Visualization and characterization of  AD is characterized by progressive loss of memory and cognitive abilities causing severe interference in daily life of patients and is diagnosed, albeit not in a definitive manner, by careful examination of the patient based on live history, neurological exams, cognitive tests and brain imaging. Definitive diagnosis is achieved post-mortem when the two main histopathological hallmarks of the disease are revealed. They are represented by extracellular deposits of the 2. 4. On the other hand, ex-vivo single plaque imaging presents challenges for standard investigation techniques. Histological sectioning provides good resolution and staining to unambiguously recognize 7. It provides contrast in soft tissues, down to the cellular level with minimum sample preparation and volumetric visualization11. Thanks to its capability to extract virtual slices of the tissue matching the histology resolution, XPCT is gaining importance in pre-clinical investigations as a 3D virtual histology technique, providing quantitative data also in pathological conditions13. In particular, accumulation of 15. Several transgenic mouse models have been developed in order to mimic AD pathology and facilitate the investigation of pathological mechanisms and therapeutic efficacy. These models are characterized by the presence of AD-related human mutations in the APP gene alone or in combination with mutations on the presenilin 1 or 2 genes, all involved in the release of the 21 and AD mice22. However, cerebellum involvement in AD has been attracting much attention recently, due to new evidence showing that cerebellar neuronal cell loss and synaptic alterations are linked to the onset of motor and cognitive alterations24. In addition, the cerebellar cortex presents a highly ordered structure both at macroscopic examination as well as at cellular level, providing an interesting base to explore the properties of plaques as a function of the surrounding tissue, and of the cytoarchitecture of that specific brain area. By exploiting the volumetric information provided by XPCT, plaque morphology as well as orientation in space has been assessed. We found the formation of plaques elongated in shape, with a log-normal distribution of the volume and with a specific orientation depending from the area of the cerebellum where they are localized. In particular, plaques in hemisphere and vermis are oriented mainly perpendicular to the transverse and sagittal planes, respectively. Our findings support the current knowledge about the morphology of AD plaque in cerebellum and the idea that surrounding tissue plays a fundamental role into their development. Moreover, a striking similarity with the plaques of the human cerebellum is found, suggesting that amyloid aggregates in humans develop through similar processes.Alzheimer’s disease (AD) is the most common form of dementia affecting about 50 million people worldwideAn APP/PS1 murine brain and an age-matched wild-type (WT) brain were compared by XPCT. The micron scale resolution achieved allows to distinguish 25. Compact and bright clusters located in the molecular layer are also seen, which are completely absent in the tissue of the WT mouse  shows a log-normal distribution, as shown by the plot of log(P(V)) in comparison with a discrete gaussian distribution with the same mean and standard deviation  in the hemisphere cortex allow a better view on plaque morphology and their arrangement in space. In particular, plaques, marked in blue, are easily detected and their elongated shape appears evident in the top rendering of Fig. The low average sphericity found for In hemisphere, plaques oriented almost perpendicularly to transverse plane have been qualitatively observed. Remarkably, this behavior is confirmed by quantitative analysis. According with the scheme shown in Fig. rest see . The ori28. There is a large interest in imaging of 29. Plaques are easily detected in phase contrast volumes of murine cerebellum as hyper-intense clusters. In particular, plaques with a dense core surrounded by a corona are observed31. The higher density of the core may be explained considering the presence of metals inside the plaque32. Remarkably, inspection of XPCT slices reveals that plaques are concentrated in molecular layer while they are not observed in others layers of the cerebellar cortex. This has been confirmed by histology showing, in addition, few smaller plaques hardly detectable with XPCT in both granular layer and white matter. The difference with XPCT findings can be explained considering the higher grey level value of granular layer in XPCT compared to the molecular one. Thus, a similar grey value hinders plaque detection in the granular layer. Furthermore, plaques in granular layer may be mistaken with Purkinje cells when located at the interface with molecular layer due to similar size and grey value. Differences in the prevalence of plaques between the granular and molecular layer might be related to differences in the structural layers of the cerebellum related to the numbers and size of neuronal cell bodies, axons, dendrites, and terminals33. Irrespective of the origin of amyloid protein in AD brain, the morphological diversity of the deposits could conceivably be related to differences in the size of neurons and/or their processes33. Moreover, the smaller size of plaques outside the molecular layer might be related to a different timing in plaque evolution. It could be speculated that 17. To our best knowledge, no observation of such a peculiar shape is reported for other regions of the brain, suggesting a fundamental role in plaque development of the highly symmetric structure of the cerebellar cortex. Segmentation of plaques has been a fundamental step to support our qualitatively observations and to fully exploit the volumetric information provided by XPCT. In this work, a semi-automatic approach has been used involving human intervention to select a region of interest around plaques and then segmenting with an intensity threshold. This approach has the disadvantage of human intervention being time consuming and thus limiting its application to small volumes. Moreover, Purkinje cells at the boundary of granular and molecular layers were also segmented in order to provide a comparison in sphericity with respect to plaques. In this case a shape-based segmentation approach has been used based on the almost perfect round shape of the cellular body34. Despite the highlighted limitations, the segmentation procedure adopted in this work allowed to extract hundreds of plaques providing a reliable statistic on plaque volume and sphericity. We found a log-normal distribution P(V) of plaque volume. In general, a log-normal distribution of clusters size is frequently found in nucleation and growth processes and it is compatible with a growing rate proportional to cluster size itself36. Remarkably, a log-normal distribution has been also specifically reported for the distribution of surface area of senile human Alzheimer’s plaques in cerebral cortex determined through histology and it agrees with a model where plaque growing rate is proportional to the plaque volume39. An increase in growing rate proportionally to the volume supports a structural model of the plaque as a non-compact aggregate where growing process is not limited to the surface, in agreement with other findings40. This observation also correlates with a low PET signal found in the cerebellum and explained by diffuse nature of 41. Moreover, the peculiar elongated shape of plaques, confirmed by the distribution of sphericity, suggests that a strong constraint in plaque growth is imposed by the surrounding tissue. Supporting this hypothesis, we observed that plaques are aligned in different directions in hemisphere and vermis. In the former, they are normal to transverse plane, while in the latter they are almost normal to sagittal plane, despite with a less strict distribution. Thus, a change in tissue arrangement is reflected in a change of plaque orientation. This observation supports the idea that plaque evolution is driven by the surrounding tissue21. In particular, the compact and ordered arrangement of the cerebellar tissue may physically limit the available space forcing plaques to grow along a specific direction. Further studies are currently on going to investigate plaque morphology also in different brain regions, exploiting 3D visualization provided by XPCT.The presence of deposits of 43. Mice were all drug and behavioral test naive and the experiments were all conducted during the light cycle. All animals were housed in a SPF facility in groups of 4 in standard mouse cages containing sawdust with food (2018S Harlan diet) and water ad libitum, under conventional laboratory conditions  and a 12/12 hour light/dark cycle (7:00 am–7:00 pm). The research protocols were reviewed and approved by the Mario Negri Institute Animal Care and Use Committee, that includes members specific for ethical issues, and the Italian Ministry of Health. The Mario Negri Institute for Pharmacological Research adheres to the principles set out in the following laws, regulation, and policies governing the Care and Use of Laboratory Animals: Italian Governing Law ; Mario Negri Institutional Regulations and Policies providing internal authorization for persons conducting animal experiments ; the NIH Guide for the Care and Use of Laboratory Animals (2011 edition) and EU directives and guidelines (EEC Council Directive 2010/63/UE). The statement of Compliance (Assurance) with the Public Health Service (PHS) Policy on Human Care and Use of Laboratory Animals has been reviewed (9/9/2014) and will expire on September 30, 2019 .The APPswe/PS12 to drastically reduce bleeding. Brains were removed, placed in paraformaldehyde for 24 h, and stored in 70% EtOH at 4 °C until analysis. After XPCT data acquisition the brains of the wild-type and APP/PS1 mice were sectioned along both coronal and sagittal planes (20 μm thick) and incubated for 1 h at room temperature (RT) with 10% normal goat serum (NGS) then overnight (O/N) at 4 °C with primary antibodies . After incubation with the appropriate biotinylated secondary antibodies , immunostaining was developed using the avidin-biotin kit (Vector Laboratories) and diaminobenzidine  as chromogen.One mouse for each condition (APP/PS1 and WT) has been used for XPCT experiments. Both mice were killed under CO45. The brain harvesting procedure was submitted to and approved by the Ethics Committee of the University of Pavia in the context of the InveCe.Ab study46. Joining the donation program is a personal decision, and complete awareness is needed.For a comparison with human histopathology, a case of severe amyloid deposition was selected from the Abbiategrasso Brain Bank (ABB) series. The histology section comes from a case of 84-year-old woman who died with a clinical diagnosis of Major-NCD(dementia) due to probable Azheimer’s Disease. She presented a typical clinical onset with memory impairment and progressive involvement of other cognitive domains. During the intermediate phase of the disease, she also manifested behavioral and psychotic symptoms. Neuropathological examination revealed widespread amyloid deposition corresponding to a Thal 5 stage , associated with meningeal amyloid angiopathy, intermediate Alzheimer TAU pathology (Braak IV TAUopathy), limbic sinucleinopathy and limbic TDP-43 deposition and moderate basal ganglia small vessel disease. The neuropathological definite diagnosis of this case is dementia due to multiple aetiologies with severe and widespread amyloid deposition . In line with our institution’s Human Research Ethics Committee and the BrainNet Europe’s Code of Conduct, the ABB performs its activities following ethical standards30. Phase contrast tomography measurement was performed with 17 keV monochromatic radiation in free space propagation mode with a propagation distance of 5 cm. Images were detected with a PCO edge camera , in Villigen, Switzerland52. Segmentation process is composed of two steps. The first requires user intervention. Exploiting the projection of maximum intensity value of a small number of slices plaques are easily identified by visual inspection and a circular ROI is created around it. This procedure avoids creating large ROIs also for small plaques increasing the risk of including other features from the tissue and blood vessels. The procedure is then repeated for each plaque and each group of projected slices. Thus, cylindrical VOIs with different diameters and height depending the number of projected slices are created around each identified plaque. Finally, the histogram of grey values of voxels included in all VOIs is calculated. In the histogram two different peaks can be easily detected and assigned to background of the tissue and plaque, respectively. Then, an intensity-based segmentation extracts binary mask of plaques8. We note that even if a small blood vessel is found in the extracted VOIs, the large difference in brightness compared to plaques prevents its inclusion in the segmented features  and sphericity (plaques and Purkinje cells) of each binary connected region. The sphericity is calculated as the ratio between the surface area of a sphere with the same volume of the given binary region and the surface of the region itself. A perfect sphere has 54. Final images have been created with ImageJ and Inkscape56.Quantification of volumetric data was performed with custom routines developed in Matlabures see . In addiSupplementary Information


Sharing the benefits of laboratory accreditation could help more countries within sub-Saharan Africa overcome existing challenges to achieve accreditation and robust quality systems. This study investigated the impact of ISO/IEC 17025 accreditation on the performance of an NMRA Quality Control laboratory to provide evidence of improved quality compliance within a low-resource setting.The number and severity of nonconformities generated during an audit of a medicine testing laboratory indicates its level of quality compliance. Quality standards are established and maintained to ensure the reliability of laboratory test reports. The National Medicines Regulatory Authority (NMRA) Quality Control laboratories assess the quality of medicines used by the populace as part of their regulatory function. Although countries desire to have reliable medicine testing facilities, accrediting a national laboratory to international standards poses financial and technical challenges for many low-income countriesPre- and post- accreditation audits of nonconformities for management and technical requirements of the ISO/IEC17025:2005 standards were evaluated from a Quality Control laboratory in the National Agency for Food and Drug Administration and Control (NAFDAC), located in Nigeria, West Africa. The following research questions were addressed: “does accreditation impact the adherence to quality standards?” and “does accreditation decrease the severity of nonconformities in Quality Control laboratory audits?”2 = 74, p-value = 9.99e-05, r = 0.67). Further examination of audits from the years 2013 through 2018 audits also revealed a reduction in the number of nonconformities . A reduction in the number of major observations and a decrease in the severity of nonconformities was also observed.Statistical analysis of the pre- to post- accreditation audits from the years 2013 through 2017 revealed a significant decrease in the total number of nonconformities (χA higher level of quality compliance was exhibited for the laboratory during the post-accreditation years. Overall, ISO/IEC 17025 accreditation of the NMRA Quality Control laboratory resulted in improved reliability of test reports and enhancement of the laboratory quality system.The online version contains supplementary material available at 10.1186/s12913-020-05934-8. Institutions that conduct medicines testing apply international standards to improve the processes in their Quality Control laboratories. Laboratory accreditation can help ensure the quality of analytical reports generated by such testing facilities. In West Africa, like many parts of the world, the Quality Control laboratories of the National Medicines Regulatory Authorities (NMRA) have the task of verifying the quality and purity of medicines. Government and private institutions submit pharmaceutical products for testing to the national laboratories for regulatory compliance and other purposes. Accreditation of NMRA Quality Control laboratories provides credibility to the results generated by these government agencies and helps build practices that support the reliability of data across multiple accredited laboratories .To attain this international recognition, laboratories develop their quality procedures using standards such as ISO/IEC 17025 and guidance from the World Health Organization (WHO), namely ‘Good Practices for Pharmaceutical Quality Control Laboratories’ (GPPQCL) . The ISOTesting laboratories develop and maintain their systems through accreditation to international quality standards. A laboratory’s accreditation confirms its competence to carry out tests or calibrations aligned with recognized standards . EstabliIn a similar manner, educational and public health institutions that offer testing facilities also seek national and international recognition for competence in services they provide by attaining accreditation. The following reports from higher education institutions (HEI) provide an evaluation of the HEI levels of preparedness to attain ISO/IEC 17025 accreditation. Putri and colleagues, from Sebelas Maret University in Indonesia, conducted a gap analysis for the institution’s testing laboratories . SimilarIn further efforts that endorsed accuracy of test reports through accreditation or certification, several researchers examined laboratories handling biological samples. The Korean certification body reported that the four-year variance index score was significantly different between laboratories with or without accreditation . Their rAfter review of several studies, however, a paucity of literature related to the medicines’ regulatory space was observed. This region of the world encounters several barriers and cultural challenges in implementing international standards . BurdickDespite the studies noted above, there are limited reports on the performance of Quality Control laboratories in sub-Saharan Africa. The purpose of this study, therefore, was to examine empirical data and evaluate the impact of ISO/IEC 17025 accreditation on a Central Drug Control Laboratory (CDCL) of the National Agency for Food and Drug Administration and Control (NAFDAC), Nigeria’s NMRA.1. Show that the accreditation process influenced laboratory performance.2. Determine if some types of nonconformities (major and minor) improved more, or less than others.3. Determine if there was evidence of a significant post-accreditation change in the number (#) of nonconformities (2017 vs. 2018).The present study was descriptive and comparative in design. It evaluated pre- and post- accreditation audit nonconformities (NCs) for management and technical requirements of the ISO/IEC 17025:2005 Standards. Due to the limitation of publicly available data, a convenience sampling method was used to select an NMRA Quality Control laboratory in West Africa with ISO/IEC 17025 Accreditation. Data in audit reports from a pre-accreditation year (2013), were compared to two post-accreditations years (2017 and 2018). Parametric methods were used to compare the numbers and types of non-conformances obtained from the laboratory audit reports reviewed. Certified true copies of the audit reports were provided by the Quality Assurance unit of the laboratory. The goals, hypothesis, and research questions for the study were:ISO/IEC 17025:2005 Accreditation improves the quality management systems in a NMRA Quality Control laboratory.Does accreditation of a Quality Control laboratory lead to improvement in the adherence to quality standard (ISO/IEC 17025:2005).0: number (#) of nonconformities post-accreditation audits = number (#) of nonconformities pre-accreditation auditsHA: # of nonconformities post-accreditation audits < # of nonconformities pre-accreditation auditsHDoes accreditation decrease the severity of nonconformities, as defined by major/minor observations or citations, in quality control laboratory audits?0: # of major/minor nonconformities (post-accreditation audit) = # of major/minor nonconformities (pre-accreditation audit)HA: # of major/minor nonconformities (post-accreditation audit) < # of major/minor nonconformities (pre-accreditation audit)HIn preparing the NAFDAC CDCL for accreditation, the United States Pharmacopeia Promoting the Quality of Medicines team (USP PQM) used the 2005 standard to assess the status of the laboratory in 2013 . ThereafThe laboratory was accredited in eleven test scope areas in 2015, with an increase to sixteen areas of scope in 2018. Details of the test scope areas can be found at the International Laboratory Accreditation Cooperation (ILAC) website .The instrument used for the laboratory assessment was the ISO/IEC17025:2005 Standard . The docRecently, many ISO standards were reviewed to align with the requirements of the ISO 9001 ‘parent’ document and a current version of the ISO/IEC 17025 standard was published to replace the older 2005 version. Several authors –37 have For ISO/IEC 17025:2005, all activities related to legal, financial and other administrative responsibilities in running a laboratory are contained in the management requirements, while those tasks that affect how tests are conducted are contained in the technical requirements. Audits are commonly used to assess the compliance level of a laboratory and include the identification of nonconformities when the laboratory fails to meet standard requirements –41. NoncUnlike an accreditation audit, post- accreditation assessments might not include a detailed check for each item in the 25 clauses of ISO/IEC 17025. This risk-based approach for most post-accreditation audits is based on an evaluation of the internal audit reports from the laboratory quality system. The high-risk areas identified from the internal audit reports provides areas of focus for the current audit.Laboratory audit reports from assessments performed by ISO/IEC 17025 certified auditors were evaluated and secondary data analysis was conducted on one pre-accreditation report from 2013 and two post accreditation reports from the years 2017 and 2018. At the time of this study, though CDCL has undergone other audits, the three reports used for the analysis were the only ISO 17025:2005 standard assessments they had undertaken. All reports were obtained from the Quality Assurance unit of the NMRA. The laboratory audit reports were evaluated by grouping the identified nonconformities into the corresponding ISO/IEC 17025 clauses for management [4.1–4.15] and technical [5.1–5.10] requirements. The nonconformities were also categorized as major, minor and OFI (opportunity for improvement) as documented in the audit reports. The number of pre- and post-accreditation nonconformities recorded in the audit reports were analyzed. Effect sizes were used to compare the direction and relative magnitude that the independent variable  had on the dependent variable (pre- and post-accreditation accreditation number of nonconformities).The numbers and severity of nonconformities in each year audit report were counted manually, and tables of the total counts were created. The following assumptions were made: each year’s findings were drawn from a normally distributed population of audit observations and the number of nonconformities in the three audit reports have a common variance.p-value, robust to small cell-counts, compared the total nonconformities between pre- to post-accreditation years. All statistics in our study were computed using Microsoft Excel 2010 edition, R studio [Analysis of Variance (ANOVA) was used to test the null hypothesis that all group means are the same, or the number of nonconformities were the same for the pre- and post-accreditation years at 95% confidence interval, α = 0.05. A Chi-square contingency test with simulation-based R studio  and effeR studio .Does accreditation of a Quality Control laboratory lead to improvement in the adherence to a quality standard (ISO/IEC 17025:2005)?ISO/IEC 17025:2005 standard has 25 clauses; activities related to legal, financial and other administrative obligations in running a laboratory are contained in the management requirements while tasks related to testing activities are contained in the technical requirements. Table p-value . Moreover, the high coefficient of determination, R2, indicated that the reduced total number of citations was attributable to the difference between the pre- and post-accreditation audits.As shown in Table p-value, robust to small cell-counts, was used to compare the total nonconformities between pre- to post-accreditation years and determine which audit years had significant changes. Total nonconformities between ‘2013 vs 2017’, ‘2013 vs. 2018’ and ‘2017 vs. 2018’ audit reports were compared. As shown in Table 2 = 73.96, p = 9.99e-05, α = 0.05); similarly, the decrease from 2013 to 2018 was significant . There was no significant difference, however, in the total number of nonconformities post-accreditation from 2017 to 2018 .A Chi-square contingency test with simulation-based p-values (Table r = 0.67), similar to the 2018 audit  as shown in Table Following the observed decrease in nonconformities Figs.  & 2 and es Table , effect p-values obtained for both scenarios, there was evidence to reject the null hypothesis that there was no difference in the number of nonconformities pre and post- accreditation. Therefore, the alternative hypothesis was retained since the numbers of nonconformities were lower than in the pre-accreditation era. Furthermore, the slight increase in the number of nonconformities in the post-accreditation years’ audit from 2017 to 2018, see Table 2 = 2.33, p = 0.198, α = 0.05) and suggests the laboratory had similar citations in the post accreditation years.Following the significant Does accreditation decrease the severity of nonconformities in Quality Control laboratory audits?In considerations for severity, the citations that have the greatest potential to negatively impact the quality of laboratory testing are termed major. Minor nonconformities have lower risk outcomes, while other observations are termed opportunity for improvements (OFI). The Tables S10, S11, S12, and S13  as shown in Table p = 0.2722, α = 0.05), which suggests that the mean number of nonconformities post-accreditation years were the same . Examination of the second research query—does accreditation decrease the severity of nonconformities? —the Chi-squared contingency evaluation between audit years, showed a significant decrease in severity of nonconformities in the post- accreditation years.Attaining a laboratory accreditation comes with several costs, including facility upgrades, procurement of new equipment, calibration and maintenance schedules, as well as building capacity for personnel. These costs often form barriers for lower-resource settings to attain international standard requirements. The NMRA in the current study received support from the USP PQM program , sponsorThe NAFDAC laboratory faced other hurdles common to the region, such as political and cultural challenges. Tene and colleagues (2018) highlighted similar barriers faced by African countries in adopting ISO standards. These bulwarks include weak institutional frameworks, poorly managed-donor-funding, and low technical capacities . DespiteAlthough the observed increase in the number and severity of nonconformities from 2017 to 2018 were not significant, it was expected that these indices should have continually declined, in contrast to the audit reports. This will be an area of focus for further research for subsequent years’ audit trends.Overall, the accreditation process improved the laboratory’s performance as evidenced by a significant decrease in the number of nonconformities from 2013 to later years 2017 and 2018. Furthermore, there was a reduction in the severity of nonconformities after laboratory accreditation. In summary, the laboratory quality management systems improved after ISO/IEC 17025 accreditation, consistent with previous research conducted in other types of laboratories , 43, 52.Admittedly, a lower sampling error would have occurred with a larger sample size. In addition, a random sampling would have been performed if the researchers had more options of publicly available data from other NMRAs Quality Control laboratory audits. At the time of this study, though this NMRA laboratory had undergone several audits from various inspection bodies, they were audited only thrice with the ISO/IEC 17025:2005 standard. Since there are variations in the requirements of different laboratory standards, this study assessed only the audit reports related to the ISO/IEC 17025:2005 standard. The three audit reports reviewed, however, covered all clauses under this standard against which the laboratory’s performances were measured.The present study aimed to investigate the impact of ISO/IEC 17025 accreditation on the quality management systems of an NMRA quality control laboratory as a means of improving the reliability of analytical reports emanating from testing activities of such labs. The study provided some evidence of improved quality compliance from a laboratory in a lower-resource setting after attaining ISO/IEC 17025:2005 accreditation. Therefore, the accreditation process improved the laboratory performance.Also, laboratory accreditation requires commitment from upper level management to fulfill most of the requirements in the ISO/IEC 17025 standard. Together with other technical components, the major and minor nonconformities in a laboratory testing environment can be significantly reduced.The following recommendations are provided for future consideration. Mutual recognition of laboratory Quality Control reports within NMRAs in sub-Saharan Africa would enhance harmonization buy-in within the region and positively impact the cost and timeliness of medicines regulation. Common recognition of analytical reports should be fostered among member countries which attain international accreditation for their NMRA Quality Control laboratories.Collaboration within the sub-region may have produced more robust results if more countries’ NMRA Quality Control laboratories released their results  for pre- and post -accreditation evaluation of the ISO/IEC 17025 audits.Additional file 1: Table S1. Management Requirements 2013 Audit Summary. Evaluation of Quality Management System Based on ISO/IEC 17025 Standard. Date (s) of Inspection / Audit- June 17-22, 2013. Table S2. Technical Requirements 2013 Audit Summary. Evaluation of Quality Management System Based on ISO/IEC 17025 Standard. Date (s) of Inspection / Audit- June 17-22, 2013. Table S3. Summary for Evaluation of Quality Management System Based on ISO/IEC 17025 Standard 2013. Table S4. ISO/IEC 17025 Surveillance Audit - Date (s) of Inspection / Audit- December 14-15, 2017. Table S5. Summary for ISO/IEC 17025 Surveillance Audit 2017. Table S6. ISO/IEC 17025 Mock Audit - Date (s) of Inspection / Audit- September 24-26, 2018. Table S7. Summary for ISO/IEC 17025 Mock Audit (24-26 September 2018). Table S8. Management Requirements Nonconformities (NCs) (Pre - Post Accreditation). Table S9. Technical Requirements Nonconformities (Pre - Post Accreditation). Table S10. Types of Management Requirements Nonconformities ) Pre-Post Accreditation. Table S11. Types of Technical Requirements Nonconformities ) Pre-Post Accreditation. Table S12. Summary of the Severity of Types of Nonconformities ) Pre- and Post-Accreditation. Table S13. Summary of Severity of Nonconformities (NCs) Pre-Post Accreditation. Figure S1. Management Requirement Nonconformities Decreased Pre - Post Accreditation. Figure S2. Decline in Technical Requirement Nonconformities Pre - Post Accreditation. Figure S3. Management Requirement Nonconformities (NCs) Decreased Pre - Post Accreditation. Figure S4. Decrease in the Types of Management Requirement Nonconformities ) Pre-Post Accreditation. Figure S5. Decrease in Technical Requirement Nonconformities ) Pre-Post Accreditation. Figure S6. Impact of Accreditation on Severity of Nonconformities (NCs) Illustrates Decline Pre-Post Accreditation Years.


As highlighted by European statistics, the employment of donor oocytes is a growing option for women who cannot make use of their own gametes. As the potential recipients are continuously increasing in number, a donor programme which satisfies this demand is mandatory. Improvements in cryopreservation techniques, like oocyte and embryo vitrification, have led to the overcoming of the sequence of stimulation-retrieval-transfer both from a spatial and a temporal point of view, with the development of cryobanks of oocytes permitting crossborder donation. However, while some studies report comparable success when using vitrified and fresh oocytes we still need to investigate whether the use of fresh oocytes give higher live birth rate than cryopreserved ones, when the same number of oocytes are given. The performance of embryo cryopreservation, conversely, seems to be more reliable. A novel approach based on the shipment of frozen sperm from the recipient’s country to the oocyte donor’s one, where fresh oocytes are inseminated and the resulting embryos frozen and transported back to the referring IVF centre to perform a frozen embryo transfer may be a good strategy. We believe that the use of frozen embryos from fresh oocytes could be associated with a higher cumulative live birth rate per cycle, while favouring personalised oocyte recipient care with a flexible number of oocytes assigned and limiting the burden of travelling abroad. In vitro fertilization (IVF) with donor oocytes is an increasingly widespread therapeutic option for women who cannot make use of their own oocytes . There cEuropean statistics highlight that there is an increasing need for donated oocytes. Among 39 countries in Europe offering ART, a total of 56,516 egg donation cycles were reported in 2014, with a steep increase in treatment numbers since 2013 (+ 40.4%) .As the number of potential recipients is continuously increasing, the major difficulty in establishing a donor oocyte program is the limited availability of donor subjects. Donor recruitment is challenging, since it depends on many factors . Donors usually need to be younger than 35 years old to reduce the risk of chromosomal abnormalities in oocytes . MoreoveMany countries do not allow donors to be compensated financially, except for a reasonable reimbursement of their expenses and this will not make easy to promote the need of donating oocytes among the general female population. A possible solution to the lack of donors could be oocyte sharing, which means that women undergoing IVF for infertility could donate some oocytes to a recipient. Oocyte sharing, however, raises several ethical and medical concerns. A “shared” oocyte donation programme is limited by the very low number of oocytes available for donation and by the quality of the oocytes, which depends largely on donor’s age and, in the case of donation by an infertile woman, on her infertility etiology. Furthermore, IVF patients often wish to maximise their chances of pregnancy by using a significant number of their own oocytes and by cryopreserving supernumerary embryos for their own use .Unlike other countries with more restrictive laws, oocyte donation has been regulated in Spain since 1988. Spain is the world leader in organ donation and transplantation; this success is due to several factors, such as adequate legislation, widespread public campaigns, a systematic approach to potential donations in hospitals, the presence of a donation coordinator, and a social conscience that promotes organ donation, in which oocytes are included. The procedure is compensated with a flat-rate amount that takes into consideration several factors . This amount, which is about 1,000 euros per donation (irrespectively of oocyte number), is the same in every clinic and has been suggested by the national bioethics committee. The donor’s identity is protected by anonymity and the greatest possible phenotype similarity between the donor and the recipient may be offered by an extensive donor bank of different nationalities which enables the matching of the phenotypical features of patients of different ethnicities.While in Spain  the donor’s identity is protected by anonymity, in many other countries the debate about disclosure of donor conception is not new  While maWhen gathering and analysing data concerning ART regulations and policies in the European Union, it is easy to come to the conclusion that a lot of disparities  exist among countries and even within countries. These disparities often represent obstacles for accessing ART and can, indeed, contribute to inequalities between patients accessing reproductive health care services. Reproductive health could therefore be a fertile ground for so called “health tourism” or, in this specific case, “reproductive tourism.”. “Cross-border reproductive care” refers to patients travelling from their home country to another one in order to get fertility treatment through ART. The main drivers for fertility tourism are legal regulations, or lower price and higher success reported in foreign countries when compared to the home country. In countries in which donors’ availability is still insufficient to cover the therapeutic demands, patients could therefore be referred abroad for treatment.www.iss.it/pma, data from 2014 to 2017). Nevertheless, Italian patients and clinicians face the problem of very limited availability of oocyte donors at a local level. Every year thousands of couples travel abroad to seek treatments with heterologous gametes  treatments reported, which in 2014, for the first time, exceeded that of fresh transfers .The revolution we are witnessing with the development of cryopreservation techniques is the freedom to move away from the standard sequence of ovarian stimulation-retrieval-transfer, to which we were previously bound, in order to provide the best chances of success . Oocyte However, while some studies report comparable success rate when using vitrified and fresh oocytes , this doCompared to oocytes, the performance of embryo cryopreservation seems to be more reliable as it shows reduced centre-dependence since results published in the literature are very consistent . BecauseRecently, a novel approach based on frozen embryos instead of frozen oocytes to satisfy the increasing request of crossborder oocyte donation has been proposed by our group  Figure .Unlike the routine of most cryobanks ,it allowIn our recent article based on 630 patients undergoing oocyte donation cycles with frozen embryos from fresh oocytes, the live birth delivery rate was 30.6% after the first embryo transfer. The conservative and optimistic live birth delivery rate after the second frozen embryo transfer were 39.3% and 48.7%, respectively. Importantly, after the first and the second embryo transfer, the percentage of patients who still had at least one cryopreserved embryo was 85.3% and 73.1%, respectively .Recently, another Italian experience but this time based on the shipment of vitrified eggs has been published . In thiswww.iss.it/pma)  programme seems to provide several advantages over the shipment of vitrified oocytes. We think that the use of frozen embryos from fresh oocytes could be associated with a higher cumulative LBR per started cycle when compared to a similar number of vitrified oocytes; this may be due to true loss of developmental ability of vitrified oocytes, imperfect warming techniques in the receiving centres and the difficulty of shipping a tailored number of assigned oocytes to the patient’s needs. In the last reports of the Italian IVF registry, data relative to the pregnancy rate with the use of vitrified donor oocytes have been reported. In 2017, 3089 such cycles were performed (more than twice the cycles performed in 2015) with a pregnancy rate per couple treated of 36.0% (.it/pma) . The strA recent updated analysis of our clinical activity including more than eight hundred patients showed a live birth rate per transfer as high as 37.2% after the first embryo transfer . Of noteThe use of fresh oocytes enables personalised care for the oocyte recipient, with a flexible number of oocytes assigned. This personalisation is more complicated than the procedure carried out with fixed pre-prepared packs of oocytes, and this is probably the reason why cryopreserved oocyte donation cycles may perform more poorly than fresh donor oocytes. When using frozen oocytes, in fact, a standard number of 6-8 oocytes is usually assigned to a couple. In our approach, on the contrary, clinicians have the possibility to personalise the number of oocytes, for example if a male factor or low fertilisation risk is assessed. In our already published experience , we repoIn any case, shipment of both gametes and embryos has ultimately very positive aspects from a patient’s perspective, as it leads to easily accessible treatment in the patients’ own cultural and medical context, and to the reduction of expenses for traveling abroad.There have been a few criticisms towards this approach. First, it has been suggested that the use of frozen embryos is associated with decreased pregnancy rates compared to the one reported for oocyte donation cycles (when based on fresh embryo transfer) in the principal national and international IVF registries . FurtherAccording to our national data collected from 2015 to 2017, there has been a widespread take-up of TOD, indirectly confirming its efficacy: in this period almost 5,000 and more than 7,000 cycles using frozen embryos from fresh oocytes and vitrified donor oocytes respectively were initiated . It is iTransportation of eggs and embryos between different international laboratories has permitted an increased accessibility to oocyte donation in countries with low availability of donors. In our experience, many couples have declared that they would have never travelled abroad for an ART treatment due to high costs of flights and hotels, insufficient knowledge of foreign languages, and low experience of travelling abroad. For these couples, the success rate of 40-50% with TOD must be compared to 0%, which is resulting from the non-adherence to the egg donation programme . From thOf course, TOD may be based on fresh or frozen eggs; so we propose to define “fresh transnational oocyte donation” (fTOD), the programme based on shipment of sperm and retrieval of frozen embryos, and “vitrified transnational oocyte donation” (vTOD), the one based on importation of vitrified eggs .We can h


Functional normalization increased the concordance of methylation values among technical replicates in both CSF and blood. SVA improved the asymptotic behavior of the test of association in a simulated EWAS under the null hypothesis. To determine the suitability of blood as a surrogate for CSF, we calculated the correlations of adjusted methylation values at each CpG between blood and CSF globally and by genomic regions. Overall, mean within-CpG correlation was low (r < 0.26), suggesting that blood is not a suitable surrogate for global methylation in CSF. However, differences in the magnitude of the correlation were observed by genomic region  and orientation with respect to nearby genes . In conclusion, the correlation analysis and QC pipelines indicated that DNA extracted from blood was not, overall, a suitable surrogate for DNA from CSF in aSAH methylomic studies.One challenge in conducting DNA methylation-based epigenome-wide association study (EWAS) is the appropriate cleaning and quality-checking of data to minimize biases and experimental artifacts, while simultaneously retaining potential biological signals. These issues are compounded in studies that include multiple tissue types, and/or tissues for which reference data are unavailable to assist in adjusting for cell-type mixture, for example cerebral spinal fluid (CSF). For our study that evaluated blood and CSF taken from aneurysmal subarachnoid hemorrhage (aSAH) patients, we developed a protocol to clean and quality-check genome-wide methylation levels and compared the methylomic profiles of the two tissues to determine whether blood is a suitable surrogate for CSF. CSF samples were collected from 279 aSAH patients longitudinally during the first 14 days of hospitalization, and a subset of 88 of these patients also provided blood samples within the first 2 days. Quality control (QC) procedures included identification and exclusion of poor performing samples and low-quality probes, functional normalization, and correction for cell-type heterogeneity via surrogate variable analysis (SVA). Significant differences in rates of poor sample performance was observed between blood (1.1% failing QC) and CSF (9.12% failing QC;  The epigenome-wide association study (EWAS) approach has emerged in recent years as a hypothesis-free method for investigating the associations between epigenetic marks, such as DNA methylation, and human phenotypes. Challenges pertaining to the cleaning and processing of methylomic data persist, including issues related to sample quality, controlling for cell type heterogeneity, comparing methylomic profiles across tissue types, and modeling dynamic changes in methylation over time . Here, wM-values , were assessed for over 450,000 cytosine-phosphate-guanine (CpG) sites. QC analyses of methylation data were performed in the R statistical computing environment using the following packages: minfi  and blood. This study investigated CSF samples collected longitudinally from 279 patients during the first 14 days of hospitalization, and blood samples from 88 of these individuals collected within the first day of hospitalization. Methylomic profiles were obtained using a genome-wide array, from which methylation levels, quantified as beta-values  and s: minfi , ENmix  and a Fisher grade (measure of hemorrhage burden) > 1. Informed consent was obtained from the participant or their legal proxy using a protocol approved by the University of Pittsburgh Institutional Review Board. Exclusion criteria included a history of debilitating neurologic disease or subarachnoid hemorrhage due to arteriovenous malformation, trauma, or mycotic aneurysm.Daily CSF samples were collected for the first 14 days after aSAH from an external ventricular drain placed as standard of care and DNA extracted using the Qiamp Midi kit . Venous blood was collected within the first day of hospitalization and DNA was extracted using a simple salting out procedure. All DNA was stored in 1X TE buffer at 4°C.This study included 279 aSAH patients. For the CSF samples, we targeted days 1, 4, 7, 10, and 13 post-aSAH, and substituted samples ±1 day when target days were unavailable. Blood samples collected within the first day of hospitalization after aSAH were included in this study for 88 of the 279 participants.The severity of aSAH was assessed at hospital admission by Fisher grade  employinThe Illumina  Infinium HumanMethylation450 BeadChip platform was used to assess the methylation levels at over 450,000 CpG sites in the samples. Methylation data collection was performed by the Center for Inherited Disease Research (CIDR) of Johns Hopkins University. Each BeadChip, hereafter referred to as a plate, consists of eight chips of 12 samples arranged in a layout of six rows by two columns. This enables 96 samples to be run on a single plate. To avoid plate effects, all blood samples were assayed together on a single plate. CSF samples were placed across 11 plates using several strategies to reduce the impact of technical artifacts. First, all longitudinal samples from the same patient were included on the same chip within the same plate so that longitudinal changes in methylation were not obscured by chip and plate effects. Second, row and column positioning of samples from the same patient were carefully assigned to available positions within a chip so that longitudinal changes in methylation were not confounded with row and column effects. Third, cases and controls for DCI were balanced within chips using a checkerboard pattern so that DCI was not confounded with row, column, chip, or plate effects  were categorized as low-quality samples. These, along with outliers in total intensity or beta value distribution were removed from our subsequent analyses . Specifically, we performed functional normalization, an extension of quantile normalization, which makes use of the control probes on the array to regress out unwanted variation in the methylation data . Whetherp-values > 0.01 or with fewer than three beads) in more than 1% of samples. Differences in the number of CpGs passing quality filters was tested using McNemar’s test.After normalizing the data, we removed CpG sites from our analysis due to: (1) overlap of methylation probes with known polymorphic sites (which can cause biased methylation assessments), (2) probes located on the sex chromosomes  , (3) croBlood has a mixture of cell types and DNA methylation-based references have been established for blood cells. Therefore, to estimate the proportions (cell counts) of each cell type, we employed Houseman’s reference based method  using thp-values for SVA-adjusted and unadjusted EWAS scans under the null hypothesis were qualitatively compared to determine effect of SVA on genomic inflation. We measured inflation/deflation using the genomic inflation factor (λ), which is defined as the ratio of the empirically observed to expected median of the distribution of the test statistic.Owing to the lack of reference methylation data for cell types found in CSF after an aSAH event we employed surrogate variable analysis (SVA) to perform reference-free adjustment for cell-type heterogeneity across the samples in blood and CSF data. SVA, as implemented in the sva R package , simultaM-values at each qualifying CpG site were adjusted for age, sex and the surrogate variables , and were then used to calculate correlation coefficients between the blood and CSF profile across samples. In addition to these within-CpG correlations, we also calculated within-individual blood-CSF correlation coefficients across the methylome for each of the 70 patients who had data for both tissue types, using the same adjusted M-values as above. To mitigate the potential batch effect resulting from our design that blood and CSF samples were assayed on separate plates, we chose to compare blood and CSF by calculating their correlation which is invariant to systematic shifts in mean and scale, rather than by directly comparing their absolute M-values.We compared the methylation profiles of individuals with blood samples collected within the first day after hospitalization and CSF samples collected at days 1, 4, 7, 10, and 13. We used 65, 64, 65, 61, and 47 subjects to compare the methylation profiles of blood and CSF at days 1, 4, 7, 10, and 13, respectively to facilitate individual level comparison. For this comparison, following the approach of p = 0.003). A total of 1,012 methylation profiles  were measured from CSF samples collected longitudinally from 279 aSAH patients. Additionally, 92 methylation profiles  were measured on blood samples in a subset of 88 of these patients; the majority of these blood samples (77) were sampled between zero and 2 days post-hospitalization . QC analM-values, before and after functional normalization, for samples profiled together on a plate (p = 0.015) (p = 0.153).After removing low-quality samples, we performed functional normalization to reduce probe type (Infinium Type I vs. Type II) and batch  effects. The reduction in chip, row, and column effects can be visualized in the distribution of  a plate . Row eff= 0.015) . For thep < 2.2 × 10–16 for all), indicating that CSF samples may yield somewhat lower-quality methylation data, as is also evident in Individual probes were filtered out of analyses for reasons pertaining to probe design such as overlap with any single nucleotide polymorphisms (SNPs) and with cross-reactivity with off-target genomic positions  in the individual, which is known to cause increased proliferation of B cells in blood, bone marrow and other lymphoid tissues . Samplesλ) improved from 1.11 to 0.98 in the set of blood samples, and improved from 0.73 to 0.99 in the set of CSF samples within 2 days after hemorrhage  were generated for the set of blood samples, and 13 SVs were generated for day 1 CSF samples. Fifteen, 15, 14 and 10 surrogate variables were generated for CSF samples for days 4, 7, 10, and 13 respectively. To determine the benefit of SV-adjustment, we interrogated its effect on CpG site association tests under the null model of no association by simulating a dummy binary phenotype similar to the distribution of DCI and performing EWAS, with and without including SVs as covariates. The behavior of the test statistic better followed the null distribution after SV-adjustment, as shown in quantile-quantile plots . Specifimorrhage  and likemorrhage , 3. GenoM-values between the two tissue types obtained from aSAH patients. Specifically, we compared the methylation profile of blood collected within 48h of hospitalization versus CSF samples collected at days 1, 4, 7, 10, and 13 post rupture, respectively. Following our long-term goal of understanding the methylomic changes occurring across tissues after aSAH, we explored the suitability of peripheral blood collected within the first day of hospitalization as a surrogate for the normally less accessible longitudinally collected CSF based on the within-CpG correlation of adjusted p < 0.001 for all), with islands and shores showing greater positive correlation than shelves and seas  environment and its proximity to the hemorrhagic location. We note that the overall low within-CpG correlation is not at odds with the observed strong within-individual correlation. The within-CpG correlation is more pertinent to downstream analysis where the methylation level at individual CpGs are compared between groups . One possible limitation of our correlation analysis is that blood and CSF samples were measured on different plates. However, correlation coefficients have the property of being invariant with linear changes and therefore our results are not likely to be confounded by the blood-CSF plate batch effect.This study benefited from several strengths including the a plate design aimed at reducing confounding of experimental effects with technical artifacts within each tissue, thorough and rigorous application of data QC procedures, pairing of blood and CSF samples from the same patients, and assessment of methylomic profiles in a novel tissue type (post-aSAH CSF) that captures the CNS environment post-aSAH. Despite these strengths, limitations of the current study include limited statistical power to resolve the intra subject differences among the samples that may ultimately pose challenges in using this dataset for future EWAS studies. Additionally, the cell composition of CSF may vary over time after hemorrhage, which would also affect the methylation levels. Thus, longitudinal analyses of post-aSAH samples are challenging as cell-type heterogeneity may be confounded with days post-injury. Overcoming these challenges will be necessary to accomplish goals such as identifying genes whose changes in methylation after injury are predictive of recovery outcomes.In conclusion, this study is one of the first attempts to investigate DNA methylation at the genome scale in a sample of aSAH patients, as well as one of the first to measure methylation in CSF. Our analysis protocol showed that methylomic profiles can be obtained from CSF for use in EWAS analysis and that QC steps can improve the analysis by eliminating low-quality data points and reducing biases and experimental artifacts. Likewise, we show that blood, while readily accessible, is not a sufficient surrogate for the methylomic status of CSF. Our study lays the groundwork for more comprehensive analyses in the future, where efforts to understand methylation profiles in aSAH patients, and changes that occur post-injury, may ultimately lead to the discovery of biomarkers of clinical utility in predicting patient recovery.phs001990.v1.p1.The data can be accessed through dbGAP: The studies involving human participants were reviewed and approved by University of Pittsburgh Institutional Review Board. The patients or participants provided their written informed consent to participate in this study.YC was the principal investigator of the project. YC, DW, and JS conceived and designed the study. AA, DL, TK, JS, and DW performed the experiments and statistical analysis. AA, JS, and DW contributed to the initial writing of the manuscript. EC contributed to the clinical investigation of the research. All authors reviewed, edited, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracranial hypotension is a disorder characterized by low cerebrospinal fluid (CSF) pressure typically caused by loss of CSF. Although some mechanisms account for the CSF leakage have been elucidated, spinal canal stenosis has never been reported as a pathological cause of intracranial hypotension.C1-C2 sign is a characteristic imaging feature, which indicates CSF collection between the spinous processes of C1 and C2, occasionally observed on magnetic resonance imaging (MRI) in patients with intracranial hypotension.A 58-year-old man was presented to our institute with complaints of posterior cervical pain persisting for 3 months, along with numbness and muscle weakness of extremities. A fat suppression T2-weighted image of MRI illustrated fluid collection in the retrospinal region at C1-C2 level, and an 111In-DTPA cisternoscintigram clearly revealed the presence of CSF leakage into the same region. The MRI also showed stenosis in spinal canal at C3/4 level, and a computed tomography (CT) myelogram suggested a blockage at the same level. We gave a diagnosis as intracranial hypotension due to the CSF leakage, which might be caused by the spinal canal stenosis at C3/4 level. Despite 72 h of conservative therapy, a brain CT showed the development of bilateral subdural hematomas. We, therefore, performed burr-hole drainage of the subdural hematoma, blood-patch therapy at C1/2 level, and laminoplasty at C3–4 at the same time. Improvement of symptoms and imaging features which suggested the CSF leak and subdural hematoma were obtained post-operatively.The present case suggested the mechanism where the CSF leakage was revealed as fluid collection in the retrospinal region at C1-C2 level. Increased intradural pressure due to the spinal canal stenosis resulted in dural tear. CSF leaked into the epidural space and subsequently to the retrospinal region at C1-C2 level, due to the presence of spinal canal stenosis caudally as well as the vulnerability of the tissue structure in the retrospinal region at C1-C2 level. Thus, our theory supports the mechanisms of previously reported CSF dynamics associated to C1-C2 sign, and also, we suggest spinal canal stenosis as a novel etiology of intracranial hypotension. Intracranial hypotension has gained increasing recognition over the past 20 years –4, as a C1-C2 sign is a characteristic imaging feature, which indicates CSF collection in retrospinal soft tissue between the spinous processes of C1 and C2 levels, occasionally observed on magnetic resonance imaging (MRI) in patients with intracranial hypotension –10. An iWe have recently encountered a patient with intracranial hypotension in association with spinal canal stenosis as its potential etiological factor, and were presented with a characteristic imaging with the C1-C2 sign. Moreover, we performed combined therapies which include burr-hole drainage of the subdural hematoma, blood-patch therapy and laminoplasty. Here, we report suggestive strategies of diagnosis and treatment of intracranial hypotension.A 58-year-old Japanese man was presented to our out-patient unit complaining of posterior cervical pain. The pain firstly appeared 3 months before the first contact to us without any particular triggers such as trauma. He was relieved from the pain when lying flat; however, he kept working as a plumber and the pain got worse over the time period. A brain computed tomography (CT) was performed 1.5 months after the onset in another clinic and it revealed bilateral subdural effusion  score , as wellA fat suppression T2-weighted MRI without gadolinium enhancement of the cervical spine illustrated a fluid collection in the soft tissue spaces of the retrospinal region at C1-C2 level Fig. . Further72 h of conservative therapy, including bed rest, intravenous fluid hydration, muscle relaxants and non-steroidal anti-inflammatory drugs, was ineffective at improving his symptoms, and the re-examination of brain CT showed progression of the bilateral subdural effusions to bilateral subdural hematomas Fig. .Fig. 5BAfter reviewing of his imaging data and confirmation that no other cause presented in development of subdural hematoma along with careful discussions, we gave a diagnosis of intracranial hypotension due to the CSF leakage from C1-C2 level, which might be caused by spinal canal stenosis at C3/4 level. We, therefore, performed burr-hole drainage of the subdural hematoma, blood-patch therapy at C1/2 level and laminoplasty at C3–4, at the same time. In the laminoplasty, CSF flew out from the epidural space subsequently to the opening of lamina, and after sufficient decompression, the CSF flow was reduced in the operation field, without detection and restoration of dural tear (Video). In the blood-patch therapy, an epidural catheter was inserted from the surgical field at C3–4 level to C1-C2 level under x-ray fluoroscopy observation. Approximately 5 ml of autologous blood sampled from patient’s peripheral vein was injected to the epidural site at C1-C2 level and the blood-patch therapy was achieved.Postoperative response was significantly favorable. His symptoms completely improved and the imaging features that suggested CSF leak and intracranial hypotension disappeared Fig. . He discAdditional file 1. Microscopic pictures of the laminoplasty. Legend: On the left side of the screen indicates the cranial side and the right side indicates the caudal side. CSF flew out from the epidural space subsequently to the opening of lamina, and after sufficient decompression, the CSF flow was reduced in the operation field, without detection and restoration of dural tear.We report a case of intracranial hypotension which was potentially caused by spinal canal stenosis with C1-C2 sign on MRI fat suppression T2-weighted image. The C1-C2 sign in the present case deserves special attention. This sign is typically seen as a focal area of fluid-like signal intensity on MRI and as a CSF collection between the spinous processes of C1 and C2 on CT myelograms or a cisternoscintigrams. These imaging features are found in 12–67% of the patients with intracranial hypotension , 13, 16.Interestingly, though, it should be noted that C1-C2 sign does not necessarily denote the exact site of CSF leakage; hence, it has also been called as C1–C2 false localizing sign , 10. SchAnother novel finding in the case is that spinal canal stenosis could be a pathological cause of CSF leakage and hence, intracranial hypotension. The suggested mechanism from the present case is that the spinal canal stenosis and straining could lead to increased intradural pressure which eventually resulted in the dural tear. Subsequently, the CSF leaked into the epidural space and later escaped to the retrospinal region at C1-C2 level, because of its vulnerability; and dysfunction of CSF pressure regulation in cervical subarachnoid space contributed by the obstruction of the caudal epidural space resulting from spinal canal stenosis. Thus, our theory could fit and support the mechanism of CSF dynamics in the mechanism of C1-C2 presentation advocated by Schievink et al. , and theAdditional file 2.


Objective: Investigating the relevance of the incomplete circle of Willis (COW) to the plaque wall distribution in the atherosclerotic middle cerebral arteries (MCAs) through utilizing high-resolution magnetic resonance imaging (HR-MRI), and its potential clinical impact.Methods: This hospital-based study enrolled consecutive adult patients with acute ischemic stroke or transient ischemic attack, who received a 3.0T Achieva MR system scanning. The COW completeness was evaluated on MR angiography imaging, including anterior (A) and posterior (P)-COW sections. The MCA plaque wall distribution was assessed on HR-MRI. The occurrence of perforator infarction was detected on diffusion-weighted imaging.Results: Among 87 patients (mean age = 62.39 ± 11.64 years old) with atherosclerotic plaques in the MCA M1 segments, the incomplete COW types were more prevalent than the complete COW type . The incomplete A-COW had more inferior but fewer ventral plaques of MCA atherosclerosis than the complete A-COW, while the incomplete P-COW had fewer inferior MCA plaques than the complete P-COW. Moreover, symptomatic MCA plaques causing perforator infarctions were more likely to locate on the superior wall.Conclusion: Our findings suggested that the COW completeness could influence the vessel wall distribution of the MCA plaques, among which the superior plaques of symptomatic MCA atherosclerosis was associated with branch occlusive disease. Intracranial atherosclerosis (ICAS) is the main etiological factor of ischemic stroke (IS) , the neuHigh-resolution magnetic resonance imaging (HR-MRI) can serve as a non-invasive vessel wall imaging technique in delineating the direct morphology of intracranial atherosclerotic plaques , 13. DesIn this study, adult subjects were consecutively recruited from our single institution (2016–2018). The patients were included, if they met the following criteria: (1) first-ever acute IS or transient ischemic attack (TIA) within 7 days; (2) any intracranial large-artery atherosclerotic stenosis confirmed by magnetic resonance angiography (MRA) or digital subtraction angiography (DSA); (3) MCA atherosclerotic plaques detected by HR-MRI; (4) at least one atherosclerotic risk factor, including hypertension, hyperlipidemia, diabetes and smoking; and (5) good image quality for detecting plaques. The subjects were excluded, if they had any evidence of (1) non-atherosclerotic stenosis ; (2) cardioembolism ; (3) coexistent moderate to severe internal carotid artery (ICA) or extracranial carotid artery (ECA) stenosis; (4) no MCA atherosclerotic plaque detected by HR-MRI, and (5) contraindications to MRI. The flow chart for the subject inclusion was presented in The imaging protocols of both 3D Time-Of-Flight (TOF) MRA and transverse 3D T1-weighted (T1w) Volumetric ISotropically Turbo spin echo Acquisition (VISTA) sequences were described in our previous MRI study .3, acquired resolution 0.6 × 0.6 × 1.0 mm3, reconstructed resolution 0.5 × 0.5 × 0.5 mm3 utilizing zero filling, repetition time (TR) 1,500 ms, echo time (TE) 36 ms, SENSE factor 1.5 (phase-encode direction), echo spacing 4.0 ms, TSE + startup echoes 56 + 6, and scan duration 6:51 min. The 3D TOF MRA sequence was also performed, the parameters of which included FOV 200 × 200 × 56 mm3, acquired resolution 0.4 × 0.6 × 0.7 mm3, TR/TE 23/3.5 ms, and scan duration 3:07 min.A 3T Achieva MR system with an 8-channel head coil  was used in this study. All the subjects were scanned by the transverse 3D T1w VISTA sequence before and after the administration of a gadolinium-containing contrast agent  (0.1 mL/kg to each subject). The interval between the two scans was ~5 min. The T1w VISTA sequence parameters included field-of-view (FOV) 200 × 167 × 45 mmTwo experienced readers blind to the clinical parameters identified the structural integrity of the COW, according to the findings on MRA , 17. TheA plaque in the atherosclerotic MCA M1 segment was detected, if shown as eccentric wall thickening, and the thickest site on the vessel wall was > twice the thinnest point via the visual inspection . All croU-test. The relationship between the COW integrity and symptomatic MCA plaques leading to perforator infarctions was determined by Chi-squared test or Fisher's exact test. P < 0.05 was considered as statistical significance. Inter-rater reliability was tested by Cohen κ or intraclass correlation coefficient with 95% confidence intervals (CIs). A coefficient > 0.81 was regarded as excellence.Statistical analysis was conducted by the SPSS version 25.0 . Variables were expressed as mean ± standard deviation (SD) or percentage frequencies, when appropriate. The individual percentage wall distribution was analyzed for each atherosclerotic plaque, and generated the mean superior, inferior, ventral, and dorsal wall distribution of the total groups . The meaEighty seven subjects with MCA atherosclerotic plaques met the inclusion criteria in the current study. The clinical characteristics of patients were shown in Overall, 130 lesions of the MCA M1 segments were identified as atherosclerotic in this study, including 81 asymptomatic and 49 symptomatic lesions. A total of 471 image slices on HR-MRI were used for detecting plaque wall distribution in the atherosclerotic MCA M1 segments.P = 0.008, P < 0.001, P = 0.043, P = 0.008, P = 0.011, P = 0.032, The plaque wall distributions of MCA atherosclerosis between groups with and without incomplete COW subtypes were summarized in P = 0.045). As presented in P > 0.05).To further the understanding of the clinical impact of our findings, we then investigated the association between the plaque wall distribution of symptomatic MCA atherosclerosis and perforator infarcts , and betInter-rater reliability on the evaluation of the plaque detection and the COW integrity was excellent .In the current study, first, we confirmed that the incomplete COW types commonly existed in patients with MCA atherosclerosis. Second, the distinctive feature of plaque wall distribution in the MCA M1 segments could be distinguished between complete COW and incomplete COW types by utilizing HR-MRI. We found that the MCA plaques in the incomplete A-COW located on more inferior but fewer ventral walls, compared to those in the complete A-COW. Besides, the incomplete P-COW had fewer inferior plaques in the MCAs than the complete P-COW. Third, superior plaques of symptomatic MCA atherosclerosis was significantly associated with branch occlusive disease. Our observations might gain an insight into the cerebral vascular mechanisms of MCA atherosclerosis.The structural variations of the COW have existed in the normal populations since the initial formation of the COW . In a prPrevious research suggested that the MCA plaques were mainly distributed on the inferior and ventral walls, and the asymptomatic MCA atherosclerotic lesions had more inferior plaque wall orientation . Our stuIn this study, we also explored the possible clinical impact of our findings. The results confirmed the prior research reporting that the superior plaques in the symptomatic MCAs were robustly related to the occurrence of branch occlusive disease via occluding the perforator orifice . On the Limitations should be considered in the current study. First, a selection bias could be introduced in this study. Subjects with chronic IS and TIA were excluded to avoid any confounding factors, as the process of the intracranial atherosclerotic plaques might be affected by time course, medical therapy, and lifestyle changes . Second,The incomplete COW types were prevalent in patients with MCA atherosclerosis. The incomplete A-COW was associated with more inferior but fewer ventral wall plaques in the atherosclerotic MCA M1 segments, while the incomplete P-COW was found in relevance to fewer inferior MCA plaques. Besides, the occurrence of branch occlusive disease was correlated with the superior wall plaques of symptomatic MCA atherosclerosis. Our observations may highlight the hemodynamic function of the incomplete COW structures on the pattern of plaque wall distribution in MCA atherosclerosis, but the clinical impact still requires further investigations.The datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by the Joint Chinese University of Hong Kong-New Territories East Cluster Clinical Research Ethics Committee (The Joint CUHK-NTEC CREC). The patients/participants provided their written informed consent to participate in this study.JL analyzed imaging data and drafted the manuscript. LZ and W-JY recruited patients. C-YS-T participated in analyzing imaging data. TL participated in study coordination and recruited patients. X-YC conceived the study, participated in its design and coordination, and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Perilla oil (PO), rich in α-linolenic acid , is increasingly alleged to have numerous health benefits in humans. However, the current reports detailing the effects of PO on human mental health are not adequate. Therefore, in the current investigation we compared the effects of PO or placebo treatment on the mental condition of healthy adult Japanese volunteers. At baseline and after 12 months of treatment, mental health condition was assessed using the Zung Self-Rating Depression Scale (SDS) and Apathy Scale, and serum biochemical parameters were determined. From baseline to 12 months of intervention, both SDS depression and apathy scores improved significantly in the PO-administered group. Compared to those of control group, serum norepinephrine and serotonin levels after 12 months decreased in the PO-administered group. The enhanced mental state observed in PO-subjects was accompanied by LNA level increases in erythrocyte plasma membranes. Our data demonstrate that PO intake enhances blood LNA levels and may maintain healthy mental conditions in adult subjects. Numerous reports suggest that ω-3 polyunsaturated fatty acids (PUFAs) have major roles in the maintenance and sustenance of brain cognition in both animals and humans ,2,3. FatPerilla oil (PO) is considered a rich source of ω-3 PUFAs, as it is comprised chiefly of α-linolenic  and linoleic (13–20%) acid. PO also contains 12–22% monounsaturated oleic acid (OLA), and a small percentage of saturated fatty acids such as palmitic  and stearic  acid. Notably, amongst the plant seed-based oils, PO possesses the highest proportion of ω-3 fatty acids, the most notable of which is LNA -NE -NE . Hypothe3H]-NE ,65. We h3H]-NE . However3H]-NE  and rats3H]-NE , while d3H]-NE . Moreove3H]-NE ,69,70,713H]-NE , ω-3 PUFWe need to mention herein the facts that there are some limitations which deserve additional discussion. Firstly, since the participants in this study were healthy individuals without mental disturbance, their mental health parameters were mostly normal and there were no significant differences between PO group and placebo group. Δ-SDS and Δ-Apathy scores were defined as the difference between the baseline and 3 or 12 months in order to achieve statistical differences, since there were no significant differences in both SDS and Apathy scores between the placebo and PO groups. In the future, we should investigate the effects of PO ingestion on individuals with mental disorders such as depression. Secondly, we did not control for menstrual cycle or consider menopause for female subjects, and future research will need to take these into account given the influence of menstrual cycle or menopause on psychological health ,74,75.In conclusion, the current study suggests that PO administration increases blood LNA and EPA levels in the adult subjects, leading to mitigation of mental health issues in adults without having any noticeable clinical side effects. These effects may be a result of the adequate balance between serotonergic and adrenergic neuronal activity. Further investigation is necessary to comprehensively conclude the mechanism responsible for the beneficial effects of PO on mental condition.


A 64-year-old African American male, with past medical history of hypertension, depression, and seizure disorder, presented with an episode of generalized tonic-clonic seizure. He was treated for seizures, and after 48 hours seizure-free, the patient started complaining of chest tightness and troponin levels were found to be 34.71 ng/mL. No evidence of myocardial infarction was found after extensive diagnostic workup, including cardiac catheterization. We suspect alternative causes of elevated troponin including post-seizure and transient takosubo cardiomyopathy. Markers of cardiac injury are used to diagnose myocardial infarction (MI) but problems arise in cases of falsely elevated markers leading to unnecessary interventions. Cardiac markers evolved from aspartate aminotransferase (AST) to lactate dehydrogenase (LDH), creatine kinase (CK), CK-MB, and finally to troponins. Troponins are considered the most sensitive and specific markers and are composed of three isoforms: troponin T, troponin I, and troponin C. To diagnose MI, cardiac troponin should rise and/or fall above the 99th percentile combined with evidence of myocardial ischemia as suggested by one of the following:1) symptoms of myocardial ischemia;2) new ischemic electrocardiographic changes;3) new pathological Q waves;4) new ischemic regional wall motion abnormalities on cardiac imaging;5) acute coronary thrombus on coronary angiography.Causes of falsely elevated troponins should be ruled out if no myocardial infarction is present. We report a case of very significantly raised troponins following an epileptic episode. Though seizures are a known cause of raised troponin levels, such significantly high levels increase suspicion of stress-induced cardiomyopathy during an epileptic episode.A 64-year-old African American male was admitted for an episode of seizure. His seizure was treated with levetiracetam. A CT scan of his head was done since the patient had a fall during a seizure, which was negative for any acute hemorrhage or focal lesions. After 48 hours seizure-free, the patient started complaining of chest tightness. It was not associated with any diaphoresis, palpitation, nausea, vomiting, or shortness of breath. On examination, his vital signs were stable with a heart rate of 82, blood pressure of 122/82 mmHg, respiratory rate of 18, with oxygen saturation of 98% at room air. He was alert and oriented. Lungs were clear to auscultation. Cardiovascular examination revealed normal S1, S2 with regular rate and rhythm. Neurological and abdominal examinations were normal.Past medical historyPast medical history was significant for hypertension treated with amlodipine, seizure disorder on valproate and levetiracetam, and depression on sertraline 20mg.Differential diagnosisA 64-year-old male with hypertension and chest tightness raises suspicion for myocardial infarction which is very important to be ruled out immediately. Raised levels of cardiac troponin further make MI likely. Other possible differential includes aortic dissection and pulmonary embolism.InvestigationsCardiac troponin along with electrocardiography was ordered initially. Troponin was found to be significantly raised as shown in Table Electrocardiogram (ECG) showed hyperacute T waves and U waves Figure .Cardiac catheterization was done due to significantly raised troponin levels, with suspicion of non-ST-elevation myocardial infarction (NSTEMI). Coronary angiography revealed disease-free arteries without any stenosis Figure .Echocardiography was performed which showed a normal ejection fraction of 50-55%. Chest CT with contrast was done to rule out PE which was normal.ManagementThe patient was initially treated with loading doses of aspirin and ticagrelor, heparin for acute coronary syndrome (ACS) protocol was started. He was given nitroglycerin sublingually for chest pain, which helped decrease the pain. Heparin was continued for 24 hrs and since the cardiac catheterization was normal and the troponin trended down, it was discontinued.Follow upThe patient was given supportive therapy after cardiac catheterization. His aspirin, ticagrelor, and heparin were discontinued and he was treated for his seizures with loading doses of Keppra and his troponin trended down to 35.5 ng/ml. He was discharged home symptom-free and was followed up after a week in the adult clinic. His troponin was back to <0.01 ng/ml and he did not complain of any symptoms.This case presentation is an example of the fact that elevated troponin is not limited to cardiovascular pathology but a broad differential should be considered. Cardiac troponin (cTn) is a universally accepted marker for cardiac injury and plays a crucial role in the diagnosis of acute myocardial infarction. These markers of cardiac injury are regulatory proteins controlling the interactions between actin and myosin mediated by calcium . The hisBrobbey et al. reported the first case of raised troponin after a grand mal seizure without any evidence of cardiac pathology. Value of troponins ranged between 5.5 ng/mL and 6.3 ng/mL, in contrast to our case in which troponins were very highly elevated from 12.57 to 49.64 ng/mL .Falsely elevated troponins could be due to technical errors or from other causes discussed above, so other cardiac biomarkers should be ordered along with troponins to have more evidence for an invasive procedure. In the case of falsely elevated troponin, different causes should be ruled out using appropriate investigations. In our case history, examination, lab workup, and imaging revealed no evidence of any potential cause except the fact that the patient had an episode of seizure.Stress-induced cardiomyopathy, also known as Takotsubo cardiomyopathy, is also one of the causes that could account for this elevation of troponin. It is characterized by transient dysfunction of the apical portion of the left ventricle with compensatory hyperkinesia of the basal walls, thus resulting in apical ballooning that gives the heart the appearance of a Japanese octopus's trap or Takotsubo. It is triggered by emotional or physical stress and is very similar to acute coronary syndrome, although the coronary arteries are normal. Usually, cardiac enzymes are slightly increased, and serial electrocardiographic changes often consist of ST-segment elevation followed by inversion of T waves . AlthougThe decision of whether or not to perform a life-saving invasive procedure in a patient with nonspecific elevation of troponin remains very challenging. Coronary catheterization was normal and probably had decreased utilization in our case but it was a crucial step to rule out MI in the setting of chest pain and raised troponin levels. Through this case we want to highlight the importance of false-positive troponin elevation and possible association with an episode of seizure or transient stress-induced cardiomyopathy.


Introduction: Surfactant proteins (SP) have been shown to be inherent proteins of the human CNS and are altered during acute and chronic disturbances of CSF circulation. Aim of the study was to examine the changes of surfactant protein concentrations in CSF of preterm babies suffering from intraventricular hemorrhage.Patients and Methods: Consecutive CSF samples of 21 preterm infants with intraventricular hemorrhages (IVH) and posthemorrhagic hydrocephalus (PHHC) were collected at primary intervention, after 5–10 days and at time of shunt insertion ~50 days after hemorrhage. Samples were analyzed for surfactant proteins A, B, C, and G by ELISA assays and the results were compared to 35 hydrocephalus patients (HC) without hemorrhage and 6 newborn control patients.Results and Discussion: Premature patients with IVH showed a significant elevation of surfactant proteins SP-A, C, and G compared to HC and control groups: mean values for the respective groups were SP-A 4.19 vs. 1.08 vs. 0.38 ng/ml. Mean SP-C 3.63 vs. 1.47 vs. 0.48 ng/ml. Mean SP-G 3.86 vs. 0.17 vs. 0.2 ng/ml. SP-A and G concentrations were slowly falling over time without reaching normal values. SP-C levels declined faster following neurosurgical interventions and reached levels comparable to those of hydrocephalus patients without hemorrhage.Conclusion: Intraventricular hemorrhages of premature infants cause posthemorrhagic CSF flow disturbance and are associated with highly significant elevations of surfactant proteins A, C, and G independent of total CSF protein concentrations. Surfactant proteins (SPs) have been demonstrated to be inherent proteins of the CNS –3 and arPrematurely born babies have a high risk of intraventricular hemorrhage (IVH), which is presumed to arise from immature germinal matrix at the ventricular walls of the lateral ventricles. Our recent studies focusing on embryology and concerning the CNS surfactant system in hydrocephalic conditions suggest a relationship between surfactant protein concentrations and intraventricular hemorrhage . ImmaturSP-A has recently found to modulate CNS immune response of astrocytes and microglial cells –8. AdditThe aim of this study was the examination of potential alterations of surfactant protein concentrations within the CSF of neonates suffering from intraventricular hemorrhage resulting in posthemorrhagic hydrocephalus (PHHC).We investigated serial CSF specimens of 21 neonatal patients suffering from hydrocephalus due to intraventricular hemorrhage. All patients or caregivers gave their written informed consent for the scientific use of CSF samples and analysis of clinical and radiological data.n = 11), a ventricular access device for repeated tapping to remove CSF (n = 1) or an endoscopic lavage procedure plus external ventricular drainage (n = 9). When applicable, follow-up CSF was examined after 5–10 days in patients with external ventricular drainage (EVD) or Rickham-Reservoir  and at time for shunt insertion at a median of 57 days after birth . Four additional patients were treated in other institutions initially but required transfer to our center for shunt insertion due to persisting CSF disturbance. Thus, for those four patients only CSF specimen at time of shunt insertion were available.All neonatal patients required neurosurgical intervention to relieve intracranial pressure due to posthemorrhagic hydrocephalus (PHHC) either by implanting an external ventricular drainage (2 as minimum area (correlating to ~5–6 mm above the 97th percentile of Levine's ventricular index). Early interventions were performed esp. in patients that underwent neuroendoscopic lavage procedures to alleviate the posthemorrhagic inflammatory response and resolve obstructions of the aqueduct or third ventricle. Mean gestational age was 28.5 weeks (range 23.7–41 weeks) and birth weight 1,356 g . At the time of primary intervention, the mean age of the patients was 22 days. All patients suffered from Levine IVH grades II or more and developed ventriculomegaly.Indication for primary intervention due to PHHC was made either due to signs of elevated intracranial pressure  or rapid increase of head circumference. At the coronal ultrasound plane all patients presented a ventricular size of at least one lateral ventricle larger than 2 cmThe control group comprised six neonatal patients that necessitated CSF examination by lumbar puncture to rule out CNS infection. Mean gestational age was 32.3 weeks range 31–41 weeks) and birth weight 2,326 g . Furthermore, CSF samples were obtained from 35 older patients (mean age 13.5 years ranging from 0 to 65.9 years) that underwent a diagnostic workup and treatment of hydrocephalus in isolated aqueductal stenosis without hemorrhagic or postinfectious cause (HC group) (–41 weeksQuantification of surfactant protein concentrations was performed using enzyme-linked immunosorbent assays (ELISA) according to the manufacturer's manual. Commercially available ELISA kits  were used to quantify the amount of SP-A , SP-B , SP-C  and SP-G  in CSF samples. The analyses were performed using a microplate spectrophotometer (ELISA-reader) at a wavelength of 450 nm and a reference wavelength of 405 nm for measuring the absorbance. Surfactant protein concentrations in ng/ml CSF were calculated by comparison between standard series and the determined values of antigen concentration (protein concentration) according to the manufacturer's manual.Routine CSF laboratory data  were obtained to rule out infection or other inflammatory and autoimmune diseases. Patients with CSF infections were excluded from further analysis. Total CSF protein content and SP concentrations were analyzed with Pearson's correlation coefficient to rule out a direct association between total protein content and surfactant proteins.t-test and ANOVA. Thereby, the significance level was set to 0.01.Statistical analyses were performed with the SPSS Version 22  based on the Welch This study was approved by the institutional review board of the medical faculty (Ethikkommission Universität Leipzig Az 330-13-18112013).p < 0.001). There was no association between total CSF protein content and SP-concentrations with R values 0.14 for SP-A, 0.08 for SP-C, and 0.0009 for SP G, respectively .Analyses of the CSF obtained in 21 primary interventions in newborns revealed marked elevations of CSF surfactant proteins A, C, and G in PHHC patients compared to control and HC group  controls and 7 of 35 (20%) HC group specimens and only 3 (14%) PHHC patients. Therefore, SP-B results are shown in the table but not incorporated in the boxplot figures.During follow-up, SP concentrations fell over time, but did not reach control levels. SP-C fell to HC group levels after the primary intervention, whereas all other SPs still remained markedly elevated compared to HC group levels at time of shunt surgery approx. 50 days after hemorrhage . Total cn = 6) making up the control group had normal concentrations of SPs compared to pediatric and adult control individuals  at time of CSF sampling are unknown. Investigations focused on these parameters require and justify the use of animal models. Secondly, the control group and hydrocephalus group consists of older patients that SP-D was not examined due to small sample volumes that did not allow further ELISA measurements. SP-B was found in only few controls and hydrocephalus patients but was detected in very high concentrations in preterm babies after IVH. The few specimens, however, did not allow further detailed analysis, and future investigations are necessary to evaluate SP-B.A major shortcoming is the lack of surface tension analysis and lipid studies of the corresponding CSF specimen to underline the clinical significance of surfactant in hydrocephalic conditions. Reduction of surface tension by protein concentration and cellular components have been stated already in the 1940s in historical books and were demonstrated by Kratochvil and Hrncír  in 2002.In light of the multiplicity of evidence of the existence of the Cerebral Surfactant System and the importance of surfactant application in the respiratory distress syndrome of the preterm, further research may open horizons to diagnostic and therapeutic use of CSF surfactant proteins in preterm infants.Intraventricular hemorrhages of the preterm and newborn babies that cause posthemorrhagic CSF disturbance are associated with highly significant elevation of surfactant proteins A, C, and G, independent from total CSF protein concentrations.SP-A and G concentrations fell slowly over time without reaching normal values. In contrast, the reduction of SP-C levels following neurosurgical interventions occurred faster. SP-C reached levels similar to those of hydrocephalus patients that did not suffer from ventricular hemorrhage. This suggests an involvement of SPs in the acute and subacute posthemorrhagic CNS response. Further investigations are needed to elucidate potential diagnostic and therapeutic approaches to treat this specific pathology.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethikkommission Universität Leipzig Az 330-13-18112013. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. The present study included the retrospective analysis of clinical data, routine CSF examinations, and surfactant protein analysis. It was positively reviewed by the local ethics committee of University Leipzig (Ethikkommission Universität Leipzig Az 330-13-18112013).All authors reviewed the data and critically revised the manuscript. MKr, UN, UT, MKn, and CG collected CSF samples, clinical data, and routine CSF data. UN and MKr prepared all CSF samples for further analysis. JP and SS performed the ELISA analysis. MKr and CM conducted statistical analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.