Kynurenine metabolites are potential modulators of psychiatric disease. We aimed to develop a highly sensitive biochemical analysis of cerebrospinal fluid (CSF) tryptophan (TRP) metabolites, to investigate the stability of metabolites and to confirm our previous findings of aberrant CSF quinolinic acid (QUIN) and picolinic acid (PIC) in suicide attempters using this method.Ten CSF TRP metabolites were analyzed with ultraperformance LC–MS/MS. The method showed small intra- and interassay variation. Metabolites were stable following freeze–thaw cycles. A decreased CSF PIC/QUIN ratio was found in suicide attempters.The feasibility of reliably determining CSF TRP metabolites were demonstrated, including separation of the two isomers PIC and nicotinic acid (NA) and the finding of a reduced PIC/QUIN ratio replicated in suicide attempters. Metabolites of the kynurenine pathway of tryptophan (TRP) degradation see  have gaiN-methyl-D-aspartate (NMDA) receptor NAA, [13C6]NA, XA-d4. 3-HK-d3 and 3HA-d3 were obtained from Toronto Research Chemicals, Canada. KYNA-d5 and PIC-d4 were obtained from C/D/N Isotopes Inc. . Solutions for the mobile phases: water, methanol and formic acid 99% were all LC–MS grade .TRP, L-kynurenine (L-KYN), pyridine-2,3-dicarboxylic acid (QUIN), KYNA, PIC, nicotinamide (NAA), nicotinic acid , XA, 3-HK and 3-HANA – were all purchased from Sigma-Aldrich . The Internal standards (IS): TRP-d3. which had the final concentration of 50 μM in water and stored in -80°C in aliquots of 350 μl. The IS working solution was prepared by dilution of IS stock (1:5) with 5% formic acid in water and stored at 4°C until analysis.Stock solutions of all unlabeled standards  were prepared at 100 μM and TRP in 1000 μM in water and stored at -20°C. Calibrators were generated mixing all compounds in a final solution of 10 μM (TRP 100 μM). The calibrator mix was then aliquoted in volumes of 200 μl and stored in -80°C. Standard mix was thawed and serial diluted in water before each experiment. The IS stock solutions of all compounds were prepared at 5 μM with an exception of TRP-m/z for the MRM transitions of each individual analyte, along with optimal cone voltages and collision energies were determined by manual tuning using the instrument’s built-in fluidics system (Masslynx 4.1 software). A 10 l/min flow of 100 ng/ml tuning solution was introduced to the mass spectrometer in combination with an LC flow of 0.2 ml/min and a composition of 20/80 mobile phase A/mobile phase B. The MRM transition providing the highest sensitivity was chosen as quantification trace for all compounds, except for TRP where the second most intense transition was chosen. This provided better linearity of the response over the calibration range, since the concentration of TRP in biological samples are significantly higher that for the other analytes in this method. The dwell times for the MRM channels were automatically calculated by the software, giving a desired number of 15–20 data points across the chromatographic peak. The precursor/product transitions for all compounds and belonging internal standards are shown in The UPLC–MS/MS system used was a Xevo TQ-XS triple-quadrupole mass spectrometer  equipped with a Z-spray electrospray interface and a Waters Acquity UPLC I-Class FTN system . The MS was operated in electrospray-positive multiple reaction monitoring (MRM) mode. The system was operated using a source temperature of 150°C, capillary voltage of +3.0 kV, desolvation temperature 650°C, desolvation gas flow rate 1000 l/h and detector gain 1 was used. The Thirty microliter of human CSF sample, calibrator sample or Quality Control sample was mixed with 30 μl of IS working solution during 15 s in LC–MS Certified Clear Glass 12 × 32 mm vials  before transfer to an autosampler (set to 4°C) that injected 3 μl into the UPLC–MS/MS system, all samples included in the study were analyzed in duplicates.spiked-Cnonspiked/Cspiked]) and the assay precision is presented as percent relative standard deviation (STDEV [Data Range]/AVERAGE [Data Range] *100) and was calculated from repeated measurements within the same experiment  or from three different experiments running over 2 days (interassay). The matrix effect was calculated from the area of the IS in CSF (n = 6) in relation to the response in a prepared sample of pure water (n = 6), as (Area [CSF]/Area [water] - 1) *100%  of nominal concentrations. Since we are analyzing endogenous components present in biological matrices, it is impossible to obtain totally analyte-free CSF for method validation. However, charcoal-treated matrices or other artificial matrices could be used as these do not contain most of the endogenous interferences that are present in the real matrices. Thus, in the present study, selectivity for all ten metabolites was investigated by comparing chromatograms of extracted blank CSF obtained from six different human samples spiked with a mix of all ten metabolites and IS to ensure that it was free of interference at the retention time of the compounds. The linearity was tested with a calibrator mix diluted in water in concentrations ranging between 0.1 and 250 nM (for TRP concentration was 0.1–25 μM). Each calibrator concentration and human CSF sample was analyzed in duplicates. The standard curve was calculated by 1/X-weighted least squares linear regression of standard curve calibrator concentrations and the peak area ratios of analyte to IS. An S/N ratio of three and S/N ratio of ten was used for estimating LOD and LOQ, respectively. Spiked CSF in two different concentrations  was used in order to test the accuracy and precision of the assay. Accuracy is presented as percentage recovery . The freeze–thawing stability was evaluated using six human CSF samples frozen at -80°C, then thawed to room temperature for four cycles with 24 h between. In order to investigate stability before and after the first freezing cycle, we used CSF samples from four different subjects. These CSF samples were analyzed directly after lumbar puncture, before freezing and next after 24 h at room temperature before any freezing, then again after one freezing cycle . Percent stability for each analyte is given as the mean percent stability of the six individual CSF ± standard deviation (SD) at the given hour.In December 2017 and in March 2019, ten multiple sclerosis (MS) patients at the MS outpatient clinic at the Karolinska University Hospital were asked to participate in the study: patients should meet the following criteria: being 18–55 years of age. No known major somatic or psychiatric diagnoses besides MS. No psychiatric or psychotropic medication, including glucocorticoids, within 90 days of sampling. CSF from the six patients enrolled in December 2017 was analyzed following storage in -80°C until analysis (February 2018) and used for testing the stability following repeated freezing and thawing cycles as well as following storage at room temperature for up to 4 h. CSF from the four patients enrolled in 2019 was used for investigating the impact of the first freezing and thawing cycle, in other words, that the CSF was analyzed within 30 min after the lumbar puncture and before putting the samples into the freezer. The next day, following 24 h in either -80°C or at room temperature, the same samples were analyzed again.The inclusion criteria for the patient group was that they had performed a suicide attempt with a clear intent to die. A suicide attempt was defined as “situations in which a person has performed an actually or seemingly life-threatening behavior with the of jeopardizing his/her life” . The patSupplementary Table 1). Subjects were allowed to take pain killers , oral contraceptives  and nonprescription allergy medications . Lumbar puncture was carried out on all healthy controls and patients as described below. Briefly after the suicide attempt, a psychiatrist diagnosed all patients according to the DSM IV. The diagnoses were set by the psychiatrist within days after the suicide attempt and after an approximately 2-h-long structured interview using the Comprehensive Psychiatric Rating Scale [Thirteen patients  were enrolled following admission to Lund University Hospital after a suicide attempt (see above for definition). Thirteen healthy controls  were recruited via the population registry; and were prescreened via a phone interview before being invited to enroll in the study and subsequent diagnostic interviews . The stung Scale  and the All patients and controls in both cohorts underwent a general physical examination and a complete medical history was taken. We measured the subjects temperature and screened the blood for erythrocyte sedimentation rate, C-reactive protein and white blood cell count to identify any participants with infections. All measurements were within the normal reference levels for these parameters.We used a standardized protocol to draw CSF from the L4–L5 interspace, between 8 AM and 11 AM after a night of bed rest and fasting. We kept all samples on ice and immediately aliquoted and froze them at -80°C, except for the samples that were used for stability testing. The suicidal patients were psychiatric inpatients, whereas the controls and MS patients had spent the night at home. CSF from MS patients was analyzed within 2 months from collection, whereas CSF from the suicidal patients and the healthy controls was analyzed within 6–9 years after collection.The statistical analysis were performed using IBM SPSS Statistics 20.0  and graphs were performed with GraphPad Prism 8 and SPSS 25 for Mac. The nonparametrical two-tailed Mann–Whitney U-test was used to compare age, BMI and concentrations of TRP, L-KYN, KYNA, 3-HK and NAA, between healthy controls and suicide attempters. Since we have previously published, and our hypothesis is, that QUIN and the L-KYN/TRP ratio are increased and that PIC and the PIC/QUIN ratio are reduced in suicide attempters, we used one-tailed Mann–Whitney U-test when comparing the CSF levels of these metabolites as well as the ratios between healthy controls and suicide attempters. Data are expressed as mean ± SD. Spearman rank correlation analysis was used to analyze correlations between markers. Statistical significance was considered when p < 0.05.good linearity within the concentration range tested   . The corAccuracy and precision were evaluated at two different concentrations of spiked CSF, covering the range of concentrations found in CSF. Intra-assay accuracy was stable for the majority of metabolites (9/10) with small variations 85 and 112% of the target . Intra-aAs endogenous concentrations of analytes are present in matrices, evaluated CSF matrix effects were determined with IS only and the matrix factor of IS calculated, in accordance with EMA and US FDA and guidelines. Matrix effects were found to be less than 13% in all metabolites tested with an exception for 3-HK that had 23% .The stability of kynurenine metabolites were tested in CSF from six subjects with MS. Mean baseline concentrations in these six subjects were TRP (1.5 ± 0.5 μM), kynurenine (60.2 ± 30.6 nM), KYNA (2.4 ± 0.8 nM), QUIN (23.6 ± 19.7 nM), PIC (14.5 ± 3.0 nM), 3-HK (3.1 ± 1.2 nM) and NAA (22.8 ± 20.9 nM). The percent stability of TRP, kynurenine, KYNA, QUIN, PIC and NAA ranged between 89 and 113% in samples stored at room temperature for 30 min, 1, 2, 3 and 4 h . The aveThe stability of kynurenine metabolites following the very first freeze–thaw cycle was analyzed in four additional subjects with MS. Mean baseline concentrations in fresh CSF, analyzed within 30 min after lumbar puncture and before storing the samples in the -80°C freezer, were TRP (1.3 ± 0.1 μM), kynurenine (67.7 ± 57.7 nM), KYNA (2.0 ± 0.4 nM), QUIN (22.0 ± 3.6 nM), PIC (9.2 ± 4.2 nM), 3-HK (4.4 ± 3.2 nM) and NAA (26.2 ± 10.4 nM).The percent stability after one freeze–thaw cycle ranged between 93 and 104% TRP (102 ± 3 %), kynurenine (104 ± 4 %), KYNA (98 ± 2 %), QUIN (99 ± 2 %), PIC (100 ± 3 %), 3-HK (93 ± 4%) and NAA (93 ± 8 %). Storage at room temperature for 24 h decreased 3-HK levels with 25% (3.7 ± 2.5), but did not affect the levels of any other metabolite analyzed.No differences were found regarding biological sex , age  and BMI  between healthy controls and suicide attempters. Mean total SUAS score among suicide attempters were 43.8 ± 17.0.TRP and six of its metabolites were successfully detectable in all CSF samples from healthy controls and suicide attempters, see The CSF PIC/QUIN ratio was found to be decreased in the suicide attempters compared with healthy controls . Furthermore, the differences in the CSF levels of PIC and QUIN (see above) as well as the L-KYN/TRP ratio between healthy controls and suicide attempters were all in the hypothesized directions, although without reaching statistical significances .We detected a significant positive correlation of L-KYN to both KYNA (r = 0.7 p = <0.0001) and QUIN , but not to 3-HK  in CSF from all subjects . 3-HK did not show any correlation with QUIN  in this CSF material.Simultaneous detection of multiple kynurenine pathway metabolites is important to further understanding of their role(s) in different disease states. In the present study, we have developed a new protocol with high accuracy and precision for the quantification of multiple CSF TRP metabolites using UPLC–MS/MS. In addition, this method also clearly separated the two isomers, PIC and NA. Seven metabolites: TRP, L-KYN, NAA, 3-HK, QUIN, PIC, KYNA, were reliably detectable and measured in the CSF. Thus, this is the first UPLC–MS/MS study showing detectable CSF levels of PIC, at the same time visualizing the isomer NA as a clearly separate peak see . To our Several kynurenine metabolites, such as PIC, QUIN and KYNA, are suggested as putative biomarkers that may be used in the diagnostics for several diseases. As such, in order to develop robust biomarkers for clinical application, it is of critical importance to have information about the stability of the metabolites over time and following freezing and thawing. Indeed, in some clinical situations, samples may be standing on the bench for some time during the sampling procedure or in the laboratory during the biochemical analysis. Furthermore, CSF used in clinical research might be sparse, and therefore it might be useful to know whether the levels of metabolites are stable over numerous cycles of freezing and thawing. For simulation of these conditions, we explored the effects of storage in room temperature (22°C) as well as the influence of several repeated freeze–thaw cycles on the concentrations of the kynurenine metabolites. The result of the stability test clearly demonstrates that all detectable metabolites in CSF are stable up to 4 h in room temperature and do not degrade following multiple freeze–thaw cycles. However, the concentrations of 3-HK was found to be decreased following long-term, up to 24 h, storage in room temperature.In the present study, efforts were also made to investigate if the very first freeze–thaw cycle affected the concentration of the metabolites. Thus, CSF samples were analyzed within 30 min after the lumbar puncture and again following 24 h in the -80°C freezer. We found that the first freeze–thaw cycle did not affect the levels of any detected metabolite .We have previously used GC–MS to analyze the levels of PIC and QUIN in blood and CSF from several cohorts of suicide attempters and controls . Our resde novo production of these metabolites is highly dependent on the availability of kynurenine in CSF. Surprisingly, 3-HK did not follow this pattern. Furthermore, we could not find any correlation between 3-HK and QUIN, even though both these two metabolites are produced by the same rate-limiting enzyme kynurenine monooxygenase. These data encourage further investigation of the pattern of metabolism in the kynurenine pathway not only during normal conditions but also in an inflammatory state.In the present study, we also discovered a strong positive correlation between L-KYN and QUIN as well as between L-KYN and KYNA, indicating that Collectively, the method described here has several advantages over previous protocols for detecting multiple kynurenine metabolites and as such represents a valuable tool for advancing our knowledge on the role of kynurenine pathway in different disease states.Kynurenine metabolites are gaining attention as modulators and key players of several disorders, including cancer, cardiovascular, neurodegenerative, psychiatric, immunological and infectious disorders. Indeed, metabolites of the kynurenine pathway have the potential to serve as biomarkers in future clinical settings. It is, therefore, pivotal to develop a quantification method for simultaneously analysis of multiple metabolites with high precision. In addition, the stability of metabolites following several freezing and thawing cycles and/or different times in room temperature is of a importance for the future of biomarker research. In the present study, we have developed a sensitive assay for quantification of multiple kynurenine pathway metabolites. Importantly, the method clearly separate PIC from its isomer niacin. We were also able to confirm our previous report showing that the CSF ratio of two of the metabolites, PIC/QUIN, is decreased in suicide attempers. The purpose of including this small pilot of suicide attempters in the current manuscript was to validate the robustness of the biomarkers. Ongoing studies in our lab utilize large cohorts with several populations of patients and aim to find cutoff values to define the use of these biomarkers in the clinic. We believe that this method will be a corner stone for future analysis of kynurenine pathway metabolites and pave the way for the development of clinical biomarkers for psychiatric disorders, in particular suicidality.Kynurenine metabolites are modulators of and key players in several disorders.There is a need for a high sensitive and high precision method for simultaneous analysis of multiple kynurenine metabolites.An UPLC–MS/MS method was developed in order to measure ten cerebrospinal fluid tryptophan metabolites with high sensitivity and able to separate the two isomers, picolinic acid (PIC) and NIC.The method was developed following the US FDA guidelines for bioanalytical analysis testing matrix effects, accuracy and precission.The developed method had a small inter- and intra-assay variation.The linearity and the sensitivity were good, as well as the specificity of NIC and PIC chromatogram separation.All metabolites, except 3-hydroxyanthranilic acid were stable for several freeze–thaw cycles as well as for storing in room temperature up to 4 h.A reduced PIC/quinolinic acid ratio in suicide attempters was confirmed.Click here for additional data file.


Handwashing is important considering the impact of communicable diseases on the public. We aimed to identify the association between years with incidence of communicable diseases during the coronavirus disease 2019 (COVID-19) pandemic and hand hygiene in South Korea.This cross-sectional study evaluated 5 years  of data from the Korea Community Health Survey and included 1,034,422 adults. Multinomial logistic regression analysis was performed to assess handwashing frequency by year. Receiver operating characteristic analysis was used to determine the cut-off point for handwashing frequency.The always/frequently handwashing rate was 44.7%. This tendency was stronger in adults with each ascending year, with reference to 2013 . Among women, the OR of frequently/always handwashing was 3.55 times higher  in 2020 than in 2013. This OR was 2.95 among men . In influenza-vaccinated participants, the OR of frequent/always handwashing was 3.25 times higher in 2020 than in 2013 , while in non-vaccinated participants it was 3.17 . Among adults who practiced physical distancing during the COVID-19 pandemic, the OR was 1.36 times higher  with frequent handwashing, 1.64 times higher  than those who did not practice it.There was a strong tendency toward frequent handwashing over the years; the trend was even greater in 2020 during the COVID-19 pandemic. Given that communicable diseases and handwashing are closely related, it is necessary to promote hand hygiene for prevention.The online version contains supplementary material available at 10.1186/s12889-022-13951-x. The incidence of communicable diseases designated by law has tended to increase since 2013 in Korea. The total incidence per 100,000 people was 148.4 in 2013, 185.7 in 2015, 295.5 in 2017, 307.7 in 2019, and 281.6 in 2020. The most common communicable diseases were Middle East respiratory syndrome (MERS) in 2015, scarlet fever in 2017, viral hepatitis A in 2019, and coronavirus disease 2019 (COVID-19) in 2020 [By August 2021, the cumulative confirmed number of COVID-19 cases in Korea was 251 421 accounting for 0.49% of the total population . The cumHandwashing is effective in preventing communicable diseases. Proper handwashing can reduce the spread of transmissible diseases by 24%–31% and water- and food-borne communicable diseases by 50%–70% –13. HandThe awareness rate of proper handwashing among Korean adults was high at 90.0%. However, the practice rate of proper handwashing was low at 72.4% . PreviouPublic handwashing may be related to historical and cultural factors in health and hygiene. Until the 1390 s in Korea, as Buddhism was transmitted as a precept, a culture that cares about hygiene was popularly developed. Thereafter, until 1910, a culture of partial washing of the body developed, and body exposure was considered taboo owing to Confucianism . AccordiThe influence of the prevalence of communicable diseases in society to handwashing behavior may be related to the health belief model (HBM) or social learning theory. HBM is a representative theory that explains the intention of an individual’s health behavior and can be applied to understanding infection prevention behavior such as COVID-19 vaccination and handwashing. The components of HBM in the context of handwashing according to the prevalence of communicable diseases are as follows . As a peThis study aims to contribute to the prevention of public infection by understanding social handwashing in relation to communicable diseases. By focusing on the period when there are no significant changes in the campaigns or accessibility, it would be possible to understand directly the association between the incidence of communicable diseases and handwashing. In particular, we focused on handwashing in 2020 when COVID-19 was prevalent. Four common items of the handwashing questionnaire were analyzed over the 5-year investigation, and we investigated handwashing in terms of COVID-19-related variables.The research data were obtained from the Korea Community Health Survey (KCHS) of 2013, 2015, 2017, 2019, and 2020. KCHS has been performing surveys on handwashing, which is the dependent variable of this study, every two years since 2013. KCHS performed a survey on handwashing in 2020 as an exception because of the COVID-19 pandemic. The KCHS is a representative anonymous, self-reported online survey of Korean adults conducted by the Korea Disease Control and Prevention Agency (KDCA). Since 2008, the KDCA has been conducting a nationwide survey every year. KDCA published the KCHS Data Profiles as a brief report in 2015 to describe the data . The surParticipants were adults aged 19 years or older. A total of 1,034,422 adults participated in the study, consisting of 480,923 men and 553,499 women. Among the participants, 109,666 persons with uncertain responses were excluded because of missing data.The variables of interest were the years 2013, 2015, 2017, 2019, and 2020. The dependent variable, handwashing, was investigated in only the aforementioned years. The dependent variable was the total score of handwashing calculated by a questionnaire including four common questions during the survey years. The questionnaire consisted of contents related to the investigation of previous studies –32. The The covariates were demographic variables (sex and age), socioeconomic variables , a variable related to mental health (perceived stress), variables related to health behavior (current drinking and current smoking status), and medical utilization .To evaluate the association between years with the incidence of communicable diseases during the COVID-19 pandemic and hand hygiene, we conducted multiple logistic regression analysis using the PROC SURVEYLOGISTIC procedure with weight, cluster, and strata for analysis. Results included odds ratios (ORs) and 95% confidence intervals (95% CIs). For all variables, there was no multicollinearity using the variance inflation factor. To investigate the dose–response relationship, multinomial logistic regression was also performed. Results from analysis of variance and tests of independence were also valid. The trend test was conducted to identify the relationship between the independent and dependent variables. Statistical analysis was performed using SAS, version 9.4 .Table P for trend < 0.0001” was observed. Specifically, the OR for handwashing frequently was 2.49 times higher (95% CI = 2.42–2.56) in 2020 than in 2013. Concerning handwashing always, this OR was 3.61 times higher (95% CI = 3.51–3.71) in 2020 than in 2013  in 2013, and tended to increase thereafter. The incidence in 2015 was 185.7 people. MERS accounted for 0.14% of the total communicable diseases, but it had an impact on public awareness because a high fatality rate (20.5%) was recorded within a short period (46 days) in areas with high incidence  . The incThe incidence of communicable diseases increased by year, and women were more likely to wash their hands always/frequently compared with men. This is consistent with results of previous studies showing that women have better hand hygiene than men , 41, 42.People with good self-perceived health status in 2020 were more likely to wash their hands always/frequently compared with those with bad self-perceived health status in 2013. The trend was particularly evident in handwashing after going out. People following physical distancing due to COVID-19 were also more likely to always wash their hands than those not following physical distancing. COVID-19 is a highly contagious disease, and our investigation was conducted before its vaccine was released; thus, there was high anxiety about infection among the public. COVID-19 is transmitted through respiratory droplet spread; therefore, individuals need to be careful when using public transportation or crowded facilities. Handwashing has been recommended by the government as a representative prevention method for communicable diseases.To the best of the authors’ knowledge, this is the first study to identify an association between communicable diseases, focusing on COVID-19, and routine handwashing by years using national survey data of adults. Data from random cluster sampling are sufficiently representative of Korean adults.The limitations of this study are as follows. First, the causality between communicable diseases, specifically COVID-19, and routine handwashing is obscure. As this study had a cross-sectional design, it was difficult to determine causality . NeverthCommunicable diseases including COVID-19 are closely related to handwashing practices in adults, especially among women and those vaccinated against influenza. Adults are more likely to wash their hands when practicing physical distancing. Adults washed their hands with soap more frequently after going out after the outbreak of COVID-19 in 2020 than before. Given that communicable diseases and handwashing are closely related, it is necessary to promote hand hygiene for prevention. Proper handwashing is required for people at risk of contracting communicable diseases, especially COVID-19.Additional file 1.


A major barrier to developing effective therapies for LMD is the lack of cell-based or preclinical models that recapitulate human disease. Here, we describe the development of ex vivo using human meningeal cell-conditioned media. Using immunoassays and RNA-sequencing analyses of PD-CSF-CTCs, molecular signaling pathways were examined and new therapeutic targets were tested for efficacy in PD-CSF-CTCs preclinical models.CSF-CTCs were collected from melanoma patients with melanoma-derived LMD and cultured in vitro and in vivo. Global RNA analyses of PD-CSF-CTCs revealed several therapeutically tractable targets. These studies complimented our prior proteomic studies highlighting IGF1 signaling as a potential target in LMD. As a proof of concept, combining treatment of ceritinib and trametinib in vitro and in vivo demonstrated synergistic antitumor activity in PD-CSF-CTCs and BRAF inhibitor-resistant melanoma cells.PD-CSF-CTCs were successfully established both in vitro and in vivo from some melanoma patients with LMD and used as preclinical models. These models retained melanoma expression patterns and had signaling pathways that are therapeutically targetable. These novel models/reagents may be useful in developing rationally designed treatments for LMD.This study demonstrates that CSF-CTCs can be grown  For the first time, we propagated CSF-CTCs from LMD patients in vitro and in vivo.We identified therapeutically relevant target using scRNA-seq for patient-derived CSF-CTCs.PD-CSF-CTCs may be valuable to better understanding LMD biology and in rational drug design for this disease.in vitro and in vivo. Here, we describe the successful culture and expansion of PD-CSF-CTCs from melanoma patients in vitro, and in vivo using xenograft models. We identified a therapeutically tractable target (IGF1R) whose pharmacological inhibition prolonged survival using in vivo PD-CSF-CTC models of LMD. We anticipate that these reagents/models will lead to the development of rational drug design strategies for LMD.LMD is a devastating complication of several cancers with very short survival and without effective therapies. A major barrier to the development of rational drug design strategies for LMD has been the inability to propagate PD-CSF-CTCs 1 Patients with LMD have a dismal prognosis, with survival ranging from weeks to months,1–3 and no truly effective treatments exist.A complication of advanced melanoma is leptomeningeal disease (LMD), the development of tumor cells in the central nervous system (CNS) and cerebrospinal fluid (CSF).5 and we have previously performed unbiased omics-based analyses of CSF from patients with LMD.2 We found that the CSF was enriched with proteins involved in innate immunity, protease-mediated damage, and insulin-like growth factor– (IGF–) related signaling. Similarly, our single-cell analyses of LMD showed specific upregulation of several immunocyte subtypes in the CSF that were distinct from immunocyte populations in brain metastases or systemic metastases.6Recent studies have shown how cancer cells colonize the unique environment of the leptomeningeal space,in vivo from several cancers,8 and these preclinical models resembled human disease, retaining therapeutically relevant targets and making them highly valuable for drug discovery.9 To our knowledge, breast cancers are the only source of CSF-CTCs that have been cultured in vitro,10 and there are no reports of in vivo LMD models derived from PD-CSF-CTCs. Here we describe, for the first time, the isolation of melanoma CSF-CTCs from patients for in vitro propagation and in vivo LMD model development. For the first time, we successfully generated PD-CSF-CTC cultures via in vitro and in vivo expansions. The ability to grow CSF-CTCs will be a valuable asset for future development of rationally designed LMD therapies.CSF circulating tumor cells (CSF-CTCs) from liquid biopsies are useful for genetic profiling and molecular characterization of LMD, but the scarcity of these cells in CSF poses an obstacle to performing experiments that are critical for better understanding LMD biology and therapeutic development. There have been no reports of the successful propagation of patient-derived CSF-CTCs (PD-CSF-CTCs) from melanoma patients. This may reflect the challenges of growing these rare and fragile cells and the difficulty of adequately reproducing the CSF microenvironment. Human peripheral blood (PB) CTCs were successfully inoculated Our deidentified patient specimen collection protocol was approved by the University of South Florida’s Institutional Review Board . CSF was collected from various site/time points, including from lumbar puncture (LP), during an Ommaya reservoir placement surgery, from an Ommaya reservoir, and during an autopsy. CSF was immediately placed on ice and then CSF and cell pellets were separated by centrifugation at 1500 rpm for 5 minutes at 4° C. CSF was aliquoted and stored frozen, whereas cell pellets were either resuspended in human meningeal cell– (HMC–) conditioned media for culturing or cryopreserved. Patient and melanoma characteristics were also collected.11Complete methods for this procedure are detailed in HMCs were purchased from ScienCell Laboratories  and maintained in poly-L-lysine–coated T175 culture flasks containing complete Meningeal Cell Medium (MenCM)  + penicillin/streptomycin solution ). When cells reached 70% confluence, the media was centrifuged at 1500 rpm for 5 minutes, supernatant was collected for generating HMC-conditioned media to culture CSF-CTCs. Cell pellets from CSF was resuspended in a 1:1 ratio of HMC-conditioned media to complete MenCM, with additional 40 ng/ml of fibroblast growth factor (FGF) and 40 ng/ml of epidermal growth factor (EGF) . CSF-CTCs were cultured in single wells of a 96-well plate until confluent, and then transferred to larger culturing apparatus. Culture media was refreshed every 3 days. Melanoma cell line cultures details are in Cells were fixed using 4% paraformaldehyde on an 8-well chamber glass slide . Complete immunostaining procedure and computer-assisted analyses are described in 12 To facilitate rapid analysis of single-cell datasets in a user-friendly manner, the Interactive Single Cell Visual Analytics (ISCVA) tool, which we developed, was used.6 The webtool is accessible to public at http://iscva.moffitt.org. Details on quality control/cell typing methodology can be found in Single-cell RNA sequencing and analysis techniques have been described in detail by our group.4 cells/ml suspension were seeded in a 384-well plate  and treated for 72 hours with increasing concentrations of ceritinib  or trametinib .13 Inhibition of proliferation was measured by the CellTiter-Glo Luminescent Cell Viability Assay . Drug synergism or antagonism was determined using Compusyn software to calculate the combination index (CI) values using the Chou-Talalay method.14 A CI < 1 was considered synergistic, CI > 1 was antagonistic, and CI = 1 was additive. All data represent the mean of 3 independent experiments.Twenty-five microliters of 2.5 × 104 enhanced NanoLuc–labeled cancer cells into the CSF space via cisterna magna of 6- to- 8-week-old NOD scid gamma (NSG) mice  as previously described.15 Status of LMD was assessed once a week. Bioluminescence signals were detected by intraperitoneally injecting 0.1 cc of a 1:40 dilution of NanoLuc-reporter substrate  in sterile phosphate-buffered saline into the animal. Bioluminescence images (BLI) were captured using the Xenogen IVIS 200 system , and BLI analyses were performed using Living Image Software . Treatment for LMD began seven to fourteen days postinjection of cancer cells via the cisterna magna (or when LMD was first detected via BLI), mice were given 10 mg of compounds/kilogram of ceritinib , 1 mg of compounds/kilogram of trametinib (Chemietek), or both once a day via oral gavage; treatment lasted for 49 days (7 weeks). To sample CSF from xenograft mice under anesthesia, an incision approximately 1 cm long was made between the skull and C2 vertebra. A 30 g needle was inserted into the cisterna magna and CSF was withdrawn. Mice were subsequently euthanized. Institutional animal care and use committee approval was obtained from the University of South Florida (IS00005974).Cancer cells  were virally transduced with enhanced GFP–NanoLuc plasmid. To generate the LMD xenograft models, microsurgery was performed to inject 5.0 × 10Human Phospho-Receptor-Tyrosine-Kinase Array  and Human-Growth-Factor Array CI  kits were used according to the manufacturer’s instructions. The method to quantify the immunoblot is described in Histologic slides stained with hematoxylin and eosin were assessed by the pathologist, Dr. Robert Macaulay. The histopathologic assessment is described in t test. Kaplan-Meier survival curves were constructed, and the Mantel-Cox test was used to determine significant differences between cohorts. GraphPad Prism 6 software was used to calculate statistical significance.Bar graph results were reported as mean values, with error bars indicating ± standard error of the mean. The magnitude of changes between different conditions was determined using parametric paired BRAF V600E-mutated melanoma. The median overall survival after the diagnosis of LMD was 2.7 months  with all patients succumbing to LMD. The amount of CSF collected varied depending on clinical availability/patient tolerance and whether the patient consented for autopsy. For patients who had multiple CSF collections, we enumerated CSF-CTCs from the first visit. The median number of CTCs per ml was 21.2 .CSF specimens from 11 melanoma patients with LMD were collected during surgery, via LP, via Ommaya reservoirs, or during rapid autopsy . All patex vivo expansion of CSF-CTCs was possible  patients’ CTCs expanding in vitro  medium. Patient 12’s PD-CSF-CTCs originated from CSF collected during autopsy, whereas patient 9’s PD-CSF-CTCs were obtained from both LP and autopsy. We verified long-term cell cultures were melanoma by confirming the presence of BRAF V600E mutation and by staining for Melan-A, a melanocyte marker  in the CSF of immunodeficient mice (NSG)  to at least 5.0 x 104 cells before inoculation, and for the PDX model, we injected the noncultured CTCs from patient’s CSF . (n = 3) developed LMD at 20 weeks, and patient 12 mice (n = 2) at 8 weeks. Disease mice showed >15% weight loss, displayed ataxia (in vitro or in vivo.For ce (NSG) . We util 9, 15). . None ofd ataxia  and had d ataxia . H&E brad ataxia . We collin vitro and in vivo expansions, we next assessed how reflective the long-term PD-CSF-CTC cultures were of the original patient-derived cells using scRNA-seq analysis. We targeted a minimum of 5000 cells in each of the in vitro and in vivo PD-CSF-CTC cultures, and all single cells that were present in the noncultured CSF. For the analyses, we developed a 2-stage architecture analytic tool called ISCVA, through which we could process scRNA-seq data and visualize different subpopulations of cells and genes in Uniform Manifold Approximation and Projection (UMAP) and t-distributed stochastic neighbor embedding (t-SNE) format as described.6 We merged the scRNA-seq data of patients 9’s and 12’s CSF samples and their respective in vitro and in vivo PD-CSF-CTC cultures to produce a projection of how the transcriptome profiles were related  cluster stratified by individual patients (patients 9 and 12), we compared commonalities and differences in gene expression between noncultured CSF-CTCs, and y issue. . Qualitay issue. . We founMLANA,19SOX9,20ErbB3,21 and IGF1R23; this pattern is reminiscent of melanoma cells undergoing epithelial-mesenchymal transition,24–26 which can play key roles in tumor progression  found in this patient’s CSF, and filtered for melanocyte-specific gene signatures. After this process, we identified the melanocyte genes that were most enriched (average logFC in expression > 0.4) , which igression . Furthergression . Interes in preclinical wildtype melanoma models.27To determine whether PD-CSF-CTCs could be used as a tool for functional analysis of therapeutic responses in LMD, as a proof of concept, we tested IGF1R as a drug target on the basis of our scRNA-seq data . In addiBRAF V600E melanoma cell lines WM614 and the therapy (BRAF inhibition) resistant derivative, WM164R    and both13 combination of trametinib with ceritinib proved synergistic with CI’s of 0.84 for patient 12’s and 0.27 for patient 9’s PD-CSF-CTCs (In PD-CSF-CTC cultures (patients 9 and 12) and briefly propagated CSF-CTCs , we also observed a high degree of sensitivity to varying degrees of ceritinib . TrametiCSF-CTCs &B. Taken29 Using an RTK array we found that high levels of IGF1R and INSR phospho-activities were reduced upon treatment with ceritinib (IGF1R in PD-CSF-CTCs using CRISPR/Cas9. Successfully edited cells expressed green fluorescent protein (GFP), as the IGF1R cutting site was inserted with a GFP-P2A donor sequence   .ex vivo sections, we confirmed IGF1R activity to melanoma in the brain meninges by IHC (P =.027), and trametinib was effective for WM164-LMD cohort (P = .003)  . However = .003) .in vitro and in vivo propagation processes, PD-CSF-CTC cultures maintained transcriptome resemblance to that of noncultured cells. These findings also provide support that PD-CSF-CTCs are impactful tools for better understanding LMD pathology testing the efficacy of targeted therapies.Collectively, our results showed that CSF-CTC expansion was possible. Despite going through an in vitro and in vivo from patients with LMD from melanoma. These may be valuable assets to better understand the biology of LMD.LMD from melanoma is a devastating disease with a significant unmet medical need for effective treatments. The lack of PD-CSF-CTCs available for high-throughput drug screening for LMD has been a barrier to drug development. Here we showed the first successful propagation of CSF-CTCs 9 or changing the media recipe after short culturing periods.18 None of the efforts proved effective, despite our experience with growing glioma stem cells.31 Ultimately, we successfully propagated CSF-CTCs using HMC-conditioned medium and supplemented with FGF and EGF.Successful establishment of CSF-CTCs was a trial-and-error process. We attempted a variety of culturing methods, such as fractional PB-CTC culture, growing CTCs in neurosphere and hypoxic conditions,in vitro culture conditions do not recapitulate the CSF microenvironment.16 The meninges secrete a variety of trophic factors/cytokines into the CSF .32–36 Indeed, similar growth factors are present in HMC-conditioned media, which led us to speculate their importance to CSF-CTCs growth. For example, IGFBP2 modulates the bioactivity of IGF1/IGF1R, and is mainly synthesized in the choroid plexus and leptomeninges.37 It has been suggested that IGFBP2 levels in CSF may correlate with CNS malignancy.39 Similarly, IGF1 activity in melanoma LMD may determine disease prognosis.2There may be several reasons why CSF-CTCs are difficult to expand. Based on our experience, the likelihood of a successful CSF-CTC propagation relies on at least three elements. First, it is clear that standard ex vivo for functional analyses. In our experiment, we successfully developed a CDX LMD model and we are aware that it may partially (but not fully) recapitulate the biological environment of LMD.40 We further posit the use of humanized mice may also facilitate high engraftment rates.41A second contributing factor to successful CTC establishment may be the relative scarcity of CTCs in patients’ CSF samples. Low number of CTCs from CSF posed a challenge when we attempted to develop a PDX model. The collection of CSF at autopsy allowed higher volumes of CSF to be collected (and hence more CTCs) than would be acceptable clinically. However, we may still require to briefly grow CTCs 42 Unfortunately, we did not have access to primary tumor specimens to perform scRNA-seq comparison with CSF-CTCs. We may be able to use this method to predict unique subpopulation of PD-CSF-CTCs that distinguished them from the primary tumor in future studies and be used to predict which melanoma patients will develop LMD.Thirdly, it is likely that specific subset(s) of circulating CTCs are responsible for the development of LMD, and identifying these cells may allow for better enrichment of culture conditions, and successes in expansion in PDX models.in vitro and in vivo PD-CSF-CTC cultures were largely representative of CSF-CTCs before expansion. Though a small percentage (0.5%) of gene signatures were unique to noncultured CTCs in CSF, a majority of biological pathways were retained. We used this to select most commonly enriched pathway(s) shared between propagated and noncultured cells and test clinically relevant drug targets. For example, once we identified that IGF1R is enriched in CSF-CTCs, we confirmed that there was phopho-IGF1R activation in LMD samples from autopsy specimens and used IGF1R-depletion assays to confirm the requirement of growth. Hence, as a proof of concept, we attempted to target IGF1R activity in preclinical LMD model.As shown by our scRNA-seq data, 43 as it is highly protein-bound in CSF,44 with only ~1.4% (0.012 μM) unbound drug. However, given the highly disrupted blood-CSF barrier found in LMD,4 effective dosages are completely unknown for therapeutic CSF penetration. We are currently assessing other inhibitors of IGF1R signaling to determine the potential of IGF1/IGF1R as drug candidates for LMD.46 Because ErbB3 was also enriched in PD-CSF-CTCs, another strategy we explored is a dual inhibition of IGF1R and ErbB3. Since ErbB3 is sometimes regarded as “undruggable’ due to its lack of phosho-activity,47 allosteric inhibitors were recently developed to prevent ErbB3 from dimer signaling.48 In another study, bi-specific antibody for IGF1R and ErbB3 was tested in preclinical models of pancreatic cancer.49 Further investigation is currently underway in our lab to investigate ErbB3’s role in LMD.Because there are no FDA-approved/brain penetrant IGF1R inhibitors, we used ceritinib, a noncanonical drug against IGF1R as proof of principle and noted its effectiveness against melanoma cells in this LMD model with acquired resistance to MAPK inhibitors . Further functional and biochemical analyses to fully understand the mechanism of anti-IGF1R in the context of LMD are the focus of future studies. Whether ceritinib can be clinically used as an IGF1R inhibitor for patients with LMD is uncertain,We are aware of several limitations of our study. First, it is not clear how representative PD-CSF-CTC cultures are of the general population of patients with LMD from melanoma. A second limitation is somewhat conceptual: we could only generate long-term PD-CSF-CTC cultures from autopsies and, hence, this method cannot be used for “personalized LMD medicine.” If we can successfully optimize CSF-CTCs from Ommayas or LPs then we may be able to use these for individualized LMD treatment and identify, for example, the early emergence of treatment resistance and alter therapy. A third limitation is the quantity of cells per sample which limited by clinically acceptable volumes. This occurred in our scRNA-seq experiment where one CSF sample (patient 9) from an Ommaya had very small number of viable cells, which contributed to the RNA quality. This might reflect the fragile nature of CSF-CTCs and we will accumulate serial collection specimens when possible.In conclusion, propagating CSF-CTCs from patients with melanoma-associated LMD may provide a valuable resource to better understand the biology of LMD and discover new therapeutic targets via strategies like high-throughput drug screening.noac054_suppl_Supplementary_Figure_S1Click here for additional data file.noac054_suppl_Supplementary_Figure_S2Click here for additional data file.noac054_suppl_Supplementary_Figure_S3Click here for additional data file.noac054_suppl_Supplementary_Figure_S4Click here for additional data file.noac054_suppl_Supplementary_Table_S1Click here for additional data file.noac054_suppl_Supplementary_Table_S2Click here for additional data file.noac054_suppl_Supplementary_MaterialClick here for additional data file.noac054_suppl_Supplementary_LegendsClick here for additional data file.


Worldwide, many patients, including minors, seek chiropractic care. The purpose of this study was to investigate the practice characteristics of chiropractors who treat pediatric patients in Quebec, Canada.We conducted a web-based cross-sectional survey of all licensed chiropractors working in Quebec (Canada). Data were collected using an adapted questionnaire. Descriptive statistics were produced for all the variables collected.Among our 245 participants (response rate: 21%), 63% were women, and half defined themselves as general musculoskeletal (MSK) health care practitioners. Nearly all participants reported seeing 0–5 new pediatric patients/week, and the most common pediatric age group was 6–12 years old (57%). Pediatric patients were most commonly referred by family members and “word of mouth”. The respondents most frequently indicated that they strongly agreed with statements affirming their confidence in their own diagnostic capacities regarding MSK disorders with respect to all age groups as well as non-MSK disorders with regard to young teens. They reported a moderate level of agreement with similar statements concerning the diagnosis of non-MSK disorders in newborns, preschoolers, and children. Chiropractors rarely referred their pediatric patients to a nurse/family doctor or a pediatrician. When presented with potential pediatric red flags, the respondents commonly indicated that they would refer the patient to a physician in an emergency situation or for comanagement.Chiropractors in Quebec are confident in their diagnoses of pediatric MSK conditions and refer patients to physicians in the rare event of a worrisome presentation. However, some chiropractors may have expectations that are unsupported by evidence regarding the diagnosis and management of non-MSK complaints.The online version contains supplementary material available at 10.1186/s12998-022-00464-y. Patients worldwide seek chiropractic care, including children under the age of 18 years old . Recent Despite these data, the provision of chiropractic care to children is controversial, and a position statement was issued by the Canadian Pediatric Society in 2002 to warn about the effectiveness and safety of such care . Twenty Conducting a cross-sectional survey on chiropractic care in Quebec can inform current pediatric chiropractic practice trends and patterns and help promote a better understanding of the profession. This information provides a means of advancing interprofessional relationships between chiropractors and pediatric health care providers. The purpose of this study was to document the practice profile of chiropractors in Quebec who treat children below 18 years of age. More specifically, we aimed to describe the (1) demographic characteristics of chiropractors who treat children in Quebec; (2) patients’ characteristics by age group; (3) knowledge of red flags and referral patterns; (4) pediatric educational training; (5) level of certainty in the chiropractor’s clinical impressions; (6) the pediatric conditions treated; and (7) the nature of interdisciplinary collaboration between chiropractors and other primary care and allied professions.We conducted a web-based cross-sectional survey of all licensed chiropractors working in Quebec (Canada).The questionnaire developed by Siegenthaler  was testDemographic data (4 items)Practical characteristics (5 items)Collaborative practices (4 items) and referral patterns (5 items)Diagnostics and recommendations (2 items)Patient characteristics (20 items)Management of red flags (3 items)Professional affiliation and continuing education (3 items)Future avenues for research regarding the pediatric population (1 item).The final questionnaire included a consent form and 47 items focusing on eight themes  who were recruited on social media via two Facebook pages targeting chiropractors with pediatric interests in Quebec . RespondA web-based cross-sectional survey was sent to all members of the “Ordre des Chiropraticiens du Québec” (OCQ). In the province of Quebec, the mandate of the OCQ is to ensure public protection and excellence in chiropractic practice. Membership in the OCQ is a requirement to practice chiropractic within the province. To obtain an adequate portrait of Quebec’s chiropractic pediatric care and enhance the feasibility of the study, new graduates (those with less than one year of experience), practitioners without pediatric patients, chiropractors practicing outside of Quebec, or individuals without public contact information were excluded. The ethics board for research involving humans of the Université du Québec à Trois-Rivières (UQTR) deemed that an ethical certification was not warranted due to the confidential nature of the data collection process. The individuals involved were not considered to be participants for the purposes of the Tri-Council Policy Statement: Ethical Conduct for Research Involving Humans—TCPS 2 (2018), article 2.1.In July 2019, an email invitation was sent to all members of the OCQ with publicly available contact information asking them to complete our survey on the SurveyMonkey platform (1999–2021 SurveyMonkey©). Participants were also solicited on social media via two Facebook pages targeting chiropractors with pediatric interests in Quebec . ParticiDescriptive statistics (frequencies and percentages) were generated for all items included in the questionnaire. Statistical analysis was conducted using SPSS statistical software, version 27 .Among the 1175 members of the OCQ who were invited to participate in 2021, 261 participants completed our survey. The average completion time was 30 min. Sixteen participants did not meet the inclusion criteria. The final response rate was 21% (n = 245 OCQ respondents)  certification, indicating postgraduate training after passing their board examination; furthermore, 54% of participants were active members of the Quebec Chiropractic Association in Pediatrics and Perinatal Care (AQCPP), and 50.4% defined themselves as general practitioners.Regarding practice characteristics, 45.7% of the participants reported that pediatric patients visited the clinics 0–5 times per week. A total of 99.2% of participants reported seeing 0–5 new pediatric patients per week in their clinics, and the most common pediatric age group was the 6–12-year-old group (57.1%), followed by 13–17-year-old group (54.3%) and the 0–6-month-old group (34.7%). Group practice (44.1%) was the most frequent type of practice. When asked to identify the main objective of their chiropractic treatment, 47.3% of participants indicated that the main objective was to improve function. A total of 24.9% of participants held a “With regard to patterns of referral to the participating chiropractors and their sources of referral, referrals from “parents, siblings or other family members” and by “word of mouth/nonfamily members” were most common. In contrast, referrals from pediatric hospitals, psychologists, medical specialists, and other chiropractors were less frequent Table . FurtherThe conditions treated by Quebec chiropractors by age group are presented in Table Bulging or sulking fontanelles, the inability to wake up a baby, and persistent abdominal pain or abnormal breathing in a child with diabetes.Fever, pain in chest, impaired mental state or other neurological findings.Fracture or dislocation.Signs of dehydration and/or a decrease of 50% of fluid intake over a 24-hour period in a baby or child, persistent vomiting, weight loss of more than 5%.Cold or white lower limbs and/or cyanosis of the lips, febrile petechiae or purpuric rash.Suicidal ideation, neoplasms or noncircumscribed lesions.Descriptive statistics regarding the screening/management of red flags are presented in Table Exclusive chiropractic care was rarely reported as the preferred management for pediatric red flags. Exclusive chiropractic care was only reported by the majority of the respondent for the management of “tilting the head”. Chiropractors commonly reported a comanagement approach to the following clinical presentations: scoliosis > 20°, allergies, delay in motor development, muscle weakness, and chest discomfort of low to medium density.This research represents the first comprehensive study of current pediatric practices and trends undertaken in Quebec to collect the demographic characteristics of chiropractors who treat children. This information includes patients’ characteristics by group age as well as chiropractors’ knowledge of red flags, referral patterns, levels of educational training with respect to pediatric patients, certainty in their clinical impressions, commonly seen pediatric conditions and finally the nature of interdisciplinary collaboration. Overall, the demographics and practice characteristics of the chiropractors working Quebec included in our sample resemble those of chiropractors worldwide –12, 17. Furthermore, the 6–12-year-old group (57.1%) was the group most frequently treated by chiropractors in Quebec, followed by the 13–17-year-old group 54.3%). This finding may be partially explained by the fact those two groups are the groups most frequently referred by medical doctors. The scientific literature also suggests that the prevalence of MSK conditions such as back pain, headaches, sports injuries and scoliosis might be growing with age .3%. This. The thiThe majority of respondents noted they would not hesitate to seek a medical opinion when presented with a patient who exhibited challenging signs and symptoms. Their level of confidence regarding their evaluations was legitimately lower for non-MSK conditions since these conditions had not been the focus of their training and practice. However, some chiropractors treat non-MSK conditions which are not supported by evidence. Based on a limited number of high-quality studies, a recent systematic review questioned the validity of the theory that treating spinal dysfunction with manual spinal therapy has a physiological effect on organs and their functions .The results of our study indicated that the largest proportion of pediatric referrals to chiropractic services for expertise and care were from parents, siblings and other family members, followed by family physicians and nurses, with allied professionals representing the third most common source. In addition, the children most frequently referred by health professionals are those between 0–6 months and 13–18 years old. These results suggest openness to collaborative care, which might represent an opportunity to create more defined care pathways between chiropractic and medical care for pediatric patients.Even though there is a lack of evidence regarding the effectiveness of chiropractic care for pediatric patients –32, someGiven our response rate, selection criteria and the higher proportion of women in our sample than among members of the OCQ in general, it is likely that our respondents are chiropractors with a particular interest in pediatric care. Therefore, our findings might not be generalizable to all chiropractors. However, we believe that this study provides particularly relevant information regarding this subgroup of chiropractors. The validity of our respondents’ answers was not assessed. Therefore, it is possible that the information reported in this study might not perfectly represent the actual clinical practices of our respondents. Although clarity issues were not highlighted during the pilot testing of our questionnaire, the formulation of the question on the confidence in diagnostic capacity might be improved by changing the answer categories to 'I am always certain', 'I am certain most of the time', 'I am certain sometimes', 'I am rarely certain', 'I am never certain'. Data was collected over a long period of time, which included the onset of the COVID-19 pandemic, and we cannot completely rule out the possibility that the profile of practice our respondent might have changed during that time.Chiropractors in Quebec are confident in their diagnoses of pediatric MSK conditions and refer patients to physicians in the rare event of a worrisome presentation. However, some chiropractors may have expectations that are unsupported by scientific evidence regarding the diagnosis and management of non-MSK complaints.Additional file 1. Survey questionnaire.


More knowledge about the long-term impact of sperm donation is essential as the donor’s attitude towards donation may change over time. Personal and social developments may prompt a rethinking of previous actions and decisions, or even regret. Thus, the aim of this study was to explore the experiences and attitudes of men who were sperm donors more than 10 years ago.From May to September 2021, semi-structured, qualitative interviews were conducted with 23 former donors (> 10 years since last donation) from Cryos International sperm bank. Two participants were non-anonymous donors and 21 were anonymous. The interviews were conducted by phone or via video (mean 24 minutes). All interviews were recorded, transcribed verbatim and rendered anonymous. Data were analyzed using thematic analysis.The analysis showed that most men had been donors for monetary and altruistic purposes, and now considered sperm donation as a closed chapter that was ’unproblematic and in the past’. Most men valued anonymity and emphasized the non-relatedness between donor and donor conceived offspring. Knowledge about recipients and donor offspring was seen as ’damaging’ as it could create unwanted feelings of relatedness and responsibility towards them. All men acknowledged donor conceived persons’ potential interests in knowing about their genetic heritage in order to understand appearance and personal traits, but also emphasized the donors’ rights to anonymity. Potential breach of anonymity was generally considered ’highly problematic’ as it was expected to disturb their families and force a relationship on them.This study reports on former donors who might not have volunteered for research due to lack of interest or protection of privacy. The majority of men valued anonymity and clearly demarcated a line between sperm donation and fatherhood, which was enforced by not knowing about the donor offspring or recipients. Men’s reasons for becoming a sperm donor have been extensively researched, pointing to mainly financial and altruistic incentives ,2. HowevInvestigating the long-term impact of having been a sperm donor is particularly relevant since the donor’s attitude towards donation may change over time in response to developments in their individual situation and in society as a whole. Changes in personal circumstances, such as having children of their own and/or current partner’s attitude towards donation, may prompt a rethinking of previous actions and decisions, or even regret . FurtherThe aim of this study was to explore the experiences and attitudes of men who were sperm donors more than 10 years ago.The study was conducted with an explorative design based on semi-structured, qualitative interviews .anonymous donation, the identity of the donor is not known by the recipient or the offspring and the offspring will not be able to receive any further identifiable information from the sperm bank. In non-anonymous donation, the identity of the donor is not known by the recipient or the offspring, however, the offspring will be able to receive the name and latest address on the donor by request to the sperm bank after they reach 18 years of age. Neither anonymous nor non-anonymous donors are able to receive identifying information regarding the DCPs.The study was conducted in the context of Danish legislation on treatment of assisted reproduction, that up until 2007 only allowed anonymous sperm donation. However, since 2007, sperm donors have had the option of anonymous or non-anonymous donation. In www.cryosinternational.com). From February to November 2021, former donors were contacted in batch by the Cryos Research Team to be invited to participate in a range of studies, including this one. The inclusion criteria were: 1) > 10 years since last donation, and 2) available contact information. Approximately 200 former donors met these criteria, and in total 77 responded to contact. Recruitment for the present study took place from May to September 2021, and all former donors who responded to Cryos Research Team contact during that period were consecutively invited to participate in the interview study. As a result, a total of 39 former donors were invited to participate and 29 consented to being contacted by the first and second author, who were primary researchers in the study and not affiliated with the sperm bank. They provided potential participants with additional information and, upon consent, an interview appointment was arranged. Out of 29 potential participants, two declined participation, and four could not be reached, thus leaving a sample of 23 former donors.Participants were recruited via Cryos International sperm bank at four branches across Denmark (All interviews were conducted by phone (n = 21) or video (n = 2) and lasted on average 24 minutes (range: 9 to 47 minutes). All interviews were conducted by SL and SB, who are both experienced in qualitative interviewing. The interviews were guided by a semi-structured interview guide informed by current qualitative literature and the interdisciplinary team of authors. Topics in the interview guide and examples of questions can be found in The material was analysed using reflexive thematic analysis ,14. All https://en.nvk.dk/how-to-notify/what-to-notify). The study was approved by the Danish Data Protection Agency . Informed oral consent was obtained and recorded prior to all interviews.The study was presented to the Central Denmark Region Committee on Health Research Ethics (J. No. 172/2022). According to Danish legislation, research using questionnaires and interviews that do not involve human biological material (§14(2) of the Committee Act) interview studies are exempt from approval from the Committee on Health Research Ethics Second, all men mentioned the altruistic opportunity to help people struggling with infertility. Thus, being a sperm donor was considered by them as a win-win situation where monetary and altruistic purposes could be conveniently and meaningfully combined. For three men, however, altruism was the main motivation. They had all experienced the distress of infertility first-hand :Well, I witnessed how my sister and brother-in-law struggled to have children. I saw first-hand how miserable they were. And when [sperm bank] opened in [city of residence at the time] then I just thought… this is a way where I can maybe make a difference for someone. (Chris)An additional motivation for some men was curiosity about their sperm quality. Being approved by the sperm bank and having acceptable sperm quality was a source of satisfaction, or even pride, that also provided peace of mind regarding own future fertility:I was motivated by several factors, but honestly, I was curious about sperm quality… if it was good enough. I guess it was a bit of a vanity thing… (Daniel)Finally, a main motivation and (for many) the very foundation for even thinking about becoming a donor was the opportunity to be anonymous. Many stressed that the option of anonymity and their confidence in the sperm bank’s ability to uphold that anonymity was essential for their decision to become donors:For me it was essential that it was anonymous. I imagine that very few would donate if you couldn’t do it anonymously. (Joe)Only by being anonymous can you put it away again and say: I am donating something, ok, and I will never know any more about it. Or risk being confronted with it in the future. (Kevin)Anonymity was often described as a way to protect their future self from unwanted and unpredictable consequences. Two men had chosen to be non-anonymous donors, as they thought that offspring should have right to contact if interested. One of them had been motivated by the possibility of reproducing his genes:I think it’s exciting to know that future generations carry my genes. I think it’s something deep in our biology, that all humans want to multiply. (Michael)Generally, the men had felt well-informed by the sperm bank during their time as a donor; and, in hindsight, had not required any further information or support during or after it finished. The reasons for stopping being a donor varied. A few had their contract cancelled by the sperm bank and a few were unhappy with changes in remuneration, but for the majority it was simply no longer convenient . At the time of the interview, only one participant expressed regret for having been a donor. He had been informed that a diseased child had been born from his donation and expressed feelings of guilt towards the recipients and child. However, today, most men still felt positive about, or even proud of, having been a sperm donor.’closed chapter’ that belonged to the past; not a secret, but simply not relevant to their current lives. For these men, many of the interview questions guide were answered with ’I haven’t thought about it’ or ’I don’t know’, but many tried to offer more explanation. However, it was obvious that having been a sperm donor did not impact their current lives. Many had now settled in family life and considered donating part of their youth:During interviews, most men (including one non-anonymous donor) reported they hardly ever thought about having been a sperm donor. It was described as a I still feel good about it [having been a sperm donor]. But I am also somehow happy that it’s history. Because now I have my family, I have a life that is quite different from back then… when I was the young bachelor type. (Matt)Thus, donating sperm was understood as a temporally bounded event that belonged to the past. For a few men, however, the boundary was less clear and their time as a donor was still present in their current lives. For example, one non-anonymous donor often thought about and looked forward to being contacted by offspring, while an anonymous donor had actively searched for recipients and offspring. Except for the latter, all men were content with their original choice of donor status (non-anonymous or anonymous) and found it important to uphold their contractual rights and obligations towards the sperm bank, the recipients and donor offspring.When addressing their perceived, potential relationship or relatedness to donor offspring, many men found it difficult to articulate or define, often asking the researcher to repeat or re-phrase the questions. Most men simply maintained that there was no relationship at all:There is no relationship. At all. That was essential for me going into this… I mean, of course there is the ’good cause’ and all, but it wasn’t my primary motivation and I have no relation to… It was a job. I thought of it as a job and that is all [laughs]. (James)It’s like an organ or… I guess it’s a bit cynical, but…. […], It’s something you pay for, when you want to have a child. I give something, they get it, that’s all there is to it really. (Jacob)’out in the world’, and some would joke about how ’lucky’ or ’poor’ this donor offspring was. Most men made the distinction between genetic origin and fatherhood, and the latter only extended to their relationship to their ’own children’. It was emphasised that fatherhood is a status and a privilege that is socially and emotionally achieved rather than genetically determined:However, when explicitly asked about relatedness, many men acknowledged that they potentially shared genes with DCPs It’s not just a matter of genes. It’s raising, protecting, and caring for a child that defines parenthood to me. Not the genes. And I passed the ’raising, protecting and caring’ part on to those who received the donation. (Chris)To claim the status of fatherhood thus entailed protecting, bringing up and being a part of the children’s lives. For the fathers in the sample, all concern and loyalty were firmly directed at their own children, and they stressed that they in no way should or could be perceived as having a father-like relatedness to offspring.’damaging’:At the time of the interview, sixteen men had children of their own. For some, fatherhood had led them to consider the wellbeing of potential offspring; but they also emphasized how such knowledge could be When I was younger, I didn’t care [about the fate of offspring]. But today, I have children of my own and I realise how dependent they are–on love and good surroundings. So, in some ways I am concerned [for DCP], and there is a sort of relief that I don’t know who they are. […] If one of the children were born into a deeply dysfunctional family, then that would make me upset. I would feel responsible. Therefore, it’s better not to know. (Alex)’a bit unhappy’ with not having the sole rights to the man’s gene pool:The men (fathers and non-fathers) expressed how knowing about their donor offspring and recipients could create unwanted feelings of relatedness and responsibility towards them. This was something that they wanted to protect themselves and their families from. Most men had informed their current partner about their past as a donor and generally, the partners were reported to be overall accepting. A few men described their wives as a Back then it wasn’t a problem, but over the years…I don’t know. After we got our own kids, she started thinking about it differently… that I might have a few more out there. (Tyler)This could indicate that some partners attached meaning to genetic relatedness and that the past donorship threatened the exclusivity of their genetic relatedness. The potential meaning and importance of genetic relatedness was addressed by several men, who acknowledged that such information might be of importance to DCPs. Many men understood genetics to be an explanatory factor in understanding both appearance and personal traits. While maintaining that the recipients were, and would always be, the DCP’s parents, many donors accepted that knowing about genetic inheritance could be important to some DCPs:I can definitely relate to that. Particularly when you’re a teenager and figuring things out. Who am I? Where do I come from? You’re looking for answers, and I understand why one might be interested in biological origin. And maybe some explanations… about appearances, but also reactions and behaviors. I think it’s really interesting. (John)As such, several men expressed sympathy towards some DCPs’ desire to meet a donor but felt that their rights to anonymity and the protection of his family should carry more weight.All men reported to trust the security of their data at the sperm bank but also acknowledged the ever-increasing possibilities for online investigations and searches where DCPs could be successful in identifying siblings and/or the donor. Generally, the donors were not well informed about, or interested in, the presence of online ancestry databases, voluntary registers, or Facebook communities; a finding that resonates well with the donor’s lack of interest in contact with potential offspring. However, many men also mentioned that the (unlikely) risk of unwarranted contact from an offspring was an inherent uncertainty in sperm donation:I know that sometimes they manage to find the father, even if he’s anonymous. If it happens, it happens. I’ll deal with it. (David)’highly problematic’ or ’very unfortunate’, while others described offspring-initiated contact as a matter of ’crossing that bridge when I get there’. The men who had been anonymous donors had generally not given offspring contact much thought and had not planned for what to do in that unlikely event:Many men clearly stated that breach of anonymity would be I haven’t thought about it. I guess it’s one of those things where you can’t really know until it happens. It’s a difficult question and I can’t predict my response. (Chris)The main concern regarding breach of anonymity was that offspring contact would create an unwanted disturbance in their current lives and families. Fathers expected to be particularly concerned about the response of and impact on their children, none of whom knew at the time of the interview that their father had been a sperm donor, mainly because they were still quite young:I also have a family to take care of and that’s probably… Like, I think I could probably deal with it OK, but what about the rest of the family? […] It will create an imbalance in the family when they have to deal with it too. And that would be the hardest for me, that it would disturb their everyday life. (Joshua)’force’ a personal relationship onto the man. Many former donors imagined how being confronted with an ’actual, living person’ would probably make it difficult not to get engaged in that person’s life, even if they were initially not interested in it:Another concern was that offspring-initiated contact would I wanted to be anonymous, because… well, if someone came around, I just think… I think I’m this loving person and I could really just end up seeing them as family. And they already have a family, I know, but I think I couldn’t help think that we were family too. That’s why I chose to be anonymous. (Joe)Several men brought up, that if a donor offspring had gone through such troubles to circumvent anonymity, then they must be very much in need of answers and the men expected to feel morally obliged to respond. While some men imagined that setting boundaries for this relationship or contact could be difficult, others felt that they could maintain very clear boundaries if a donor offspring unexpectedly made contact:OK, so let’s go have a cup of coffee. Somewhere neutral. And let’s talk through it all, could be interesting. And then, that would be the end of it. Like, I am your genetic origin and we can talk about that. But we are not going to discuss me being your dad. ‘Cause I’m not. (Alex)’be a decent person’ and respond to a human being who had gone through a lot of trouble while still protecting his family and clearly place them as top priority.Such boundaries would allow the man to A few men described a fleeting interest in having contact with their donor offspring, mainly due to curiosity about similarities in appearance and personality traits. A couple of men had done a little online research but stopped themselves:I am curious about it and I thought that if I could maybe find them [DCP] without making contact… It seemed very easy. But then, I guess, knowing about them would create an imbalance. I’d risk my everyday life just to satisfy a curiosity. Not worth it. (Justin)’romanticizing’ the potential relationship with donor offspring . The other, a non-anonymous donor, were excited about the future, potential relationship and imagined himself being like ’an uncle’. Even so, the men were mindful of the potential vulnerabilities and risks of contact . Nevertheless, this uncertainty was outweighed by their desire for meeting their donor offspring.Two men, both without children, explicitly expressed interest in meeting their offspring due to curiosity about their lives, their appearance and personal traits. One of them, an anonymous donor, described sometimes This study provides insights into the experiences of former donors who generally described their donation as a temporally bounded transaction, that belonged in the past and, for most, had little impact on their current lives. The men generally acknowledged DCPs’ interests in knowing about their genetic origin, but emphasized their non-relatedness to offspring. Most of the men had not thought much about a potential breach of anonymity but were mainly concerned that it would disturb their families and force them into a relationship and feelings of responsibility towards their donor offspring.Overall, our findings are remarkable in that they provide insights into the—often underrepresented—perspective of anonymous donors who are disinterested in identity release and DCP contact. As noted by Kirkman and colleagues , most renot want information about DCPs, which also resonates with the findings in the present study. Interestingly, these differences in characteristics are often discussed with an implicit positive bias towards the older and more settled type of man opting for open donor status. For example, in their investigation of personality characteristics of men who became donors in a system allowing only non-anonymous donors, Sydsjö and colleagues https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  4 Jan 2023Response to decision letterDear Sónia Brito-Costa, Academic Editor, PLOS OneFirst, we wish to thank the reviewers for insightful and very constructive comments on our manuscript. We have revised the paper in accordance with the reviewers' suggestions and believe it has improved the manuscript.Below we address the comments and explain how we have aimed to accommodate them in the revision of the manuscript. All reference to line numbers are in the track changes version of the revised manuscript.We hope that the revision meets your expectations and look forward to hearing from you.On behalf of the authors,Best regards,Stina LouJournal requirements 1. Please ensure that your manuscript meets PLOS ONE's style requirements.  - We have revised the manuscript in accordance with PLOS ONE's formatting requirements, including title page, tables and manuscript headings (PLOSOne_formatting_sample_main_body.pdf (storage.googleapis.com)2. Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Please include your amended statements within your cover letter; we will change the online submission form on your behalf.- Updated Funding Statement: This study was funded by Cryos International  and the Novo Nordisk Foundation (Grant No. NNF16OC18722 to IV). Staff at Cryos International assisted in recruitment of participants for the study, but the funders had no role in the data collection, analysis and interpretation of results.3. Thank you for stating the following in the Competing Interests section:http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared.“SL, SB, MDT and IV declare no conflict of interest. AP, GP and SWA are members of the Cryos External Scientific Advisory Committee. GP has received honorarium for this. ABS is an employee of Cryos International.” Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” ? Please develop that part ie how many in total were available >10 years, if not all there is a need for mor information regarding the criteria for being recruited.  - Good point. We have added more information on the recruitment process, L121-283. My second concern is about the semi structured questions. How were they used? Were all these questions asked to all of the participants? - The same guide was used for all interviews, but the extent of probing to different topics varied between respondents. We have added this information to the manuscript, L140-414. It would improve the understanding if you describe a little more (few examples) how your process of doing reflexive thematic analysis was performed/developed regarding from the main text from the interviews?  - Yes, transparency is important! We have now revised the section and added more examples to our process of doing reflective thematic analysis, L154-67 The analysis was based on interviews 9-47 minutes (mean 24). 9 minutes are very short to get substance for an analysis. We completely agree. And that was also an argument for continuing recruitment until we had a relatively large sample to ensure sufficient substance in the analysis. For some of these men, having been a donor was completely unproblematic and not something they were used to articulating (which is an interesting finding in itself). Therefore, with a few men, the interviews were short, because they mainly provided one-syllable answers and/or did not respond to/recognize some of the topics and potential problems, that we asked about. Luckily most interviews were longer and with more substance. 5. You base your results on 21 anonymous and separate 2 non-anonymous donors answers. Why did you include only 2 non-anonymous, or why did you include these two at all? The main message may be more clear if focus and discussion only were about anonymous donors. As you write (and participants answers show) the non-anonymous donors think differently.  - Reviewer 1 touches an important point that we have also discussed in the research team several times. However, we decided to 'keep' the non-anonymous donors, because they provide an interesting juxtaposition to our main findings. 5. Interesting discussion that add insight about donors long-term perspectives.  - Thank you!Reviewer #2 1. The abstract should be improved, as it is not particularly appealing to the potential reader. I also emphasize the need to correct the second sentence of its "methods" section and improve the conclusions;  - Sorry about that second sentence! We have thoroughly revised the abstract to make it more appealing, L34-71.2. Although in Denmark this type of study does not require the statement of an Ethics Committee, this statement is required by PLOS One "if the study involved human participants", so the authors, if they intended to submit their study to this journal, should have read the submission rules and prepare theirr project accordingly. The decision of whether or not to accept the publication without fulfilling this requirement will, of course, be at the discretion of the Editor;  - We have uploaded an ethics statement with the submission. Also, we have revised the ethics statement to clarify that the project has been presented to the ethics committee, L173-753. Despite being a qualitative study, I believe that the designation in the results, repeatedly, of terms such as "many", "for the majority", "a few", "several" and "most" should be replaced by the number of participants that we are actually mentioning in each case.  - There are very divergent attitudes towards the use of numbers in qualitative research. We believe that numerical values can be misleading in qualitative research, particularly if they are read as 'evidence' for a particular finding. A qualitative analysis should describe a pattern or an overall theme. Within this analysis, data points that appear less frequently may also have great value, for example to shed light on varied points of view. We hope that reviewer #2 can accept this explanation for why we have chosen not to add specific numbers to the manuscript 13 Jan 2023Experiences and attitudes of Danish men who were sperm donors more than 10 years ago; a qualitative interview studyPONE-D-22-21878R1Dear Dr. Lou,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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For more information, please contact Kind regards,Sónia Brito-Costa, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 19 Jan 2023PONE-D-22-21878R1 Experiences and attitudes of Danish men who were sperm donors more than 10 years ago; a qualitative interview study Dear Dr. Lou:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Sónia Brito-Costa Academic EditorPLOS ONE