ability to select specific donors for their trial. In these cases, we recommend selecting different healthy donors, employing an adaptive clinical trial design in which donors are cycled after they have clinical failures (as described previously in ), and peCycling healthy donors in adaptive trialsAs donors change through the course of an adaptive trial, clinicians may elect to select their donors randomly or to more rationally cycle through donors . “DifferDonors may also be selected to span the range of “healthy” microbiomes in a given population. For example, clinicians may pick a "median" healthy donor who is similar to existing healthy reference microbiomes , 55, or Discovery-based retrospective analyses of completed FMT trialsIn these exploratory FMT clinical trials, discovering microbiome characteristics which are differentially associated with FMT response may be a valuable secondary endpoint , identifWe performed a simulation to determine the likelihood of a retrospective analysis to identify donor-derived bacteria associated with different patient responses to FMT. We performed this simulation for multiple FMT trial set-ups and outcomes (i.e. number of FMT responders and non-responders). We used existing microbiome datasets to model different effect sizes, where we use “effect size” to mean the number of bacteria which are differentially abundant in donor samples given to patients who did and did not respond to FMT. Case-control datasets were used to model the microbiome data and various effect sizes, with a large effect represented by an infectious diarrhea dataset , a mediuIn cases where the microbial signature for FMT response is expected to be large (i.e. the difference in donor stools given to FMT responders vs. non-responders is as large as the effect of diarrhea effect on the microbiome), we found that small FMT trials would recover most of the top hits in the majority of cases. The power to detect associations decreased as FMT response rates became less balanced (i.e. response rates different from 50%), and in these cases trials would need to include up to 50 patients in the FMT arm to recover the key mediating taxa. For both medium and small effect sizes, however, prohibitively large FMT arms would be needed to recover most key mediating taxa regardless of FMT response rate. We found that when the microbial signature for FMT is equivalent to the effect of diseases like colorectal cancer on the microbiome, at least 100 patients are needed in the FMT arm to recover at least half of the most truly differentially abundant genera for most FMT trials. This simulation was performed at the genus-level, and underestimates the power needed to identify associations at finer taxonomic levels . In cases where strain-level biology is involved, FMT trials will need even more patients to be powered to find significant associations in retrospective analyses.These results suggest that successful secondary analyses of microbiome data from FMT trials will require either very large FMT arms, investigating more targeted hypotheses, meta-analyses, or additional sample collections. For example, clinicians may consider pairing donor and patient samples or collecting longitudinal patient samples to increase power to make discoveries , 61. Thea priori hypotheses to determine which microbiome-mediated disease model may underlie their indication of interest, and then select donors accordingly. By rationally choosing donors during the FMT trial design, clinicians will increase the likelihood of successful FMT trials in diseases in which donor heterogeneity affects patient response. Our power simulation analysis also suggests that specific plans for retrospective analyses of the microbiome data generated should be developed during trial design, with targeted hypotheses of interest and sample collection plans tailored accordingly. Otherwise, exploratory analyses of single FMT studies are unlikely to make new discoveries. Paired with adaptive clinical trial designs, FMT trials with rationally-selected donors will become an important tool in advancing translational microbiome research and clinical treatment to improve and save patient lives.The framework presented here encourages clinicians to leverage their clinical experience, existing microbiome research and published datasets, the increasing availability of screened healthy donor stools, and partnerships with bioinformaticians and microbiologists to more efficiently translate microbiome-based interventions into clinical impact. Clinicians can apply their existing knowledge and As a conceptual framework for approaching rational donor selection in practice, this work has significant limitations. Many logistical and scientific considerations  involved in performing FMT trials are not addressed in this framework, and should be considered during the trial design in addition to donor selection. FMT trials are also inherently limited by the material that they use: donor stool. It is unclear to what extent stool will be sufficient to address diseases in which regional differences in gut microbiota composition are involved (e.g. mucus-associated microbes implicated in IBD), and to what extent analyzing fecal samples provides a representative view of the entire GI physiology. Furthermore, while 16S rRNA-based microbiome profiling may be a good first-pass analysis when more extensive functional data  is not available, it is limited in its ability to provide insight on functional or strain-level associations. Further work will be required to address which measurement modality is optimal for performing rational donor selection in exploratory FMT clinical trials, while recognizing that these pilot studies are often constrained for resources.Prevotella than North Americans, it might be advisable to source donors locally to better match the expected healthy state of the patients [As FMT-specific clinical trial design methodologies become more developed, many additional questions will need to be addressed. Some of these key questions relate to choosing healthy donors: what defines a “healthy” donor, and when and how should that definition change? Some screening criteria, like excluding certain known pathogens, will apply to all donors. Beyond these, however, there is little consensus on what defines a “healthy” microbiome. Consider a clinician carrying out an FMT trial in an African setting: given that healthy Africans from across the continent are known to have more patients , 62, 63.patients , 64, 65.On the patient side, comorbidities, lifestyle, and dynamic disease manifestations present additional challenges and opportunities to improve donor selection and FMT clinical trial designs. How should comorbidities be incorporated into donor selection? Patients with multiple disease processes may be dominated by one disease model or may exhibit a combination of models, perhaps affecting which donors would be optimal for their specific case. For example, a person whose condition involves both acute dysbiosis and community-level dysfunction might respond well to any healthy donor, or may require a more complex combination of total community replacement along with enrichment for community function. Relatedly, diseases that change manifestations over time may benefit from employing different donor selection strategies over the course of a longitudinal FMT trial. Additionally, although there have been no serious adverse events related to FMT material in either clinical practice for rCDI or in clinical trials across adults or pediatrics, could some donors further reduce the probability of adverse events in at-risk patients? Finally, how should other sources of heterogeneity like lifestyle, diet, and medication usage be incorporated into rational donor selection? In cases where FMT is combined with other microbiome-targeted interventions like prebiotics or dietary changes, could some donors have synergistic effects with these paired interventions and lead to greater clinical success?To ensure that FMT reaches its full potential to improve and save patient lives, clinicians should think critically about how their FMT trials can be designed for maximal impact. Applying new approaches like rational donor selection will decrease the number of false negative clinical trials which fail even though they could have succeeded with a more optimal donor. Furthermore, by developing targeted hypotheses, post-trial analysis plans, and associated sample collection schema alongside the core FMT trial design itself, the number of basic scientific discoveries that are made from each trial will significantly increase. As FMT expands beyond rCDI and microbiome-based therapeutics are developed to target a range of diseases, novel methods and approaches tailored to the unique challenges and opportunities presented by FMT will be critical to ensuring the advancement of translational microbiome science and beneficial impact on patient lives.Raw fastq data files were downloaded from the European Nucleotide Archive using the following accession numbers: Jacob et al 2017 , PRJNA38E. ventriosum, E. hallii, and E. rectale from our analyses as these species-level taxa were not present in our data and do not comprise one genus with conserved butyrate production . The ge10.21228/M8RM32.Metabolomics data was generated as described in . and dowFor donors with multiple samples, we considered the mean metabolite abundances across all sampled time points. We identified three short chain fatty acids (SCFAs) in the data  and the major primary (cholate and chenodeoxycholate) and secondary (deoxycholate and lithocholate) bile acids. Lithocholate abundances were available for both C-18 negative and HILIC negative modes; we considered only the C-18 negative data to match the other bile acids. Bile acid conversion rates were calculated as in . Donors We performed a simulation to determine the power of FMT trials to retrospectively find associations between donor bacterial abundances and FMT response. We used case-control gut microbiome datasets from MicrobiomeHD  to modelWe modeled different FMT clinical trial designs and outcomes by varying the number of total patients in the trial and the percent of FMT responders . For each of these designs, we subsampled the correct number of case samples to represent FMT responders and control samples to represent non-responders from the original datasets. We identified significantly differentially abundant genera with the ‘kruskalwallis’ function from scipy.stats.mstats (scipy v. 1.1.0) as genera with q < 0.05 after multiple hypothesis testing correction with the multipletests function (method = ‘fdr_bh’) from the statsmodels.sandbox.stats.multicomp package (statsmodels v. 0.9.0). We then counted how many of the top genera identified through the signal-to-noise ranking were identified as significantly different as a proxy for the power to detect effects.https://github.com/cduvallet/donor-selection/. Data were downloaded from original sources as described above.Code to reproduce all of these analyses and figures can be found at S1 FigDonors are ordered along the x-axis in the same order for all plots. The respective patient response status is indicated through the bar color .(PNG)Click here for additional data file.S2 FigDonors are ordered along the x-axis in the same order for all plots. The respective patient response status is indicated through the bar color . Note: Butyricicoccus was not present in any of the donor samples.(PNG)Click here for additional data file.S3 FigDonors are ordered along the x-axis in the same order for all plots. The respective patient response status is indicated through the bar color .(PNG)Click here for additional data file.S4 FigThe difference was calculated by subtracting the patient’s total abundance of butyrate producers from the total abundance in their respective donor sample. Butyrate producers were identified as described in the Methods.(PNG)Click here for additional data file.


The optimum granulocyte colony-stimulating factor (G-CSF) treatment for cancer patients after being treated with cytotoxic chemotherapy remains unknown. Therefore, a systematic review and Bayesian network meta-analysis were performed to assess the efficacy and tolerability of 11 G-CSF drugs on patients after chemotherapy. A total of 73 randomized controlled trials (RCTs) containing 15,124 cancer patients were included for the final network meta-analysis. Compared with pegfilgrastim, there were a higher risk with filgrastim for incidence of febrile neutropenia (FN) , and a higher risk with short-acting G-CSF (S-G-CSF) biosimilar and lenograstim for incidence of bone pain (BP) . Mecapegfilgrastim, lipegfilgrastim and balugrastim were best G-CSF drugs in reducing FN . S-G-CSF biosimilar, empegfilgrastim, and long-acting G-CSF (L-G-CSF) biosimilar were best G-CSF drugs in reducing severe neutropenia (SN) . Mecapegfilgrastim, balugrastim, lipegfilgrastim and L-G-CSF biosimilar were best G-CSF drugs in reducing BP . Mecapegfilgrastim, lipegfilgrastim and balugrastim might be the most appreciate G-CSF drugs with both good efficacy and tolerability when treating cancer patients after cytotoxic chemotherapy. These complications lead to chemotherapy delay, dose reduction, and increased risk of infection2. Patients with these complications need to be treated with antibiotics and hospitalization3, which indirectly increases the cost for care of these patients4. Furthermore, the condition could deteriorate and lead to death as a result of FN and/or SN after chemotherapy5.Febrile neutropenia (FN) and severe neutropenia (SN) are the most common and serious complications of cancer patients after treatment with cytotoxic chemotherapy6. The guidelines of National Comprehensive Cancer Network (NCCN) recommend primary prophylaxis with G-CSF when the risk of FN associated with chemotherapy regimen is greater than 20%7. Filgrastim was the first short acting G-CSF drug approved for treatment of neutropenia by the United States Food and Drug Administration (FDA) in 1991. Subsequently, a number of new G-CSF drugs have been invented for the treatment of neutropenia worldwide. Long-acting G-CSFs (L-G-CSFs) are PEGylated forms of short-acting G-CSFs (S-G-CSFs) with decreased elimination and increased half-life in serum after subcutaneous injection. Moreover, some of these new G-CSF biosimilar drugs are not as glycosylated as filgrastim8. Since the structure and mechanism of drugs differ, the effect of different G-CSFs remains unclear.Granulocyte colony-stimulating factors (G-CSFs) promote the growth of neutrophils, decrease the incidence of FN and SN, shorten the time of hospital stay, reduce the severity and duration of neutropenia, decrease the risk of infection, and improve the tolerance to cytotoxic chemotherapy6. Patients might give up treatment due to severe adverse events. The incidence and degree of bone pain after the injection of different G-CSF drugs are diverse9. Although some reviews on the difference of several G-CSF drugs have been reported11, these reviews did not include sufficient studies and samples, trials that assessed new G-CSF drugs, or a complete list of G-CSF drugs. The effect of G-CSFs and the optimum choice remains unclear.Bone pain (BP) is the most frequent adverse event associated with G-CSF drugs12. In the present study, a Bayesian network meta-analysis was performed to compare the major 11 G-CSF drugs  in terms of efficacy (FN and SN) and tolerability (BP) in the treatment of patients after cytotoxic chemotherapy. This aimed to summarize the direct evidence obtained from the results of randomized controlled trials (RCTs), in order to provide reliable information for guiding clinical treatment decisions.Since there is no evidence from head-to-head trials, pairwise meta-analysis for mixed treatment comparisons between multiple medical interventions appears to be impossible. The Bayesian network meta-analysis, which combined direct and indirect evidence to obtain an estimated effect value, has been considered to be a statistical method for mixed multiple trial data comparisons, when a head-to-head trial is not available82 of the 73 RCTs from 1991 to 2018 were included for the final network meta-analysis , SN (I2 = 91%), and BP (I2 = 56%). This heterogeneity might have been introduced by the variation that resulted from the multiple types of tumors, since there were approximately five kinds of tumors in these seven trials. Since the sample size for every specific kind of tumor in these trials containing multiple types of tumors that was too small, it was difficult to implement an effective subgroup analysis. In the sensitivity analysis, no significant heterogeneity change was observed after removing studies from the analysis.A traditional direct pair-wise meta-analysis was performed, as shown in Table P < 0.05) reduced the incidence of FN and SN, while balugrastim and L-G-CSF biosimilar reduced the incidence of SN. Although the difference was not statistically significant (95% CI contains 1), a reduction in the incidence of FN and SN was observed when empegfilgrastim, lenograstim, leridistim, mecapegfilgrastim, and pegteograstim were compared with placebo. The reason may be because the number of trials included was too small. In terms of the incidence of BP, S-G-CSF biosimilar and lenograstim significantly led to more than pegfilgrastim . The incidence of BP by filgrastim, S-G-CSF biosimilar, lenograstim, and pegfilgrastim was significantly higher than placebo. However, there was no difference between other G-CSF drugs in the incidence of BP. By contrasting direct with indirect evidence using the node-split method, the network analysis did not reveal any statistical inconsistency with regards to FN, SN and BP.Figure Figure In the present network meta-analysis, the efficacy and tolerability of 11 different G-CSF drugs for cancer patients after chemotherapy in 73 RCTs containing 15,124 patients were evaluated using FN, SN and BP as indicators. It was found that pegfilgrastim was better than filgrastim in reducing FN, and more tolerable than S-G-CSF biosimilar and lenograstim in terms of the incidence of BP. In terms of both efficacy and tolerance, mecapegfilgrastim, lipegfilgrastim and balugrastim might be the most efficacious and tolerable among G-CSF drugs.83, FN was chosen as the primary outcome of the G-CSF drug treatment and a crucial indicator to evaluate the efficacy of G-CSF drugs. In the present study, it was found that compared with placebo, most of the G-CSF drugs could reduce the risk of the incidence of FN, except for empegfilgrastim, leridistim, and pegteograstim. While leridistim might have an opposite effect, although the effect was not statistically significant. The network meta-analysis revealed that there was no difference or inferiority among the tested G-CSF drugs, except for filgrastim and pegfilgrastim in FN (filgrastim vs. pegfilgrastim OR [95% CI]: 1.63 [1.07–2.46]). Filgrastim, pegfilgrastim, lipegfilgrastim and lenograstim reduced the incidence of FN in cancer patients undergoing chemotherapy compared with placebo. Lipegfilgrastim appeared to lead to a greater reduction in the incidence of FN, when compared to pegfilgrastim and filgrastim, although the difference was not statistically significant. These findings were consistent with the previous observations84. In accordance with previous reports, pegfilgrastim was more effective than filgrastim in reducing the incidence of FN88. SN is also another important evaluation indicator of G-CSF drug efficacy. Filgrastim, pegfilgrastim, lipegfilgrastim, S-G-CSF biosimilar, mecapegfilgrastim, and lenograstim reduced the incidence of SN in patients undergoing myelosuppressive chemotherapy based on direct and indirect evidence. All these results indicate that compared with placebo, most of the tested G-CSF drugs were effective to prevent the incidence of FN and SN.Since FN is the main and severe adverse event for many chemotherapy regimens, and is intimately associated with chemotherapy-related mortality89, and is an indicator of G-CSF drug tolerance. Filgrastim , lenograstim , pegfilgrastim  and S-G-CSF biosimilar  led to a higher incidence of BP, when compared with placebo. Lenograstim  and S-G-CSF biosimilar ]) led to a much higher incidence of BP than pegfilgrastim. However, the level of incidence of BP widely varied among the RCTs of G-CSF drugs, which might have resulted from the differences in race of patients, stage and type of tumors, chemotherapy regimens, and definition of BP. These results suggest that patients might have different tolerances to different G-CSF drugs.BP is one of the most common adverse events associated with G-CSF drug treatmentEven though there was no difference in efficacy among the tested G-CSF drugs and tolerability among patients to these G-CSF drugs in the pair-wise meta-analysis, the comparative ranking of these 12 G-CSF drug treatments suggest that mecapegfilgrastim, lipegfilgrastim and balugrastim might be more effective than leridistim, filgrastim and S-G-CSF biosimilar in preventing the incidence of FN, and S-G-CSF biosimilar, empegfilgrastim and L-G-CSF biosimilar might be more effective than filgrastim and pegfilgrastim in preventing the incidence of SN. In terms of BP, mecapegfilgrastim, balugrastim, lipegfilgrastim and L-G-CSF biosimilar might be more tolerable for patients, when compared to other G-CSF drugs. Those results indicate that mecapegfilgrastim, lipegfilgrastim and balugrastim might be the most efficacious and tolerable G-CSF drugs, and might provide a guideline for the selection of G-CSF-drugs for patients after chemotherapy.90. Although no inconsistency was found in FN, SN and BP through the node-split method in the main network analysis, the direct and indirect meta-analyses revealed contradictory results in terms of the comparisons between filgrastim vs. S-G-CSF biosimilar and filgrastim vs. L-G-CSF biosimilar. This mutually exclusive result could be explained as follows90: (1) if the direct evidence of the pair-wise meta-analysis was true, the comparison between other G-CSF drugs in indirect evidence of the network meta-analysis might overstate or understate the efficacy and tolerance; (2) if the indirect evidence was true, significant intrinsic heterogeneity might exist in the comparison among filgrastim, S-G-CSF biosimilar and L-G-CSF biosimilar. A low or moderate heterogeneity was observed in the pair-wise meta-analysis, indicating that the direct pair-wise meta-analysis was true.Caution should be taken in interpreting the results, since there might be inconsistencies between the direct and indirect comparisons. These inconsistencies might have resulted from the different characteristics of trials, such as the study design, definition of indicators, inclusion criteria of subjects, and method of implementation, as well as the difference in identifying the external effect on the mean effect of the specific comparison between the network meta-analysis and pair-wise meta-analysis methodsAlthough the present study is the first network meta-analysis to comprehensively assess clinically and commonly used G-CSF drugs, it should be acknowledged that there were some limitations with the present analysis. First, many factors correlated with neutropenia after chemotherapy were not analyzed, such as the duration of neutropenia, duration of SN, depth of the absolute neutrophil count (ANC) nadir, time to recovery of ANC, FN-related hospitalization, and other toxic or side effects of G-CSF drugs. Second, in most of the included trials, the report for FN, SN and BP was incomplete, which caused some of the G-CSF drugs to be ruled out for comparison in terms of SN and BP. Third, trials on some G-CSF drugs were too few to be assessed. For example, merely one trial on mecapegfilgrastim has been reported to date. Fourth, the definition of BP and other indicators varied among these studies. Furthermore, the dose of G-CSF drugs also varied across the studies. These might be the source of heterogeneity and inconsistency. Fifth, the outcomes might only apply to developed countries, since some G-CSF drugs are not available on the market in many developing countries.In summary, based on the present network meta-analysis, evidence suggests that compared with placebo, most of the tested G-CSF drugs are not different in terms of efficacy and tolerability, except for pegfilgrastim, which is more effective than filgrastim in reducing FN. Furthermore, pegfilgrastim is more tolerable for patients, when compared to S-G-CSF biosimilar and lenograstim, in terms of BP. Mecapegfilgrastim, lipegfilgrastim and balugrastim might be the most appreciate G-CSF drugs, which have both better efficacy and tolerance. It is noteworthy that more large-scale RCTs would be required to further confirm the efficacy and tolerance of the G-CSF drugs observed in the present study. The benefit-risk ratio of these G-CSF drugs still deserves to be further explored.84 and PRISMA network meta-analysis extension statement91. RCTs on 11 G-CSF drugs  for cancer patients after cytotoxic chemotherapy were searched in PubMed, Embase, Cochrane Library, Cochrane Collaboration Central Register of Controlled Clinical Trials, American Society of Clinical Oncology, and ClinicalTrials.gov up to the 8th of October 2018, without language restrictions. The terms included “balugrastim”, “empegfilgrastim”, “filgrastim”, “Neupogen”, “G-CSF biosimilar”, “lenograstim”, “leridistim”, “lipegfilgrastim”, “mecapegfilgrastim”, “pegfilgrastim”, “Neulasta”, “pegteograstim”, “GCPGC”, “rhG-CSF”, “PEG-rhG-CSF” and “Pegylated Recombinant Human Granulocyte Colony Stimulating Factor”  guidelinesRCTs that compared at least two different G-CSF drugs (placebo-controlled included) in all kinds of cancer after chemotherapy were included. These trials should report the data on FN, SN, and/or BP in cancer patients after the use of G-CSF drugs. Non-randomized controlled trials, non-interventional studies, retrospective studies, or trials that contained only one treatment (single-arm) were excluded. Furthermore, studies that included healthy volunteers, but not cancer patients who received chemotherapy, were also excluded.92 was independently used for the quality assessment and evaluation of risk of bias by the same authors. The key characteristics of each study were recorded, which included: the first author’s name and year of publication, country, study design, patient characteristics, chemotherapy regimens, dose and protocol of treatment, and outcomes . All data for the study characteristics and clinical responses were summarized in a structured table to ensure consistency. All the disagreements were resolved by discussion and consensus with a third author (Y. Li).Study selection, data extraction and review, and quality assessment were independently performed by two authors (Y. Wang and L. Chen), according to the predefined criteria from eligible studies. The Cochrane Collaboration’s tool for assessing risk of bias9/L, with an oral temperature of ≥38.0 °C. SN was defined as ANC < 0.5 or 1.0 × 109/L. If both data of both grade 3 and 4 bone marrow suppression (ANC < 0.5 and 1.0 × 109/L) were reported in a study, the data of the ANC < 0.5 × 109/L was used with priority for analysis, because grade 3 had lesser clinical significance, and was not always reported in the included studies.The incidence of FN after cytotoxic chemotherapy within two weeks was taken as the primary indicator of efficacy of G-CSF drugs, the incidence of SN was taken as the secondary indicator of G-CSF drug efficacy, and BP was taken as the primary indicator for the tolerability of G-CSF drugs. FN was defined as an absolute neutrophil count (ANC) of <0.5 or 1.0 × 10P < 0.05 indicated significant heterogeneity) and I2 statistic. If I2 = 0–25%, it is designated as low heterogeneity, if I2 = 25–50%, this was designated as moderate heterogeneity, if I2 = 50–75%, this was designated as high heterogeneity, and if I2 = 75–100%, this was designated as extremely high heterogeneity. According to the Cochrane handbook, heterogeneity can be accepted when I2 ≤ 50%93. Pair-wise meta-analysis was performed using Review Manager 5.3  and STATA 12.0  statistical software.Pair-wise meta-analysis was carried out for FN, SN and BP to compare the corresponding interventions. The random effects model for pair-wise meta-analysis was used to account for the heterogeneity. The heterogeneity among different trials was estimated by Cochran’s Q-test  were collected or calculated from combing the direct evidence, and the significance was assessed by P < 0.05, or the 95% confidence interval (CI) did not contain 1. The best efficacious and tolerant regimen was confirmed by ranking the included G-CSF drugs according to the OR for each G-CSF drug compared with placebo, and assessing the probability. Inconsistencies in the present study were assessed by comparing the direct evidence with indirect evidence from the network meta-analysis using the node-split method95.Random-effects models were applied for the network meta-analysis. Bayesian network meta-analysis was used to combine the collected data. The Bayesian network meta-analysis was performed with WinBUGS version 1.4.3 . Random effects models were used to incorporate the effects from different studies, while heterogeneity within the comparison was evaluated in a relatively conservative and appropriate mannerA sensitivity analysis was performed by determining whether there was statistically significant heterogeneity in the meta-analysis after studies were randomly removed from the others.Supplementary


Neuroinfections are a significant medical problem and can have serious health consequences for patients. Their outcome, if not fatal, can be associated with permanent residual deficits. Cerebrospinal fluid (CSF) examination is commonly used for meningitis confirmation. Fatty acids (FA) are precursors of lipid mediators with pharmacological activity. They actively modulate inflammation as well as contribute to its resolution. Therefore the aim of this study was to determine the FA and selected endocannabinoids (ECB) content in the CSF obtained from patients with bacterial (BM) and viral meningitis (VM) using chromatographic techniques. A significantly lower level of saturated FA was found in patients with BM and VM as compared to controls. There was a significantly higher concentration of long-chain monounsaturated FA and polyunsaturated n-6 FA in the CSF obtained from patients with neuroinfection. Moreover, a significant reduction of n-3 FA in CSF obtained from patients with BM and VM was demonstrated. The highest amount of ECB was detected in the CSF of patients with VM: eicosapentaenoyl ethanolamide (1.65 pg/mL), docosahexaenoyl ethanolamide (655.5 pg/mL) and nervonoyl ethanolamide (3.09 ng/mL). Results indicate the participation of long-chain monounsaturated and polyunsaturated FA and their derivatives in the inflammatory process and likely in the process of resolution of inflammation during neuroinfection. It seems that the determination of the FA and ECB profile in CSF may be a valuable biomarker of health and may allow the development of new pharmacological strategies, therapeutic goals and fatty acids supplementation necessary in the fight against inflammation of the central nervous system. Despite progress in the prevention and treatment of infectious diseases, central nervous system (CNS) infections are a persistent problem in modern medicine. Neuroinfections account for significant worldwide morbidity and mortality, and are observed throughout the globe in every age group from neonates to older adults. They are characterized by a variety of symptoms ranging from fever and headaches to the most serious consequences including increased intracranial pressure and irreversible damage to the nervous system, which, if not fatal, can be associated with permanent residual deficits (Leibovitch and Jacobson Fatty acids (FA) and their derivatives are important mediators in the human immune system. The intensity of the inflammatory process is influenced by changes in the metabolism of eicosanoids, endocannabinoids (ECB) and other lipid mediators . The BBB provides structural as well as functional protection by maintaining homeostasis of the CNS microenvironment, protecting the nervous system against harmful factors, and also allowing selective transport of substances between blood and cerebro-spinal fluid (CSF). The BBB is therefore a barrier to pathogens, but also creates unique conditions for the operation of the host immune system. Selective BBB permeability is associated with a different structure of the capillary endothelium, forming a physical barrier. Endothelial cells are connected with each other through adherens junctions (AJs) and tight junctions (TJs), which have no gaps or pores, therefore substances present in the plasma can overcome the endothelium barrier only by means of transcellular transport  (6 women and 13 men), 19 patients with viral meningitis (VM) (9 women and 10 men), and 19 patients (8 women and 11 men) in whom meningitis was excluded, who were hospitalized at the Department of Infectious and Tropical Diseases, Jagiellonian University Hospital in Krakow, Poland. All patients had signed a filled informed consent to participate in the examination.The diagnosis of meningitis was based on history, physical examination and laboratory testing. Meningitis was confirmed or excluded after CSF examination, in which cytosis and the level of protein, glucose and white blood cells (WBC) were assed. Blood tests included WBC with differential leukocyte count and C-reactive protein (CRP), the tests were performed according to generally accepted standard methods. Moreover, each sample of CSF underwent microbiological assessment. Exclusion criteria included any other acute or chronic inflammatory diseases. Samples from each group were obtained from lumbar punctures for medically indicated reasons. All samples came from CSF left over in the main laboratory. Blood tests, demographic and clinical information were recorded using standardized data collection forms. The study was conducted in accordance with the Declaration of Helsinki 1975, and approved by the Jagiellonian University Ethics Committee .v/v) . Two microliters of internal standards were used throughout: eicosapentaenoyl ethanolamide (EPEA-d4) and docosahexaenoyl ethanolamide (DHEA-d4), at concentration 10 μg/mL . Samples were vortexed for 30 s and centrifuged for 10 min at 1000 x g. Organic phases were collected and dried under a stream of nitrogen. The residue was dissolved in 40 μL of acetonitrile (Merck).One mL of CSF were acidified with 50 μL of 0.01% formic acid . Ten microliter of 0.001% butylated dihydroxytoluene  was added to prevent lipid oxidation. Lipids were extracted with chloroform–methanol solution  with a J&W DB-23  column, as described earlier  was carried out with 14% BFLiquid chromatography was performed using an Agilent 1100 LC system . Chromatographic separation was carried out with a Thermo Scientific BDS HYPERSIL C18 column . The advance column, with its pre-column , was set at 40 °C with a mobile phase flow rate of 0.3 mL/min. The gradient elution mobile phases consisted of formic acid (0.02 mol/L) in acetonitrile and formic acid (0.02 mol/L) in water. MS/MS analyzes were performed on an Applied Biosystems MDS Sciex  API 2000 triple quadruple mass spectrometer equipped with an electrospray ionization (ESI) interface. ESI ionization was performed in positive ionization mode. The multiple reaction monitoring (MRM) mode of the dominant product ion for each ECBs was used. A comparison of the paired ion  and LC retention times with standards served to confirm the identification of ECBs in the samples. Arachidonoyl ethanolamine (AEA), AEA-d4, 2-ariachonoylglycerol (2-AG), eicosapentaenoyl ethanolamide (EPEA), EPEA-d4, docosahexaenoyl ethanolamide (DHEA), DHEA-d4 and nervonoyl ethanolamide (NEA) standards were purchased from Cayman Chemical. An ion pair was 348 > 62 for AEA, 379 > 269 for 2-AG, 346 > 62 for EPEA, 372 > 62 for DHEA, 409 > 62 for NEA, 350 > 62 for EPEA-d4 and 376 > 66 for DHEA-d4. Data acquisition and processing were performed using Applied Biosystems Analyst version 1.4.2 software as described previously (Gdula-Argasińska and Bystrowska 25) and upper (Q75) quartiles. Normal distribution of variables was checked using the Levene test. Differences between study groups were determined using the one-way ANOVA and Scheffe post hoc test, or Kruskal-Wallis test if normality was not observed. Analysis of similarities between content of the FA in CSF of studied groups was performed using clustering methods to identify homogenous groups of cases. Ward’s method with Euclidean distance matrix was used to join objects into clusters such that the variance within a cluster is minimized and to maximize the significance of differences between clusters. Calculations were performed using STATISTICA 13 , and statistical significance was defined as p ≤ 0.05.All data are presented as means ± standard deviation (SD) or medians and lower , DHEA (655 pg/mL) and NEA 3.09 ng/mL) were detected in CSF from VM patients when compared to control , C20:2 , C20:3, C20:4  and C22:2 (adrenic acid) was observed in CSF from VM patients. DGLA, which is product of GLA conversion by elongase 5 (ELOVL5), can be enzymatically transformed to several metabolites with anti-inflammatory properties , lipid mediators can be produced along enzyme pathways that involve multiple cells, including microglia, in a process known as trans-cellular biosynthesis  and Streptococcus pneumoniae directly affect BBB by producing spore-forming toxins  adhere to the endothelium and functions to assist in taking certain substances (eg. hormones) from the blood and transferring them to the CSF, as well as in the opposite direction  and nuclear factor kappa B (NF-ĸB) transcription factors (Gdula-Argasińska and Bystrowska


Idiopathic intracranial hypertension, space-flight associated neuro-ocular syndrome (SANS), and glaucoma are conditions that are among a spectrum of cerebrospinal fluid (CSF)-related ophthalmologic disease. This implies that local CSF pressures at the level of the optic nerve are involved to variable extent in these disease processes. However, CSF pressure measurements are problematic due to invasiveness and interpretation. The pressure measured by a lumbar puncture is likely not the same as the orbital CSF pressure. It is believed this is at least in part due to the flow restrictive properties of the optic canal. To investigate CSF flow within the orbit, a model for CSF dynamics was created using three medium-sized pigs. Contrast was administered through a lumbar subarachnoid space access. The contrast front was imaged with repeated computed tomographic (CT) imaging. Once contrast entered the orbit, rapid, sequential CT imaging was performed until the contrast reached the posterior globe. Head tilting was performed to highlight the role of gravitational dependence within the subarachnoid space. The primary circulating fluid of the central nervous system is the cerebrospinal fluid (CSF). Produced in the choroid plexus within the lateral, third and fourth ventricles, the fluid takes a circuitous route through the spinal column and throughout the cerebral cortex. The optic nerves themselves are surrounded by CSF enclosed by the dura mater, or the optic nerve sheath. Therefore, the CSF and its flow is important in the context of ophthalmologic disease. For example, idiopathic intracranial hypertension (IIH), an uncommon disease that primarily affects young, obese women, is characterized by increased intracranial pressure in the absence of a secondary or identifiable cause. With the development of papilledema, swelling of the optic nerve, the condition can lead to significant vision loss and blindness. Cerebrospinal fluid pressure (CSFP) has re-emerged as a possible risk factor for glaucoma. Space flight-associated neuro-ocular syndrome (SANS), a condition that has affected astronauts returning from prolonged microgravitational exposure, can produce optic nerve swelling, hyperopia, and globe flattening .One major problem in the investigation of CSF-related ophthalmologic diseases is the difficulty in studying this variable. The gold standard for CSFP measurement is a lumbar puncture, a procedure that is uncomfortable and is not without risk. Further, the pressure measurement is performed in the lateral decubitus or prone position, in which humans do not spend much of their lifetime, unless bed-ridden, in a position similar to this. This is important since the relationship between the lumbar CSFP and intracranial pressure is not always equivalent. In the decubitus position, it has been shown that the CSFP in the lateral ventricle is similar to the pressure measured by lumbar puncture . HoweverFurther complicating our understanding of CSFP in ophthalmic disease is the knowledge that the fluid conductivity between the intracranial compartment and the orbital CSF space, or the perioptic subarachnoid space (POSAS), may not be congruous. Studies by Morgan et al. –6 and HoFeola et al. , in 3 eyBoye et al.  used an Therefore, the creation of a suitable animal model to investigate CSF dynamics is important. Pigs are docile and easy to handle and have previously been recognized as suitable for research of orbit surgery due to similarities to humans . The pursuc scrofa (30–50 lbs) were used as the model for this work. The study was approved by the institutional IACUC and performed in conjunction with and under direct observation of the Division of Laboratory Animal Medicine personnel. The studies were carried out in strict accordance with the National Institutes of Health guide for the care and use of Laboratory animals . Anesthesia and sedation were administered to minimize discomfort and distress. A lumbar puncture was performed in the animal in the prone position with its legs flexed to separate the intervertebral spaces. A line was drawn between the most cranial aspect of the bilateral wings of the ileum. A 22-gauge 1.5-inch needle was used to access the subarachnoid space. Variable doses of iopamidol , were delivered into the subarachnoid space after confirming entry with computed tomographic (CT) imaging. Doses were different between the pigs in an effort to find a concentration that permitted rapid migration of the contrast front to the orbital CSF space. Repeat CT imaging using an orbital imaging protocol was performed to visualize the movement of the contrast agent. If contrast was not migrating, the animal was placed in Trendelenburg position and re-imaged. Contrast and positioning were adjusted on a case-by-case basis as trial-and-error. Once contrast was observed to migrate rostrally, serial CT scans were performed until contrast was visualized abutting the globes. Images were reconstructed using both bone and soft tissue windows to 0.6 mm slices. After the first pig, all other pigs had their head tilted in order to visualize differential contrast migration as a further function of dependence. The position of the contrast front was determined by image analysis through OsiriX MD v 10.0 . Images were primarily analyzed in a coronal view, defined by a plane that was tangential to the optic nerve. A region-of-interest (ROI) was placed at the optic canal and the end of the obvious contrast front. When a superior and an inferior aspect of the nerve sheath were highlighted by the contrast front, a separate ROI was placed at the initiation of the nerve from the optic canal and at its termination in the inferior or superior location, respectively. A soft tissue reformatted scan was used to determine the distance between the optic canal and the termination of the nerve at the globe for the right and left eyes, respectively. This value was used to determine the linear percentage of filling of the nerve : Contrast (Iopamidol 41% concentration) was administered into the CSF space in the lumbar spine. Serial CT imaging identified contrast accumulation in the lumbar spine without migration. After 30 min, the pig was placed in Trendelenburg position (~15°) and the animal was re-imaged. Another 14 min later, the pig's hind region was raised up to ~30°. Contrast eventually migrated into the chiasmatic cistern at 55 min after contrast administration and 22 min after body re-positioning. After serial scanning, ending at 64 min, no contrast was observed entering the perioptic subarachnoid space, even though the contrast was situated in the chiasmatic cistern.Pig #2 : Contrast was administered at a higher concentration (Iopamidol 76%) compared to the pig #1 trial. After injection of contrast, the hind region of the pig was minimally elevated to an acute angle of nearly 5–10° and the head of the pig tilted laterally, and serial CT imaging was performed. Contrast was observed to migrate rostrally through the spinal column to the prechiasmatic cistern with entry into the perioptic subarachnoid space in the dependent position at 14 min . Six minPig #3 : Administration of contrast of iopamidol 76% via lumbar puncture was performed. The hind region of the pig was similarly elevated as before with the head rotated with the right orbit ~1 cm below the left on the horizontal axis. Serial CT imaging was performed, and contrast was observed to reach the chiasmatic cistern. Contrast was visualized to have entered into the dependent optic nerve and migrated 82.4% of the length of the nerve at 55 min post-injection. The contralateral, non-dependent nerve had 37.6% entry of the inferior aspect of the nerve and ~31.3% entry into the superior aspect of the same nerve. At 58 min, there was complete filling of the dependent perioptic subarachnoid space, and 59.2% inferior and 36.8% superior filling of the non-dependent nerve sheath , Table 2The head was rotated to convert the non-dependent nerve into the dependent space and serial imaging ensued. At 63 min, the first scan after head tilt, 75.1% of the inferior and 61.7% of the superior aspect of the nerve were filled. Seven minutes later, the nerve had been completely filled inferiorly, although the front did not migrate much further on the superior aspect of the perioptic subarachnoid space ~61–70%; . There wThis porcine model allows for the imaging of contrast migration through the subarachnoid space as a function of time without the restriction of radiation exposure from repeated CT imaging. The model was able to provide information about contrast migration into the orbital CSF space. Preliminary results revealed features of contrast-CSF migration as well as limitations of studying CSF flow with a heavy contrast agent. In the absence of eye movement, it takes at least 6 min for contrast to migrate down the perioptic subarachnoid space to the globe, appreciated in two of the pigs. Eye movement would be expected to accelerate CSF and contrast movement. There is a dependent component to CSF migration at least as studied by this poorly diffusible contrast agent. Our results suggest that flow of contrast preferentially accumulates at 1/4th to 1/3rd down the length of the optic nerve upon its initial entry into the orbit. It is interesting to note that the contrast front from Killer's study, identifying reduced flow in the POSAS of NTG patients, was similar to that seen in this study in a pig model . Based oFurthermore, we suggest four hypotheses that may explain the observed phenomenon:CSF is incompressible; hence, its volume flow rate due to clearance into the venous system  must be The slowdown of the contrast velocity may also be explained by an increase of the rate of reabsorption of CSF in the region where contrast accumulates, which reduces the CSF velocity.The third possible explanation is that the CSF flow may be time-dependent, and the flow rate reduces by the time the contrast reached 1/4th to 1/3rd down the length of the optic nerve. The reduction of the CSF velocity may be explained by a gradual increase of hydraulic resistance to the CSF flow. For example, Rodbard  studied The fourth possible explanation is based on interaction between flows of a denser contrast and a less dense CSF. For example, due to local CSF circulation the rate of contrast front propagation may not be representative of CSF velocity, and CSF may bypass or channel though the accumulating contrast layer.The optic canal is nearly 10 mm in length, and the subarachnoid space, if patent, can be <1 mm. Additionally, the optic nerve SAS is characterized by numerous trabeculae and septae , 19. In k is the permeability, ΔP is the pressure difference, A is the cross-sectional area, μ is the dynamic viscosity of the fluid, and l is the length of the optic canal.where The viscosity of iopamidol at a concentration of 200 mgl/ml (41%) at 37°C is ~2.0. Compared to the 0.7 mmPa viscosity of cerebrospinal fluid at 37°C, theoretical flow is reduced nearly 65%. At a concentration that of Iopamidol 300 mgl/ml (61%), the flow is reduced more than 85%. Therefore, it is important to account for the fact that contrast-based studies, such as Killer's  and thisThere are a number of limitations of this study. The first is that the porcine model is not an ideal example of human CSF relationships given that the animal is a quadruped with vastly different positional and dependent pressure relationships between compartments. Even if otherwise healthy, anesthetized animals are not representative of truly normal, healthy physiology. This is particularly important in that anesthesia limits eye movement, an important contributor to CSF flow in the orbit. Further, for the migration of contrast in a human CT myelogram, a certain amount of positioning, either by the patient or by the table, is required. In the anesthetized pig, the only motion of the animal itself is from induced breathing. Therefore, all movement is performed by external mechanisms. Another major limitation is the small numbers of pigs used for the investigation. This is due to the nature of invasive research in an animal model. However, we believe the successful procedures provided the information being sought out. The amount of contrast that was eventually administered in the CSF space was not recorded. Initial injections were needed to ensure the correct entry into the subarachnoid space, and therefore the overall contrast that was eventually injected was unable to be accurately measured. We did not feel that reporting estimates was appropriate.The use of gadolinium (Gd) and MRI was considered. However, the need to develop this model in conjunction with the eventual need for invasive interventions, we felt that CT would offer the greatest access. Manipulation within the magnet would also be limited due to constraints from the coil. Furthermore, the susceptibility artifacts associated with this region could severely limit our ability to visualize the contrast front. We do anticipate exploring the magnetic resonance visualization of the model more fully in the future.Hounsfield units were not used to determine true contrast fronts, although due to the partial volume effects in such a small space as the perioptic subarachnoid space, the ability of Hounsfield unit determination would be diminished. The ROIs for the optic nerve were determined in a linear fashion for overall simplicity and to account for potential eye movement. Therefore, the natural orbital redundancy and mild tortuosity of the optic nerve was not accounted for by this technique. There may also be other ways of having administered contrast into the intracranial CSF space which may have increased the yield of success for acquiring the CSF space in all pigs. One example is the work of Kaiser et al. , althougLastly, it should be reiterated clearly that radiographic examinations involving intrathecal contrast are not accurate representations of CSF flow, especially as described in this model. For reasons described above the relationships examined herein should be appreciated in the context of the significant deviations of normal physiology, fluid properties, and dynamics introduced by contrast, anesthesia, anatomy, and species.In conclusion, we present the development of a novel porcine model for studying CSF flow in the neural axis and the orbit. Based on the preliminary data, we surmise that CSF flow in the porcine perioptic subarachnoid space is slow in the absence of eye movement, and likely much slower than measures of CSF in other commonly tested parts of the central nervous system, like the cerebral aqueduct . As specAll datasets generated for this study are included in the manuscript/supplementary files.The animal study was reviewed and approved by University of North Carolina at Chapel Hill IACUC.DF: design of study, conduction of experiment, data analysis, and author of manuscript. OK, BT, AK, and LG: data analysis and contributor to manuscript. RS: data analysis. XZ, DO, and CZ: conduction of experiment. YL: design of study, conduction of experiment, data analysis, and contributor to manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The purpose of this study is to explore donor relatives’ experiences of the medical interventions enabling organ donation, as well as to examine the donor relatives’ attitudes towards donating their own organs, and whether or not their experiences have influenced their own inclination to donate.The experiences of donor relatives were explored via in-depth interviews. The interviews covered every step from the deceased family member being struck by a severe bleeding in the brain till after the organ recovery, including the medical interventions enabling organ donation. The interviews were analysed through qualitative and quantitative content analysis.Brain death and organ donation proved to be hard to understand for many donor relatives. The prolonged interventions provided after death in order to enable organ donation misled some relatives to believe that their family member still was alive. In general, the understanding for what treatment aimed at saving the family member and what interventions aimed at maintaining organ viability was low. However, most donor relatives were either inspired to, or reinforced in their willingness to, donate their own organs after having experienced the loss of a family member who donated organs.There is a need for greater transparency regarding the whole chain of events during the donation process. Yet, having experienced the donation process closely did not discourage the donor relatives from donating their own organs–but rather inspired a willingness to donate. This indicates an acceptance of the medical procedures necessary in order to enable organ donation after death. The purpose of the medical care at an intensive care unit (ICU) is mainly to ease pain, to cure and to save lives. Nevertheless, some patients cannot be saved and death is inevitable. A limited number of patients then have the possibility to contribute to saving the lives of other patients by donating their own organs after death.In this article, we describe the experiences of donor relatives with regard to medical treatment transitioning from being provided in order to save the life of a patient to being provided in order to make organ donation possible. Taking part of the donor relatives’ unique experiences of the circumstances surrounding organ donation offers us the opportunity to evaluate and improve the management of the delicate donation process.… to establish whether taking steps, before death, to facilitate organ donation would be in the patient’s best interests” [All care and treatment provided within our medical health care should be provided with the informed consent of the patients and with the best interests of the individual patient in mind . This raterests” . Hence, terests” , 5. Thisterests” –7. This terests” .Brain death, defined as the brain having lost all brain functions, totally and irreversibly, has been the criteria for death since 1987 in our county Sweden. Though this concept of death is widely accepted, it sometimes complicates the understanding of death. In the situation of organ donation, the patient dies while treated with a ventilator, which means that the dead patient looks alive although being dead. However, continued ventilator treatment is the prerequisite for donation after brain death, to maintain organ viability.Eurobarometer conducted in 2009, which is a survey commissioned by the European Commission's Directorate General SANCO, that included 26,788 European citizens in the 27 European Union Member States, as well as 3,504 in the candidate countries and the Turkish Cypriot Community, the main reasons for people not wanting to donate organs were fear of manipulation of the body and distrust in the system [In order to maintain public trust in the donation and transplantation system, clarity and transparency of all the actions taken during the donation process is fundamental , 9. Accoe system . Hence, e system . In our e system . Furthere system . HoweverThough the trust for the health care, as well as the willingness to donate is high in our country, we are still struggling with relatively low donation rates, and people are still dying while waiting for an organ , 11. HowOne subgroup of the general public who is well acquainted with organ donation due to their presence during the donation process and who has witnessed the medical interventions enabling organ donation, is the donor relatives. Consequently, this group has a unique insight into the procedures at our ICUs and can therefore provide us with important knowledge of the interventions enabling organ donation.The aim of this study is to explore donor relatives’ experiences of the medical interventions enabling organ donation, as well as to explore the donor relatives’ attitudes towards donating their own organs, and whether or not their experiences have influenced their own inclination to donate.In this interview study we included relatives of patients who died due to a total brain infarction and who donated their organs. The families in the study were all relatives of patients at the same Neuro Intensive Care Unit at a University Hospital, in Stockholm Sweden, during the years 2001–2004 . The donAn ethics application was sent to the regional ethics committee in Stockholm. The ethics committee stated that the research was not covered by the Ethics Review Act. This, as the research was carried out after the participants had given due consent to the treatment. Therefore, no formal decision was made by the ethics committee, but the authors received a recommendation from the committee in which the committee stated that it did not find any obstacles for the study to be carried out (ref number 04-615/5). Nevertheless, when planning and conducting a study like this one there are several ethical considerations that need to be made. The authors’ ethical considerations are described in the appendix.To guarantee coverage of the same topics with all participants, data were collected using a semi-structured interview guide with open-ended questions (see interview guide). The interviews covered every step from the deceased family member being struck by a life-threatening brain haemorrhage, to the stay at the ICU including the whole donation process, to the hospital follow-up and the nearest future after that. The interviews also included questions about the donor relatives’ own decision-making process regarding donating their own organs. The interviews were conducted by the first author, who is an experienced medical social worker specialised in organ donation and transplantation. The interviewer had had no prior contact with the included donor relatives.All interviews but three were conducted in the participants’ homes, the location was chosen by the participants. The interviews were carried out in Swedish and they were recorded and transcribed verbatim. All interviews were conducted with the informed consent of the participants. All participants spoke Swedish fluently, 20 of them came from Sweden and one from Finland. None of the participants expressed any strong religious beliefs in the interviews. About one third of the participant had some sort of higher education. For further demographics of the participants and interview information, see In order to fulfil the aim of this study, the methodologies used in the analyses are both qualitative and quantitative content analyses –15. InduTo fulfil the aim of the study, we also needed to understand whether the donor relatives’ decisions on organ donation were influenced by the donation process in conjunction with the loss of a family member who donated organs. This led us to the fourth main category, “the donor relatives’ own decision-making process”. All main- and subcategories that derive from the qualitative content analysis are illustrated in “more insightful and meaningful results than would using either approach alone” [In order to count the donor relatives’ own decisions regarding organ donation, a quantitative content analysis was made , 16 withh alone” .To help organize the extensive material, the areas of interest were extracted with the help of the qualitative software programme ATLAS.ti . The useThe final analysis was made by the first author/the interviewer. However, one of the co-authors—an experienced qualitative researcher, Professor and Social Worker—also read all the interviews and made an independent analysis. A comparison of the co-author’s and the interviewer’s analysis was then made. In cases of differing analyses, a discussion of how to interpret the findings, codes and categories, took place, until a shared understanding was reached and both authors felt satisfied with the interpretation deriving from the discussion. To test the credibility and the confirmability a continuous dialogue took place within the research team, and between the interviewer and the co-author during the whole process of analysis , 13, 18.The point of time at which the relatives perceived their family member to have died varied widely: from the time when the family member initially collapsed to the actual donor operation.immediately when the family member first collapsed, for example at home or at work or in the ambulance on the way to the hospital: “…I sat there by the ambulance and then I kind of understood, oh…is it this bad… Because I saw that it was just, that she wasn’t there so to say. No, I think that she was dead already then.”Some believed that death had occurred brain death being determined or when they were formally informed of the declaration of death: “…And then the second time, well they said that now we have done the next test and there was still no reaction. And then they declared him dead and also set a time…”Others considered death to have occurred during the process of when they were asked about organ donation: “But then I remember that this nurse came (donation nurse), and then, then I understood…”For other donor relatives the insight came in connection with the donor operation: “…I didn’t see her after the operation, or wait… I did see her when she was dead…”, “Well yes, we knew that it would be over then (after the operation), that he would be dead then…”. These relatives often believed that death occurred when the heart stopped beating: “That someone is dead, that is when the heart stops working… So that…that is an easy thing to know… “.Some even believed that the family member died uncertain of the time of death or who talked about it in a contradictory manner. The staff’s communication with the unconscious or, in some cases, the dead patient, most likely contributed to the uncertainty conveyed by some relatives regarding the patient’s condition: “…The staff were speaking to him all the time…it was kind of amazing, the respect towards a man who really almost could be seen as dead, but well, you never know…”,”Well, they were speaking to him…until the very end…”. Some also stated that they received vague information about the declaration of death from the staff: “She didn’t say it explicitly–dead–but that there was no reaction up here in the brain. And then one can figure out that it must be something like that.” There was only one relative who did not completely trust the reliability of the procedures to declare death: “…what if they didn’t do it correctly. One has read, sometimes one reads about things…”. We also found relatives who in this demanding situation had psychological difficulties accepting the loss and this made it hard to understand that death had occurred: “She isn’t dead at all, and I became somewhat aggressive to him (the physician)… Because the last thing I saw of her, was that she still was alive…ehhh…she was alive when I left the emergency room and now all of a sudden she was dead, no, I couldn’t get it into my head. She couldn’t be brain dead—she is alive. That’s just the way it is.”Finally, we found relatives who were The only aspect commented on by almost all relatives regarding the medical interventions necessary for organ donation, was the ventilator treatment. However, the understanding of the purpose of the ventilator varied widely.the ventilator was keeping their family member alive—even after the diagnosis of brain death. These relatives were often uncertain about when death had occurred. Some even thought that their family member still was alive at the time of the actual organ recovery: “He was alive thanks to the machines, so without the machines he would have died and then they cannot pick anything from him.” Even though the ventilator made it hard to fully grasp the concept of death, most relatives understood that there was no hope for recovery despite the use of the ventilator, because: “the brain cells were dying”, “nothing worked in the brain”, “there was nothing to do about it”,” there was no return” and “the end was near”.Some relatives indicated that they thought indeed was dead despite the ventilator and they mentioned that the ventilator was kept on anyway: “to keep the body going”, “to keep the body functions going”, “to keep everything possible going”. However, they were not specific about this being done for the sake of the organs. Another group of relatives were well aware of the necessity to keep their family member on the ventilator with the sole purpose of enabling organ donation in the sense that a high quality of the organs needed to be maintained: “to keep a high quality of the lungs”,”to be able to use the heart and all that”,”so that the organs would not be destroyed” and”the organs needed to be fresh”. Some also knew that one of the main factors for the quality of the organs was their access to oxygen: “they needed to keep him breathing to not damage the organs”, “it was the organs that needed oxygen so that they could be used”.Other relatives expressed that their family member prolong the ventilator treatment to make an organ donation possible, to gain time to ask about organ donation, to prepare for organ donation, and to conduct the donor operation:”I do not know for how long–but enough to be able to donate”,”it was just for the sake of the organ donation—otherwise they would have switched it off much earlier”. Generally, the prolonged treatment was not commented on as something that caused discomfort—only two relatives indicated some discomfort: “She was still connected to all the tubes and she was full with cables everywhere, and swollen, unpleasant in that way, very unpleasant.”, “It seems like they just awaited death, one almost wondered why she had to go there.”Some also mentioned the necessity to actually before one ends up on the operating table—there are a lot ofpreparationsthat need to be done”, “they givefluidsand various other things to keep the organs fresh”. Instead, the vast majority of the donor relatives put forward that they were very pleased with the overall medical care but the analysis showed that they mostly could not distinguish between which treatment that aimed at recovery and which interventions that aimed at enabling organ donation.The donor relatives hardly talked about any other medical interventions given in order to enable organ donation than the ventilator care. The only statements we found which reflect any other type of interventions were: ““Perhaps they did not give a hundred percent on this.”, “Look, here we have a chance, we might not be able to save him…”, “The efforts were not the same then, it was already a done deal apparently…”.Three relatives feared that their family member did not receive the optimal medical treatment because of the possibility of organ donation: Unspecific information: The information given by the staff regarding the procedures surrounding the operation was often vague or they did not receive any information according to the donor relatives: “no they did not tell me anything, I mean, it is their thing…”. Some relatives say that they were given information but that they cannot recall any of it; others remember a few details concerning the operation, often that the body would be restored after the operation: “Well they told us what they would do and then restore …that they would make it look nice.”did not wish additional information. They were satisfied with the knowledge they already had concerning organ donation and put forward that they did not need any more details: “…you have your thoughts on so many things, when you sit there, ehh…and a close family member just passed away… It was brief when she told us about it (the donor operation). But I thought that it was enough what she said. It is one thing if you don’t know anything about organ donation, but I mean, well everyone knows.”Some did not want to discuss the operation. This was stated in a manner to indicate that the donor operation was something they would rather not think about: “And how they did it, the donation, for me personally, it isn’t interesting! …No, I don’t want to know, then it becomes too much delving, because then you start digging into things.” Among these relatives we also found individuals who expressed uneasiness when considering the operation and who also said that they had felt uncomfortable seeing the body after the donation.Others clearly stated during the interviews that they Specific information: When the relatives refer to more specific information, it mainly concerns the organs; i.e. what organs can be donated and what organ is most often needed by patients waiting for an organ: “Well, she told us which organs one can use, cornea and… I don’t know them all, but there are so many… But after all, I guess she took the organs that people… the organs that are used most often, what is the expression, longest waiting list.” “They try to take as much as they can, that is in good shape.” Some had also received information about the specialised surgeons involved and about the comprehensive logistic planning that the operation requires.positive and/or respectful: “It is just like a, ehh, surgery. So they treat you like in any surgery… well like in any operation. With respect as well…” or; something neutral and/or with an emotional distance: I don’t see it as surgery you know, they were only going to pick them (organs) up and it is a rather complicated thing…” and finally; as something negative and/or connected with uneasiness: “How is it done, when they do the autopsy and all that? When they, when they take the organs and then… I don’t want them to desecrate her in any way, if she in some sense still would have the ability to feel or something like that, that I don’t know. But… well you know, one fantasises about her lying on a cold table and then they start cutting and carving, it mustn’t be like that…”.The donor relatives’ reflections on the donor operation can be described in three distinct positions; as something a positive view of the body post operation: “… that they were going to use the eyes sounded a bit nasty. But when we later took farewell of her one could not notice it at all. She looked so nice…” or; a neutral view of the body post operation:“No, she looked very tidy, but I don’t know if it was good or bad to see her… Yes… well… I’d rather remember her the way she looked when she was alive. But it is just something that you should do, take farewell…” and finally; a negative view of the body post operation: “That experience, I didn’t like that experience after the operation. Well, one collapses at once, when they have removed it all… She looked so collapsed.”The donor relatives’ reflections on the state of the body after the donor operation can be described in the three following positions; positively affected; some had even changed a previously negative decision to a positive one. Having experienced the donation process made the relatives more knowledgeable about organ donation, and they afterwards knew that they themselves were not too old or too sick to donate. Some had also been inspired by knowing how many recipients had been helped thanks to their dead family member’s donation, and for some the pride they felt at their dead family member’s donation had inspired or reinforced their own willingness to donate. The one relative who had been negatively affected, decided to change her previously positive decision in protest against a chain of events during her husband’s stay at the ICU, including treatment mistakes made during the medical care of her husband which she believed to have contributed to his death. In addition, she also thought that her husband’s arm had been exposed in a TV-news report (media exposure) about organ shortage.Of the relatives who described their own willingness to donate as directly affected by their dead family member’s donation (n = 9), all but one stated that their intention had been willingness to help others in need, or with a belief that it was the right thing to do. They stated that if you are willing to receive an organ, you should also be willing to donate. Some donor relatives had knowledge of the fact that the need for organs by far exceeds the number of organs available (organ shortage), or they had knowledge of other facts concerning organ donation through their employment in medical health care. Even though those donor relatives often had made up their minds a long time ago, many stated that they now felt even more convinced and strengthened in their decision to donate. Hence, they went from an often instinctive decision, to a more conscious choice to donate.The decisions of most of the relatives regarding organ donation, however, were not directly influenced by their dead family member’s donation (n = 12). Instead, their minds had often been made up long before and in many cases the basis for their decision had been related to a feared that his organs would be taken before death and the other wanted his burial according to old traditions, with the body intact.After having experienced the loss of a family member and the subsequent donation, the majority of the relatives remained willing to donate Click here for additional data file.S2 Text(DOCX)Click here for additional data file.


The mechanical obstruction of cerebrospinal fluid (CSF) flow can lead to very high hyperproteinorachia. In myeloma, tumoral clivus obstruction can cause such high proteinorachia, associated with paraprotein detection in the CSF in absence of intrathecal synthesis. The mechanical obstruction of cerebrospinal fluid (CSF) flow can lead to very high hyperproteinorachia. In myeloma, tumoral clivus obstruction can cause such high proteinorachia, associated with paraprotein detection in the CSF in absence of intrathecal synthesis. We describe a myeloma patient with impairment of consciousness, associated with profoundly increased proteinorachia without signs of meningitis. We suspect this was related to mechanical obstruction caused by tumoral clivus infiltration rather than paraprotein intrathecal synthesis.The presence of neurological symptoms in a patient relapsing after multiple myeloma (MM) may evoke either hypercalcemia, CNS infection, or MM with CNS involvement (CNS MM). The latter is rare: Approximately 1% of MM patients develop CNS MM. It is associated with poor prognosis, life expectancy in this case being 4‐6 months, thus requires rapid clinical intervention. The standard criterion for the diagnosis of CNS MM is based on cytology, which lacks sensitivity.2A 64‐year‐old man with kappa free light chain MM was admitted to the intensive care unit of a tertiary referral hospital in February 2018 for acute dyspnea, fever, and confusion.2 and autologous stem cell transplantation 48 hours later. He presented pneumonia and sepsis 2 weeks after stem cell transplantation. He received a consolidation regimen 3 months after  then maintenance treatment by lenalidomide during 6 months. The patient showed partial response to treatment according to the international response criteria. The patient also displayed thrombocytopenia related to bone marrow plasma cell infiltration and drug toxicity. Iterative platelet infusions did not correct thombocytopenia.Diagnosis of MM was made in 2015, when he presented multiple lytic bone lesions, without renal failure, and 23% bone marrow plasmacytosis. The patient had an international scoring system (ISS) staging of 1. Cytogenetic was not studied at time of diagnosis, but would later reveal no high‐risk cytogenetic features (no t(4‐14) nor del17p). He received initially a lenalidomide‐dexamethasone‐ixazomib induction regimen during four cycles, followed by high dose melphalan at 200 mg/mIn April 2017, the patient underwent surgery for an L2 vertebra fracture. He relapsed afterward in May 2017, October 2017, and January 2018. These three successive relapses were treated with lenalidomide‐dexamethasone‐daratumumab, with pomalinomide‐dexamethasone, and with bortezomib‐adriamycin‐dexamethasone, respectively.3 ; platelets 11.000/mm3 ; Ca2+ 1.90 mmol/L or 76 mg/L ; proteins 60 g/L; creatinine 126 µmol/L or 14.25 mg/L ; LDH 2227 IU/L; albuminemia 32 g/L; ammonium 69 µmol/L; IgG 2.37 g/L; IgA 0.78 g/L; IgM 0.08 g/L; serum kappa free light chain (FLC) 2290 mg/L; and lambda FLC 3.5 mg/L. Blood gases under 9 L O2 were as follows: pH 7.52; PaCO2 36 mm Hg; PaO2 78 mm Hg; HCO3− 29 mmol/L; lactate 1.0 mmol/L. He soon required mechanical ventilation. A pulmonary CT scan showed bilateral pure ground‐glass opacities. All tests for infection and autoimmunity were negative. Bronchoalveolar lavage was negative. He received daily platelet transfusions, which resulted in a transient improvement. Intra‐alveolar hemorrhage was suspected.In February 2018, soon after the last relapse, he presented confusion during two weeks but no focal neurological features. Blood test results were as follows: hemoglobin 11 g/dL ; white blood cell count 3.530/mm3; RBC 1600/mm3; proteins 44.6 g/L ; glucose 5.6 mmol/L or 1.0 g/L; Cl‐ 107 mmol/L; and LDH 824 U/L. The remarkably elevated proteinorachia was further investigated, including by protein electrophoresis and immunofixation in paired serum and CSF samples. The CSF exhibited a profile similar to that of the serum with all protein fractions in their usual proportions, with a CSF/serum albumin ratio of 1.30. Given the similar electrophoretic aspect between CSF and serum, the research of oligoclonal bands in the CSF was not performed. The kappa monoclonal FLC was present in the same proportion as in the serum  were negative. The EEG under propofol was normal and reactive. However, in addition to already known diffuse vertebral infiltration, cerebral and spinal MRI showed an infiltrating lesion of the clivus spreading to the cavernous and sphenoidal sinuses and internal carotid sheathing , in the absence of radiological signs of intracranial hemorrhage. Blood uptake during lumbar puncture could be ruled out since only a few red blood cells were present in the CSF sample.Two mechanisms, not mutually exclusive, may be envisaged to explain such elevated nonselective proteinorachia: an increase in the passage of serum proteins into the CSF or a decrease in protein clearance in the CSF. Traditional views consider that the CSF is mainly produced by the choroid plexus and to a lesser extent at the blood‐brain barrier. Water, solutes, and cells then flow through the ventricular system and/or the extracellular space of the brain into the subarachnoid space, from where the CSF flows out of the CNS through the arachnoid villi into the dural venous sinuses. Sheaths around the cranial nerves provide an alternative pathway for CSF outflow, predominantly through the cribriform plate, into the lymphatic system of the nasal mucosa, draining the deep cervical lymph nodes.The most frequent interpretation of an increased protein concentration in the CSF is the so‐called “protein leakage” from the serum characterizing barrier impairment. Actually, it has been shown convincingly that a reduced CSF turnover is in most cases able to explain the presence of increased levels of blood‐derived proteins in the CSF. A reduced CSF flow may be due to a lower CSF production rate , or a lower outflow on account of local mechanical , inflammatory or tumoral obstruction of the CSF outflow pathways.Considering this case, it was believed at first that this patient presented intrathecal synthesis of FLC, given the very high concentration of FLC in the CSF. If the participation of such intrathecal synthesis in the elevated paraproteinorachia cannot be formally discarded in this case, it cannot either entirely explain the strikingly high and unselective proteinorachia. Rather more, impairment of the CSF outflow through the cribriform plate and cranial nerve sheaths might explain the markedly elevated proteinorachia and the neurological symptoms. In addition, although measurement of FLC in the CSF can provide useful information concerning suspected plasma cell dyscrasia with CNS involvement,4Very high proteinorachia can occur in myeloma patients without meningitis. In this case, we suspect that it was related to a mechanical obstruction caused by tumoral clivus infiltration, rather than intrathecal synthesis. Clonal free light chain was increased in the CSF, but to the same extent as in the serum.The authors have no conflict of interest to declare.JRL, AG, and LG: managed the patient. YC, BL, and PA: performed the analysis. YC, JRL, PA, and LG: wrote the manuscript. All authors reviewed and approved the final version of the manuscript.