Altered proteome profiles have been reported in both postmortem brain tissues and body fluids of subjects with Alzheimer disease (AD), but their broad relationships with AD pathology, amyloid pathology, and tau-related neurodegeneration have not yet been fully explored. Using a robust automated MS-based proteomic biomarker discovery workflow, we measured cerebrospinal fluid (CSF) proteomes to explore their association with well-established markers of core AD pathology.n = 48) or impaired cognition (n = 72). LC-MS quantified hundreds of proteins in the CSF. CSF concentrations of β-amyloid 1–42 (Aβ1–42), tau, and tau phosphorylated at threonine 181 (P-tau181) were determined with immunoassays. First, we explored proteins relevant to biomarker-defined AD. Then, correlation analysis of CSF proteins with CSF markers of amyloid pathology, neuronal injury, and tau hyperphosphorylation  was performed using Pearson’s correlation coefficient and Bonferroni correction for multiple comparisons.Cross-sectional analysis was performed on CSF collected from 120 older community-dwelling adults with normal  ≥ 0.38). We identified 50 additional CSF proteins associated with CSF tau and 46 proteins associated with CSF P-tau181 . The majority of those proteins that showed such associations were brain-enriched proteins. Gene Ontology annotation revealed an enrichment for synaptic proteins and proteins originating from reelin-producing cells and the myelin sheath.We quantified 790 proteins in CSF samples with MS. Four CSF proteins showed an association with CSF AβWe used an MS-based proteomic workflow to profile the CSF proteome in relation to cerebral AD pathology. We report strong evidence of previously reported CSF proteins and several novel CSF proteins specifically associated with amyloid pathology or neuronal injury and tau hyperphosphorylation.The online version of this article (10.1186/s13195-018-0397-4) contains supplementary material, which is available to authorized users. Proteome alterations have been identified in a multitude of pathologies, such as cancer, metabolic disorders, and brain diseases . Several1–42], total tau, and hyperphosphorylated tau [P-tau]) linked to the main hallmarks of AD pathology, such as amyloid plaques and neurofibrillary tangles, can complement clinical examination for the diagnosis of AD  (SODC), AT1A3, pyruvate kinase PKM (KPYM), and L-lactate dehydrogenase B chain (LDHB). Those nine proteins were associated with tau and/or P-tau181. Pathway enrichment analysis using the KEGG database did not yield any significant results (data not shown).In Fig. 1–42 levels, 50 proteins with CSF tau, and 46 proteins with CSF P-tau181 levels. The CSF proteins related to Aβ1–42 were different from those associated with tau or P-tau181.In the present study, we used MS-based shotgun proteomics to measure the CSF proteomes of 120 older adults and investigate broad CSF protein relationships with core AD pathology. Overall, human CSF proteome coverage was composed of 790 proteins. Four CSF proteins were associated with CSF Aβ1–42. Using LASSO logistic regression, we observed that CSF proteins could significantly increase the classification accuracy of non-AD versus AD CSF biomarker profiles as compared with models based only on clinical parameters and the presence of the APOE ε4 allele. Nonetheless, those statistical models relying on CSF proteins might be overfitted and should be interpreted with caution; class imbalance also affected their strict performance. Overall, with both exploratory analyses, we identified specific CSF proteome alterations that are related to AD pathology and may provide novel mechanistic insights. Assessing the whole sample and the subgroup of subjects with cognitive impairment, we could decipher the strong contribution of some CSF proteins, such as SMOC1 and 1433Z  to elaborate further on the involved mechanisms. Most of the correlations of CSF proteins were with CSF tau and P-tau181 . Unbiased classification based on markers of cerebral amyloid and tau pathology and neuronal injury has been proposed for use across the clinical stages . We firs181 Fig. , suggest1–42 in our study , but mainly in association with amyloid pathology [We found that CSF Aβsue Fig. , and thiathology . Our res1–42, tau, and/or P-tau181 levels in our study  . Of the or P-tau , confirmAltogether, our shotgun MS-based proteomic approach  was confUsing an MS-based proteomic workflow, we have quantified a number of CSF proteins in 120 older adults with normal cognition and with cognitive impairment. We report strong evidence of known and new CSF proteins related to amyloid pathology, neuronal injury, and tau hyperphosphorylation. Although we confirmed several previous findings of CSF proteins related to AD pathology, our work reveals a large number of additional CSF proteome alterations involving in particular reelin-producing cells and the myelin sheath.Additional file 1: Supplementary Methods.Table S1. Demographics and clinical characteristics of subjects removed from the statistical analyses. Table S2. Non-AD versus AD CSF biomarker profile group comparison after selection in all subjects of 26 proteins with LASSO. Table S3. Non-AD versus AD CSF biomarker profile group comparison after selection in subjects with cognitive impairment of 18 proteins with LASSO. Table S4. Correlation of CSF proteins with CSF Aβ1-42. Table S5. Correlation of CSF proteins with CSF tau. Table S6. Correlation of CSF proteins with CSF P-tau181. Table S7. Group comparisons of CSF protein measurements for AD versus non-AD CSF biomarker profiles in all subjects. Table S8. Group comparisons of CSF protein measurements for AD versus non-AD CSF biomarker profiles in subjects with cognitive impairment. Figure S1. Box-plots of CSF proteins  for positive and negative CSF profiles of AD pathology in all subjects and subjects with cognitive impairment. Figure S2. Pairwise correlation heatmap of the 26 CSF proteins selected with LASSO for classification of non-AD versus AD CSF biomarker profiles for all subjects. Figure S3. Pairwise correlation heatmap of the 18 CSF proteins selected with LASSO for classification of non-AD versus AD CSF biomarker profiles for subjects with cognitive impairment. Figure S4. Correlations of CSF neurogranin and neuromodulin with CSF tau and P-tau181. Figure S5. Chord diagram of the relationships of 59 CSF proteins with CSF tau, P-tau181, and/or Aβ1-42. Figure S6. Venn diagrams of CSF proteins with significant group comparison differences between AD versus non-AD CSF biomarker profiles and those correlating with CSF Aβ1-42, tau, and P-tau181. Figure S7. Venn diagrams of CSF proteins selected with LASSO to classify non-AD versus AD CSF biomarker profiles and those correlating with CSF Aβ1-42, tau, and P-tau181. (DOCX 2575 kb)


Fiber Bragg grating (FBG) measuring bolts, as a useful tool to evaluate the behaviors of steel bolts in underground engineering, can be manufactured by gluing the FBG sensors inside the grooves, which are usually symmetrical cuts along the steel bolt rod. The selection of the cut shape and the glue types could perceivably affect the final supporting strength of the bolts. Unfortunately, the impact of cut shape and glue type on bolting strength is not yet clear. In this study, based on direct tension tests, full tensile load–displacement curves of rock bolts with different groove shapes were obtained and analyzed. The effects of groove shape on the bolt strength were discussed, and the stress redistribution in the cross-section of a rock bolt with different grooves was simulated using ANSYS. The results indicated that the trapezoidal groove is best for manufacturing the FBG bolt due to its low reduction of supporting strength. Four types of glues commonly used for the FBG sensors were assessed by conducting tensile tests on the mechanical testing and simulation system and the static and dynamic optical interrogators system. Using linear regression analysis, the relationship between the reflected wavelength of FBG sensors and tensile load was obtained. Practical recommendations for glue selection in engineering practice are also provided. Bolt supporting technology has been widely used in a variety of geotechnical engineering applications ,2,3,4. TGiven its unique high accuracy, excellent electromagnetic immunity, durability, and distributed measurement characteristics, the fiber Bragg grating (FBG) sensor has been widely applied as a perceived element and transmission medium to study the behavior of rock bolts or anchors by many studies in the field of underground engineering . Each tyFor a steel bolt, one common approach for installing the FBG sensors is to glue them inside the symmetrical grooves along the bolt rod, and then refill the groove using silica gel to protect the FBG sensors. Then, the FBG measuring bolt is manufactured for field applications to obtain the stress distribution and its variation along the bolt. However, the depth and shape of grooves cut within the bolt undoubtedly reduce the strength of the bolt itself, which may have a direct impact on the long-term monitoring accuracy of FBG sensors, especially given the complexity of the underground environment. In addition, the application of various types of glue material may also affect the monitoring accuracy of the FBG measuring bolt. Unfortunately, in the abovementioned literature, all factors interfering with the FBG monitoring sensors have not yet been properly investigated.In this paper, the effects of groove shape on bolt strength were analyzed based on tensile experiments on conventional rock bolts. Following that, the stress distribution in an anchor bolt with different groove shapes was numerically analyzed by ANSYS, a continuum mechanics simulator. Furthermore, four commonly used glues and their effects on FGB monitoring sensors were assessed. Based on these results, the optimal groove shape and glue material are proposed to improve the monitoring performance of FGB bolts in underground engineering. Note that although the main engineering background in this research aims at understanding the impact of groove shape and glue material on FBG measuring bolts, the findings are also transferable to other strain gauge-based measuring bolts that also require cutting grooves and glue material.The tensile tests were performed by using the mechanical testing and simulation (MTS) electrohydraulic servo testing machines, as shown in All specimens were cut from steel bolts that were used in underground coal mines. The length of the specimens was 250 mm, and the diameter was 22 mm. The specimens were classified into six categories based on groove shape. To rule out the size effect of various grooves, the six specimens aimed to achieve roughly the same cutting area for different shapes with a laser-cutting machine. Two independent specimens for each type of groove shape were manufactured and numbered to reduce sampling bias. The groove shapes and groove sizes for the specimens are shown in The tensile test process used closed-loop control positioning. The tensile tests for the specimens were initiated at a constant loading rate of 5.1 mm/min under normal temperature and pressure conditions. The stress–strain responses of the specimens were recorded during the tensile loading process, and the frequency of data collection was 1.0 Hz. Note that the actual loading length of 110 mm was determined by deducting the length of the two ends held by the machine. The specimens were loaded until the specimen broke into two pieces. The images of the tensile tests are shown in The tension strength and displacement relationships for all 12 tested specimens are shown in The yield strength, ultimate strength, and failure strength for these specimens with different groove shapes are summarized in After the tensile load passed yield strength, the tensile load continuously increased, which resulted in the realignment of the internal grain structure of the specimens. This widely observed strain hardening behavior reflected the decrease in the specimen’s ability to resist further deformation despite the increasing tensile strength. After a period of yielding, the tensile load reached the peak strength and the internal structure of the specimen broke at around 80% of peak strength, which terminated the experiment. The average peak strength of the no-groove specimen was 213.5 kN. In comparison, the average peak strength dropped by 4.22% for both the U-groove with a chamfer and the trapezoidal groove, which was the lowest among all specimens with grooves. More than a 5% reduction in peak strength was observed in U-groove. By the time tensile load exceeded the peak strength, the lateral size of the specimen shrank dramatically. This necking phenomenon occurred shortly after the specimen broke. The breaking tensile load of the no-groove specimen was 166.5 kN, whereas it was nearly 8% lower for U-groove specimen.The experimental results suggest that bolts with a trapezoidal groove have the least strength deterioration compared with the other groove shapes, which should be recommended for the manufacturing of FBG-based monitoring bolts.To understand the stress distribution in bonded bolts with different groove shapes under tensile loading conditions, the following numerical models were developed in ANSYS. Note that numerical models developed in this section aimed to reproduce fully loaded bolts at actual working conditions, which are bonded to the surrounding rock, rather than a free state boundary, as shown in the tensile tests above. The bolt model was developed using 7400 SOLID, 95 elements, and 32,611 nodes. The material parameters used for this study were: model length 110 mm, diameter 22 mm, Young’s modulus for the bolting steel 20 GPa, and Poisson’s ratio 0.3. The boundary conditions of the bonded bolt were as follows: the x and z displacement were restrained; a fully fixed bottom surface; and the groove and top surfaces were free. The external stress applied to the model top surface was 119 kN. Model geometries . Finally, a protection plastic thin tube was placed on the optical fiber except the optical grating part, and the grooves were filled with 704 silica to encapsulate and protect FBG sensors. The 704 silica is a single-component adhesive material with good adhesion, high strength, and no corrosion, which has excellent electrical insulation, sealing performance, moisture resistance, and anti-seismic and aging resistance. Note that the characteristics of FBGs were as follows: FBG length 10 mm, fiber type was SMF-28C fiber, FWHM BW ≤ 0.3 nm, reflectivity ≥ 85%, acrylate recoat, and FC/APC connector. The completely packaged specimens with jumper connections are shown in The tensile test of the packaged specimens was performed using an MTS system and static and dynamic optical interrogators (SDOI) system. The SDOI system is composed of the static and dynamic optical interrogators, an industrial personal computer, and corresponding software, which can be used to record the specific reflected wavelength of FBG sensors embedded in a bolt with a trapezoidal groove. The general information about the interrogator is shown in The relationship curves between the tensile loads and the reflected wavelength of the FBG sensors with different glues are shown in From the above measurement results, when the tensile load on the bolt exceeded its yield strength, the bolt was expected to experience a significant amount of tensile displacement. Since FBG sensors have relativity limited ability to resist tension deformation, this non-compatible deformation that occurred between the bolt and the FBG sensor may lead to the malfunction of these monitoring sensors. Therefore, FBG sensors can provide accurate results only if the recorded load range is within the yielding strength of the bolt being monitored. However, the malfunction of FBG sensors can be seen as an indicator that bolts have serious internal damage, which may be used in engineering to diagnose bolt integrity.The accuracy of using FBG-based sensors to measure tensile load on bolts is highly reliant on the linear response of the reflected wavelength to tensile deformation, which was only observed in the elastic stage before reaching the yielding point. Therefore, for the purpose of bolt tensile load monitoring, a sound and robust linear relationship between tensile load and reflected wavelength is critically important in FBG-based monitoring of bolts. The elastic range of the four tested specimens during their tensile tests were linearly fitted using regression method as shown in R2 > 9.87) was obtained for all four specimens, the selection of glue material had various impacts on the wavelength–tensile load relationship. The specimens No. 001–No. 003 all presented a constant and reliable linear relationship with a correlation coefficient over 0.995. Conversely, the linear curve of specimen No. 004, which used H814-epoxy resin glue, fluctuated over its elastic period, showing the lowest correlation. In addition, the linear slope of the reflected wavelength using this glue was the smallest among all samples, which indicates the measured tensile load would be much more sensitive to wavelength change compared with the other three. A tiny wavelength change may result in a large variance in measured tensile load. Therefore, in engineering practice, we recommend selecting glues with robust linearity and proper sensitivity response to the reflected wavelength, for instance, glue types No. 001 and No. 002 in this research.Although a relatively high correlation  measured stress is not the true stress state in a no-groove bolt, but an elevated stress state induced by the specific shape of grooves. Four different types of glues normally used in practice were also tested to further understand their role in affecting FBG measuring bolts, according to the test results, we recommend that, among four tested glues, the modified acrylate glue is the optimal glue material for manufacturing the FBG measuring bolt.


A 37-year-old female, a known epileptic, presented to the neurology clinic with a seven-day history of persistent bilateral headache not improving with analgesics. Her neurological and systemic examinations were unremarkable except for right optic disc edema. Magnetic resonance imaging (MRI) brain and magnetic resonance venography (MRV) were normal but her cerebrospinal fluid (CSF) opening pressure was 280 mm of water with a CSF white cell count of 214. The patient showed improvement following treatment with intravenous antibiotics and acyclovir. She returned a week later with double vision and blurring in both eyes. Examination showed bilateral sixth nerve palsies and bilateral optic disc edema with left fundal hemorrhages. The spinal tap was repeated again, which showed a CSF opening pressure of 500 mm of water and the white cell count was 48. Extensive investigations for etiologies were mostly unrevealing. The patient was started on acetazolamide and topiramate combined with a large-volume therapeutic CSF tap. She continued to improve subsequently and was at the baseline functional state at three months, with complete resolution of hemorrhages and optic disc edema. Idiopathic intracranial hypertension (IIH) may present with persistent abnormal CSF with a high white cell count. Therefore, this must be diagnosed with caution, as it may be misdiagnosed and wrongly treated for other causes. Idiopathic intracranial hypertension (IIH) is predominantly seen in young, obese females and characterized by headache, visual blurring, and tinnitus with bilateral papilloedema on examination . Its annThere are many other causes of intracranial hypertension, including drugs , hereditary conditions, and vitamin deficiencies or excesses . IdiopatA 37-year old female presented to the neurology clinic with a seven-day history of diffuse, persistent, and bilateral headache not improving with analgesics. There was no history of fever, loss of consciousness, blurring of vision, or gait abnormality. The patient was a known epileptic and was compliant with her anti-epileptic medication. On examination, her Glasgow Coma Scale (GCS) was 15/15, with normal speech and comprehension. She was afebrile and hemodynamically stable. Her cranial nerves examination was normal, except for right optic disc edema. The sensorimotor and systemic examination was unremarkable.The patient was admitted to Shifa International Hospital, Islamabad, for further workup and management. Magnetic resonance imaging (MRI) of the brain Figures -2 and maLumbar puncture revealed a CSF opening pressure of 280 mm of water and a white cell count of 241, with 90% lymphocytes and 10% neutrophils. CSF proteins and glucose were only mildly deranged (Table The patient was started on intravenous (IV) antibiotics and IV acyclovir for a presumptive diagnosis of meningoencephalitis. She improved with treatment and was discharged home after five days on IV antibiotics and antiviral to complete the rest of the course at home.However, the patient returned after one week with double vision and blurring in both eyes. Examination revealed bilateral sixth nerve palsies and bilateral optic disc edema with left fundal hemorrhages Figures -6. The rOn follow-up after 10 days, the patient manifested some improvement in vision and diplopia. On examination, her ocular movements were normal, however, with persistent bilateral optic disc swelling and left fundal hemorrhages. On this instance, the patient’s CSF pressure was 40 mm of water and white cell count of 95, predominantly lymphocytes (90%) with normal CSF glucose and proteins  for Mycobacterium tuberculosis and fungal organisms were negative. There were no malignant cells seen in the CSF. Neoplastic and inflammatory diseases were ruled out with the help of chest X-rays, ultrasound abdomen and pelvis, blood tests, including CBC, ESR, HIV serology, RPR, TSH, TPHA, ANA, and extractable nuclear antigen (ENA) antibody profile.Despite extensive investigations for all possible causes of abnormal CSF, we could not find any other ongoing disease process to explain this presentation. Subsequently, her improvement with standard treatment for IIH could be considered a confirmation for this being IIH with abnormal CSF -8. The lIdiopathic intracranial hypertension can present with persistently abnormal CSF and high white cell count. Although this is extremely rare, one should be mindful of such a possibility. This should not, however, discourage from directing all efforts to seek alternative etiologies.


Analysis of this mutant demonstrates that rps9 disruption leads to impairment of erythrocyte maturation, resulting in anemia. In addition, the overall phenotype including the anemic state is p53-dependent in rps9 mutants. Furthermore, this anemic state can be partially relieved by the treatment of L-leucine, and dexamethasone, which have been previously used in rescuing the phenotype of other ribosomal protein mutants. Finally, by comparing the phenotype, we show that there are considerable differences in morphology, cytomorphology, and hemoglobin levels for four ribosomal protein mutants in zebrafish. Based on the observed difference, we suggest that the level of anemic severity correlates with the delayed status of erythrocyte maturation in zebrafish models.Ribosome is a vital molecular machine for protein translation in the cell. Defects in several ribosomal proteins including RPS19, RPL11 and RPS14 have been observed in two types of anemia: Diamond Blackfan Anemia and 5q- syndrome. In zebrafish, deficiency of these ribosomal proteins shows similar anemic phenotype. It remains to be determined if any other ribosome proteins are similarly involved in regulating erythropoiesis. Here we generated mutations in zebrafish  For rps9 mRNA synthesis, coding sequence was amplified using cDNA and cloned into pCSDest2 using gateway technology (Thermo Fisher), then mRNA was transcribed after plasmid linearization using SP6 mMESSAGE Kit (Ambion). Rescue experiment was performed by injecting 400 pg rps9 mRNA to the embryos at one cell stage. For gRNA synthesis, DNA template was amplified using gene specific oligo (rps9 guide F: TAATACGACTCACTATAGCGTATTGGAGTGCTGGATGGTTTTAGAGCTAGAAATAGC), then gRNA was transcribed in vitro using MAXIscript T7 kit (Ambion) and purified using MicroRNA Isolation Kit (BioChain USA). 200 pg of Cas9 mRNA and 100 pg of gRNA were mixed and injected into embryos at the one-cell stage. For genotyping, PCR products were amplified from genomic DNA and subjected to electrophoresis using 8% TBE-PAGE gel with the following primers: rps9 Fwd: 5′-TTCCTCGTCAATGAGGCCAT-3′ and rps9 Rev: 5′-CTTTGCACATGTAGTTAGCA-3′. PCR products showing positive bands were further cloned and sequenced.pT3TS-nCas9n plasmid  for zebrafish was linearized with Embryos were anesthetized with tricaine, then immersed in PBS/40% FBS (Hyclone) mixed with a final concentration of 5 mM EDTA (Corning). Blood cells were collected by stabbing the heart using a micro-injection needle. Collected cells were transferred on the slides and air dry. After fixing in the methanol for 5 s, cells were soaked in Wright-Giemsa staining solution for 15 s. Finally, slides were rinsed with deionized water. Images were taken with AxioPlan2 after air dry.rps9 was cloned into pUC19 plasmid (Takara) using rps9-probeF: 5′-GGTACCTGTTCGAGCCTGACACGGAC-3′ and rps9-probeR:5′-TAATACGACTCACTATAGCAAGCACTGCTGAGTCCCT-3′. Probes for other genes were used as previously described .Embryos for TUNNEL staining were stored as described in whole mount For o-dianisidine staining, embryos were collected and fixed at room temperature for 2 h using 4% paraformaldehyde (PFA) in PBS buffer after dechorionation. After the removal of PFA by PBS wash, embryos is incubated in the o-dianisidine (Sigma) staining solution in the dark for 30 min followed by PBS wash. Embryos were then bleached for 20 min. Finally, embryos were immediately imaged after washed by PBS.p53, mdm2, and p21 are previously described  from pool of 30 embryos according to the manufacturer’s directions. cDNA was made using ProtoScript II Reverse Transcriptase and random hexamer primers (New England Biolabs). Quantitative PCR was performed on Stratagene Mx3005P (Agilent Technologies) using Luna Universal qPCR Master Mix (New England Biolabs). Each experiment was performed in triplicate. Primers for https://doi.org/10.25387/g3.9948260.All data generated or analyzed during this study are included in this article and its supplementary information files. Figure S1-S2 contains protein alignment and gene targeting illustration. Figure S3-S7 and Movie S1-2 contain blood related information. Figure S8 contains vessel related information. File S1 contains a full description of these 8 supplementary figures. Supplemental material available at figshare: rps9 gene is highly conserved between human and zebrafish and their proteins share 95% of amino acid similarity . By targeting rps9 with CRISPR/Cas9, three mutant lines were generated. All these three mutant lines had a small deletion in the third exon of rps9 locus. Specifically, two frame-shift mutant lines, la491rps9 and la490rps9, have alleles predicted to encode truncated polypeptides and one mutant line, la489rps9, has an allele producing a protein with one amino acid deletion and a leucine changed to histidine . Three mutant lines were outcrossed to wildtype fish and maintained for further experiments.Located in chromosome 16, la489rps9/rps9 mutant line. When la491rps9/rps9 and la490rps9/rps9 adult fishes were in-crossed, embryos presumably with homozygous mutation started to show notable abnormality from 21 hr post fertilization (hpf). By 24 hpf, aplasia was seen in the head in rps9-deficient embryos, indicated by small eyes and enlarged hindbrain ventricles  with morphological defects all had the identified indels in the rps9 locus . This study confirmed a null deletion in the rps9 gene is associated with the observed phenotype of la491rps9 and la490rps9.No notable abnormality was observed in offspring from in-crossing the ntricles  and 1A’.ntricles . From 48ntricles . The hom mutants . To verirps9, we examined its expression by whole-mount RNA in situ hybridization. At 24hpf, the wild type sibling showed strong expression throughout the body while the mutant displayed a dramatic reduction in expression levels , implying that deficiency in rps9 was responsible for the anemic phenotype. To evaluate numbers of red blood cells, we mated rps9 mutant line into the LCR2:EGFP transgenic fish line, whose red blood cells were tagged with EGFP. A moderate decrease of the EGFP positive cells was revealed between the sibling and mutant embryos at 24hpf, and the decline became significant at 48 hpf . Likewise, the development of hematopoietic stem cell is not affected as there was no considerable change for the expression level for scl and c-myb, markers for the hematopoietic stem cell emergence . However, expression of rag1 decreased significantly, suggesting that lymphoid development is disturbed .To investigate the effect of rpl11 and rps14 shares a similar pattern between the mutant and wildtype sibling control, suggesting that the regulation for other ribosomal protein genes is unaffected upon the deficiency of rps9 .Finally, the expression pattern for two other ribosomal protein genes was also detected to check the expression change for other ribosomal protein genes. The expression of The morphological defect is usually associated with an abnormal increase in the levels of apoptosis. To determine the level change of apoptosis, we conducted a TUNNEL staining for both the mutant and wildtype sibling control. An elevation of the apoptosis level was indeed detected at all time points analyzed, confirming that morphological defect is likely caused by increased activity of apoptosis .p53 activation has been demonstrated in several ribosome protein mutants, and knockdown of p53 can alleviate the phenotype . This suggests that the anemia phenotype is largely dependent on the p53 pathway during both 48 hpf and 3 dpf in zebrafish rps9 mutants.The connection between elevated apoptosis and e embryo . By injenjection . Furthernjection . Of noterps9 mutation is identified in patients.L-leucine, and dexamethasone have been previously shown to alleviate anemia in ribosomal deficiency models . This rerps19, rpl11, and rps14 have been previously reported in zebrafish . A moderate disruption of intersomitic vessel formation was observed at 24 hpf in the mutant, implicating that rps9 might be involved in the vessel formation in zebrafish. Similar to this observation, it was reported that rps29 mutation also caused the disruption of vessel formation (rpl11 mutation seemed not to cause any significant change of vessel morphology (rps9 and Takayasu disease or ribosomal deficiency and vascular integrity in general.Through studying genetic mutation in zebrafish, we demonstrated that rps9 mutant is stronger than rps14 mutant. Erythroid failure of rps9 mutant appeared to be p53-dependent as early as 2 dpf. However, in rps14 mutant, erythroid failure was demonstrated to be p53-dependent at 3 dpf (Anemia in rpl11, rps14, rps9 and rps19. This raises the question why rpl11 deficiency develops defects later than the deficiency in the other three ribosomal proteins. A recent study suggests that ribosomal stress is mediated by free ribosomal proteins, in particular by free RPL11, to bind MDM2 and subsequently activate P53 (p53 activation induced by ribosomal protein disruption in zebrafish models has a great impact on the development of anemia, so it is possible that the other three mutants have more free RPL11 than rpl11 mutant, thus accelerating P53 activation and showing more severe phenotype. Further experiments are needed to test this hypothesis.Comparison of four zebrafish ribosomal protein mutants revealed that severity of morphological and anemic phenotype ranks in the order of


Interleukin-6 (IL-6) contributes to the development of immune-mediated complications after allogeneic stem cell transplantation. However, systemic IL-6 levels also increase during granulocyte colony-stimulating factor (G-CSF) mobilization of hematopoietic stem cells in healthy donors, but it is not known whether this mobilization alters systemic levels of other IL-6 family cytokines/receptors and whether such effects differ between donors. We examined how G-CSF administration influenced C-reactive protein (CRP) levels (85 donors) and serum levels of IL-6 family cytokines/receptors (20 donors). G-CSF increased CRP levels especially in elderly donors with high pretherapy levels, but these preharvesting levels did not influence clinical outcomes . The increased IL-6 levels during G-CSF therapy normalized within 24 h after treatment. G-CSF administration did not alter serum levels of other IL-6-familly mediators. Oncostatin M, but not IL-6, showed a significant correlation with CRP levels during G-CSF therapy. Clustering analysis of mediator levels during G-CSF administration identified two donor subsets mainly characterized by high oncostatin M and IL-6 levels, respectively. Finally, G-CSF could increase IL-6 release by in vitro cultured monocytes, fibroblasts, and mesenchymal stem cells. In summary, G-CSF seems to induce an acute phase reaction with increased systemic IL-6 levels in healthy stem cell donors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell grafts are used for allogeneic stem cell transplantation (ALLO-SCT) . This G-The increased risk of graft-versus-host disease (GVHD) for patients receiving G-CSF-mobilized stem cells has been explained by the increased number of donor T cells in these grafts . HoweverOptimal selection of the healthy stem cell donor is essential for outcome after allogeneic stem cell transplantation, and among the well-characterized donor risk factors are major histocompatibility complex mismatches, female donor for male patient, donor age, and Killing Immunoglobulin-like Receptor genotype . As descIL-6 depends on gp130 for transmembrane signaling, and C-reactive protein (CRP) production is mainly driven by classical IL-6 signaling (dependent on membrane-bound IL-6 receptors) whereas trans-signaling (dependent on soluble IL-6 receptors) seems less important . G-CSF i+ cells on the day of stem cell collection was 51.2 × 106/L (range 15.3–160.7). Age was the only factor associated with reduced level of circulating CD34+ cells (Spearman’s rho −0.420 p < 0.01). Their serum CRP levels were generally low with 75% having CRP level <2 mg/L and 50% below the lower limit of detection (1 mg/L). However, CRP levels were significantly higher  after four days of G-CSF therapy. Those patients with relatively high pretherapy CRP level  also had significantly higher CRP level than the others during G-CSF therapy . Finally, the donor CRP levels before and during G-CSF therapy were not associated with risk of acute GVHD or overall survival of the stem cell recipients. Only pretherapy CRP levels (but not CRP levels during the G-CSF therapy) showed a weak but significant correlation with the levels of circulating CD34n = 20, p < 0.001) and were even higher in graft supernatants. However, IL-6 levels normalized within 24 h after apheresis . We investigated the IL-6 cytokine family in more detail for an unselected subset of 20 healthy donors . Serum samples were then collected before and during  G-CSF therapy, immediately after and 24 h after apheresis. Graft supernatants were also analyzed. The levels of IL-6 family members were determined for all samples c. Low ILAs can be seen from Graft levels were significantly higher than the postapheresis peripheral blood levels especially for IL-31 and OSM, whereas the differences between graft and serum levels for sIL-6R and CNTF reached only borderline significance . The ratp-value = 0.02), but this correlation was absent during G-CSF treatment  when the CRP levels showed a significant correlation with serum OSM levels . Finally, age showed a significant association with peripheral blood CD34+ cell level at the time of harvesting, but the CD34+ cell levels did not show significant associations with the levels of any IL-6 family cytokines/receptors at any of the investigated time points. The systemic IL-6 and CRP levels showed a significant correlation before G-CSF therapy  cells together with the levels of CD34+ cells for our healthy stem cell donors  also varied considerably, especially for NK cells, but also for B cells and monocytes. We investigated the graft composition and the peripheral blood levels of total T cells, CD4l donors . The per+ and CD8+ T cells, B cells, and CD34+ cells; an additional inverse correlation was observed between sIL-6R and circulating CD4+ T cells. Finally, total T cell levels in the blood were also correlated with the CNTF levels. These observations suggest that IL-6 family mediators, and especially gp130/IL-6R, are involved in the trafficking/mobilization of immunocompetent cells during G-CSF therapy of healthy donors. We investigated the associations between the levels of circulating immunocompetent cells and the systemic (serum) levels of each individual IL-6 family cytokine or CRP . The graft composition will then reflect an overall effect of G-CSF therapy and the leukapheresis procedure. CRP levels then showed significant inverse correlations with the graft numbers of CD8U test; p = 0.03); this was the only significant difference that was detected. Finally, the donor age did not show any significant associations with graft or peripheral blood levels of immunocompetent cells. We compared the peripheral blood and graft levels of the various immunocompetent cell subsets for the donor subsets identified in the clustering analysis presented in IL-6 is released by immunocompetent cells and various stromal cells during acute infections in response to danger-associated or pathogen-associated molecular patterns recognized by Toll-like receptors (TLRs) ,29. HoweWe investigated the effects of G-CSF on the IL-6 release by monocytes in the presence the TLR agonists Pam3CSK4 (TLR1/2), LPS (TLR4) or Flagellin (TLR5), R837 (TLR7 > TLR8), and R848 (TLR7/8). Based on initial dose–response experiments we investigated the G-CSF effects in the presence of two different concentrations for each agonist, both concentrations being lower than the concentrations needed for induction of maximal IL-6 release. These results are summarized in We also examined the effect of exogenous G-CSF on IL-6 release by two fibroblast cell lines derived from different individuals and tissues and by MSCs derived from a healthy individual. The G-CSF effect was tested in the presence of three TLR agonists: Pam3CSK4 1 ng/mL (TLR1/2 agonist), LPS 5 ng/mL (TLR4 agonist), and Flagellin 10 ng/mL (TLR5 agonist). Both fibroblast cell lines showed increased IL-6 release in the presence of G-CSF. Increased IL-6 release by fibroblasts release was demonstrated in six independent experiments; it was detected early during culture as well as later when cells were close to confluence, and a strong effect was especially seen in the presence of Flagellin b. FinallTaken together these results suggest that both immunocompetent and stromal cells contribute to the G-CSF induced IL-6 response in healthy individuals, but their contribution possibly differs between various individuals and also seems to depend on the microenvironment of the cells as illustrated by the different G-CSF effects in the presence of various TLR agonists.Previous studies suggest that healthy stem cell donors are heterogeneous with regard to the effects of G-CSF on donor immunoregulation and the number, as well as the functional status, of immunocompetent graft cells ,19,20,21+ graft cells associated with decreased relapse risk [Several recent studies have described associations between graft composition and post-transplant outcome, e.g., high CD8pse risk  and incrpse risk . The firpse risk . Howeverpse risk . Previous studies suggest that G-CSF-induced stem cell mobilization in healthy individuals has a clinically negligible effect on CRP levels with most donors still having CRP levels below 2 mg/L after G-CSF administration ,35. In cIL-6 and CRP levels are usually highly correlated ; this waWe observed an association between the G-CSF induced acute phase reaction and OSM levels. OSM is released by various immunocompetent cells; it can initiate acute phase reactions and is also involved in tissue repair ,42. The OSM is also regarded as a disruptor of epithelial barrier functions, it is a biomarker for active inflammation in rheumatoid arthritis and increased levels are also reported in allergic rhinitis, psoriasis, and asthma . It seemThe peripheral blood levels and the corresponding graft amounts of immunocompetent cells showed a wide variation between healthy donors , and theThe levels of several circulating immunocompetent cell subsets showed an association with the systemic levels of sgp130 that serves as an important modulator of IL-6 signaling through its binding to soluble IL-6R . This obWe also investigated whether G-CSF could increase IL-6 release by in vitro cultured cells. IL-6 can be released by several immunocompetent as well as mesenchymal cells , and in We investigated monocyte/fibroblast/mesenchymal stem cell release of IL-6 in an experimental model based on serum-free  culture medium; this was use to minimize the risk of having TLR ligands in the medium. Our model is thus based on the presence of one ligand, whereas we would expect several endogenous TLR ligands to be present during physiological conditions. For these reasons we would emphasize that these results should be interpreted with great care and additional studies in other experimental models are needed to characterize, in greater detail, the effect of G-CSF IL-6 release by such cells.Fibroblasts express a wide range of TLRs, and we also observed increased IL-6 release for both fibroblast cell lines in the presence of various TLR agonists. The constitutive IL-6 release by MSC was also increased by G-CSF. Taken together these observations suggest that various cells contribute to the IL-6 response during G-CSF therapy. This is similar to the IL-6/CRP responses during infections where both immunocompetent and mesenchymal cells contribute to these responses . Several observations suggest that immunoregulatory events early after stem cell transplantation are important for the outcome after ALLO-SCT, especially the risk of GVHD, for example, the need for early initiation of GVHD prophylaxis and the association between pretransplant conditioning, post-transplant G-CSF therapy, and risk of post-transplant outcome . FurtherAll studies were approved by the Regional Ethics Committee III, University of Bergen, Norway . Only matched related donors  mobilized with G-CSF 5 μg/kg twice daily were included. The donor and patient characteristics are given in + cells was sufficient. Samples were collected before and after 4 days of G-CSF therapy, immediately after leukapheresis, and approximately 24 h after start of leukapheresis. Graft supernatants were also collected. Samples were centrifuged at 1310× g, transferred onto cryotubes within 2 h after sampling, and stored at −70 °C until analyzed. Bio-Plex kits were used to analyze the levels of soluble mediators , using the Luminex®200™ Bio-Rad platform. CRP was analyzed immediately after sampling by an immunoturbidimetric method ; the lower detection limit being 1 mg/L.Stem cell collection was commenced after four days of G-CSF if the number of circulation CD34+ T cells, CD4+ and CD8+ T cell subsets, B cells (CD19+), and NK cells (CD16+CD56+) by using a FACS Canto II flow cytometer . The data were analyzed using FlowJo software version 10.2 . The monocyte levels were determined by multi-angle polarized scatter separation (MAPSS) optical flow cytometry .Peripheral blood and graft levels of immunocompetent cells were analyzed by flow cytometry. Briefly, peripheral blood mononuclear cells and graft cells were cryopreserved in DMSO and stored in liquid nitrogen until analyzed ,51. The Samples were collected from healthy blood donors at Haukeland University Hospital. Monocytes from healthy donors were isolated from gradient-separated peripheral blood mononuclear cells (PBMCs) by negative selection using the human Monocyte Isolation Kit II . The isolation was performed according to the manufacturer’s instructions. Flow cytometric analysis verified that the purity was ≥95%. The Hs27 skin fibroblasts  and HFL1 fetal lung fibroblasts (ATCC CRL153) were also examined together with mesenchymal stem cells (MSC) derived from a healthy individual . Cells were cultured with each of the TLR agonists Pam3CSK4 , lipopolysaccharide (LPS) , Flagellin , R837 , and R848  , with and without G-CSF 50 ng/mL . Monocytes  were incubated in RPMI 1640  with TLR-agonists ± G-CSF for 24 h before harvesting of supernatants. Fibroblasts  were incubated in Dulbecco’s Modified Eagle’s Medium (Sigma) for 24 h before TLR agonists/G-CSF were added and supernatants harvested 24 h later. MSCs  were also incubated for 24 h in mesenchymal stem cell medium alone  for 24 h before TLR-agonists/G-CSF were added and supernatants harvested 24 h later. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2. Supernatants were stored at −80 °C until IL-6 analysis . These mediator analyses were performed in duplicates, and the variation between duplicates was generally less than 10%. p-values < 0.05.Statistical analyses of clinical variables were performed using Stata Version 14  and Graphpad Prism . Differences were regarded as statistically significant when


Time-resolved kinetics lead us to conclude that CT excited states relax to a ground-state intermediate with a time constant of ~3 µs, followed by a structural relaxation to the original CN-Cz2:PO-T2T configuration within ~14 µs.The lack of structural information impeded the access of efficient luminescence for the exciplex type thermally activated delayed fluorescence (TADF). We report here the pump-probe Step-Scan Fourier transform infrared spectra of exciplex composed of a carbazole-based electron donor (CN-Cz2) and 1,3,5-triazine-based electron acceptor (PO-T2T) codeposited as the solid film that gives intermolecular charge transfer (CT), TADF, and record-high exciplex type cyan organic light emitting diodes . The transient infrared spectral assignment to the CT state is unambiguous due to its distinction from the local excited state of either the donor or the acceptor chromophore. Importantly, a broad absorption band centered at ~2060 cm The development of exciplex-type hosts for thermally activated delayed fluorescence organic light-emitting diodes is hindered by a lack of structural information for these donor:acceptor blends. Here, the authors report the pump-probe Step-Scan Fourier transform IR spectra for a D:A exciplex host. The core of TADF molecules lies in their small singlet–triplet energy splitting, ΔET−S (T1 − S1 in energy), which leads to efficient repopulation from the emission forbidden triplet states (T1) to the allowed singlet states (S1) via thermal activation8. To fulfill this criterion, the correlation of two open shell electrons in the triplet state has to be reduced, so that the electron exchange energy can be minimized. This requires large separation and hence weak coupling between two adjacent singly occupied molecular orbitals. In one approach, the molecular identity can comprise an electron donor (D) and an electron acceptor (A), for which the highest occupied molecular orbital (HOMO) of donor and the lowest unoccupied molecular orbital (LUMO) are spatially separated. Upon photoinduced charge transfer (CT), HOMO and LUMO are virtually not or limited overlapped, leading to the reduction of electron exchange energy and hence a small ΔET−S9. OLEDs based on intramolecular TADF emitters with >30% external quantum efficiencies (EQE) have been reported11. However, the challenges for the stability of TADF-based OLED devices have just begun14. The sophisticated molecular designs complicate the syntheses, which might hamper advances in optimization.Lighting materials with thermally activated delayed fluorescence (TADF) have been attracting considerable attention in the organic light emitting diodes (OLEDs)15. The recent usage of exciplex-type host for phosphorescent/fluorescence OLEDs becomes popular16. The associated energy transfer is a Förster type, so that the doped fluorescent emitter can be in a very low concentration, rendering advantage for OLEDs on lower turn-on voltage and smaller roll-off17. Recently, Adachi and coworkers reported organic long persistent luminescence based on intermolecular TADF molecules, which is also expected to trigger the blossoms of new intermolecular TADF systems18. However, this facile approach still encounters challenges in accessing highly efficient exciplex systems21. One of the fundamental issues lies in the lack of structural information on the D/A exciplex. Unfortunately, the structure of such a purported exciplex remains elusive today, and that lack of knowledge impedes advances in exciplex-relevant OLEDs.In another approach, via simply mixing D and A, the intermolecular CT, and hence TADF generation, showed prospects for the future due to its versatility in providing various combinations of D and A materials extensively developed for their use in hole- and electron-transporting materials, respectively22, which are codeposited as a solid film. The cyano group on the CN-Cz2 offers two advantages. First, the -CN strong vibrational absorption at 2200–2500 cm−1 serves as a marker for transient Fourier-transform infrared spectroscopy (FTIR). Secondly, the electron-withdrawing feature of CN group can subtly modulate the energy levels of donor and hence the exciplex emission.We report herein the pump-probe Step-Scan Fourier transform IR spectra of exciplex composed of a new carbazole-based electron donor (D: CN-Cz2) and a well-known 1,3,5-triazine-based acceptor (A: PO-T2T)The cyano-contained donor CN-Cz2 Fig.  was syntΦPL) for CN-Cz2:PO-T2T film was measured to be 55% in air  to the end of delayed fluorescence emission (τ2 = 3.39 µs) at 300 K.As shown in Fig. Lmax of 77,120 cd m−2 at 10.0 V (4152 mA cm−2) with CIE coordinates of  , and 47.5 lm W−1 (PE). At a high luminance of 1000 cd m−2, the device retained a high efficiency of 14.7% (EQE) at 3.4V, which is record-high performance among the exciplex-type cyan OLEDs21. The OLED performances with different CN-Cz2:PO-T2T ratios are showed in Supplementary Fig. The mixtures of CN-Cz2:PO-T2T were introduced as EML in a device structure shown in Supplementary Fig. see Fig. . The max2 substrate to prepare a film. A fourth harmonic (266 nm) of an Nd:YAG laser was used as the pump pulse, which was synchronized with the Step-Scan FTIR spectrometer and overlapped with the IR probe beam at an angle of ~45o. The sample thickness and roughness were crucial; the thickness was typically maintained at ~1 µm to have absorbance of ~5–10 at 266 nm. The roughness was kept at ±10 nm to avoid spatial variation.We then employed an unprecedented approach in attempts to probe the exciplex structure that remains elusive for all TADF molecules. To achieve this aim, we applied time-resolved IR spectroscopy using Step-Scan Fourier transform technology. Details of the experimental set-up and methodology are elaborated in Supplementary Fig. −1) and b (1700–3000 cm−1) at a delay time of 1.6 µs. The vibrational peaks showing positive absorbance were ascribed to the IR spectra of CN-Cz2, PO-T2T, and CN-Cz2:PO-T2T (1:1) in the excited state. Those vibrational peaks having negative absorbance were accordingly associated with IR spectra of the ground-state recovery. Note that, due to the limitations on laser duration, jittering and system response, the early dynamics of a few tens of nanoseconds could not be resolved. Therefore, the acquired temporal IR spectral evolution here was mainly associated with the long-lived (>50 ns) transient species. For individual CN-Cz2 and PO-T2T, due to their lack of any TADF, the observed µs transient IR peaks should be ascribed to those of the local excited (LE) state in the triplet manifold. As for the transient IR spectrum of CN-Cz2:PO-T2T, careful analyses of Fig. −1 in CN-Cz2:PO-T2T (1:1) were new, and they were distinct from those of either CN-Cz2 or PO-T2T. Detailed transient IR data are summarized in Supplementary Table We first performed a steady state IR measurement for CN-Cz2, PO-T2T, and CN-Cz2:PO-T2T (1:1). The results shown in Supplementary Fig. d b 1700–000 cm−1 −1, as shown in Fig. −1  (delay component) and T1(CT) states, which are also subject to mutual cancellation of intensity due to the rise-decay relationship. This makes full IR spectral assignment impractical.The 3D plots (absorbance–frequency–time) for CN-Cz2:PO-T2T 1:1) transient IR spectra are depicted in Fig. :1 transi−1 centered at ~2060 cm−1  and PO-T2T  weakly bound by intermolecular static force25, so the charge carrier was subject to significant polarization, generating polaron-pair  and CN-Cz2:PO-T2T (2:1) solid films 22. For NPNPB/PO-T2T the results of higher polaron-pair absorption frequency than that of CN-Cz2:PO-T2T may indicate the more stable and hence higher efficiency of the polaron-pair formation that is subject to the subsequent polaron dissociation, resulting in the quench of emission. This trend may offer us a clue to explain the lower emission yield in the exciplex-type TADF molecules in common. Further support of this viewpoint is given by the comparative transient FTIR study of a number of highly emissive intramolecular TADF cases. As shown in Supplementary Figs. 12−1. This is rational because the strong bound excitons lead to negligible polaron-pair formation in the intramolecular TADF cases.Perhaps the most significant result for the transient IR of CN-Cz2:PO-T2T 1:1) lies in the observation of a broad band extending from 1700 to 3000 cmm−1 Fig. . This brm−1 Fig.  with tha:1 lies i−1 in Fig. 1(CT) and/or T1(CT) states (vide supra). We thus carefully scrutinize those positive IR bands that are in proximity of the negative IR peaks and found that they undergo similar ~14 µs relaxation time constant but with a decay behavior. For example, the kinetics of positive 1508 cm−1 peak show a decay time of 14.9 µs /T1(CT) IR spectra, the formation of intermediate is proposed to be in the ground state. Moreover, owing to a rise time of ~3.3 µs that is identical with the TADF decay time, the intermediate should be populated via the TADF process. As shown in Fig. 11(CT) state, the charge induction may reorganize the CN-Cz2  and PO-T2T  exciplex configuration for energy minimization. Accordingly, the recombination of polarons , which upon vertical Franck–Condon transition, gives S0(LE)’ in the ground state. The S0(LE)’ state is then subject to further structure relaxation back to the initial CN-Cz2:PO-T2T configuration  study to probe CN-Cz2 and PO-T2T codeposition film, so that the possible grain domain can be assessed. Deduced from GIXD for the CN-Cz2 and PO-T2T 1:1) codeposited film, Figure :1 codepo1 and T1 states are depicted in Fig. 1 (or T1) state are calculated to have 0.12 and 0.13% (0.13 and 0.13% in T1) contribution from PO-T2T and CN-Cz2, respectively, supporting the slight overlap between HOMO and LUMO for efficient TADF. Conversely, for the type II orientation  state are fully separated (null overlap). Therefore, the type I exciplex configuration well matches the experimental observation, i.e., the results of TADF and GIXD. We further calculated the associated IR spectra based on the type I exciplex configuration  obtained experimentally. These vibrations show characteristics in that both molecules exert aromatic C = C stretching in combination with bending motions simultaneously due to the exciplex formation. Note that these IR peaks, together with their relaxation dynamics, have been elaborated above. On the basis of the optimized exciplex S1(CT) structure, we then performed a S1(CT) → ground state vertical transition, followed by full geometry optimization to obtain a local minimum S0(LE)’ ground state shown in Fig. 0(LE)’ state is expected to be weakly bound via van der Waals interaction and tends to relax back to initial CN-Cz2:PO-T2T configuration, which, in a qualitative manner, supports the above proposed kinetics during a TADF cycle.We then performed the ab initio computational approach. Our methodology for calculation to obtain the putative exciplex configuration is elaborated in the supporting information. According to the above in-plane intercalation-like configuration, we considered two displaced orientations for CN-Cz2 and PO-T2T exciplexes, for which the -CN group in CN-Cz2 is toward or away from PO-T2T, specified as type I and II interaction, respectively  consisting of a cyano-contained carbazole-based electron donor CN-Cz2 and an acceptor PO-T2T codeposited as the solid film. We then snapshot the transient IR spectrum of this highly efficient exciplex. Equally important is the observation of polaron-pair absorption spanning the 1700–3000 cm6% consisAdditional synthesis, crystallographic, spectroscopic data, and other support material are provided in the www.ccdc.cam.ac.uk/data_request/cif.The data that support the findings of this study are available from the corresponding author on reasonable request. The X-ray crystallographic coordinates for the CN-Cz2 reported in this article has been deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition number CCDC 1547184. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


To determine the diagnostic and prognostic significance of neurofilament light chain (NfL), TAR DNA‐binding protein 43 (TDP‐43), and total tau (t‐tau) in cerebrospinal fluid (CSF) and plasma of patients with amyotrophic lateral sclerosis (ALS) and to investigate whether the combined use of those biomarker candidates can improve their diagnostic performance.This was a single–center, prospective, longitudinal study. CSF and plasma samples were collected at the time of enrollment from a discovery cohort of 29 patients with ALS and 29 age–matched controls without neurodegenerative disease. In a validation cohort, there were 46 patients with ALS, and 46 control (not age‐matched) patients with motor weakness resulting from neuromuscular diseases. NfL, TDP‐43, and t‐tau levels in CSF and plasma were measured using ultrasensitive single molecule assay (Simoa) technology.The following findings were reproducibly observed among the discovery and validation cohorts: increased levels of CSF NfL, plasma NfL, and CSF TDP‐43 in ALS compared with control groups; shorter survival associated with higher levels of CSF and plasma NfL. When the CSF NfL and CSF TDP‐43 levels were combined, the areas under the ROC curves (AUC) were slightly improved relative to AUCs for each biomarker alone.CSF and plasma NfL may not only serve as diagnostic biomarkers but also provide a measure of disease progression. CSF TDP‐43 is also useful as a diagnostic biomarker of ALS, but has no prognostic value. The combined use of CSF NfL and CSF TDP‐43 may be a useful biomarker for the diagnosis of ALS. The most promising biomarker for amyotrophic lateral sclerosis (ALS) is neurofilament light chain (NfL) at present. Elevated levels of NfL in cerebrospinal fluid (CSF) and plasma/serum have been reported in patients with ALS compared with controls; moreover, they were associated with poor outcomes.N = 17), Guillain–Barre syndrome (GBS: N = 18), multifocal motor neuropathy (MMN: N = 6), and inclusion body myositis (IBM: N = 5). . Informed consent from patients was obtained when possible and also from the nearest relative. Study procedures were designed and performed in accordance with the Declaration of Helsinki. The discovery cohort consisted of 29 individuals with possible, probable, or definite ALS and 29 age–matched controls. The control group participants had nonneurodegenerative diseases and presented with no neurological symptoms. All participants of the discovery cohort underwent CSF and plasma collection. The sample size of the discovery cohort was set according to the effect size of previous biomarker studies.All measurements of the biomarkers were carried out on a Simoa HD‐1 Analyzer  using of commercially available kits.For detailed information of measurements, sample preparation, and statistical analyses, see Data P = 1.000) or sex (P = 0.7840) between the ALS and control groups in the discovery cohort. In the validation cohort, the median age of the control group was significantly younger than that of the ALS group (P < 0.0001), while there was no significant difference in sex between the two groups (P = 0.3696).The demographic characteristics are summarized in Table P = 0.0035, Fig. P < 0.0001, Fig. P = 0.0299, Fig. P < 0.0001, Fig. P = 0.0178, Fig. P = 0.1062, Fig. The concentrations of TDP‐43, NfL, and t‐tau in the samples from the discovery cohort are summarized in Figure According to ROC analysis of the discovery cohort, CSF NfL generated the highest area under the curve (AUC) value . Such a significant CSF‐plasma correlation was also identified in the control group    Fig. B. Neithe63) Fig. A and C.P = 0.0248, Fig. P = 0.0207, Fig. P = 0.0124, Fig. P = 0.003; CSF NfL, HR = 7.727, P = 0.002), while the association between CSF t‐tau and survival did not reach significance .All members of the ALS group in the discovery cohort were included in log‐rank analysis Fig. . NineteeP = 0.0087, Fig. P = 0.0031, Fig. P < 0.0001, Fig. n = 42) and control (n = 24) groups, consisting of individuals aged no younger than 60. In these advanced age groups, comparisons between groups regarding biomarkers showing significant differences between the groups based on raw data  were conducted. Significant elevation of CSF TDP‐43 and CSF NfL and plasma NfL levels in the ALS group compared with those in controls was preserved .Not all patients with ALS in the validation cohort were included in the log‐rank analysis due to missing samples. We performed survival analysis involving 20 ALS patients with plasma biomarker data and 41 ALS patients with CSF biomarker data Fig. B. The hiRegarding the CSF TDP‐43, CSF NfL, and plasma NfL markers that showed significant elevation in the ALS compared with control groups for both discovery and validation cohorts, we calculated composite parameters of the products of CSF NfL × CSF TDP‐43, of CSF NfL × plasma NfL, and of plasma NfL × CSF TDP‐43 Fig. . In bothP = 0.0137, Fig. P = 0.228, Fig. P = 0.0006, Fig. P = 0.0002 and P = 0.0193, respectively). Those significances were still preserved after age‐adjustment . Meanwhile, there was no significant difference in survival between the high– and low–level groups based on TDP‐43 and t‐tau levels in plasma and CSF  analyses for the optimal composite parameters of the discovery and validation cohorts.Click here for additional data file.Data S1. Sample collection and preparation, measurement of NfL, TDP‐43, and t‐tau, and statistics.Table S1. Clinical information and concentrations of biomarkers in the control and ALS groups of the discovery cohort.Table S2. Clinical information and concentrations of biomarkers in the control and ALS groups of the validation cohort.Table S3. Measurements of the internal controls and interassay concordance.Click here for additional data file.


The extension of donor eligibility criteria represents one of the possible ways to increase the organ shortage, thus decreasing the waiting time for kidney transplantation. Expectedly, this strategy is associated with a growing number of more technically demanding living donor nephrectomy procedures requiring careful assessment, and sound surgical experience in order to avoid intraoperative complications.After a thorough evaluation through preoperative imaging, we performed a hand-assisted left laparoscopic living donor nephrectomy in a 56 year-old overweight patient with history of prior abdominal surgery, harboring a left-sided inferior vena cava (IVC).This case describes our comprehensive approach in this complex surgical scenario to preserve donor safety and provide an optimal kidney graft. Actually, the final decision on the donor selection, surgical approach, and kidney graft laterality often relies on the team involved and the accumulated experience of their members rather than on a well established protocol .Herein, we describe our comprehensive approach to an overweight living kidney donor harboring a left-sided IVC, and multiple adhesions secondary to previous abdominal surgery, aiming to provide a rationale for adequate decision-making and counseling, thus empowering surgical safety and optimal kidney graft retrieval.This study has been reported in line with the SCARE criteria .22) and showed a previous history of cholecystitis and morbid obesity treated with open cholecystectomy and gastric bypass, respectively. She was cleared after full medical assessment, and a careful review of preoperative Computed Tomography angiography (CTA) revealed an infrarenal IVC located at the left side of the Aorta, crossing the midline to its right side at the level of the ostium of a single left renal vein in which both adrenal and gonadal veins drained  at 4 months follow-up.3The chance of getting off the kidney transplant waiting list for the almost 95,000 patients with end stage renal disease awaiting a deceased donor organ in United States was estimated in 1:8 for the year 2018 . Living Since Ratner et al.  descriptHand-assisted LDN was introduced aiming to combine the advantages of the laparoscopic technique with the quicker and safer kidney retrieval of the open technique (provided by the tactile feedback) . DespiteRenal vascular anomalies are rare, but if unexpected may represent an important concern in candidates for living kidney donation given its potential for donor and organ damage (vascular injury) in unexperienced hands. Hence, preoperative assessment of pertinent vascular anatomy by means of high resolution cross-sectional imaging is crucial to avoid potential complications, resulting advisable in every potential candidate to better select the most appropriate operation strategy .Left-sided inferior vena cava (LIVC) is caused by the persistence of the left supracardinal and regression of the right supracardinal veins occurred during the complex process of embryogenesis (6–10th weeks of gestation), and represents the second most common anatomical anomaly of the IVC (0.04%–0.5% of population). In the adult form, the left infrarenal portion of the IVC crosses anterior to the aorta at the level of the ostium of the left renal vein, joins the right renal vein, and remains right-sided in its suprarenal portion. However, different degrees of regression and associated ureteric anomalies creates a wide variety of different configurations . Recent Nevertheless, the debate on the safety, efficacy, and potential advantages of choosing the right or left kidney for LDN is still open . In this4The extension of the donor eligibility criteria will provide an increasing number of complex vascular cases with previous abdominal surgery. To our knowledge, this is the first case presenting a minimally invasive approach to an overweight living donor with LIVC and simultaneous intra-abdominal adhesions. Careful preoperative evaluation, extended adhesiolysis, wide vascular exposure for adequate visualization, and meticulous hilar dissection were key aspects in avoiding intraoperative complications for the donor and obtaining an optimal kidney graft. Furthermore, both donor and recipient had a successful outcome in terms of renal function.“Enrico ed Enrica Sovena” Foundation, Rome, Italy.This work was supported by a grant of the The case report is exempted from ethical committee.Written informed consent was obtained prior to surgery from both donor and recipient.Giuseppe Serena .Javier González .Leonardo E Garcia (Drafting Article).Giselle Guerra .Mahmoud Morsi .Gaetano Ciancio .Since this is a case report, not a research study, registration is not indicated.Gaetano Ciancio MD, MBA, FACS.Not commissioned, externally peer-reviewedThe authors of this manuscript have no conflict of interest to disclose.


We aimed to assess the relationship between levels of a cerebrospinal fluid (CSF) marker of pericyte damage, soluble platelet-derived growth factor receptor β (sPDGFRβ) and CSF markers of blood-brain barrier (BBB) integrity  and disease pathology  in Alzheimer’s disease (AD).n = 23) in a separate neurologically normal group for which the CSF/serum albumin ratio had been determined.sPDGFRβ and albumin were measured by sandwich ELISA in ante-mortem CSF from 39 AD and 39 age-matched controls that were grouped according to their biomarker profile . sPDGFRβ was also measured in matched serum and CSF samples (p = 0.0038) and correlated positively with albumin , total tau  and phosphorylated tau  in AD but not in controls. CSF sPDGFRβ did not correlate with Aβ42. Serum and CSF sPDGFRβ were positively correlated  in the independent neurologically normal CSF/serum matched samples.CSF sPDGFRβ level was significantly increased in AD (We provide further evidence of an association between pericyte injury and BBB breakdown in AD and novel evidence that a CSF marker of pericyte injury is related to the severity of AD pathology.The online version of this article (10.1186/s13195-019-0534-8) contains supplementary material, which is available to authorized users. Alzheimer’s disease (AD) and vascular dementia (VaD) together account for most cases of dementia. Cerebral hypoperfusion, neurovascular uncoupling and blood-brain barrier (BBB) breakdown are defining features of VaD, but there is now compelling evidence they are also major contributors to cognitive decline and disease pathology in the early stages of AD . PerPDGFRβ is shed from human pericytes (but not vascular smooth muscle cells) when they are cultured under hypoxic conditions (1% oxygen for 48 h) or exposed to Aβ peptides . Cell meNation et al.  reported181P and Aβ42  had previously been determined using commercially available sandwich enzyme-linked immunosorbent assays (ELISAs) . All AD patients had abnormal CSF levels of the core AD biomarkers , while controls had normal levels . The cut-off values used in this study are in line with current clinical practice and closely resemble those outlined by Hansson et al. [APOE status were not recorded, and matching serum samples were not available for this cohort.CSF sPDGFRβ and albumin were measured in 39 AD cases and 39 controls for which CSF t-tau, p-taun = 23) spanning a larger age range (23–84 years). The CSF/serum albumin ratio had previously been determined by an immunoturbidimetric albumin method on Elecsys . The demographics of this cohort are presented in Table sPDGFRβ was, however, also measured in an independent CSF and matched serum sample cohort from neurologically normal controls n = 3 spanninCSF and serum sPDGFRβ levels were measured by sandwich ELISA . CSF samples (100 μL undiluted) and serum (diluted 1 in 40 in proprietary dilution buffer supplied with the kit) were loaded. Standards, samples and blanks were added in duplicate. Absorbance was read at 450 nM in a FLUOstar OPTIMA plate reader . Reproducibility reported in the datasheet indicates an inter-assay CV < 12% and intra-assay < 10%, with spike recovery between 90 and 110% for serum, plasma and cell culture media. PDGFRβ concentration in samples was calculated by interpolation against the standard curve for each case, derived from serial dilutions of recombinant PDGFRβ . The mean values from duplicate measurements are presented.CSF albumin level was measured by sandwich ELISA (Cat no 108788) . CSF samples were diluted 1 in 2000 in proprietary dilution buffer supplied with the kit. Standards, samples and blanks were added in duplicate. Absorbance was read at 450 nM in a FLUOstar OPTIMA plate reader . Albumin concentration in each sample was interpolated from a standard curve derived from serial dilution of recombinant human albumin (200–3.125 ng/mL). The mean values are presented.t tests or ANOVA with Bonferroni post hoc correction was used for comparisons between groups, and Pearson’s test was used to assess linear correlation. Data values outside the 99% confidence interval of the linear regression line were considered to be outliers and removed prior to statistical analysis. The analyses used SPSS version 16  and GraphPad Prism version 6 . p values < 0.05 were considered statistically significant.As the CSF sPDGFRβ and albumin levels were normally distributed, unpaired two-tailed p = 0.0038)   but not in controls   Fig. c. Inspecr = 0.50, p = 0.0017) but not in controls   but not in controls   or AD cohort   or AD  groups   Fig. a. CSF sP85) Fig. b.Fig. 2We have confirmed that CSF sPDGFRβ is increased in AD, to an extent that correlates with CSF albumin level (a marker of BBB leakiness) and with the neurodegeneration biomarkers CSF t-tau and p-tau. We have also shown that the level of sPDGFRβ in CSF correlates with that in serum in an independent cohort of matched CSF/serum samples. Our findings support recent reports of pericyte injury and BBB leakiness in AD, as demonstrated in histological and biochemical studies of autopsy tissue  and in bF7/F7 mice, which have disrupted PDGFRβ signalling, have a more pronounced vascular phenotype associated with vascular loss and BBB leakiness [+/− heterozygous mice were crossed with Tg-APP mice, there was accelerated Aβ accumulation and clinical disease progression [Post-mortem studies have confirmed pericyte loss and BBB leakiness in AD , 10. Redeakiness , and whegression , supportgression .Compromised BBB integrity that can be demonstrated in the hippocampus in normal ageing and is exacerbated in MCI, as revealed by advanced dynamic contrast-enhanced MRI, is associated with elevated CSF sPDGFRβ . Nation APOE genotype [The mechanisms of pericyte injury in AD remain unclear, but both Aβ and hypoperfusion are probably contributors. Aβ peptides at supraphysiological concentrations were shown to be toxic to human brain pericytes , dependegenotype . The toxgenotype . In a bigenotype . The preAPOE genotype, that would be of interest for further analysis. This is also an observational cross-sectional study and does not provide clues as to the timing and regional changes in pericyte loss in relation to onset and progression of disease. The extent of vascular burden within our cohort is also unclear, and as in other clinical studies, it is likely to include a spectrum of AD and mixed dementia cases. Other variables that related to the collection of the samples and might theoretically impact on the measurements  were also not available for analysis. Nonetheless, our data together with recent studies suggest that markers of vascular dysfunction, including pericyte loss and BBB leakiness, are related to cognitive impairment in MCI and disease pathology in AD and can potentially be monitored by analysis of CSF and possibly also serum. The inclusion of vascular biomarkers as part of an AD research framework, as suggested by Sweeney and colleagues [This study has several limitations including the small number of AD cases and controls and the paucity of clinical details relating to the de-identified AD cases, such as dementia severity, disease duration and lleagues , would iAdditional file 1:Figure S1 CSF-sPDGFRβ is not altered in relation to age or gender. (a) Scatterplot showing no statistically significant relationship between CSF-sPDGFRβ level and age in AD (red) and control (green). The best-fit linear regression line and 95% confidence interval for each group are superimposed. (b) Bar chart showing CSF-sPDGFRβ level in control and AD group stratified for gender. No significant differences were observed. Bars represent the mean ± SEM. (DOCX 209 kb)


Cerebrospinal fluid (CSF) from extra-ventricular drainage (EVD) systems is routinely analysed to diagnose EVD–related bacterial meningitis. We investigated the effect of time delay and sample processing on cell count and basic biochemistry results in EVD CSF to define optimal turnaround time and whether manual and automated cell counting are comparable in such samples.In total, 32 EVD CSF samples were analysed. Baseline testing included cell counting  and biochemistry analyses . Manual cell counting was also performed at intervals of 61-90 and 91-150 minutes from baseline in the residual sample. Biochemistry analyses were performed in samples before and after centrifugation at baseline and at 91-150 minutes interval.At 91-150 minutes total cell count (P < 0.001), large lymphocytes (P = 0.007), neutrophils (P < 0.001) and phagocytes (P = 0.006) obtained by manual counting decreased and the number of disintegrated cells count increased (P = 0.016) compared to the baseline values. Considering method comparison, proportional difference between methods for all cell (sub)groups was obtained, whereas polymorphonuclears also showed the constant difference (y = 11.21 + 1.22x). Compared to centrifuged CSF, lower concentration of glucose and lactates were obtained in uncentrifuged samples (P < 0.001) at baseline.Manual cell counting should be performed within 60 minutes as any delay can alter results. The same counting technique should be used to obtain longitudinally assessable results. Biochemistry tests are stable in uncentrifuged CSF up to 2.5 hours. Cerebrospinal fluid (CSF) assays are an integral part of diagnostics of neurological diseases, especially in the emergency neurological patients. In such cases, it is imperative that the results are available as soon as possible to assist in diagnosis and further patient treatment.Extraventricular drainage (EVD) system is indicated for the management of patients who suffered subarachnoid or intracranial haemorrhage, hydrocephalus, shunt malfunction or have a tumour  H56-A guidelines on analysis of cellular components in body fluids recommend that cells should be natively counted using a counting chamber. Cell counting should be performed as soon as possible because of rapid According to the consensus protocol for the standardization of CSF collection, the CSF samples obtained by lumbar puncture (LP) should be processed as soon as possible but the document also acknowledges that processing of CSF samples within an hour is not a common practice in most laboratories. Thus, the main recommendation is that sample processing should be done within a time delay of 1.5 hours (± 30 minutes)  for these samples. Additionally, we have compared automated and manual counting methods to investigate whether both methods could be performed interchangeably in EVD samples.This prospective experimental study was performed from October 2016 to January 2017 in Department of Laboratory Diagnostics, University Hospital Centre Zagreb, Croatia. Consecutive CSF samples collected from EVD systems using standard collection procedure of adult patients from neurological intensive care units, and delivered to the laboratory for routine processing in the morning hours (from 7 to 11 am) with sufficient sample volume (> 2 mL) were included  and red blood cells (RBC) were counted in native CSF using the Fuchs-Rosenthal chamber. Total cells were differentiated to small and large lymphocytes, neutrophil granulocytes, phagocytes and disintegrated cells and results were expressed as a cell number/3x106/L.The cell count was also performed in Body fluid (BF) mode on the Sysmex XE5000 haematology analyser . The cell count in BF mode included: white blood cells (WBC), red blood cells (RBC), polymorphonuclear cells (PMN) and mononuclear cells (MN). Because of the limited sample volume, counting in Body fluid mode was not repeated at delayed time intervals. White blood cells parameter corresponds to TC in the manual count. The results were expressed as a cell number/3 x10Glucose, lactate and total protein concentrations were measured on Cobas 6000 c501 biochemistry analyser  with standard methods, using reagents GLUC3 (enzymatic reference method with hexokinase), LACT2 (enzymatic method with lactate oxidase) and TPUC3 (precipitating method with benzethonium chloride), respectively . For each analyte, quality control was performed daily in two levels using PreciControl ClinChem Multi 1 and PreciControl ClinChem Multi 2 , and Precinorm PUC and Precipath PUC controls . Coefficients of variations in the tested time period were within analytical requirements for imprecision.post-hoc test to test whether the differences in biochemistry tests between centrifuged and uncentrifuged CSF are associated with the TC or RBC count. Comparisons of automated and manual cell counting methods were performed using Passing-Bablok regression, presented with regression equation and 95% confidence intervals (95% CI) for slope and intercept, and Bland Altman analysis. The following cell subgroups were compared: WBC and TC, PMN and neutrophil granulocytes, MN and the sum of small and large lymphocytes. A P value < 0.05 was set as a level of statistical significance. Statistical analysis was performed using MedCalc Software v.17.4.4 .Data were tested for normality using the Shapiro-Wilk test. Data did not follow a normal distribution and were presented as median (interquartile range (IQR)). Statistical differences were tested using the Wilcoxon rank sum test for paired samples. Spearman correlation was performed as a A total number of 32 CSF samples collected from EVD system were included in the study. Median (IQR) of tested analytes at each time interval and sample processing status are summarized in Tables 1 and 2Throughout the tested period regarding the manual counting, the number of RBCs (P = 0.202) and small lymphocytes (P = 0.167) remained unchanged compared to the baseline values. Already, at 61-90 minutes interval, TC count (P = 0.033), neutrophil granulocytes (P = 0.046) and phagocytes (P = 0.020) decreased compared to the baseline counting. Finally, at 91-150 minutes interval, large lymphocytes decreased (P = 0.007) and the number of disintegrated cells increased (P = 0.016) compared to the baseline count .6/L and a sample with 632,320/3 x106/L RBCs. These samples had extremely high cell counts, which would influence Passing-Bablok regression analysis. Automated and manual methods for cell counting showed: the proportional difference for WBC  x] . Bland AThe results of biochemical tests in uncentrifuged EVD CSF samples were available very quickly with a median time of 16 min (IQR 6-28) from sample admission. Median time to results was 27 min (IQR 22-37) for centrifuged samples. However, compared to the centrifuged EVD CSF samples assayed within 60 minutes of sample admission, uncentrifuged samples had lower glucose (P < 0.001) and lactate (P < 0.001) concentration. Glucose (P = 0.006) and lactate (P < 0.001) concentrations were also lower in uncentrifuged CSF samples compared to centrifuged samples at the 91-150 minutes interval . DiffereCerebrospinal fluid samples from EVD system are partially different from CSF samples obtained by lumbar puncture and represent a special category of biological material. The stability of different analytes in such samples is not well established. We investigated the effect of time delay and sample processing on cell count and basic biochemistry results in these samples.Our results showed that a relatively short delay in cell counting (61-90 minutes) caused a statistically significant fall in total cell count and granulocyte count by manual method. These results are in accordance with previously reported results for CSF obtained by LP that indicated that the delay in WBC counting could produce false negative results in 40% of samples with pleocytosis and as high as in 86% of the samples with a mild elevation in WBC count (in vitro study suggest that the effect of RBC contribution on lactate concentration becomes significant only after 6 hours (Manufacturer recommends measuring glucose in CSF immediately as such samples can be contaminated with bacteria and can often contain other cellular constituents. Otherwise, storing the sample at 4 °C or - 20 °C is recommended (Analysing uncentrifuged EVD CSF samples is unacceptable because glucose and lactate concentrations were significantly lower compared to centrifuged samples. This effect was present regardless of time delay and was not associated with total cells or red blood cells count. Therefore, faster TAT cannot be achieved by omitting centrifugation, even in samples with low cell counts.Due to increased use of automated methods for cell counting in CSF, especially in emergency services, we analysed EVD samples at the baseline by both manual and automated methods, to establish if they could be used simultaneously and/or as a substitute for each other. Contrary to previously reported studies comparing CSF samples collected by lumbar puncture, an automated and manual method for cell counting proved not to be comparable when assaying CSF collected from EVD in our study (Limitations of the present study include small sample size and uneven distribution of the measurements that could influence the comparison of the manual and automated methods of cell counting in EVD samples. This could also be the reason for the disagreement with the results of other studies performed in CSF collected by lumbar puncture. However, due to the difficulty in sampling CSF from EVD, limited amount of the collected material, and the relatively long duration of the study, we consider that the included sample size is representative for the studied population. Furthermore, the information on time passed from the sampling to the laboratory was not available, and we were only able to investigate the effect of time delay in EVD samples within the laboratory. Due to the insufficient CSF volume, we were unable to test the effect of time delay in more time intervals. Therefore, we have limited our study design to test only the effect of 2.5 hours time delay on biochemistry analytes and compared automated and manual cell counting only at the baseline.In summary, our results confirmed that delay in cell counting in EVD CSF samples causes a significant fall in total cell count and granulocyte count. Therefore, we recommend TAT for cell number analysis of maximum 60 minutes. Glucose, lactate and total proteins are stable in uncentrifuged EVD CSF samples meaning the eventual delay in sample processing would not influence analytical results, but samples have to be centrifuged prior to biochemistry analysis. Results of manual and automated counting methods are not comparable for EVD samples. It is therefore imperative to always use the same counting technique to obtain results which could be correctly longitudinally assessed.