Medulloblastoma (MB) is the most common pediatric embryonal tumor. Accurate identification of cerebral spinal fluid (CSF) dissemination is important in prognosis prediction. Both MRI of the central nervous system (CNS) and CSF cytology will appear false positive and negative. Our objective was to investigate the added value of preoperative-enhanced T1-weighted image-based radiomic features to clinical characteristics in predicting preoperative CSF dissemination for children with MB.n=60; internal validation cohort, n=24). A set of cases between December 2018 and February 2020 were used for external validation (n=40). The children with normal head and spine magnetic resonance images (MRI) and no subsequent dissemination in 1 year were diagnosed as non-CSF dissemination. The CSF dissemination was manifested as intracranial or intraspinal nodular-enhanced lesions. Clinical features were collected, and conventional MRI features of preoperative head MRI examinations were evaluated. A total of 385 radiomic features were extracted from preoperative-enhanced T1-weighted images. Minimum redundancy, maximum correlation, and least absolute shrinkage and selection operator were performed to select the features with the best performance in predicting preoperative CSF dissemination. A combined clinical-MRI radiomic prediction model was developed using multivariable logistic regression. Receiver operating curve analysis (ROC) was used to validate the predictive performance. Nomogram and decision curve analysis (DCA) were developed to evaluate the clinical utility of the combined model.This retrospective study included 84 children with histopathologically confirmed MB between November 2006 and November 2018 (training cohort, One clinical and nine radiomic features were selected for predicting preoperative CSF dissemination. The combined model incorporating clinical and radiomic features had the best predictive performance in the training cohort with an AUC of 0.89. This was validated in the internal and external cohorts with AUCs of 0.87 and 0.73. The clinical utility of the model was confirmed by a clinical-MRI radiomic nomogram and DCA.The combined model incorporating clinical, conventional MRI, and radiomic features could be applied to predict preoperative CSF dissemination for children with MB as a noninvasive biomarker, which could aid in risk evaluation.The online version contains supplementary material available at 10.1186/s12957-021-02239-w. Medulloblastoma (MB) is the most common embryonal tumor located in the posterior cranial fossa. It usually affects young children before the age of 9 years . ApproxiAt present, the standard treatment for MB is maximum resection followed by risk-adapted adjuvant chemoradiotherapy. Depending on the absence or presence of adverse prognostic factors including age younger than 3 years, anaplastic histopathologic subtype, CSF dissemination, and residual tumor greater than 1.5 cm in diameter, the child with MB will be stratified into an average or high-risk group . AlthougBoth MRI of the CNS and CSF cytology will appear false positive and negative. On the one hand, MRI of the CNS is not able to detect early dissemination, while on the other hand, the equivocal findings such as enhanced meningeal thickening and nerve roots clumping may be misdiagnosed as dissemination , 6. BecaMRI is the modality of choice for risk stratification in children with MB. It can provide more information than conventional MRI features evaluated by radiologists, because images are not data mined . To the Radiomics is an approach that is able to extract high-throughput quantitative features from medical images . In theoThis retrospective study was approved by the Institutional Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine , and therefore, informed consent was waived.The inclusion criteria included (i) the availability of preoperative head MRI with diagnostic quality, (ii) availability of spine MRI with diagnostic-quality performed pre- or postoperative but before adjuvant therapy, (iii) children without CSF dissemination confirmed by head and spine MRI required follow-up results for 1 year, and (iv) without any previous treatment. The exclusion criteria included (i) insufficient head and spine MRI quality, (ii) without 1-year follow-up results for children without CSF dissemination, (iii) previous treatment, and (iv) equivocal findings on spine MRI such as enhanced meningeal thickening and nerve roots clumping.Children with pathologically confirmed MB were reviewed between November 2006 and November 2018. The children met the including and excluding criteria were randomly distributed to the training and internal validation cohorts according to a 7:3 ratio. A set of cases between December 2018 and February 2020 were used for external validation of the prediction model.Clinical features, including age, gender, and histopathologic subtype  of all children with MB, were collected via medical records.The workflow of this retrospective study is displayed in Fig. All of the children underwent pre- and postoperative contrast-enhanced T1-weighted head MRI using a 3.0-T MRI scanner  with an 8-channel head coil. The contrast-enhanced T1-weighted images were obtained with a slice thickness of 4 mm and a matrix of 512 × 512. Children also underwent contrast-enhanced spine MRI, which was given either preoperative or postoperative but before adjuvant therapy. Axial and sagittal-enhanced T1-weighted images were obtained with a thickness of 3 mm and gap of 0 mm. The detailed parameters for head and spine MRI are provided in The contrast material was administrated with a dose of 0.1 mmol/kg . Children that were unable to remain motionless during the MRI examination were sedated with chloral hydrate (0.5 mg/kg).All of the preoperative head MR images were reviewed by two pediatric radiologists . Discrepancies were resolved by consulting with a third pediatric neuroradiologist (Y.H.L) with 30 years of experience. A set of conventional MRI features were evaluated, including the location of the tumor, contrast enhancement pattern, intratumoral necrosis, hemorrhage, calcification, peritumoral edema, and the minimal apparent diffusion coefficient (minADC) value. A description for these features is provided in The enhanced T1-weighted peri-surgical head and spine MR images were used for CSF dissemination assessment. Children with normal head and spine MR images and no subsequent dissemination at the 1-year follow-up were regarded as the non-CSF dissemination group. CSF dissemination was manifested as intracranial or intraspinal nodular lesions and leptomeningeal enhancement. Children with equivocal findings on head and spine MR images were excluded.www.itksnap.org). The peritumoral edema and surrounding vessels were carefully avoided. The segmentations were examined by another pediatric neuroradiologist (M.L with 20 years of experience). Both were blinded to the CSF dissemination status.The delineation of MB and radiomic extraction is shown in Fig. By using an in-house software Analysis Kit , a total of 385 radiomic features were extracted automatically from the VOI including histogram parameters, volume and shape parameters, Haralick features, gray-level co-occurrence parameters, and gray-level run-length matrix parameters.Differences in clinical and conventional MR imaging characteristics between the children with and without CSF dissemination in the training, internal, and external validation cohorts were assessed. A clinical prediction model was built using the features with significant differences.In the training cohort, minimum redundancy and maximum correlation (mRMR) and least absolute shrinkage and selection operator (LASSO) were performed to reduce dimension and select the radiomic features with the strongest CSF dissemination-related correlation. The radiomic score (rad-score) of each child with MB was calculated by adding all of the products of the selected features and their corresponding coefficients, after which a radiomic prediction model was built. A combined clinical-radiomic prediction model was developed using multivariable logistic regression, which incorporated selected clinical features and rad-scores. A clinical-radiomic nomogram was then constructed in the training cohort.The receiver operating characteristic (ROC) curves were used to evaluate the predictive performance of the clinical, radiomic, and combined models in both the training and two validation cohorts. The area under the curves (AUC), sensitivities, specificities, and accuracy were calculated. And then, the Hosmer-Lemeshow test was performed to assess the goodness-of-fit of the combined model. Decision curve analysis (DCA) was implemented to determine the clinical usefulness of the prediction models in the training cohort at different threshold probability.t tests or chi-squared tests according to the type of the data. The optimal values of the ROCs were determined using Youden’s index. Delong’s test was used to assess the differences in the AUC values between the clinical and clinical-radiomic combined models.The differences of clinical and conventional MRI characteristics between the training and two validation cohorts, as well as between children with and without CSF dissemination in their respective cohorts were evaluated using independent http://www.Rproject.org). The ROC curves were performed using the “pROC” package. Multivariate logistic regression was plotted with the “rms” package. The Hosmer-Lemeshow test was conducted using the “Resource Selection” package. DCA was developed with the function of “dca.R”. P < 0.05 was set as statistical significance.All statistical analyses were performed using the R software package . None of the children experience extra-CNS metastases. Forty-four children (35.6%) were confirmed to have CSF dissemination by head and spine MRI evaluation. Five children had only intracranial dissemination while 39 children had both intracranial and intraspinal dissemination. Eighty children without CSF dissemination were identified by MRI evaluation and follow-up information.P=0.028) in the CSF dissemination group than in the non-CSF dissemination group.The clinical features of the children in the training and two validation cohorts were displayed in Table In the training cohort, nine radiomic features with the strongest CSF dissemination features were selected using mRMR and LASSO shown in Fig. Rad − score =  − 0.557 × Kurtosis−0.502 × ShortRunHighGreyLevelEmphasis _ AllDirection _ offset7 _ SD−0.538 × ClusterShade _ AllDirection _ offset7 _ SD−0.444 × RunLengthNonuniformity _ angle0 _ offset1−0.909 × Inertia _ angle45 _ offset1−0.248 × Inertia _ angle0 _ offset4−0.026 × RunLengthNonuniformity _ AllDirection _ offset1−0.204 × GLCMEntropy _ angle135 _ offset7−0.17 × Intensity/Variability − 0.735The combined clinical-radiomic predictive model was developed using multivariate logistic regression analysis. The AUC of the value of the combined model was 0.89. And the AUC of the clinical model was much lower (0.67), which indicated that radiomic features could improve performance. The results of the combined predictive model in the training and two validation cohorts were shown in Table The CSF dissemination predictive performance of combined prediction model was also robust when applied to the internal and external validation cohorts with an AUC of 0.87 and 0.73, respectively. The Hosmer-Lemeshow test demonstrated that the goodness-of-fit of the combined model was high in both the training and two validation cohorts Fig. . DCA revn=124), we developed and validated a clinical-radiomic combined model for preoperative prediction of CSF dissemination in children with MB. The nomogram combined the age of the children and a preoperative-enhanced T1-weighted image-based radiomic signature. The results demonstrated that compared with the prediction model built using clinical features alone, the radiomic signature could improve the predictive performance of CSF dissemination. The AUC was increased from 0.67 to 0.89 in the training group. The sensitivity, specificity, NPV, and PPV were relatively higher both in the training and two validation cohorts. The higher sensitivity indicated that the combined model could identify children with CSF dissemination accurately. These children should undergo examinations with a higher specificity, such as CNS, MRI, and CSF cytology to evaluate the dissemination staging.In the present study with a relatively large case group (Children with CNS dissemination should be given aggressive therapy, as prognosis is usually poor, no matter what the molecular subgroup is. It is, therefore, paramount to establish a method to accurately diagnose CNS dissemination. Traditionally, diagnosis of CSF dissemination encompassed comprehensively analyzing the combined results of CNS, MRI, and CSF cytology , 13, botIn this retrospective study, CSF cytology results were not obtained. Therefore, the diagnosis of CSF dissemination depended on head and spine MR images. Children without CSF dissemination were identified by negative head and spine MRI and outcome at the 1-year follow-up. This diagnosis strategy is consistent with the literature determining the status of CSF dissemination by using overall survival . The equRadiomics can evaluate the intratumoral heterogeneity and predict prognosis by extracting high throughput quantified features using medical images. A previous study indicated that when the prediction accuracy of the radiomic signature was constructed by using 24 radiomic features from preoperative computerized tomography images for lymph node metastasis in patients with colorectal cancer, the concordance index was 0.773 . In anotIn CNS tumors, radiomics is mainly applied to differential diagnosis of tumors, World Health Organization grading, molecular profiling, and prognosis prediction in the adult population –19. HoweAccording to previous reports, the CSF dissemination probability of medulloblastoma has a close relationship to molecular subgroups. For example, the prognosis of children with group 3 is very poor and prone to early metastasis . Some MRIn the present study, the results demonstrated the added value of radiomics to clinical and conventional MRI characteristics in predicting CSF dissemination for pediatric MB. The higher sensitivity and accuracy of the combined model compared with the clinical model alone is encouraging. It is likely that the probability of CSF dissemination could be calculated using the proposed combined model nomogram.The CSF dissemination prediction model included the clinical variable of age, as group 3 tumors are prone to occur in younger children and infants . RegardiThere are several limitations to the current study. First, due to the small sample size of the cohort, useful conventional MRI features for the predictive model could not be used because of selective bias. The association between conventional MRI features and CSF dissemination in children with MB should be further explored in a large-scale study. Second, due to the retrospective nature of the study, an external validation from another institution was not performed. A multi-center prospective research with different MRI scanning protocols is needed to evaluate the generalization of our predictive model. Third, only enhanced T1-weighted images were used in this radiomic research. Multiparametric MRI including T2-weighted images should be implemented into the predictive model to improve its robustness. Finally, we were not able to obtain CSF samples in the majority of their patients to identify M1 disease. But because of technical or sampling problems, CSF cytology may fail to detect the tumor cells. A more precise technique called circulating tumor DNA should be used in further study to improve the performance of the predictive model.In summary, our preliminary study demonstrated that a preoperative-enhanced T1-weighted image-based radiomics could improve the prediction accuracy of CSF dissemination in children with MB. A nomogram constructed based on the radiomic signature and selected clinical features may provide more useful information for clinical decision making.Additional file 1..


Due to the development of novel equipment for the acquisition of two‐dimensional serial images and software capable of displaying three‐dimensional (3D) images from serial images, the accurate 3D reconstruction of organs and tissues has become possible.Based on published studies, this review summarizes techniques for the 3D reconstruction of the testis cords/seminiferous tubules, with special reference to our method using serial paraffin sections and 3D visualization software.The testes of mice, rats, and hamsters of various ages were 3D reconstructed and species and age differences in the structures of the testis cords/seminiferous tubules were analyzed. Our method is advantageous because conventional paraffin‐embedded normal and pathological specimens may be utilized for the 3D analysis without the need for complicated and expensive equipment.By further decreasing the time and labor required for the procedure and adding information on molecular localization, the technique for 3D reconstruction will contribute to the elucidation of not only the structures, but also the functions of various organs, including the testis. The testes of mice, rats, and hamsters of various ages were 3D reconstructed and species and age differences in the structures of the testis cords/seminiferous tubules were analyzed. Our method is advantageous because conventional paraffin‐embedded normal and pathological specimens may be utilized for the 3D analysis without the need for complicated and expensive equipment. However, tissue sections obtained from three‐dimensional (3D) organs and tissues only provide two‐dimensional (2D) information. It is very important, but often challenging, to reconstruct missing 3D information in attempts to match the shape observed in a tissue section to the original 3D structure. Manual 3D reconstruction from serial sections is possible, but has seldom been used in morphological studies, possibly due to the extensive amount of labor required. In recent years, with the development of new technologies for the acquisition of 2D serial images and software capable of displaying 3D images from serial images, the accurate 3D reconstruction of organs and tissues has become possible.Confocal microscopy,In addition to the development of imaging technology, the advent of software that visualizes a 3D image from serial 2D images has been important for accurate 3D reconstruction.2Mammalian testes consist of two compartments: the interstitial compartment and seminiferous tubule compartment.A growing body of evidence indicates that region‐specific morphological and functional differences exist within the testis. For example, we recently revealed that the sites at which spermatids initially occur in the postnatal mouse testis are preferentially distributed in the upper‐medial areas of the testis close to the rete testis,3The 3D structure of the whole testis was initially reported in the early 1900sA study by Clermont and Huckins in 1961 on the 3D structure of the rat testis cords/seminiferous tubules was the most impressive among the reconstruction studies using testis serial sections at that time.http://bio3d.colorado.edu/imod/), a set of image processing, modeling, and display programs,Combes et al. reconstructed the mouse testis cords on E12.25‐E15.5.Nel‐Themaat et al. investigated the structure of the testis cords in Sox9‐enhanced green fluorescent protein knock‐in mice by confocal microscopic imaging without reconstruction and by the 3D reconstruction of serial histological sections.Silva et al. recently investigated seminiferous tubules using micro‐CT.en bloc and fixed in Bouin's solution overnight at room temperature. Serial 5‐µm‐thick sections at intervals of 50 µm were obtained by cutting the specimen longitudinally in parallel to the plane involving both the testis and epididymis using a microtome and stained in the basement membrane with PAS‐H to mark the outlines of seminiferous tubules. Sections were digitized using a whole‐slide scanner  with a 20‐fold objective lens, and the resulting digital images of sections were visualized using viewer software . The extraction of the PAS‐H‐stained basement membrane in digital images was performed using ImageJ software  and Adobe Photoshop 2020 software (Adobe Inc.). After extraction, images were converted into gray scale in the JPEG format with Adobe Photoshop 2020 software. Using Amira 6.3.0 software, serial images were automatically aligned with rigid algorithms followed by manual adjustments, and the inside of the outlines of a selected tubule was filled with a particular color using threshold processing and traced from section to section. This procedure, called semiautomatic segmentation, was repeatedly applied to all seminiferous tubules with different colors, and they were then subjected to 3D rendering. To draw the core lines of seminiferous tubules, individual traced tubules in cross sections were shrunk concentrically and reconstructed into a thin tubule, in which the core lines were drawn using the same software. The software sometimes failed to distinguish closely apposed different tubules from the branched portions of a single tubule and drew wrong lines, partly because alignment was slightly inaccurate due to the distortion of sections. This error was corrected manually by tracking the reconstructed core lines in all serial sections. While the method described above was the standard, we also made some modifications to it depending on the specimens and fixatives. For example, small and soft specimens, such as embryonic gonads, were fixed in modified Davidson's fluid or 10% formalin neutral buffer solution, a milder fixative than Bouin's solution, and the basement membranes were immunostained for marker proteins, such as laminin, to enable more accurate semiautomatic segmentation. On the other hand, large specimens, such as the adult hamster testis, were sometimes insufficiently fixed, even with Bouin's solution, making PAS‐H staining of the basement membrane not sufficiently clear to allow for semiautomatic segmentation. In such cases, we adopted manual segmentation by marking the outlines of seminiferous tubules from section to section. Using these methods, we reconstructed the testis cords/seminiferous tubules of mice, rats, and hamsters of various ages using serial paraffin sections.We recently reconstructed whole testis cords/seminiferous tubules using serial sections and Amira 6.3.0 (Visage Imaging GmbH), high‐performance 3D reconstruction software.4Figure Figure In P21 mice, the zigzag convolutions of seminiferous tubules became more constant in direction. The cranial turns of convolutions were in contact with the tunica albuginea, whereas the caudal turns occupied the central areas of the testis. Overall, all seminiferous tubules formed funnel‐shaped networks that tapered toward the caudal direction, and caudally located networks surrounded the preceding cranially located networks from the bottom and outside with the appearance of stacked paper cups. The acrosome, a special structure of the spermatid that appears for the first time in the P21 mouse testis, was visualized with lectin histochemistry and superimposed onto the testis and seminiferous tubules at P21 Figure . AcrosomIn adult mice, the total length of seminiferous tubules per testis was 86‐, 27‐, and 2.2‐fold larger than those in E15.5, E18.5, and P21 mice, respectively  based on the deep learning of pattern recognition will overcome this issue by automating both the segmentation and tracing steps. We have started analyzing the 3D structure of the human testis using this AI technology.Furthermore, as already discussed, region‐specific morphological and functional differences within the testis may represent region‐specific gene expression that is regulated by particular molecules produced inside or outside the testis. The histochemical localization of various bioactive molecules and their receptors may be superimposed on the 3D‐reconstructed testis structure, similar to the lectin that recognized acrosomal molecules in our previous study.The authors declare no conflicts of interest. The present animal study was approved by Kanazawa University , the Tokyo Medical University Committee (R2‐0045), and the Animal Research Committee of St. Marianna University School of Medicine (1712012).


Age was associated with a lower ECD of 6 ± 2 cells/mm2 per year (p = 0.001). Female ECD was 189 ± 44 cells/mm2 higher than male ECD (p < 0.001). Pseudophakic eyes had 378 ± 42 cells/mm2 less compared with phakic eyes (p < 0.001). Cause of death did not affect the ECD. Of note, 55% and 38% of corneas harvested on the second and third postmortem day, respectively, and 45% of corneas from donors older than 80 years were still suitable for transplantation. Conclusions: In the context of a growing need for donor corneas, we do not recommend limiting donor age and collection time to 24 h or excluding oncology donors, as is the practice in many countries. Therefore, we propose a mathematical model for better donor preselection.Background: In Germany, more than one-third of donor corneas harvested are not suitable for transplantation. We evaluated the factors associated with the usability of donor corneas. Method: Data from 2032 consecutive donor corneas harvested at the Rhineland-Palatinate Eye Bank in Mainz, Germany, were retrospectively analyzed. Factors of interest were age, sex, lens status, cause of death, cardiopulmonary resuscitation (CPR), death-to-explantation-interval (DEI), and the influence of these factors on the proportion of discarded donor corneas. Factors associated with endothelial cell density (ECD) were analyzed in a linear regression mixed model. Results: Higher donor age, male gender, pseudophakic lens status, and longer DEI were associated with significantly reduced ECD. With respect to DEI, the estimated cell loss was 7 ± 2 cells/mm In 2018, more than 9100 keratoplasties were performed in Germany, twice as many as 10 years earlier in 2008 ,4, the gSome studies also showed an association between male sex and lower ECD ,16, whilIn Germany, the death-to-explantation-interval (DEI) is limited to a maximum of 72 h. In some studies, an increased postmortem interval was associated with chronic endothelial cell loss , whereasSo far, the influence of donor characteristics and especially their combination on the quality of the harvested corneas has not been satisfactorily determined. The main objective of this analysis was to investigate whether and to what extent donor characteristics influence the suitability of corneas for transplantation.In this regard, we investigated the influence of age, sex, lens status, DEI, CPR, and different causes of death on ECD. Another objective of this study was to create a mathematical model to predict the corneal suitability for transplantation aimed at saving eye banks’ resources.The Eye Bank Rhineland-Palatinate, Mainz, Germany, recruits donors within the University Medical Center of the Johannes Gutenberg University Mainz and from eight cooperating hospitals and institutions in Rhineland-Palatinate. After evaluation of the donor´s medical history with regard to the inclusion and exclusion criteria, an interview is conducted with the donor´s physician. If all criteria are met, consent must be obtained from the donor´s next of kin.All data in this retrospective study were obtained from deceased donors; therefore, no ethics vote was obtained in this study.The exclusion criteria for corneal donation of a deceased person at the Eye Bank of the University Medical Center in Mainz are shown in 2 and <2000 cells/mm2 are discarded as optical grafts but can still be used as emergency or tectonic grafts. The specific exclusion criteria for corneal donor grafts at the Eye Bank of Rhineland-Palatinate are shown in The cadavers are kept in the stable temperature of 4 °C, and their eyes are closed. We do not use any artificial tears or ointments to keep them humid.®, B. Braun SE, Melsungen, Germany) into the fornices, the excess is used for a generous disinfection of the ocular environment. After 5 min of exposure time and rinsing with 0.9% NaCl-solution, 0.5 mg/mL gentamicin eye drops  are applied to both fornices. After covering the face with a sterile drape and applying the eyelid retractor, the conjunctiva is lifted with forceps and opened circularly with ophthalmic scissors. Excision of the scleral corneal button is performed with a trephine centered around the cornea; if necessary, the incision is completed with a pair of scissors. The remaining choroidal tissue is removed with blunt, curved scissors. The cornea is then transferred to a storage container, and the procedure is repeated for the other eye. The lens status is assessed and documented during excision.In situ excision of a scleral corneal button is now the standard technique in the Eye Bank of Rhineland-Palatinate, whereas whole globe enucleation is used only in a few exceptional cases. After application of 5 mL 7.5 iodine . Organ culture is performed in a closed system at a temperature of 34 °C room air. Before transplantation, the cornea is transferred to transport medium  supplemented with fetal calf serum (Biomchrom GmbH). The duration of organ culture is limited to a maximum of 34 days .During cultivation, the following tests are performed:ECD measurement is performed between the third and fifth day after explantation. For the ECD measurement, the cornea is placed in a well of a 12-well cell culture plate filled with 0.9 % NaCl. The plate is placed on an inverted phase-contrast microscope and a video camera is used to view the endothelium in the optical region of the cornea as a live image with 10× magnification (Nikon Eclipse TE 2000-S with 1/3 “CCD camera (charge-coupled device)). For evaluation, at least 3–4 still images from the central and paracentral areas are manually created and saved. The size of the saved areas varies depending on the image quality. The image creation and further evaluation is done using a special software  from the company RHINE-TEC GmbH, Duesseldorf, Germany). The measurements of ECD is performed by three experienced employees of the eye bank.2), at least one additional ECD measurement is performed before transplantation. In this study, the first ECD measurement forms the basis of our evaluations since every cornea—whether suitable or later discarded—is subjected to this measurement. If the corneal graft meets the quality standards  is performed to detect abnormalities in corneal morphology  that would affect the corneal suitability for transplantation. Corneas that are not suitable for penetrating keratoplasty may be considered for posterior lamellar keratoplasty.Between the third and fifth day of storage, the corneas are placed in fresh culture medium. From the previous medium, the samples are transferred to two sets of microbiological culture medium , penicillinase (BD) is added to both the aerobic and anaerobic culture medium, and Fastidious Organism Supplement  is added to the aerobic culture medium. All cultures are sent to the Department of Medical Microbiology and Hygiene at the University Medical Center Mainz and are incubated for 14 days. The earliest information about the microbiological status is on the seventh day of incubation, the earliest possible date for transplantation. The characteristics of the donor such as age, gender, cause of death , and cardiopulmonary resuscitation (CPR) received are documented according to the donor´s medical history recorded by the eye bank personnel. DEI is calculated using the time of death from the donor death certificate and eye bank records of processing dates and times. The lens status  is first assessed at explantation.By default, both donor corneas are included in the evaluation. The results are compared with the results of the same tests performed on the right eye data only to ensure that the data are not biased.2; (3) cornea discarded for other reasons, e.g., ECD<2000 cells/mm2 during corneal culture, positive serology, or microbial contamination.In this study, we evaluated associated factors with the continuous variable ECD as a parameter for good corneal quality. In addition, we analyzed three scenarios related to corneal quality: (1) cornea suitable for transplantation; (2) cornea discarded immediately after explantation due to low ECD<1500 cells/mmDescriptive analysis included calculation of relative and absolute frequencies of categorical data and mean and standard deviation for approximately normally distributed data. To investigate associations with ECD, a linear mixed model was used. The model was constructed hierarchically with the donor as the subject. Lens status, sex, cause of death , CPR, age, and DEI were entered as independent variables. For the final simplified model, all non-significant independent variables were removed. 2; Group 3, corneas discarded for other reasons. The Pearson chi-square test was used to examine whether there was a difference in graft usability for the groups for some categorical data . For variables with cell frequencies <5, the Fischer exact test was used. To avoid clustering of alpha errors, the local significance level was set to 0.008 to achieve a global significance level of 0.05 (Bonferroni correction).To identify the possible effects of donor characteristics on corneal suitability for transplantation, we first performed exploratory testing. Of all the noted reasons for discarding the cornea, we considered ECD to be the most informative for corneal quality/suitability. For the categorical analysis, we chose to divide corneas into corneal treatment subgroups as follows: Group 1, corneas suitable for transplantation; Group 2, corneas discarded immediately after explantation because of low ECD<1500 cells/mmBinary logistic regression was used to investigate the association between donor characteristics as independent variables and corneal usability as dependent variable (suitable for transplantation—discarded). For this reason, hierarchical analysis was used to fit competing models, the best of which was selected by comparing the chi-square statistics (Omnibus test of Model Coefficients). The final model included age, DEI, lens status, sex, and CPR. Analysis of the residuals using Cook’s distance, leverage , standardized residuals, and DFBeta revealed no evidence of outliers or influential cases.For all statistical tests, we used SPSS software .A total of 2032 consecutive corneas of 1019 donors harvested between 2014 and 2016 at the Eye Bank of Rhineland-Palatinate in Mainz, Germany, were included in this study. In 1013 cases, both corneas were donated, while only the right cornea was explanted in five donors and only the left cornea in one donor.Of the donors, 585 were male  and 431 were female . The most common cause of death was cardiovascular disease (39.2%), followed by cancer (35.2%) and sepsis (15.9%); 3.6% donors received CPR .2, 20.4% of corneas had an ECD < 1500 mm2, and 18.6% 1500 > ECD < 2000 mm2 . Other characteristics of the included eyes are described in Of all retrieved corneas, 52.4% had an ECD ≥ 2000 cells/mmNine hundred five harvested corneas (44.5%) were not suitable for transplantation, and 46.7% of the male and 41.6% of the female donor corneas were discarded. Of the pseudophakic donor corneas, 58.1% and of the phakic corneas, 36.4% were not suitable for transplantation .2, followed by positive serology and ECD < 2000 cells/mm2 .The causes for discarding the cornea are shown in The most common cause of discarding the cornea was low ECD, which decreased proportionally with donor age. The suitability of corneal tissue according to donor age is shown in The mean DEI was 30.7 ± 16.4 h and ranged from 1 to 72 h. The proportions of corneas suitable for transplantation according to the different DEIs are shown in p < 0.001), sex (p = 0.006), and received CPR (p = 0.002) corneal usability . Phakic lens status, female sex, and CPR positively influenced corneal tissue usability. There were no significant differences in corneal usability with respect to causes of death .The chi-square test revealed significant differences between lens status (p < 0.001) and between DEI and ECD . Pearson correlation analyses showed a significant negative correlation between age and ECD , p < 0.001) less compared with phakic eyes. For aphakic eyes, the results were not significant (p = 0574) and had a wide confidence interval with positive and negative values . Fixed effects estimation shows that corneas from pseudophakic eyes had −375 cells/mm2 compared with corneas from female eyes. Corneas from male donors had a reduced ECD of −188.5 cells/mm2 indicates that corneas lost 6.2 cells/mm2 per year. Age had a significant effect on ECD. The fixed effects estimate of −6.3 cells/mm2 indicated that corneas lost 7.3 cells/mm2 per hour that elapses between death and explantation. DEI had a significant effect on ECD. The fixed effects estimate of −7.3 cells/mmp < 0.001). The large standard deviation (454 ± 124 cells/mm2) implies that there are serious differences between individuals and that other donor characteristics must also be considered as influencing factors. The variable donor, as a random effect, was also significant . The mean ECD values in this study are lower than those obtained in other studies such as Schaub et al. , but the donor population differs in terms of the lower proportion of pseudophakic eyes and the higher proportion of female donors compared with our study. Compared with other studies showing a negative influence of pseudophakia on corneal ECD [2 less in pseudophakic eyes), although a direct comparison with other studies is not possible because of different study designs. In our study, 35.8% of all corneas were from pseudophakic eyes. In the donor age groups of 80–89 years and 90–99 years, 53% and 69% of the eyes were pseudophakic. The mean ECD of pseudophakic eyes was significantly lower than that of phakic eyes  and a mean ECD of 1839 ± 586 cells/mm2 in donors aged 90 years and older (n = 147). The donor age-related ECD loss in our study (6.2 cells/mm2/year) is comparable with the results reported by McGlumphy et al. (5.2 cells/mm2/year). It needs to be discussed whether an age restriction should be applied to corneal donors because of the negative influence of aging on ECD. In our department, as in many others, corneas from old donors are considered necessary to meet the high demand for grafts. Remarkably, as many as 45% of corneas harvested from donors older than 80 years and 49% of corneas harvested from donors older than 90 years were suitable for transplantation. In addition, there are other positive aspects of older donors that should considered, such as the easier preparation and intraoperative handling of the Descemet endothelial complex for DMEK.Age is certainly the best studied factor influencing corneal quality. In Germany, there is no age limit for corneal donation. We found that age has a significant negative impact on ECD results. Interestingly, our results show a lower ECD and a wider standard deviation compared with other studies. Gain et al. reported a mean ECD of 2135 cells/mm80 years . Our stu2 less than those from female donors, as also found in other studies [In our study, donor sex was found to have a significant effect on ECD, with corneas from male donors having an average of 189 cells/mm studies ,16. Howe studies ,17,18, s studies ,32. To the best of our knowledge, the possible influence of CPR on corneal ECD and quality has not been investigated in previous studies. Interestingly, the ECD of corneas from donors who received CPR was significantly higher than that of donors who did not receive CPR. However, this difference could be explained by age because the mean age of all donors who received CPR was 70.5 years, whereas the mean age of all donors who did not receive CPR was 74.7 years. It is conceivable that CPR is more likely to be performed in younger donors with higher ECD than in older, multimorbid patients, some of whom may decline CPR (for example by living will). Nioi et al. proposed the use of the iVue spectral-domain OCT system for in situ postmortem corneal examination in animals . Napoli In summary, the aim of our analysis was to identify factors that significantly influence the suitability of donor corneas for transplantation. Age, male sex, longer DEI, and pseudophakic lens status negatively influenced ECD and thus graft usability. Cause of death did not appear to be relevant to donor quality. To meet the growing demand for donor tissue, we do not recommend limiting the donor age or collection time to 24 h or excluding oncologic or septic donors, as is done in many countries. We propose a model that can be used to predict the “usability” of the donor based on all known parameters to reduce the number of unnecessary declarations and to economize this process.This model takes into account the factors that most influence the quality of the corneas harvested, such as donor age, sex, DEI, and lens status. This model provides an estimate of ECD based on factors that can be determined prior to corneal harvest. However, the donor itself and the general random effect also influence the ECD. Nevertheless, our model predicts a trend for expected ECD. In practice, this may be useful when a qualitative consideration becomes necessary. Occasionally, the situation arises where corneas could be collected from two donors simultaneously, but human or organizational resources only allow corneas to be collected from one donor in the allowed period. In this case, the model could be used to predict corneas with a higher probability of high ECD based on donor-dependent factors.


The majority of adults and children with meningitis and encephalitis have unknown etiologies fostering universal admission, prolonged length of stay, empirical antibiotic and antiviral therapies. A qualitative multiplexed nucleic acid-based in vitro diagnostic test (FilmArray) is helping clinicians identify organisms and improve clinical outcomes.CSF) with WBC >5 cells/mm3, and leftover CSF were available for testing. All CSF specimens underwent testing with the Biofire® Film Array Meningitis Encephalitis (FAME) panel. This multiplex PCR tool utilizes 0.2 ml CSF sample to identify the presence of 14 viral, bacterial and fungal pathogens in 1 hour.Patients presenting between July 5, 2018, to April 26, 2021, with meningitis or encephalitis, cerebrospinal fluid (Haemophilus influenzae (8.3%), HHV6 (8.3%), Streptococcus pneumoniae (8.3%) and HSV1 (4.1%)]. The addition of the FAME panel to the standard of care evaluation increased the known etiologies from 119 (41.7%) to 197 pathogens (69.1%), (p< 0.05). 52 patients (18.2%) underwent a repeat LP . Empirical antibiotic and antiviral therapy were given to 89% and 63% of patients, respectively.Of 5291 CSF specimens screened, 285 (5.3%) met the criteria for meningitis or encephalitis and underwent FAME testing. The majority were adults , male , White , immunocompetent  and  had meningitis presentation. Median age was 41 years (0 mo-100 yrs) and  were admitted to the hospital. Median duration between CSF collection and viral PCR result as 13 hrs for Enterovirus, 31 hrs for HSV, and 57 hrs for VZV. The FAME panel detected a pathogen in 103 patients (36.1%) of whom 76 (73.7%) had a viral etiology. 166 (58.2%) patients were discharged with unknown etiology of whom FAME was positive in 24 (14.4%) [VZV (37.5%), HSV2 (16.6%), Enterovirus (16.6%), A rapid multiplex PCR decreases the proportion of unknown etiologies and has the potential to decrease length of stay and the use of empirical antibiotic and antiviral therapies. Testing with the FA ME panel resulted in pathogen detections not previously recognized and for which treatment is recommended.Rodrigo Hasbun, MD, MPH, Biofire (Speaker’s Bureau) Rodrigo Hasbun, MD, MPH, Biofire  Involved: Self): Consultant, Research Grant or Support


Podarcis raffonei is an island endemic that survives only on three tiny islets, and on the Capo Grosso peninsula of the Vulcano island, thus is among the European vertebrates with the smallest range and one of the most threatened by extinction. This species is declining due to competition and hybridization with the non-native lizard Podarcis siculus, but a regular monitoring program is lacking. Here we assessed the size and status of the Capo Grosso population of P. raffonei on Vulcano. In September 2015 we captured 30 individuals showing the typical brown phenotype of P. raffonei, while one single male showed a green phenotype, apparently intermediate between P. raffonei and the non-native Podarcis siculus. In May 2017, only 47% of 131 individuals showed the typical brown phenotype (P. raffonei-like) and 53% showed the green phenotype (P. siculus-like). Based on N-mixture models and removal sampling the estimated size of the Capo Grosso population was of 800–1300 individuals in 2017, being similar to 2015; available data suggest that the total range of the species could be as small as 2 ha. The frequency of individuals with the typical P. raffonei phenotype dramatically dropped between two samplings with a parallel increase of individuals displaying the green phenotype. Observation on outdoor captive-bred individuals demonstrates plasticity for colouration in P. raffonei individuals from Capo Grosso, with several individuals showing the typical brown pattern in September 2017 and a green pattern in March 2021. Non-exclusive hypotheses, including hybridization with P. siculus and plasticity in colour pattern of P. raffonei, are discussed to explain the phenotypic shifts of the P. raffonei population of Capo Grosso. While genomic evidence is required to reach conclusions and investigate eventual hybridization, it is urgent to undertake a programme for the monitoring and management of this lizard.The Aeolian wall lizard  Monitoring of populations is an essential conservation task. Frequent monitoring is necessary to determine natural fluctuations, to detect declining trends, to ascertain the conservation status of populations and species and to ensure the effectiveness of eventual management measures –4. This Monitoring programs are often limited by accessibility. Difficult access makes surveys more expensive and less feasible, thus the least accessible areas rarely are the focus of regular monitoring, and biodiversity surveys often remain concentrated in the most accessible areas , 8, 9. TPodarcis raffonei  is among the species with the smallest geographic distribution . We performed repeated surveys on this population, documenting shifts in the frequency of phenotypes, and providing the first insights on potential processes. Finally, through the analysis of individuals kept in captivity, we showed how individual phenotypes can significantly change through time, complicating assessments of temporal trends of the species.Given the rapidity of the decline of The study does not involve human or non-human primates. Lizards were manipulated according to international safety standard approved by the Italian Ministry for the Environment. Research activities were authorized by the Italian Ministry for the Environment .Podarcis raffonei is closely related to the Sicilian wall lizard P. waglerianus [Podarcis raffonei is currently reported from only four localities within the Aeolian islands: three tiny islets , and the Capo Grosso peninsula of the Vulcano island [Podarcis raffonei is a medium-sized lizard, with a brown or dark-brown dorsal coloration. According to the species description in the "Fauna d’Italia", P. raffonei individuals can be identified by "the presence of evident dark markings on the throat "; colour of back generally brown with small dark dots and only occasionally olive hints, in males only [The Aeolian wall lizard lerianus , 38 fromlerianus . Podarciles only  Fig 2)..Podarcisles only , 25. Indles only , 25.Podarcis raffonei has suffered a dramatic decline. In the 1950’s-1970’s, this lizard was abundant in several areas of the Vulcano island [P. raffonei in two localities , whereas the introduced Italian wall lizard, P. siculus, was abundant and widespread across the island [P. raffonei have been detected in Vulcano, with the exception of the Capo Grosso peninsula [2; roughly 100 x 50 m) peninsula, connected to the Vulcano island by a narrow isthmus with vertical cliffs, which makes the cape highly inaccessible and only weakly connected with the main island. Such isolation was assumed to hamper the colonization by P. siculus, so that until recent years P. raffonei was the only lacertid inhabiting the cape and its population was deemed to remain stable [During the last decades, o island . Capula o island  performee island . After 2eninsula , 28 whereninsula . As a con stable , 28.P. raffonei was calculated on the basis of the orthophotos of the Italian Geo-portal (http://www.pcn.minambiente.it/mattm/) . The surface of the sites was used to calculate the extent of occurrence of the species . However, the lowest-altitude portions of all sites (< 10 m above sea level) are heavily washed by waves [Information on the surface of the four localities with by waves . We thusby waves . This waby waves . The surP. raffonei on Vulcano. In both occasions, adult lizards were captured by noosing, measured, sexed, and photographed. In 2015, only one capture session was performed , and individuals were immediately released. In 2017, five capture sessions were performed (2–6 people per session). Species identity of captured individuals was assessed on the basis of morphological features  and spring 2017 (end of April-beginning of May) we carried out field surveys in the Capo Grosso peninsula  in order to estimate the size and the status of the last population of features . Podarcithe back . Previouthe back . We alsoN-mixture models [N-mixture models were performed on the basis of four linear transects, each repeated 2–3 times, where we performed visual encounter surveys and we counted the number of active adult lizards. Furthermore, all the captured males were temporally transported in terraria for behavioural and ecophysiological tests and for conservation purposes (unpublished data), thus it was possible using removal sampling [In 2017, we estimated the adult population size of the Capo Grosso population using two approaches: e models  and remosampling  for an isampling . Tail tiAcheta domesticus) and water was supplied ad libitum. Pictures of the dorsal and ventral sides of the lizards were taken every 6–9 months to monitor possible colour changes due to seasonality, except during the 2020 lockdown related to the COVID-19 pandemics.A subset of individuals  showing the typical brown coloration collected during the 2017 sessions was kept and maintained under captive-breeding conditions at the Fondazione Bioparco of Rome (former zoo) for conservation actions. Individuals were hosted in an outdoor facility designed and built especially for containing lizards, preventing their escape and the entry of possible predators or other lizard species form the outside. Spontaneous grasses were left free to grow inside the cages and were managed in order to maintain enough space for lizards to bask. The habitat inside the cages remained the same during captivity period. In winter, heating lamps were provided to simulate natural conditions . Lizards were fed with crickets  in approx. 8 man-hours of sampling . All individuals showed the typical dorsal and ventral pattern of raffonei  with theraffonei .P. raffonei, whereas 76% of individuals showed some features that do not match the typically described phenotype of P. raffonei. These individuals showed a robust body, green dorsal pattern, white or pale orange ventral pattern with dark spots on the throat . Individuals with the green phenotype were larger than the brown ones ; furthermore, a significant interaction between sex and phenotype suggested that the size difference between individuals with the typical brown phenotype and the green phenotype are particularly strong for males . The removal method estimated a total population size of 538 males. The number of captured males (63) was small compared to the total population size estimated by the two models, thus confidence intervals for this estimate are not available. Assuming a balanced sex ratio, as generally observed for P. raffonei [During visual encounter surveys performed in replicated transects in 2017, we detected 85 lizards. The total population size estimated by raffonei , this woraffonei .Forty individuals with the typical brown coloration were captured and maintained under captive conditions for conservation purposes. All the 40 lizards were brown in September 2017 at the first assessment after the capture, and were brown or greyish in February 2018, June 2018, and December 2019. In March 2021, all the 13 surviving individuals  showed the green phenotype .Podarcis raffonei is probably the European reptile with the smallest range (<2 ha). Within such a small range, the Capo Grosso peninsula has a key conservation importance for this species. First, Capo Grosso is the largest site of known occurrence of P. raffonei  populations. As such, the population in Capo Grosso is expected to retain a significant portion of the evolutionary potential of the species [P. raffonei also in two other areas of the Vulcano island , i.e. Vulcanello and Gran Cratere , but more recent surveys based on morphological identification did not confirm the occurrence of P. raffonei in these areas, and only detected the invasive P. siculus [P. raffonei population, accounting for approx. 50% of all the individuals of this species.The Aeolian wall lizard, raffonei  and repr species . Our dat species . The cap siculus . Thus ovP. raffonei phenotype has dramatically changed in this two-year period. In 2015 we only detected one single male with the green dorsal pattern. Similarly, D’Amico, Bastianelli (28) surveyed the Capo Grosso population in March 2016, and only reported the occurrence of P. raffonei. Conversely, in 2017 approx. 50% of individuals did not show the typical P. raffonei features. The morphological features of these individuals match the description of hybrids between these two species [P. raffonei and P. siculus observed by Capula in Vulcano were confirmed to be hybrids based on allozyme analyses [Podarcis lizards can be extremely complex [While the total number of lizards estimated to occur on Capo Grosso has roughly remained stable from 2015 to 2017, the frequency of individuals with the typical  species , 34. Som complex , and the complex , therefoP. raffonei, it seems that the recent arrival of P. siculus-like individuals in Capo Grosso has placed at risk the population of P. raffonei from this site, with a 50% decline of individuals with the typical phenotype in less than two years. However, the interpretation of these data is complicated by the strong phenotypic plasticity observed in some of the captured individuals, which showed the typical brown phenotype in August 2017 and a green phenotype in March 2021. Several non-exclusive hypotheses can be formulated on the processes driving the changes in the in the frequency of P. raffonei-like phenotypes on Capo Grosso. These include decline of P. raffonei due to habitat change and competitive exclusion by P. siculus, or, instead, plasticity in colour pattern of P. raffonei. Habitat remained similar between 2015 and 2017, therefore, it is unlikely that these changes are determined by habitat modifications as suggested for the observed decline of the species in the main island of Vulcano [P. siculus possibly also involving hybridization, and phenotypic plasticity of P. raffonei are the two most likely explanations of the observed phenotype variation [P. raffonei by P. siculus was formulated by Capula [P. siculus in the Aeolian Archipelago, following its introduction, and the relictual distribution of P. raffonei in three tiny islets and a small portion of the Vulcano island. In the same studies, introgressed allele of P. raffonei were detected in 25% of the analyzed individuals of P. siculus on Lipari and a high percentage (~15%) of hybrids between the two species was found on Vulcano, suggesting that also hybridization with P. siculus might have played a role in the extinction of P. raffonei from Lipari and in the observed decline on Vulcano [If the phenotype alone is used to estimate the actual frequency of  Vulcano . Competiariation , 47. They Capula , 39 to e Vulcano , 39. HybP. bocagei, P. siculus and P. waglerianus [P. raffonei. Individuals of P. raffonei from Vulcano island have always been described with brown coloration [P. raffonei can show coloration shifts through time, and adults could have a green pattern during spring that make them extremely similar to previously described hybrids between P. raffonei and P. siculus [P. raffonei can also explain the significant change in the relative frequency of brown (P. raffonei-like) and green (P. siculus-like) phenotypes observed at Capo Grosso during the study period. We highlight that the morphological identification of these lizards has always been extremely challenging. In fact, populations currently belonging to P. raffonei were initially described as subspecies of P. siculus (populations from La Canna and Scoglio Faraglione) or P. waglerianus (populations from Strombolicchio and Vulcano), and only subsequent genetic analyses clarified that these populations did not belong to either of the two species, and instead they represent a distinct species [Phenotypic plasticity of coloration in lacertid lizards is an understudied topic , 48. Whilerianus , 49, 50,loration . However siculus , 34. The species , 39. Thi species –53.P. siculus jointly played in determining the phenotypic changes observed in the population of P. raffonei of Capo Grosso. In order to disentangle between these processes and to evaluate their relative contribution, a comprehensive genomic assessment of these individuals is currently in progress, with the aim of providing an accurate assessment on the occurrence of hybridization and understanding the evolutionary processes underlying the occurrence of individuals with ‘green’ phenotypes in Capo Grosso. Such a genetic assessment requires the analysis of a very large number of loci sampled across the whole genome, given that a limited number of markers does not allow to correctly quantify introgression in recently admixed populations [The hypotheses of competition/hybridization with invasive lizards and phenotypic plasticity are not mutually exclusive. It is thus possible that both plasticity in colour pattern and hybridization with ulations , and eveulations , 55, 56.P. raffonei individuals are actually present is urgently needed. Furthermore, the removal of P. siculus is potentially a useful strategy, but is complicated by the challenges of species identification in the field, and by the difficulty of capturing lizards with approaches that do not harm the native fauna in a very complex landscape where lizards can easily escape. Without a clear conservation strategy, this population may become extinct in a few generations and, given that P. siculus has already been introduced into multiple islets [P. raffonei, but also identify long-term strategies to allow the persistence of this endemic species in the Aeolian Archipelago.In conclusion, this study documents the complexity of assessing long-term trends in microendemic species living in poorly accessible areas, particularly when they show complex patterns of intraspecific variation and plasticity. While robust genomic evidence takes time to be, a simple principle of caution recommends immediate management actions and of standardized monitoring protocols. Accurate genomic screening of populations to identify areas where e islets , the sam


Cerebrospinal fluid is a key biological fluid for the investigation of new potential biomarkers of central nervous system diseases. Gas chromatography coupled to mass-selective detectors can be used for this investigation at the stages of metabolic profiling and method development. Different sample preparation conditions, including extraction and derivatization, can be applied for the analysis of the most of low-molecular-weight compounds of the cerebrospinal fluid, including metabolites of tryptophan, arachidonic acid, glucose; amino, polyunsaturated fatty and other organic acids; neuroactive steroids; drugs; and toxic metabolites. The literature data analysis revealed the absence of fully validated methods for cerebrospinal fluid analysis, and it presents opportunities for scientists to develop and validate analytical protocols using modern sample preparation techniques, such as microextraction by packed sorbent, dispersive liquid–liquid microextraction, and other potentially applicable techniques. Modern differential diagnostics of a wide variety of diseases and pathologies is not complete without analyzing the composition of biological fluids of the body. The most available and studied fluids are blood and urine. Less studied, as well as less available, is cerebrospinal fluid (CSF). CSF performs a number of important physiological functions in the brain and spinal cord, providing metabolic processes between the blood and the brain. Chemical compounds of different structures can penetrate from the blood through the blood–brain barrier (BBB) into the CSF, and then into the brain cells, subsequently affecting the functioning of the central nervous system (CNS) .The routine laboratory study of the CSF composition is usually aimed at the diagnosis of acute infectious diseases of the CNS such as meningitis and encephalitis. The current interest in the study of the CSF composition is due to the rapidly increasing number of neurodegenerative, mental, and other slowly progressive—and in most cases, incurable—diseases. Despite the advances in science and medicine, treatment of these diseases is directed at reducing the symptoms, but not at eliminating the cause of the disease, since the etiologies of most of the common diseases, such as multiple sclerosis ,4,5,6,7,One of the modern concepts in the medical community is the concept of “microbiota–gut–brain” connection. Previously, it was thought that the brain affects the functioning of the body “unidirectionally”, but recent research studies clearly indicate that the composition and function of the gut microbiota have an equal impact on the brain and the CNS. Numerous studies have shown that the composition of the microbiota in healthy volunteers differs from that in patients with various mental, neurodegenerative, and chronic diseases ,16. The The main fact that limits the active study of the composition of the CSF is its inaccessibility. A lumbar puncture is required to obtain the CSF that carries the risk of side complications . The mosDuring the diagnostic lumbar puncture, the volume of the sampled CSF is 2.0–5.0 mL . Subsequent routine analysis includes the determination of cytosis, microbiological studies, and biochemical analysis . Since the total volume of the CSF in an adult is 125–150 mL, any actions aimed at the additional CSF collection or procedure must be justified and approved by the local ethics committee of the institution. For scientific purposes, carrying the CSF analysis out in the residual volume of the sample after clinical and laboratory studies, which is usually about 0.5–1 mL, is highly desirable. Despite all mentioned limitations, the investigation of the CSF composition—in particular, of that of the healthy donors—is an important and crucial issue for the discovering of the new biomarkers.The limited available sample volume requires the use of modern efficient sample preparation techniques and sensitive analytical methods for the determination of metabolites, most often at the trace level. Such methods are chromatographic ones, namely gas (GC) and high-performance liquid chromatography (HPLC) in combination with different mass-selective (MS) detectors (single quadrupole (Q), time-of-flight (TOF), including high resolution equipment and tandem MS/MS or Q-TOF) .Nontargeted metabolic profiling analysis is the initial stage in the search for metabolites that distinguish patients with pathology from patients of the control group/healthy volunteers. The most common task is to identify as many chromatographic peaks in the sample as possible using the total ion current mode ,10,23,24Based on the information provided, the following requirements for the CSF analysis method can be formulated: -the use of the minimum (less than 0.5 mL) volume of the CSF for one analysis;-the quantitative analysis of several target components at the trace level in one analysis.Gas chromatography is the most common method for the analysis of volatile compounds in biological samples. Correctly selected sample preparation conditions lead to the quantitative determination of different classes of chemical compounds in one chromatographic analysis, which meets the above requirements. When comparing the capabilities of GC and HPLC, the lower cost of the GC equipment and the simpler selection of the parameters of the analysis are worth noting. Despite the wider possibilities of the HPLC in the analysis of nonvolatile high-molecular-weight compounds, gas chromatography is the “gold standard” for the tasks of metabolomics—in particular, the determination of low-molecular-weight compounds . Thus, twww.csfmetabolome.ca (accessed on 11 June 2021). From a total of 308 metabolites detected, 53 were identified using nuclear magnetic resonance (NMR) spectroscopy, 41 using GC–MS, and 17 using LC–MS [A large-scale study of the CSF composition  was carrng LC–MS . Later wng LC–MS . As noten = 9), which was performed using GC–MS/MS. Individual plots of postprandial samples of 150 CSF hydrophilic metabolites were positioned similar to the corresponding plots of preprandial samples. The postprandial effects had a far lower impact compared with interindividual variations [The authors also noted the presence of biological variability in the concentration of metabolites between individuals in average ±50%, and in some cases ±100%. This phenomenon should be considered by researchers who study potential biomarkers in the CSF . A largeriations . Thus, bImportant results were obtained describing different effects of preanalytical factors on stability of the proteomic and metabolomics profiles of the CSF. These factors were a 30/120 min delayed storage after the CSF collection at room temperature as the potential delays in the clinic, storage at 4 °C as the time that samples remain in the cooled autosampler, and repeated freeze–thaw cycles. The delayed storage factor led to the increased levels of 49 metabolites, which were analyzed using nontargeted GC–MS, and explained by metabolic processes that occurred because of the remaining white blood cells. The author’s recommendations are to remove white blood cells by the CSF centrifugation immediately after collection, use liquid nitrogen for the snap-freeze of the supernatant for storage at −80°C, and avoid freeze/thaw cycles. Samples should not be left in the autosampler for more than 24 h .The chemical composition of low-molecular-weight CSF metabolites justifies its study using GC–MS in case of compliance with the necessary requirements for sampling, storage, and selection of appropriate and sensitive methods of sample preparation, which will provide quantitative determination of the target components at the required level, taking their potential biological variability among individuals into account.Different groups of authors have made attempts for the metabolic profiling of the CSF using more sensitive types of MS detectors than those described in Application of two-dimensional GC–TOFMS led to the identification of 91 metabolites out of over 1200 detected. Sensitivity was achieved using cryogenic modulation, which concentrated analyte fractions transferred from the first (a 10 m × 0.18 mm I.D. Rxi-5ms column with film thickness of 0.18 µm) to the second column (a 1.5 m × 0.1 mm i.d. BPX-50 column with film thickness of 0.1 µm) . The metTwo groups of authors explored a similar multicomponent biomarker approach ,26 usingCareful attention should be paid to the results of the study, in which metabolic profiling was performed using not common configuration of GC with an orthogonal-accelerated TOFMS with atmospheric pressure chemical ionization interface. A distinctive feature of the chemical ionization (CI) is a softer ionization at the energy not exceeding 5 eV, compared to the electron ionization at 70 eV commonly used in GC–MS systems, which provides less fragmentation of the precursor ion. Evaluation of the analytical parameters  using model solutions led to the successful determination of the 25 different compounds . The applicability of this technique for the CSF metabolic profiling was demonstrated and resulted in 21 identified compounds from more than 300 detected .N-acetylserine, and serine [Tandem GC–MS/MS was applied for the metabolic analysis in pediatric patients and revealed 180 metabolite derivatives in the CSF samples. The main metabolites were 2-ketoglutaric acid, pyridoxamine, tyrosine, 2-propyl-5-hydroxypentanoic acid, 1,5-anhydroglucitol, 2-aminobutiric acid, 2-ketoisocaproic acid, 4-hydroxyproline, acetylglycine, methionine, d serine . Fatty ad serine .l-alanine, l-asparagine, l-glutamic acid, l-glutamine, l-isoleucine, l-leucine, l-lysine, l-methionine, l-phenylalanine, l-proline, l-serine, l-threonine, and O-phosphoethanolamine) were detected using GC–MS in the rat CSF [l-aspartic acid, xylitol, ribitol, 2-keto-d-gluconic acid, 1,4-butanediamine, d-fructose, myoinositol, glucaric acid, hexadecanoic acid, scyllitol, gulose, heptadecanoic acid, linolelaidic acid, trans-9-octadecenoic acid, oleic acid, octadecanoic acid, N-acetyl-d-glucosamine, d-glycero-d-galactoheptitol, galactitol, 5-phenylvaleric acid, benzeneacetic acid, and 1H-indole-2-carboxylic acid) [Metabolomic research studies are also in demand—specifically, studies in animals. A highly sensitive headspace solid-phase microextraction–GC–MS technique was successfully put into practice for the detection of 25 volatile constituents in rabbit CSF after intranasal administration of Asari Radix et Rhizoma frequently used in traditional Chinese medicine . Numerouic acid) .Most of the described investigations required from 25–50 µL ,34,36 toThree investigations appeared to be promising in solving these issues. The analysis with modified vial design and sample workup procedure became applicable to small volume of the CSF (10 µL), and 50 µL of the final mixture was used for GC–MS analysis. The modified vial design reduced the required volume of the insert from 500 to 200 µL, and the smaller amount of derivatizing agent resulted in a reduction to 50 µL of the total volume of the mixture for the analysis. This approach had similar number of metabolites as in the analysis of >100 µL of the CSF, i.e., 73 identified compounds from 93 detected peaks, and was successfully applied for the metabolic profiling of the rat CSF . ApplicaMetabolic profiling resulted in most cases in a number of compounds or groups of compounds that successfully distinguished the compared groups of patients or patients and healthy donors. These compounds are candidate biomarkers for a various type of diseases, including inflammatory demyelinating, neurodegenerative, oncological, infectious, mental, genetic, vascular, and epilepsies .As mentioned in the Introduction section, after metabolic profiling and various statistical and bioinformatics approaches, it is necessary to develop a selective method for the detection of a specific compound or group of related compounds. At this stage, modifications of sample preparation techniques are required to reduce the matrix effect and coextraction of nontargeted compounds.Validation of the developed analytical methods for the quantitative evaluation of the potential biomarkers in a biological matrix is critical for the successful conduct of nonclinical and clinical studies, and it ensures that the obtained data are reliable. Validation includes the evaluation of a number of parameters, such as linearity, lower limit of detection and quantitation (LLOD and LLOQ), selectivity and specificity, sensitivity, accuracy, precision, recovery, and stability of the analyte in the matrix. However, full validation requires a large amount of the biological matrix. In the case of rare matrices, the validation can be performed on a pooled sample from several persons or a model solution that has a chemical composition similar to that of the matrix. We did not manage to find fully validated methods for the determination of the analytes in the CSF, which were performed according to the FDA or EMA guidelines. Several methods, such as recovery, LOQ and LOD values, and linearity will be discussed in this section . However2PO4, 26 µM NaHCO3, 2 µM MgSO4, 2 µM CaCl2, 10 µM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 10 mM glucose and bubbled with a carbogenic mixture (95% v/v O2 and 5% v/v CO2)) was used for the validation of this method for 16 amino acids  according to the Eurachem guidelines. Arginine and histidine were not analyzed because of the thermal instability of their derivatives. The evaluated analytical parameters, such as LOD 0.01–4.24 µM, LOQ 0.02–7.07 µM, intraday and interday precision values, recoveries, and linearity allowed the authors to determine all 16 amino acids in the human CSF samples (n = 16) at the level higher than LOD values, which indicated that the developed analytical method is applicable to solving the task of the quantitative determination of free amino acids in the CSF.The presence and levels of free amino acids in the CSF can be indicators of neurological diseases ,47,64,77Tryptophan is one of the most important amino acid for CNS function. Its metabolism occurs in two main ways: the indole and kynurenine pathways. The indole metabolism is divided into the serotonin  via 5-hydroxytryptophan (5-HTP) and the microbial pathways. 5-Hydroxyindole-3-acetic acid (5-HIAA) or 5-hydroxyindole-3-ethanol (5-HTOL) appears as a result of the serotonin metabolism. The microbial pathway leads to the formation of metabolites containing an indole ring, for example, tryptamine and several indole-containing acids (indole-3-acetic (3IAA), indole-3-propanoic (3IPA), indole-3-carboxylic (3ICA), and indole-3-lactic (3ILA) acids).5-HT is a neurotransmitter, and its related indole derivatives from serotonin pathway are involved in physiological and pathological responses, which are associated with many neurological diseases ,58,59,60In contrast to the described method with classical SPE, a modern microextraction by packed sorbent (MEPS) with C18 was applied for the determination of the indole-containing acids  using only 40 μL of the CSF. The pooled CSF samples were used for the validation and the following parameters were evaluated (according to the FDA guidelines): linearity, recovery, LOD 0.2–0.4 µM and LOQ 0.4–0.5 µM, accuracy, precision, selectivity, and carryover effects. Despite the satisfactory results, only 3 IAA was detected in CSF samples of the patients with the CNS diseases .Several studies describe an importance of the changes in the concentration of the kynurenine pathway metabolites, particularly pyridine-containing quinolinic, picolinic, and nicotinic acids, which are involved in the inflammatory and apoptotic processes associated with the CNS neuronal cell damage and death ,52,53,66n = 138) from neurosurgical patients (n = 84) with different CNS pathology using MEPS and traditional liquid–liquid extraction (LLE). The validation  according to FDA guidelines was performed for both MEPS and LLE, demonstrating the equal possibilities of these sample preparation techniques. Similar results were achieved using 40 µL of the CSF sample for MEPS instead of 200 µL for LLE [Phenyl-containing acids acetic, homovanillic, and 3-(4-hydroxyphenyl)lactic acids), which are mostly microbial metabolites of the tyrosine and phenylalanine, were detected in the CSF samples  is a naturally occurring neurotransmitter and a precursor to gamma-aminobutyric acid (GABA), glutamate, and glycine in certain brain areas. The postmortem examination of the influence of temperature and time storage to in vivo production of GHB was evaluated using traditional LLE and silylation. The validation was performed using 50 μL of the CSF and resulted in LOD 0.5 mg/L, LOQ 0.6 mg/L, and interday and intraday accuracy ≥91%. GHB concentration changes were affected both during postmortem interval in the dead body and during in vitro storage .GABA is the chief inhibitory neurotransmitter and plays an important role in various neurological and mental disorders, in which both elevated and decreased concentrations in CSF may occur. A sensitive, selective, and accurate SID GC-ECNI-MS method for the determination of free and total GABA was developed using 500 μL of the CSF, derivatization in aqueous solution with methylchloroformate, extraction with ethyl acetate, and derivatization of the dried residue with pentafluorobenzylbromide in acetonitrile and triethylamine (for free GABA). Total GABA determination included hydrolysis with sulphosalicylic acid during 24 h. The following analytical parameters were evaluated: LOD <0.005 µM and interassay and intra-assay variability for both free and total GABA. The applicability of the method was successfully demonstrated for the determination of free and total GABA in patients suffering from succinic semialdehyde dehydrogenase deficiency before and during specific treatment . This meN-acetylaspartic acid, a neuron-specific marker that is identified in multiple sclerosis [Different polyunsaturated fatty acids are the components of neuronal and glial membrane phospholipids and participate in the development of Parkinson’s  and Alzhclerosis .Neuroactive steroids are steroids synthesized de novo in the CNS and play a central role in neuronal processes . Allopre2-isoprostanes (F2-IsoPs) and F4-neuroprotanes (F4-NPs) are compounds formed in vivo from the nonenzymatic free-radical-catalyzed peroxidation of essential fatty acids, primarily arachidonic and docosahexaenoic acids, respectively. Since CNS is characterized by a high level of polyunsaturated fatty acids and significant oxygen demand, considering its weak antioxidant defenses, it is also rather liable to oxidative damage caused by reactive oxygen or nitrogen species. Imbalance between free radicals and antioxidants, so-called “oxidative stress”, plays a crucial role in neurodegenerative disorders. F2-IsoPs and F4-NPs, being products of lipid peroxidation, can be biomarkers of oxidative stress and neurodegenerative diseases. The literature indicates that levels of F2-IsoPs and F4-NPs in CSF and brain tissue are elevated in case of such disorders as Alzheimer’s disease [F disease ,82,83,84 disease . For sen disease , and the disease , but forProstanoids, which include prostaglandins and thromboxanes, are the metabolites of the enzymatic pathways of arachidonic acid. These compounds have similar chemical structures but different biological and therapeutic effects. Simultaneous assay of these compounds in the CSF was developed, which included extraction with octadecyl silica gel and two-step purification with silicic acid gel chromatography . Some reThe validated analytical procedures for different metabolites of arachidonic acid are required because of the high demand in their evaluation in different neurodegenerative disorders.Abnormalities in carbohydrate metabolism are of interest in mood disorders studies, as a possible relationship between diabetes and major depression has been shown. Glucose, which serves as an energy source for cells, is conversed to fructose via the polyol pathway with sorbitol being an intermediate compound formed during this two-step process. The CSF sorbitol levels were investigated in patients with bipolar and unipolar mood disorder, and sorbitol concentrations were higher in the CSF of depressed subjects compared to normal controls . SorbitoIndomethacin is a nonsteroidal anti-inflammatory drug, mainly known for its ability to inhibit cyclooxygenase, which is responsible for the prostaglandins production catalysis. Indomethacin is often prescribed to treat inflammation and pain caused by rheumatic and orthopedic diseases or surgery. To determine its concentrations in 250 μL of CSF, a SPE sample preparation technique was applied followed by derivatization with pentafluorobenzylbromide and GC–NICI–MS. The method provides moderate analytical characteristics  with LOQ 0.1 ng/sample. The CSF indomethacin levels in healthy children were found to be 0.2 and 5.0 ng/mL after administering it intravenously .GC–MS was also used for determination of another drug: ELND005 (scyllo-inositol), an endogenous inositol stereoisomer. This drug could be used for Alzheimer’ disease treatment, and its pharmacokinetic behavior in the CSF after oral administration was of interest. A traditional combination of protein precipitation, LLE, and derivatization was used for sample treatment, and validation resulted in LLOQ 0.4 μg/mL  .Analysis of the CSF could also be beneficial for toxicology studies. For instance, morphine can be found in human biological samples and tissues due to the ingestion of heroin or codeine, since these compounds are both metabolized to morphine, or because of exposure to morphine itself. To distinguish whether it was heroine or morphine administering, determination of 6-monoacetylmorphine is often used. However, it converses to morphine rather rapidly, and its concentrations in blood may be lower than the LOD of the method used for 6-monoacetylmorphine determination. Several studies suggested that 6-monoacetylmorphine persists in the CSF and some other human biological samples when compared to blood. 6-Monoacetylmorphine, free morphine, and free codeine levels were investigated in the CSF samples in 25 heroin deaths. The sample pretreatment procedure included such steps as SPE and derivatization and was combined with GC–MS analysis. The method’s LOQ for 6-monoacetylmorphine was 0.001 mg/L; linearity and precision were evaluated. 6-Monoacetylmorphine levels were 6.6 times higher on average in the CSF samples than in blood .Analysis of the diethylene glycol and its potential metabolites  is required because of the human poisoning during misformulation into pharmaceutical products. Sample preparation for acid metabolites from 100 μL of the CSF included traditional LLE and silylation. Sample preparation for diethylene and ethylene glycol from 250 μL of the CSF included extraction and derivatization with pentafluorobenzoyl chloride with the following analysis by GC–NICI–MS. The LOQ values were 0.05–1.0 µg/mL; accuracy and linearity were evaluated .Different low-molecular-weight compounds are required to be detected in the CSF for the diagnosis of the CNS diseases. The literature data analysis revealed the absence of fully validated methods, and it presents opportunities for scientists to develop and validate analytical protocols using modern sample preparation techniques.A small sample volume is one of the main criteria for the analysis of CSF. There are interesting approaches to sample preparation of biological fluids for GC–MS analysis, which use the principle of miniaturization.The method of homogenous liquid–liquid microextraction (HLLME), compared to the classical LLE using significant volumes of organic solvents, is based on the extraction of polar organic compounds from aqueous matrices, including biofluids, with small volumes (microliters) of water-miscible organic solvents. For the phase separation, the salting-out effect is often used, followed by centrifugation. The volumes of biological fluids are usually up to hundreds of microliters and the volumes of polar solvents are often several times less; the weighed portion of the salting-out agent is tenths of a gram. The achievable LOD of analytes are nanograms and tenths of nanograms per milliliter . The metDispersive liquid–liquid microextraction (DLLME) is based on the extraction of analytes with a microemulsion followed by the phase separation. Usually, the volume of a mixture of an extracting solvent and a dispersing solvent is hundreds of times less than the volume of the analyzed solution. Analytes are extracted quantitatively at high preconcentration factors. Extraction equilibrium is established in minutes . VariousAn interesting solution is to combine the capabilities of DLLME and injector port silylation technique for the determination of polar analytes in biological matrices. Aliquots of the extract and derivatizing reagent are injected simultaneously or sequentially into a heated GC–MS injector. A gas phase reaction occurs between the silylating reagent and polar analytes at the injector temperature. This approach reduces the derivatization time (less than a minute), the possibility of derivatives decomposition, and the amount of toxic reagent and solvents used for the process is smaller .18, and/or graphitized carbon black sorbents to eliminate significantly interfering matrix components . QuEChERS, originally developed for the extraction of acidic and basic pesticides from food [QuEChERS  is a two-step process involving liquid extraction  and dispersive SPE (dSPE) using (more commonly) primary secondary amines, Crom food , is alsorom food .Micro-QuEChERS was used to analyze 148 avian blood samples collected in an environmental field study of the impact of rodenticides (applied for the treatment of common vole plague) on the wildlife. The volume of each sample was 250 μL. In combination with GC–MS/MS, this method detected the desired analytes at the level of 1.5 ng/mL . AlthougSolid-phase microextraction (SPME) is a solvent-free sample preparation technique . This meStir bar sorptive extraction was also developed as another solvent-free sample pretreatment technique, and it is actively used for isolation of low-molecular-weight components of different polarity from biological fluids ,104,105.A method of amino acids microextraction from biological fluids (including CSF), based on a combination of hollow fiber SPME and extraction with stir bar sorptive extraction—hollow fiber–stir bar sorptive extraction—was proposed . Hollow −4 to 6 × 10−3 µg/mL. Recoveries from the rat CSF samples ranged from 71.8 to 101.2%. The resulting extraction device could be used 30 times without loss of analyte sensitivity [To extract amino acids, hollow polyvinylidene fluoride fiber was used. A piece of polyvinylidene fluoride hollow fiber was sealed on the one side, and a steel rod was placed inside the fiber. A dispersion system of 0.1 g of silica microspheres in ethanol was introduced into the fiber. After removal (including evaporation) of ethanol, the fiber was sealed on the other side. Before use, the resulting hollow fiber–stir bar sorptive extraction device was washed with acetone and dried. An aliquot of the biological fluid was mixed with ethanol in a 3:1 ratio in order to reduce the surface tension of the sample to facilitate the penetration of analytes into the membrane micropores. A hollow fiber–stir bar was placed in a vial with the sample and extraction was performed while stirring under the influence of a magnetic field for a chosen time. During this time, amino acids selectively with respect to biological macromolecules penetrated from the solution of biological fluid through the pores of the membrane and were absorbed on the surface of silica microspheres due to the formation of hydrogen bonds. Next, the hollow fiber–stir bar was removed from the sample vial, dried until moisture was completely evaporated, placed in another vial, and 0.1 mL of BSTFA and 0.9 μL of a nonpolar solvent were added to extract amino acid derivatives, which should have been formed as a result of silylation. BSTFA molecules also penetrated the pores of the membrane and interacted with amino acid molecules. Derivatization was carried out in a microwave field. The resulting derivatives were extracted with a nonpolar solvent and analyzed using GC–MS. Before the next extraction, the hollow fiber–stir bar was conditioned in distilled water and acetone. The LOD of the studied amino acids ranged from 3 × 10sitivity . Due to 18, C8, silica gel), have been developed and are commercially available. A method for the determination of low-molecular-weight metabolites using a hand-operated device with hypercrosslinked polystyrene was described [One of the perspective sample preparation techniques is a MEPS  that is escribed . MEPS haescribed ,112,113—escribed ,67.The CSF samples are characterized not only by low levels of the analytes of interest, but also by small sample volumes available for analysis. Miniaturization of classic extraction methods, such as LLE and SPE which are often used for analysis of biological fluids, coupled with GC–MS presents new perspectives in the metabolic analysis of the CSF.GC–MS plays an important role in the development of the metabolic analysis of biological fluid samples. High separation efficiency and detection sensitivity, stable retention times, and reproducible mass spectra of analytes make it possible to analyze multicomponent mixtures of low-molecular-weight organic compounds of complex composition and to perform nontargeted and targeted analysis. Sample preparation based on the selective extraction of analytes with respect to interfering matrix components is often required to detect a wide range of components by this method. The complexity of the CSF analysis is caused not only by the low content of the target analytes, but also by the small volume of samples available for analysis. The miniaturization of the traditional LLE and SPE methods in combination with various options for the derivatization of polar analytes presents new possibilities in the metabolic analysis of the CSF using GC–MS.