Differences in CSF SHBG levels across groups, as well as its association with CSF YKL40, pTau181/total-Tau ratio and cognitive function were analyzed. CSF SHBG did not differ across groups, though a trend towards elevated levels in FTLD-Tau cases compared to FTLD-TDP and controls was observed. CSF SHBG levels were not associated with either CSF YKL40 or the p/tTau ratio. They, however, inversely correlated with the MMSE score , an association likely driven by the FTLD-Tau group . CSF SHBG is not a suitable biomarker to discriminate FTLD-Tau from FTLD-TDP.Biomarkers to discriminate the main pathologies underlying frontotemporal lobar degeneration  are lacking. Our previous FTLD cerebrospinal fluid (CSF) proteome study revealed that sex hormone-binding globulin (SHBG) was specifically increased in FTLD-Tau patients. Here we investigated the potential of CSF SHBG as a novel biomarker discriminating the main FTLD pathological subtypes. SHBG was measured in CSF samples from patients with FTLD-Tau ( Frontotemporal lobar degeneration (FTLD) covers a spectrum of highly heterogeneous disorders from a clinical, genetic and pathological perspective . The majMost biomarker studies performed to date have analyzed pathologically heterogeneous populations . The fewn = 85) included ante-mortem CSF samples from 52 FTLD-confirmed patients and 33 cognitive unimpaired individuals that were used as controls. FTLD cases with tau neuropathology  were selected based on autopsy  and 2 cases with corticobasal degeneration CBD tauopathy), and the presence of mutations in the MAPT gene predictive of Tau proteinopathy (n = 8) [n = 5), highly-associated to Tau pathology [n = 29) were selected based on autopsy (n = 13) or the presence of mutations predictive of TDP43 proteopathy (9 C9orf72 and 3 GRN mutation carriers) [n = 4), which is associated with underlying TDP43 pathology [The total cohort  following manufacturer’s recommendations. We first evaluated and validated the performance of the assay for CSF analysis using CSF pools . Dilution = 30) from a previous study [Levels of YKL40 were available for a subset of samples  and graphs were drafted with GraphPad Prism 9 . Between-group analyses of demographic variables were performed using the Student’s t-test or Pearson’s chi-square test after normalizing skewed data using two-step transformation. Regression and Spearman correlation analyses were performed to analyze the relationship between SHBG, age and sex. Differences in SHBG concentration between clinical groups were evaluated by general linear modeling using normalized values and including age as covariate. Diagnostic performance was evaluated using receiver operator characteristic (ROC) and areas under the curve (AUC). Partial non-parametric correlation analyses were performed to analyze the association between CSF SHBG with CSF YKL40, p/tTau ratio and MMSE score including age as covariates. Passing-Babock transformation formulas were calculated based on individuals with both Luminex and Innotest values for tTau and pTau to estimate the equivalent Innotest values for those samples measured with Luminex platform only.Analysis of demographic characteristics showed that sex did not differ between FTLD and controls. However, nominal significance indicated a higher number of females in the FTLD-Tau group and higher number of males in the FTLD-TDP group. FTLD and its pathological subtypes had lower MMSE than controls. FTLD patients had lower CSF p/tTau ratio and higher YKL40 levels compared to controls. CSF p/tTau ratio was lower in FTLD-TDP compared to both FTLD-Tau and controls . p = 0.006). Stratification into clinical groups showed that this correlation was significant in the FTLD group only . No difference in CSF SHBG levels was observed between females and males A, neithep = 0,01; p > 0.05, We next investigated the relationship of CSF SHBG with other FTLD CSF markers and with cognitive function. CSF SHBG did not correlate with YKL40 or the p/Tau ratio E–G. HoweThis study reveals that CSF SHBG, as measured by an automated ECLIA, did not differ across pathological groups, though a trend towards increased levels of CSF SHBG was observed in FTLD-Tau cases compared to FTLD-TDP and controls. CSF SHBG levels were inversely associated with cognitive function.GRN or C9orf72), share the same pathophysiological processes, which can ultimately impact the body-fluid biochemical profiles [C9orf72 mutation or with FTLD-ALS, which were not analyzed in our previous study. There was, however, no apparent pattern associated with the specific clinic-pathologic and genetic subgroups, suggesting that within group heterogeneity may not explain the contrasting findings. The FTLD-Tau group of the previous study was younger than the patients included in this study. Considering CSF SHBG increases with age, demographic differences may partly explain the discrepancies observed. Different disease stages at time of sample collection could also contribute to the difference in findings. Analyzing the effect of the disease stage on FTLD biomarkers is challenging, especially considering there is not yet an established definition of the prodromal phase of FTLD [The increasing trend of CSF SHBG levels in FTLD-Tau patients detected in this study may become significant when analyzing larger data sets. However, the CSF SHBG overlap between FTLD-Tau and FTLD-TDP will likely still be too high to be a useful discriminatory marker for FTLD pathological subtypes. This is further supported by the low and variable discriminative performance of SHBG detected in this study. The results of this study counters our previous unbiased proteomic findings in which CSF SHBG was decreased in FTLD-Tau patients compared to both controls and FTLD-TDP . Translaprofiles ,15,16,17 of FTLD . It is a of FTLD ,20,21,22Despite serum SHBG levels being often elevated in females ,23, we dWe observed that CSF SHBG was inversely associated, albeit weakly, with cognitive function, which is in line with previous findings showing higher levels of serum SHBG associated with risk of dementia or cognitive decline ,24. InteDespite the use of well-characterized samples from cases with confirmed FTLD diagnosis is a strength of this study, we did not have access to samples from patients with an underlying FUS pathology, who account for approximately 5% of all FTLD cases . We acknIn conclusion, our results indicate that, despite the fact that CSF SHBG tends to be increased FTLD-Tau, there is high overlap between controls and FTLD pathological subtypes. We showed that CSF SHBG is not a suitable biomarker to discriminate FTLD pathological subtypes using the automated immunoassay employed in this study, and thus, the quest for novel body-fluid based biomarkers deciphering FTLD pathological complexity will continue.


RBC transfusion effectiveness varies due to donor, component, and recipient factors. Prior studies identified characteristics associated with variation in hemoglobin increments following transfusion. We extended these observations, examining donor genetic and nongenetic factors affecting transfusion effectiveness.This is a multicenter retrospective study of 46,705 patients and 102,043 evaluable RBC transfusions from 2013 to 2016 across 12 hospitals. Transfusion effectiveness was defined as hemoglobin, bilirubin, or creatinine increments following single RBC unit transfusion. Models incorporated a subset of donors with data on single nucleotide polymorphisms associated with osmotic and oxidative hemolysis in vitro. Mixed modeling accounting for repeated transfusion episodes identified predictors of transfusion effectiveness.G6PD deficiency and polymorphisms in SEC14L4, HBA2, and MYO9B genes were associated with decreased hemoglobin increments. Donor G6PD deficiency and polymorphisms in SEC14L4 were associated with increased transfusion requirements in the subsequent 48 hours.Blood donor , component , and recipient  characteristics were associated with hemoglobin and bilirubin, but not creatinine, increments following RBC transfusions. Increased storage duration was associated with increased bilirubin and decreased hemoglobin increments, suggestive of in vivo hemolysis following transfusion. Donor Donor genetic and other factors, such as RBC storage duration, affect transfusion effectiveness as defined by decreased hemoglobin or increased bilirubin increments. Addressing these factors will provide a precision medicine approach to improve patient outcomes, particularly for chronically transfused RBC recipients, who would most benefit from more effective transfusion products.Funding was provided by HHSN 75N92019D00032, HHSN 75N92019D00034, 75N92019D00035, HHSN 75N92019D00036, and HHSN 75N92019D00037; R01HL126130; and the National Institute of Child Health and Human Development (NICHD).  RBC transfusions can be a critical life-saving intervention for hospitalized and chronically transfused patients. In contrast to pharmaceutical-grade drugs, there is substantial variability in hemoglobin dose and RBC quality among units of blood , 2. BlooG6PD) deficiency, an X-linked disorder affecting the ability of RBCs to handle oxidant stress, is the most common human enzymopathy, affecting approximately 400 million people worldwide  , 9–12. Iorldwide , 15. Intsed RBCs . Howeversed RBCs , 18. ThuWe identified 46,705 and 5354 patients, with 102,043 and 6168 evaluable single RBC unit transfusion episodes, in the full cohort and the RBC-Omics Study subset (in which donor genetic polymorphism information was available) , respecthttps://doi.org/10.1172/jci.insight.152598DS1) were mostly consistent with prior studies , 8 IQR, 4–15), and 6  hours before transfusion, respectively . The pos5, and 6 Because it is expected that prolonged refrigerator storage prior to transfusion is associated with an approximately 10% decrease in posttransfusion RBC recovery on average , we hypoOn multivariable analysis, donor factors predicted to increase the amount of hemoglobin in a unit , donor G6PD deficiency variants (n = 43) in male donors were strongly associated with a decreased mean ± SD hemoglobin increment in transfused recipients  compared with those with WT alleles (n = 2584) were also statistically significant  and number of additional RBC transfusions  within 48 hours of the index transfusion event.Given that a subset of the blood donors in our linked database had associated genotype data from the REDS-III RBC-Omics Study , we examD allele . Thus, tSEC14L4, MYO9B, and HBA2 , but not for HBA2  or MYO9B . In parallel, the number of additional RBC transfusions for recipients of index units from homozygous recessive donors compared with homozygous dominant donors was increased for SEC14L4 , but not for HBA2  or MYO9B .We explored the effects of other donor SNPs on transfusion effectiveness, as defined by the recipient hemoglobin increment (bilirubin increment models were not considered due to sample size limitations), in our multivariable model adjusting for donor, component, and recipient factors. We first compared allele frequency in all RBC-Omics donors with that of donors represented in our linked database to test whether there was selection bias ; the reland HBA2 . Althoug disease , 23, SECSEC14L4 again correlated with reduced hemoglobin increments, as did SNPs for Hexokinase 1 (HK1), for which mutations are associated with a nonspherocytic hemolytic anemia (Aquaporin 1 (AQP1), an integral membrane protein regulating RBC volume in response to tonicity  are not unexpected and likely represent clearance of a larger mass of damaged RBCs in units with higher doses of hemoglobin. RBC storage duration was the exception to the aforementioned correlation, with prolonged storage being strongly associated with a reduced hemoglobin and an increased bilirubin increment. This particular relationship was previously demonstrated in healthy volunteers transfused with autologous RBC units . AlthougG6PD and SEC14L4. Out of 18 loci for osmotic hemolysis examined for decreased hemoglobin increment, we observed 2 significant associations: HBA2 and MYO9B. Although SNPs in G6PD and HBA2 are associated with human disease, clinical phenotypes for SEC14L4 and MYO9B are not yet described. A sensitivity analysis of relevant donor, component, and patient characteristics did not identify any obvious differences between the alleles of these 2 SNPs . These subjects were also more likely to be recipients of multiunit RBC episodes where bleeding and additional i.v. fluids may have impacted our outcomes. In parallel with other characteristics, uncommon SNPs are also less likely to impact hemoglobin increment when occurring in multiunit transfusion events; nevertheless, they contribute to the significant variation in hemoglobin increments observed in clinical practice in conjunction with other donor and component factors. Future studies of multiunit transfusion events are needed in a controlled setting, including study of the role of platelet and plasma transfusions to demonstrate the generalizability of our findings. Although we limited analysis to pretransfusion bilirubin levels below 1.5 mg/dL to mitigate the role of patient conditions  from affecting the results, our findings may still be prone to unmeasured confounding. However, sensitivity analyses examining relative changes in creatinine levels and various pretransfusion thresholds for our outcomes did not yield different results. Due to sample size limitations, our analysis was underpowered to identify associations of uncommon donor alleles that had small effect sizes, nor were we able to examine the impact of donor genetics on posttransfusion bilirubin increments. To this end, the NIH-funded REDS-IV-P program is planning a prospective trial in chronically transfused patients with sickle cell disease, thalassemia, or pediatric cancer to confirm and expand upon the genetic determinants of transfusion effectiveness . Finally, despite the associations of donor, component, and recipient variables with decreased transfusion effectiveness, as defined by a decreased hemoglobin increment or an increased bilirubin increment demonstrated by this study, we did not explore whether decreased transfusion effectiveness is associated with patient harm other than an increased need for subsequent transfusion. Our current analyses were limited to single-unit episodes, and potential harmful effects may be greater when accounting for cumulative transfusion exposures. A recent study found that a limited set of donor and manufacturing characteristics correlated with smaller hemoglobin increments, which were then associated with additional RBC transfusion requirements in the patients  and specific outcome measures  can better address the issue of potential harm.The current study has several limitations. The retrospective and observational nature of the study design limits the ability to infer causation for the factors associated with decreased transfusion effectiveness. Nonetheless, the models confirmed previous prospective, randomized study findings of decreased hemoglobin, and increased bilirubin increments associated with increased storage duration . Furtherpatients . We simiG6PD, SEC14L4, HBA2, and MYO9B), and effectiveness measures following single-unit RBC transfusion events. The strong associations between increased storage duration and donor G6PD deficiency on transfusion effectiveness support the validity of this approach for identifying factors that influence transfusion effectiveness. These findings highlight the power of large, linked “vein-to-vein” databases that link blood donor and recipient outcome information. Furthermore, the current models expand upon previous models examining variables affecting the hemoglobin increment , component , and recipient  characteristics, including specific donor genetic polymorphisms in 4 genes , collection date and methods , as well as component manufacturing characteristics , were extracted for each RBC unit collected. Available donor genetic polymorphism data from the REDS-III RBC-Omics study  was linkAll single RBC unit transfusion episodes were included in this analysis. A RBC unit transfusion episode was defined as any single RBC transfusion from a single donor with both informative pre- and posttransfusion laboratory measures and without any other RBC units transfused in the intervening period between lab draws. The 3 outcome measures of interest were hemoglobin, total bilirubin, and creatinine increments following a single RBC unit transfusion episode. These outcomes were defined as the difference between the posttransfusion and pretransfusion levels. Pretransfusion thresholds for these measures were chosen to limit patient confounding . For pretransfusion hemoglobin, the value used was the most proximal hemoglobin measurements prior to RBC transfusion — but, at most, 24 hours prior to transfusion. For posttransfusion hemoglobin (g/dL), the laboratory measure nearest to 24 hours after transfusion, but between 12 and 36 hours following transfusion, was based on prior studies. For pretransfusion bilirubin (mg/dL), the most proximal total bilirubin measurement prior to transfusion — but, at most, 7 days prior to transfusion — was used. Furthermore, to exclude patients with preexisting liver disease, the pretransfusion bilirubin had to be below 1.5 mg/dL for the transfusion to be considered. For posttransfusion bilirubin, the most proximal laboratory measure to the transfusion — but, at most, 24 hours after transfusion — was used based on clinical trial data , 32. ForP values less than 0.05 were considered to be statistically significant. Analyses were performed in R 4.0.3   with the outcome was included in a multivariable linear mixed effects model accounting for repeated transfusion recipients. Additional a priori selected interaction terms were included in the multivariable models based on hypotheses of modifiers of clinical outcomes . Cook’s distance was used to detect outliers (Cook’s distance threshold < 0.1 was used to detect outliers) and stepwise model selection on the fixed effects (α = 0.05) was used to select the final full covariate model for each outcome. Initial models were fit on the subset of the data that had no missing observations for all covariates. Once the final model covariates were selected, the model including just those covariates was reassessed on the larger data set, excluding observations with missing values for only the selected covariates to maximize sample size. We tested for multicollinearity in the final models using generalized variance inflation factor values adjusted for the number of regressors, and we performed additional outlier assessment. Fixed marginal effects from multivariable regression models were used to correlate delta hemoglobin and bilirubin levels by week of storage duration. For the subset of recipients of RBC units from REDS-III RBC-Omics donors , we examm, 2020) , 50 and m, 2020) .Approval for data collection had been obtained from the IRB at each participating institution prior to submission as public use data to BioLINCC.NHR, SR, RC, BC, RG, SK, AM, SLS, BS, SRS MPB, and EAH conceived of and designed the study. NHR, SR, HQ, CP, FF, GP, BC, TK, SK, AM, BS, MPB, and EAH acquired and prepared the data. NHR, SR, CP, FF, GP, and EAH performed statistical analyses. NHR, SR, FF, GP, RC, BC, MG, RG, BH, JH, TK, SK, AM, SLS, BS, SRS, MPB, and EAH interpreted data. NHR, SR, and EAH wrote the manuscript. All authors contributed to the critical revision of the manuscript for important intellectual content.


Lumbar puncture is a common clinical procedure that can occasionally be difficult. Various needle guidance methods can facilitate performing this procedure, but at the expense of special expertise, equipment and facility. In the present study, we evaluated the clinical feasibility of a novel bioimpedance needle system regarding its ability to detect cerebrospinal fluid (CSF) in paediatric lumbar punctures.We performed 40 lumbar puncture procedures using the bioimpedance needle system in 37 paediatric patients, aged from 0 days to 17 months, as a part of their prescribed examinations in two university hospitals. The bioimpedance needle is similar to a conventional 22G cutting‐edge spinal needle with a stylet, except the needle and stylet are configured as a bipolar electrode with high spatial resolution. The system measures in real‐time when the needle tip reaches the subarachnoid space containing CSF. The procedure was considered successful when the erythrocyte count was determined from the obtained CSF sample.Subarachnoid space was verifiably reached in 28 out of 40 procedures (70%). Bioimpedance needle system detected CSF in 23 out of these 28 successful procedures (82%) while failed in 3 out of 28 procedures (11%). No adverse events were reported.Bioimpedance spinal needle system was found clinically feasible in paediatric lumbar punctures, and it may offer an objective and simple means to detect the time point when the needle tip is in contact with the cerebrospinal fluid. The study comprised two stages: first, to get first‐hand clinical experience in performing lumbar punctures with the novel bioimpedance needle system in small paediatric patients and to fine‐tune the tissue detection algorithm if considered relevant, and secondly, to evaluate the performance of the system in detecting CSF in paediatric lumbar punctures.2.2A patient was eligible for the study if his/her age was less than 18 months, and a lumbar puncture was required for getting a diagnostic CSF sample, or the procedure was a part of the patient's prescribed treatment plan. Written informed consent was obtained from patients’ parents before the study. The study protocol was approved by the Ethics Committee of Tampere University Hospital District. Also, the national competent authority , Helsinki, Finland) was notified about this study before commencing it. The study was carried out following the Declaration of Helsinki and Good Clinical Practice. The study is registered in ClinicalTrials.gov (NCT02792660).Exclusion criteria were a skin infection around the puncture area, bleeding tendency with thrombocytopenia, acute bleedings and problems with haemostasis, unstable haemodynamics with low blood pressure and need of inotropic agents, increased intracranial pressure with bulging fontanelles, use of ventriculoperitoneal shunt, previous back surgery or known spinal abnormalities.2.3In the Tampere hospital, all lumbar punctures were performed by an experienced paediatrician (VE), whereas in the Turku hospital, according to the hospital's practice of medical training, yet unexperienced specializing physicians performed the punctures under the supervision of an experienced paediatrician (HSo), who completed the procedure if needed.Paediatric lumbar punctures were done according to hospitals’ standard practices without general anaesthesia or sedation. For each patient, topical anaesthetic cream was applied to the puncture site and adequate time was given for the medication to take effect. Oral sucrose was used in neonatal patients before the procedure and care was taken that the patients had received paracetamol before the puncture.The criterion for a successful lumbar puncture procedure was that a proper CSF sample was obtained and the erythrocyte count (i.e. the number of red blood cells per volume) was analysed. The erythrocyte count in the sample was determined with standard cytometric methods in the hospital laboratory. In all patients and both hospitals, the third vial of the CSF sample was used for the haematological analysis.2.4TM Spinal Needle, Injeq Oy, Tampere, Finland, www.injeq.com). The stylet is electrically isolated from the needle body so that the needle tip provides a bipolar electrode and allows a highly localized, real‐time bioimpedance measurement of the tissue at the needle tip  with a stylet configured as a bioimpedance electrode (IQ‐TipTM System). The analyser employs a one millisecond long, proprietary binary pulse sequence in which most of the signal power is located at few specified frequencies within a range from 1 kHz to about 350 kHz. This low‐power signal  is continuously transmitted to the needle as an excitation signal. Bioimpedance is simultaneously measured with the needle tip electrode within less than a 1 mm3 volume of surrounding tissues and calculated from discrete Fourier transformations of voltage and current signal values at the specified frequencies  with high spatial sensitivity (<1 mm3).The stylet is connected to a bioimpedance analyser via a sterile, flexible coaxial cable, which all together make up the bioimpedance needle system  in terms of the perceived CSF detection alarms and consequent or independent actions. Also, potential safety issues and adverse events during the procedure were inquired.Raw impedance data collected during the procedure were stored in the analyser memory for further offline analysis. Results from the offline analysis were used for independent verification of the information the physicians recorded on the patients’ CRFs after completing their procedures. The physician's notes on the CRF served as the primary data regarding the system performance.The physician may have described the system performance on the patient's CRF with the following indicators:correct detection, when the system alarmed and there was CSF flow after the stylet removal and the sample was analysed,false detection, when the system alarmed but there was no CSF flow, or the obtained sample was excessively blood‐tinged and the erythrocyte count was not determined,missed detection, when the system did not alarm but there was CSF flow after the stylet removal and the erythrocyte count was determined,no false detection, when the system did not alarm and there was no CSF flow after any stylet removal.If any of the stylet removals resulted in CSF flow and the CSF sample was analysed in the laboratory verifying that the subarachnoid space was properly reached, the following four combinations of performance indicators were possible:correct detection and no false detection,false detection and correct detection,false detection and missed detection,missed detection and no false detection.If the subarachnoid space was not verifiably reached, either false detection or no false detection was the only possible performance indicators.The CSF detection accuracy and false detection rate were used as the clinical performance outcomes of the bioimpedance needle system. The CSF detection accuracy and the false detection rate were calculated with the following equations:Mean values of these clinical performance outcomes are given as descriptive data.3The first stage of the study was performed in Tampere hospital only, and it comprised 10 procedures of nine patients aged from 2 days to 17 months. Four patients were neonates . The range of patients’ body weight was from 3·5 to 11·4 kg. The second stage was performed both in Tampere and Turku hospitals and comprised 30 procedures  of 28 patients aged from 0 days (including one preterm baby) to 6 months. Twenty‐five were neonates. The range of patients’ body weight was from 1·4 to 7·8 kg.In the first stage of the study, subarachnoid space was verifiably reached in seven out of 10 procedures (70%), all in the first attempt. In these successful punctures, CSF was detected in all seven cases without false detections. In the three unsuccessful procedures, excessively bloody fluid was obtained after the stylet removal and the erythrocyte count was not determined from these samples.In the second stage of the study, subarachnoid space was verifiably reached in 21 out of 30 procedures (70%). In these successful 21 procedures, CSF was detected in 16 out of 21 (76%) and there were three false detections (14%). In eight of the nine unsuccessful punctures, excessively bloody fluid was obtained after the stylet removal and the erythrocyte count was not determined; in one case, there was no flow observed.In the pooled sample of both study stages, subarachnoid space was verifiably reached in 28 out of 40 procedures (70%). Bioimpedance needle system detected CSF in 23 out of 28 successful procedures (82%) while failed in 3 out of 28 procedures (11%). Performance indicators observed in all 40 procedures are depicted in Figure Erythrocyte counts obtained from the 28 successful procedures are plotted against the patient's age in Figure Reinsertion of the spinal needle was needed only in four out of 40 procedures (10%). In one case, the second attempt was successful, but in the two other cases, excessively bloody fluid was again obtained. In one case, no CSF sample was obtained. No adverse events were reported during the 40 procedures.The face‐to‐face comparison between the CRF data and independent offline analysis of the raw bioimpedance data indicated agreement in 38 out of 40 punctures (95%). In one case, the physician seemed to have disregarded the alarm although there was CSF flow after the stylet removal. In the other case, there was no CSF flow after the alarm, but after a small adjustment of the needle, CSF flow occurred.4The novel bioimpedance needle system was found feasible to perform lumbar punctures of neonates and infants. Its accuracy in detecting CSF was satisfactory although not quite comparable to the high accuracy observed in adult lumbar punctures using the same system. In the present paediatric procedures, the CSF detection accuracy was 82%, while in adults, it was 100%  with small electrolyte and glucose contents as well as subtle protein content. While different diseases and physiological conditions may modulate the chemical composition of CSF to some extent , priority was given to diagnostic bacteriological analysis. Those CSF samples from which the erythrocyte count was not determined, representing 30% of the procedures, may have contributed to the false detection rate as well. This is because the bioimpedance needle system can give an alarm also when the needle tip is in contact with a sufficiently conductive mix of CSF and blood. The alarm goes off when the classification algorithm finds that the measured impedance values reside sufficiently long time within the frequency‐specific ranges of impedance representing CSF. The highest evaluated erythrocyte count in the present study was 300 000 cells/µl, which indicates that approximately 1/10Blood contamination to some extent was evident in almost half of the present CSF samples analysed in the laboratory. The visual threshold for blood in CSF is as low as about 400 erythrocytes/µl (Shah et al., The present performance and safety findings from neonates and infants together with earlier findings from adults are promising and speak for the clinical feasibility of the novel bioimpedance needle system (Halonen et al., 5The novel bioimpedance needle system was found clinically feasible for lumbar punctures of neonates and infants. In those procedures, where a CSF sample was obtained and erythrocyte count was determined, representing 70% of all procedures, the CSF detection accuracy was 82%, whereas the false detection rate was 11%. The clinical utility of this novel method as an objective means to know the needle tip location in terms of the presence of CSF appears promising. Further clinical studies, preferably in a randomized controlled setting, are needed to demonstrate the benefit of the bioimpedance needle system.HS, JK, SH and TE are employees of Injeq Oy and hold stock options for tiny amounts of Injeq shares. Also, HS and TE own tiny amounts of Injeq shares. Other authors declare no conflicts of interest concerning this study, authorship and/or publication of this article.


Although initial findings proved inconclusive, it is now increasingly recognized that ALS-CSF may exert toxicity both in vitro and in vivo. Nevertheless, the mechanism underlying CSF-induced neurodegeneration remains unclear. This review aims to summarize the 40-year long history of CSF toxicity studies in ALS, while discussing the various mechanisms that have been proposed, including glutamate excitotoxicity, proteotoxicity and oxidative stress. Furthermore, we consider the potential implications of a toxic CSF circulatory system in the pathophysiology of ALS, and also assess its significance in the context of current ALS research.Based on early evidence of  Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a neurodegenerative condition that is pathologically characterized by the accumulation of ubiquitinated intracellular inclusions containing TAR DNA-binding protein 43 (TDP-43) . Clinicain vitro experiments were conducted, in which anterior horn cells were exposed to diluted serum from ALS patients , which is often considered as an ultrafiltrate of plasma, is a clear colorless liquid that surrounds most of the central nervous system (CNS). In addition to providing buoyant support and protection to the brain, it also serves a regulatory role, made possible by substantial exchange of material between CSF and interstitial fluid (ISF). In view of the close proximity of CSF to neuronal cells, it was hypothesized that the toxin could also be present in the CSF circulation. The first study to assess the toxicity of ALS-CSF thus emerged in 1981 . Measuriin vitro evidence, several in vivo studies have also been performed, demonstrating a range of pathological changes upon CSF injection. In the following sections, we elaborate on the various in vitro and in vivo observations, and discuss the potential underlying mechanisms  receptors , an antagonist of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainite receptor, could protect against CSF-induced neurodegeneration . Althougeceptors , resultseceptors . It was eceptors . Thus, gvia ionotropic glutamate receptors, increased glutamate concentrations did not correlate with the degree of induced neurotoxicity , which is expressed by astrocytes and is involved in glutamate uptake, following exposure to ALS-CSF . Variousvia a glutamate-dependent pathway could still be observed  and tumor necrosis factor alpha (TNF-α), while increasing production of nitric oxide (NO) and reactive oxygen species (ROS) . ExpressAlthough many potentially toxic factors have emerged from studying the effects of ALS-CSF on glial cells, chitotriosidase-1 (CHIT-1), an inflammatory product specific to microglia, has been proposed to play a particularly important role in CSF-mediated neurodegeneration . ProteomThere are also intriguing observations derived from co-culture studies that suggest that the glial influence may be more complex than a direct contribution to neuronal loss. Although exposure of motor neuron mono-cultures to both ALS-CSF and control-CSF resulted in neuronal death, motor neurons co-cultured with glia experienced no significant decrease in survival following ALS-CSF exposure, with administration of control-CSF even leading to an increase in cell count . As wellWhile ALS is now recognized as a proteinopathy, proteotoxicity initially received comparatively little attention as a possible underlying mechanism in both ALS pathophysiology and CSF-mediated neurodegeneration. In fact, it was found that CSF toxicity could neither be suppressed by prior heating nor by using a 5 kDa filter, thus appearing to exclude the possibility of the toxic agent being a protein . Much moin vitro findings of TDP-43 mislocalization were again observed following ALS-CSF exposure, a feature which has previously been shown to be partially reversed by vascular endothelial growth factor (VEGF) supplementation , a marker of lipid peroxidation, when raised to that of ALS-CSF, have been found to induce neuronal loss in vitro . Potentiin vitro . While t ALS-CSF . In addi ALS-CSF , immunog ALS-CSF . Finallyin vitro and in vivo models in response to ALS-CSF administration. During the 1990s, increased neurofilament phosphorylation was reported when chick spinal cord neurons were exposed to ALS-CSF (in vivo, with the change being considerably more pronounced in ventral horn motor neurons (Several pathological changes have been observed across both  ALS-CSF , a findi neurons . Despite neurons . Ultrast neurons . Mitocho neurons . While a neurons , it is av1.6 and Kv1.6 channels (In addition to cellular changes, intrathecal and intraventricular infusion of ALS-CSF in rat models were also found to result in motor impairment and other musculoskeletal features . These cchannels , althougchannels . A more channels . The celchannels . OverallIntriguingly, several neuroprotective factors have also emerged from CSF toxicity studies. VEGF, which has previously been implicated in ALS pathophysiology , has alsDespite the increasing recognition that ALS-CSF may promote neurodegeneration, with various studies having attempted to characterize its toxicity, synthesizing the current body of literature remains challenging . Most pr•What is the nature of the circulating toxin responsible for CSF-induced neurodegeneration?•What is the cellular origin of this circulating toxin?•What is/are the mechanism/s by which it triggers neurodegeneration?•Do astrocytes and microglia play a role in mediating CSF toxicity?•To what extent does CSF composition reflect that of ISF?•What is the contribution of CSF toxicity toward ALS pathophysiology?•in vivo conditions?How accurately do the models employed to study CSF toxicity recapitulate human •Could similar mechanisms explain CSF toxicity in sporadic and familial forms of ALS?It is still unclear why conflicting findings have been obtained in early toxicity studies, both in those involving serum and CSF. Studies concomitantly investigating the toxicity of serum and CSF in ALS patients have, to our knowledge, not been conducted, and could potentially reveal additional insights into their association. Furthermore, no significant correlation has to date been observed by studies assessing the possible relationship between CSF toxicity and demographic and clinical variables, suggesting that CSF toxicity may not represent a possible biomarker for disease stratification . AlthougIn spite of this, there are a number of advantages to considering the neurotoxicity of CSF in an ALS model. First, CSF toxicity, which has been observed across both sporadic and familial forms of ALS, could potentially form part of a common pathogenic pathway in ALS, thus helping to reconcile the two forms of ALS. Although its contribution in ALS pathophysiology has not yet been established, ALS-CSF toxicity appears to possess many common features, including an apparent predilection for motor neurons and an important cell non-autonomous component. As previously mentioned, the presence of a circulating toxin in CSF could also offer possible explanations for the spread of pathology in ALS . Future via a process mediated by both astrocytes and microglia (While drawing definite conclusions at this stage may not be possible, we believe, based on the numerous observations derived from past studies, that ALS-CSF contains both toxic and protective factors, and is able to promote neurodegeneration either directly or indirectly icroglia . The neuAdditional studies are nevertheless required to improve our understanding of CSF toxicity based on both its constitution and dynamics. Although proteomic and lipidomic studies have revealed altered levels of inflammatory markers as well as a different lipid profile in ALS-CSF , the litin vivo and in vitro evidence of CSF toxicity, striking similarities have been observed between CSF-mediated neurodegeneration and ALS pathophysiology. Although it would be difficult at this stage to argue for the role CSF toxicity as a driver of the disease process, we believe that the potential promise of CSF toxicity in capturing the broader pathophysiological picture of ALS may warrant its additional attention as an area of active research.From initial findings of serum toxicity to current KN and AM conceptualized the work. KN wrote the first draft of the manuscript, supervised by AM. All authors provided critical input and revised subsequent drafts, before approving the final manuscriptThe 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.


CSF-1 level was dramatically up-regulated and numerous genes downstream to CSF-1 covering tumor necrosis factor (TNF), ras-related protein 1 (Rap1) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway were significantly modulated, especially for proliferation, migration and adhesion. Moreover, the phenomenal increase of miR-21-5p and genes related to pro-tumor activity were observed in parallel. In summary, withholding of CSF-1/CSF-1R interaction would rather augment than suspend the M-CSF-driven pro-tumor activities of M2 macrophages in a long run.Macrophage colony-stimulating factor (M-CSF or CSF-1) is known to have a broad range of actions on myeloid cells maturation, including the regulation of macrophage differentiation, proliferation and survival. Macrophages generated by M-CSF stimulus have been proposed to be alternatively activated or M2 phenotype. M-CSF is commonly overexpressed by tumors and is also known to enhance tumor growth and aggressiveness via stimulating pro-tumor activities of tumor-associated macrophages (TAMs). Currently, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. However, its antitumor activity shows a limited single-agent effect. Therefore, macrophages in response to M-CSF interruption are pending for further investigation. To achieve this study, bone marrow derived macrophages were generated in vitro by M-CSF stimulation for 7 days and then continuously grown until day 21 in M-CSF absence. A selective pressure for cell survival was initiated after withdrawal of M-CSF. The surviving cells were more prone to M2-like phenotype, even after receiving interleukin-4 (IL-4) stimulation. The transcriptome analysis unveiled that endogenous  In the tumor microenvironment (TME), circulating monocytic precursors in blood are recruited to tumor site by the growth factors, such as vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), granulocyte-macrophage colony stimulating factors (GM-CSF) and M-CSF and the chemokines CCL2 and CCL5 ,2. Recru+ TAMs was also associated with the expression of M-CSF in the tumor stroma. The 5-year overall survival rate and disease-free survival rate of patients with histological grade of CD68− M-CSF− were better than their counterpart with CD68+ M-CSF+  [The pro-tumor roles of TAMs include tumor invasion, tumor angiogenesis, immunosuppression and metastasis . Many biInfiltration of monocytes/macrophages is often stimulated by tumor derived colony-stimulating factor-1 . CSF-1 not only induces macrophage precursors to mature into adherent motile baseline macrophages (M0), but also regulates macrophages survival, proliferation and differentiation ,11,12,13+ macrophages in an implanted osteosarcoma (AX cells) mice model and reduced vascularization, lymphangiogenesis and tumor growth [+ T cell infiltration, and prevent tumor growth in the K14-HPV16 transgenic mouse model of cervical carcinoma [Immunotherapy acts in a fundamentally different way in comparison with conventional therapies. Instead of destroying tumor cells directly, immunotherapy directs tumor cell killing through the immune response of the host. This mission can be achieved mainly via the effector cells of the immune system, such as macrophages . Therefor growth . An oralarcinoma . BLZ945 arcinoma .miR-21 was induced by CSF-1R signaling via PI3K activation [miR-21 in TAMs induced a proinflammatory angiostatic function of macrophage with improvement of immunostimulatory response [miR-21 blockade in TAMs also increases CD107/CD8 positive T-cells in TME. All these effects led to diminished tumor growth [Human monocyte-derived TAMs were able to be generated in vitro while culturing in conditioned media collected either from human breast adenocarcinoma cell or LK 46 lymphoblastoid cell . The exptivation . In an aresponse . Meanwhir growth .+ T cells [Hi TAMs were more sensitive to the CSF1R signal blockade (clone 5A1) bringing a higher level of cell death. They also showed that CSF1R blockade reprograms survival CD206Low TAMs to support anti-tumor interferon responses and T cell activities [Now a day, inhibition of CSF-1/CSF-1R interaction by therapeutic antibody to deplete TAMs and their pro-tumor functions is becoming a prevalent strategy in cancer therapy. Although inhibition of macrophages infiltration in tumor-bearing mice via CSF-1R blockade had a significant effect on tumor regression and survival of CD8 T cells ,25,26. H T cells ,28,29. A T cells . FurtherTo test the effect of M-CSF long-lastingly in macrophages, bone marrow mononuclear cells were stimulated with M-CSF for 7 days, then the cells were continuously grown without supplement of M-CSF from day 7 till day 21 A. By dayThe mean expression of CD206 in a cell population represented by the mean fluorescent intensity (MFI) were maintained from day 7 till day 21 A,C. Besi+/7-AAD−) starting from day 7 till day 21  contains a variety of cytokines and growth factors other than M-CSF. We were interesting in the cross-reaction of M-CSF with other stimuli, such as lipopolysaccharides (LPS), IL-4 or IL-10, on M2-like macrophages induction from cells collected from P3 and P4 fractions. Interestingly, the gene transcript of interferon regulatory factor 1 and 5 (IRF1 and IRF5), known as M1 biomarkers, were significantly downregulated in P4 (D21) compared with that in P3 (D7) macrophages while LPS was further added A,B. On tHowever, there were no significant changes between P3 and P4 macrophages in the reaction with further IL-10 stimulus. Since IL-4 and IL-10 can direct M2-like macrophages polarization, these results manifested that cells from P4 in comparison with P3 fraction were not only more resistant to re-polarization into M1 subtype, but also more preferential to go toward M2-like polarization in response to further LPS or IL-4 stimuli, respectively. These findings also attract a major concern that cytokines milieu within TME may enhance M2-like TAMs development upon M-CSF interruption. The underlying biological and molecular mechanisms for how macrophages from P4 vs. P3 fractions behaving so differently after interruption of M-CSF supplement were explored further via transcriptome profiling.We next compared the transcriptome profiling of M2 macrophages collected from P3 and P4 fractions by Next Generation Sequencing (NGS). Differential expression analysis (DEA) revealed 272 differentially expressed genes (DEGs) in P4 compared with P3  tremendously upregulated in cells from P4 compared with that from P3 fraction A. Rap1 sA previous study reported that the expression of miR-21 was induced by CSF-1R signaling via PI3K pathway activation . CompariM-CSF, was also initiated , very likely due to a consistent M-CSF autocrine signaling. These findings attract a major concern that Th2 cytokines milieu within TME containing IL-4 may enhance M2-like TAMs development, especially while the infiltrating macrophages interact with M-CSF intermittently by therapeutic CSF-1/CSF-1R antibody blockade. According to global transcriptome analysis in our study , an extraordinarily pleiotropic cytokine, is indispensable from a central role of immune homeostasis, inflammation, and host defense . IncreasmicroRNA-21 (miR-21) transcriptional regulation was demonstrated firstly by Loffler et al., who showed that miR-21 gene is induced by IL-6 in a STAT3-dependent event [miR-21 promoter. The cumulative evidences suggest that miR-21 is an oncogene conserved in vertebrates locating in 17q23.2 [miR-21 in cultured glioblastoma cells leads to the malignant phenotype by blocking expression of critical apoptosis-related genes [miR-21 is able to promote tumor growth [miR-21 were verified, mainly by the positive correlation of miR-21 and M-CSF expression in cervical cancer  [miR-21 was induced by CSF-1R signaling via PI3K pathway activation [miR-21 was equally high in cells between P3 and P4 fractions after withholding of M-CSF. In contrast, the expression level of miR-21 was significantly lower in M-CSF pretreated control (P2)  . Anothertivation . Accordirol (P2) A. This rof M-CSF A; suggesrophages .C57BL/6 female mice were purchased from the National Laboratory Animal Center  and housed in a clean and pathogen-free facility in the National Pingtung University of Science and Technology animal care facility. Eight to twelve weeks old mice were used in the experiments. The protocol was approved by the Institutional Animal Care and Use Committee of College (IACUC) of Veterinary Medicine at National Pingtung University of Science and Technology , Taiwan. Euthanasia was performed by cervical dislocation after anesthesia.2 for 7 days. After 7 days, Bone Marrow-derived Macrophages (BMDMs) were harvest by using 0.025% Trypsin–EDTA  and or continuing cultured for 21 days at 37 °C in 5% CO2 in 90% RPMI 1640  with supplemented 10% fetal calf serum  and changed every 2–3 day until 21 days.Bone marrow cells were obtained by flushing the marrow cavities of the femurs and tibias with phosphate buffered saline (PBS) (pH 7.4), containing 0.5% bovine serum albumin (BSA) . Bone Marrow-derived Mononuclear cells (BMMNCs) were isolated from the buffy coat by Ficoll-Hypaque density gradient centrifugation . BMMNCs were differentiated into macrophages by incubating in RPMI 1640  with supplemented 10% fetal bovine serum , 50 ng/mL M-CSF  at 37 °C in 5% CO4 cells/ cm2 (Cellstar 6-well plate) . Cells were classically activated (M1 phenotype) with 1 μg/mL LPS , and alternatively activated (M2 phenotype) with 10 ng/ ml IL-4  or with 10 ng/mL IL-10  for 24 h at 37 °C and 5% CO2.On day 7 in culture, the BMDM cells were washed, counted and re-plated in 2 mL RPMI 1640 media  containing with 10% fetal bovine serum  at a density of 5 × 10- and CD11b+ cells were separated by using CD11b MicroBeads . The cells were first fixed in 4% paraformaldehyde solution for 15 min at room temperature, followed by blocking with 1% BSA in PBST solution  for 30 min. Cells were then incubated with Rat biotin anti-mouse CD11b antibody (M1/70)  and Rabbit anti-mouse CD206 polyclonal antibody  for 1 h before the application of a secondary antibody , Goat anti-rabbit IgG CF594 ) for 1 h. Slides were stained with DAPI before imaging. Images were captured with a fluorescence microscope .Fluorescent immunocytochemistry (ICC) staining of CD11b and CD206 were performed on BMDMs and mounted on Cover Slips. CD11bBMDM cells were collected at various time points and were analyzed by flow cytometry for M1 or M2 cell- lineage markers. The cells were stained in PBS containing 0.5% (wt/vol) BSA . Surface receptors were stained for 30 min on ice. The identification of M1 type in BMDM was performed with Fluorescein isothiocyanate (FITC)- conjugated CD86 antibody (GL-1) (1 μg/mL). The identification of M2 type in BMDM was performed with APC-conjugated CD206 antibody (C068C2) (0.5 μg/ mL). All antibodies were obtained from Biolegend . We use unstained cells as a negative control and single-stained cells as a compensation control.4 cells/cm2) and allow cells to adhere, followed by adding 10 µL/mL of the pSIVA-IANBD probe  on cells. The cells were observed under microscope using the green fluorescence filter.BMDM cells were analyzed at various time points  by flow cytometry and fluorescent immunocytochemistry assay. The cells were then trypsinized and washed with PBS twice. Apoptosis was measured by flow cytometry using the FITC Annexin V Apoptosis Detection Kit with propidium iodide . For fluorescent immunocytochemistry assay, the cells were re-seeded into culture plates  Master Mix  following manufacture protocol. Quantitative PCR reactions were performed using a QIAGEN Rotor Gene Q Real-Time PCR. The primer sequences  used in qPCR was showed in t (-delta Ct) or 2t−ΔΔC (standardized mRNA level).The total RNA was isolated from cells using Tripure Isolation Reagent  following manufacture’s instruction. Reverse transcription was performed using iScript cDNA Synthesis Kit  for cDNA synthesis and the quantitation of gene expression was achieved from Lunahttps://dataview.ncbi.nlm.nih.gov/object/PRJNA665327?reviewer=o2ppa2v2g4db9pu1t2mh3il4i9, accessed on 24 September 2020).The purified RNA was used for the preparation of the sequencing library by TruSeq Stranded mRNA Library Prep Kit  following the manufacturer’s recommendations. Briefly, mRNA was purified from total RNA (1 μg) by oligo (dT)-coupled magnetic beads and fragmented into small pieces under elevated temperature. The first-strand cDNA was synthesized using reverse transcriptase and random primers. After the generation of double-strand cDNA and adenylation on 3′ ends of DNA fragments, the adaptors were ligated and purified with AMPure XP system . The quality of the libraries was assessed on the Agilent Bioanalyzer 2100 system and a Real-Time PCR system. The qualified libraries were then sequenced on an Illumina NovaSeq 6000 platform with 150 bp paired-end reads generated by Genomics, BioSci & Tech Co., New Taipei City, Taiwan. The datasets presented in this study can be found in online repositories  . The filmiR-21-5p . The expression level of UniSp6 (U6) was used as an endogenous control according to the manufacturer’s instructions.The cDNAs were produced from total RNA by reverse transcription using the miRCURY LNA miRNA PCR Starter Kit . The miRNA PCR primer set, based on the SYBR Green miRCURY locked nucleic acids (LNA) detection system from QIAGEN, was used to profile the expression of p < 0.05 was considered statistically significant. The analysis was performed using GraphPad Prism 7 .Data are expressed as mean ± SD. Statistical significance of differences among three or more groups were determined using One-way analysis of variance (ANOVA) or two-way ANOVA with Tukey’s multiple comparisons test. CSF-1 activation and its downstream genes reprogramming (miRNA-26a to suppress endogenous CSF-1 upregulation in cells after CSF-1/CSF-1R axis blockade may apparently be another option for combined therapy [Taking together all the results, withholding of the M-CSF supplement dramatically augments pro-tumor activity of M2 macrophages through endogenous gramming . In the  therapy .


All had tau‐PET  and CSF p‐tau181 biomarkers available. Plasma p‐tau181 and plasma/CSF p‐tau217 were available in BioFINDER‐2 only. Concordance between PET, CSF and plasma tau biomarkers ranged between 66 and 95%. Across the whole group, ridge regression models showed that increased CSF and plasma p‐tau181 and p‐tau217 levels were independently of tau PET associated with higher age, and APOEɛ4‐carriership and Aβ‐positivity, while increased tau‐PET signal in the temporal cortex was associated with worse cognitive performance and reduced cortical thickness. We conclude that biofluid and neuroimaging markers of tau pathology convey partly independent information, with CSF and plasma p‐tau181 and p‐tau217 levels being more tightly linked with early markers of AD , while tau‐PET shows the strongest associations with cognitive and neurodegenerative markers of disease progression.PET, CSF and plasma biomarkers of tau pathology may be differentially associated with Alzheimer's disease (AD)‐related demographic, cognitive, genetic and neuroimaging markers. We examined 771 participants with normal cognition, mild cognitive impairment or dementia from BioFINDER‐2 ( Tau pathology in Alzheimer's disease (AD) can be measured using neuroimaging (PET) and biofluid (CSF and plasma) markers. This study explores whether the tau biomarkers are comparable to each other or carry unique information about AD‐related demographic, cognitive, genetic and neuroimaging markers. Major scientific breakthroughs over the past decades now enable the detection of tau pathology in cerebrospinal fluid (CSF), using positron emission tomography (PET) and, most recently, in blood. Although these three biomarker modalities reflect tau pathology and are often considered interchangeable in current diagnostic and research criteria for Alzheimer's disease (AD), there are important differences between them.n = 400) and ADNI (n = 371). All had tau‐PET and CSF p‐tau181 biomarkers available, while Plasma p‐tau181 and plasma/CSF p‐tau217 were available in BioFINDER‐2 only. Concordance between PET, CSF and plasma tau biomarkers ranged between 66 and 95%. Furthermore, increased CSF and plasma p‐tau181 and p‐tau217 levels were independently of tau PET associated with higher age, and APOEɛ4‐carriership and Aβ‐positivity, while increased tau‐PET signal in the temporal cortex was associated with worse cognitive performance and reduced cortical thickness.We examined 771 participants with normal cognition, mild cognitive impairment or dementia from two cohorts: BioFINDER‐2 , while tau‐PET shows the strongest associations with cognitive and neurodegenerative markers of disease progression.et al, et al, et al, et al, et al, et al, et al, et al, The core neuropathological features of Alzheimer's disease (AD) are amyloid‐β (Aβ) plaques and hyperphosphorylated tau (p‐tau) in neuronal neurofibrillary tangles and neuropil threads  are measured  and symptomatic groups  and amyloid PET global SUVR . Furthermore, tau biomarkers were negatively correlated with CSF Aβ42/40 ratio , cognitive tests scores  and MRI measures , while only 1% were positive for tau PET (PET+) and negative for CSF p‐tau181. Tau PET was concordant with plasma p‐tau181 in 80% , with plasma p‐tau217 in 86%  and with CSF p‐tau217 in 80% of cases  in the temporal meta‐ROI and CSF p‐tau181 was 83% Fig . Of the +/PET−, 1% PET+/CSF−) and of 84% with plasma p‐tau217 , Fig +/PET− profile was more common than a biofluid‐/PET+ profile, with the exception of plasma p‐tau181.The concordance between fluid tau biomarkers ranged between 66% (CSF p‐tau217 vs plasma p‐tau181), 70% (CSF p‐tau181 vs plasma p‐tau181), 73% (plasma p‐tau181 vs plasma p‐tau217), 82% (CSF p‐tau181 vs plasma p‐tau217), 83% (CSF p‐tau217 vs plasma p‐tau217) and 95%  and Aβ pathology, whereas tau PET was more strongly associated with indicators of disease progression including lower cognitive test scores and reduced cortical thickness. When stratifying for cognitive status (i.e. CU vs MCI/dementia), this pattern was similar for CSF in CU and for PET in the symptomatic stages. Additionally, CSF was more strongly associated with MMSE and executive functioning composite score in CU individuals. These patterns of results were consistent when investigating percentual change of β‐coefficients , confirming that the L2 regularization performed as expected.Based on three sets of ridge regression models  for each AD‐related feature, we examined the possible differential associations for either tau PET or CSF p‐tau181 to other AD‐related features Fig . Ridge r42/40 ratio were among the most important predictors of tau biomarker levels for most models, but they showed tau biomarker modality specific effects. For example, the CSF Aβ42/40 ratio was an important predictor of CSF p‐tau181 levels in both CU  and MCI/dementia  groups, while for tau PET it was a more important predictor in the CU group  than in MCI/dementia . In CU, age was an important predictor for both CSF p‐tau181 and tau PET (ranks varying from #2 to #4), whereas it was not an important predictor for either tau biomarker in the symptomatic stages (ranks varying from #6 to #9). In the symptomatic stages, memory z‐score was the most informative out of cognitive tests for all models, which was more pronounced for tau PET (ranked #2 in both cohorts) than for CSF p‐tau181 (ranked #4 in both cohorts). These results are largely congruent with the information obtained using the ridge regression models.Next, we explored the association between CSF p‐tau181 and tau PET SUVR in a temporal meta‐ROI with AD‐related features using regression tree models in both BioFINDER‐2 Fig  and in AAPOE ɛ4 carriership, amyloid PET and the CSF Aβ42/40 ratio, while tau PET was more strongly associated with cognitive decline and MRI‐based atrophy compared to plasma p‐tau217  and with entorhinal cortex tau PET SUVR (in comparison with CSF p‐tau181). When comparing plasma p‐tau181 and tau PET in the temporal meta‐ROI, plasma p‐tau181 only showed a stronger association with age, while tau PET most strongly associated with all cognitive test scores and MRI measures Fig . Compareu217 Fig . Replaciu217 Fig , althougu217 Fig , with thu217 Fig . ContrarAPOE ɛ4 and Aβ positivity, while increased temporal meta‐ROI tau PET retention was more strongly associated with worse cognitive performance and reduced cortical thickness. The majority of results were consistent between the discovery cohort (i.e. BioFINDER‐2) and the replication cohort (i.e. ADNI). The data suggest that biofluid tau biomarkers are more tightly linked with early markers of AD , while tau PET showed strongest associations with cognitive and neurodegenerative markers of disease progression. Overall, the results support our hypothesis that the three tau biomarker modalities provide partially independent information.In this study, we aimed to investigate whether PET, CSF and plasma biomarkers of tau pathology in AD are comparable to each other or carry unique information about AD‐related demographic, cognitive, genetic and neuroimaging markers. First, we showed that the concordance ranged between 66% (CSF p‐tau217 vs plasma p‐tau181) and 95% (CSF p‐tau181 vs CSF p‐tau217) across all tau biomarkers, and between 80% (tau PET vs plasma p‐tau181/CSF p‐tau217) and 86% (tau PET vs plasma p‐tau217) for biofluid‐ vs neuroimaging‐based tau biomarkers. Ridge regression models showed that increased CSF and plasma p‐tau181 and p‐tau217 levels were independently associated with ageing, and et al, et al, et al, et al, et al, et al, et al, et al, 42 may yield false‐positive results in certain neurological conditions evoking a neuroinflammatory response  and for tracking disease progression  is not very sensitive and generally reveals neurofibrillary tangle pathology in Braak stage 4 or higher  and Alzheimer's disease neuroimaging initiative , including 461 cognitively unimpaired (CU) individuals  and 310 individuals diagnosed with mild cognitive impairment (MCI) or AD dementia according to NIA‐AA diagnostic criteria  and domain‐specific tests for memory, executive functioning, language and visuospatial abilities . The MP‐RAGE images underwent correction for intensity homogeneity , while multiple 1.5T and 3T MRI scanners were used in the multi‐centre ADNI study . SUVR images were created for the 90–110 min post‐injection interval using the pons as reference region. In ADNI, amyloid PET was performed using [18F]florbetapir  or [18F]florbetaben  on multiple PET scanners. SUVR values were re‐scaled onto the Centiloid scale   and Elecsys immunoassays , respectively.In the BioFINDER‐2 study, amyloid PET was performed using RO948 on a digital Discovery MI scanner . Standardized uptake value ratio (SUVR) images were created for the 70‐ to 90‐min post‐injection interval using the inferior cerebellar cortex as the reference region. In ADNI, tau PET was performed using [18F]flortaucipir and SUVR images were created for the 80–100 post‐injection interval using inferior cerebellar cortex as the reference region using a previously published approach  and 1.34 ([18F]flortaucipir) SUVR based on mean + (2 × standard deviation) uptake in elderly cognitively normal individuals and mean + (2 × standard deviation) uptake in young cognitively normal individuals, respectively  and 1.39 ([18F]flortaucipir) SUVR , as both of the methods described below needed all values to be present. An overview of the proportion of missing values is presented in Appendix Table Statistical analysis was performed using R software (Version 4.0.3). When presenting group characteristics, patient features were compared using two samples t‐tests and chi‐squared tests. Non‐adjusted Pearson correlation coefficients were calculated using the First, we examined the correlations between the different tau biomarkers and AD‐related features, as well as the degree of concordance between the PET, CSF and plasma tau biomarkers.APOE ɛ4 carriership, amyloid biomarkers , cognitive measures . We used these 95% CIs to compare the β‐coefficients of models for PET and CSF. Note that amyloid PET was not performed in cases with dementia in the BioFINDER‐2 study. Hence, amyloid PET SUVR values were not imputed (because it would introduce systematic bias), and the bootstrapped samples were taken only from cases with available amyloid PET. To compare the strength of the associations between PET and CSF tau biomarkers with the predicted AD‐features, we followed these three criteria: (i) non‐overlapping 95% CIs of the β‐coefficient of the simple models for CSF and PET , (ii) 95% CIs in the simple or combined models non‐overlapping with β = 0 for only CSF or PET (stronger association for the tau biomarker non‐overlapping with β = 0), (iii) a significant drop of the β‐coefficient from the combined model relative to the simple model for only CSF or PET . In case a feature was not significantly associated with both tau biomarkers, we did not perform the combined model. Figure 18F‐flutemetamol PET global SUVR or CSF Aβ40/42 when PET was unavailable, using previously established cut‐offs  from the models to estimate the relative importance of variables to predict tau biomarkers. The VIM for a variable is based on the average decrease of root mean squared error when it is used in the model, and higher VIM indicates that the variable is considered more important to predict the outcome. In our analysis, we present the VIMs from N = 25 bagged regression tree models, created with each of the 25 imputed datasets.Third, to identify the AD features that were most strongly associated with the tau biomarkers, we created parallel regression tree models  with continuous AD‐related patient features using the Fourth, we aimed to explore p‐tau181 in plasma, a different p‐tau isoform (i.e. p‐tau217) in plasma and CSF, and an earlier affected tau PET region  in the BioFINDER‐2 cohort only. Therefore, we conducted ridge regression models (as described above) to investigate the potentially different associations with AD features using different pairings of tau biomarkers, i.e., (i) tau PET SUVR in the temporal meta‐ROI vs plasma p‐tau181 levels, (ii) tau PET SUVR in the temporal meta‐ROI vs plasma and CSF p‐tau217 levels and (iii) tau PET SUVR in the entorhinal cortex vs CSF p‐tau181.RO and JR had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and design: RO, JR and OH. Acquisition, analysis or interpretation of data: all authors. Drafting of the manuscript: RO, JR and OH. Critical revision of the manuscript for important intellectual content: RS, AL, OS, SP, ES, HZ, PS, JLD, FB, KB, NM‐C and SJ.HZ has served at scientific advisory boards for Eisai, Denali, Roche Diagnostics, Wave, Samumed, Siemens Healthineers, Pinteon Therapeutics, Nervgen, AZTherapies and CogRx, has given lectures in symposia sponsored by Cellectricon, Fujirebio, Alzecure and Biogen, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. KB has served as a consultant, at advisory boards, or at data monitoring committees for Abcam, Axon, Biogen, JOMDD/Shimadzu. Julius Clinical, Lilly, MagQu, Novartis, Roche Diagnostics and Siemens Healthineers, and is a co‐founder of Brain Biomarker Solutions in Gothenburg AB (BBS), which is a part of the GU Ventures Incubator Program. SP has served at scientific advisory boards for Roche and Geras Solutions. JLD is an employee and shareholder of Eli Lilly and Company. OH has acquired research support (for the institution) from AVID Radiopharmaceuticals, Biogen, Eli Lilly, Eisai, GE Healthcare, Pfizer and Roche. In the past 2 years, he has received consultancy/speaker fees from AC Immune, Alzpath, Biogen, Cerveau and Roche. The other authors report no conflicts of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Table EV1Click here for additional data file.Review Process FileClick here for additional data file.


Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells. The main role of the immune system cells is to protect the body against infections, tumor cells and a broad range of ‘danger’ signals. The additional face of this function is the response to allergens and transplanted allogeneic tissues. While the latter effects are undesirable, this is an unfortunate natural consequence of the proper functioning of the immune system.graft-versus-host disease (GVHD) and pregnancy complications .,39.38,39The PCR SSP method is based on a PCR reaction with specific primers. Amplified DNA fragments are separated according to size, e.g., by agarose gel electrophoresis, then visualized with the aid of intercalating DNA dyes and then exposure to UV light. The interpretation of the PCR-SSP results is based on the presence or absence of the specific product and compared with the iterative tables and analyzed in the supplied software. The result obtained is at the low resolution level ,41,42.Real time PCR is also a very useful method. The use of SYBR labeling or specific molecular probes allows us to skip the electrophoresis stage, which makes this method easier to apply in the laboratory ,44.next generation sequencing (NGS) method has been added, thanks to which we can obtain a high-resolution result [Recently, in addition to the methods used to evaluate KIR receptors, the n result .In summary, KIR high-throughput genotyping methods are now widely available, relatively inexpensive and may increase the pool of fully characterized donors and recipients in the transplant procedures.https://www.ebmt.org, accessed on 3 July 2021), the first choice is a fully compatible family donor. The probability of having a fully histocompatibility donor is 25% [BMDW), age (a younger donor is better), CMV status, blood group compatibility and optionally permissive mismatching in HLA-DP. Donors for whom the patient has donor specific antibodies (DSA) are avoided [Treatment of patients with certain high-risk hematological diseases is only possible with stem cell/bone marrow transplantation. Selecting the best donor is a complex process that depends on many factors. The key criterion in the cell donor selection procedure for a hematological patient is histocompatibility in HLA antigens between recipient and donor. According to the algorithm adopted by the EBMT 2019 (r is 25% . Especiar is 25% . If the r is 25% . (2) Sel avoided . Differehematopoietic cell transplantation (HCT) [Given the compatibility of the HLA antigens, a fully compatible donor is the best choice, but unfortunately some patients do not have such a donor and then a non-fully compatible donor is selected, the so-called alternative donor. This is especially important with regard to the mismatch at the C locus, which is one of the ligands for KIR receptors. This mismatch may affect NK cell specific activity after transplantation . Some hion (HCT) . The effon (HCT) . NK cellon (HCT) .acute myeloid leukemia) AML after transplantation contributed to a significantly lower rate of relapses. There were no such spectacular effects in patients with acute lymphoblastic leukemia (ALL). In addition, they presented the results of an experiment carried out in a mouse model. They infused alloreactive NK cells using a protocol with reduced conditioning intensity and obtained satisfactory transplant results—reduced GvHD and relapse. In their view, even alloreactive transformed NK cells are associated with total donor chimerism and leukemia eradication [n = 114  and n = 276 , who received haploidentical stem cell transplantation (haplo-SCT). Researchers investigated the relationship between the presence of ligands in the recipient and donor for the donor KIR receptors and NK activity. Total NK cell count and anti-K562 (Lymphoblast human cell line) response were tested by examining CD107 expression levels and IFN-γ expression, among others. The final conclusion of this study was that the presence of ligands for donor KIR receptors in recipients resulted in lower levels of relapses [n = 372 with ALL and n = 344 with AML. Scientists performed KIR typing, KIR ligand mismatch, KIR2DS1 and Cen B/telomeric A mismatch in the recipient donor pairs. The aim was to find out if any variables relevant to KIR were related to relapses in children with AML or ALL. Unfortunately, they found that none of the mismatches were significantly associated with relapse or disease-free survival, considering the entire patient cohort (ALL and AML) and separately AML or ALL. There was also no relationship between the KIR variables and the results in the group of transplantation with T cell depletion. The authors, taking into account these findings, did not recommend in the donor selection a consideration of the KIR/HLA matching in children [Currently, research on the importance of the KIR/HLA relationship in the field of transplantation is still in progress and opinions in this area are disparate and widely discussed. A pioneering report published in 2002 by Velardi and colleagues proved in a mouse model and in clinical trials the importance of a KIR ligand mismatch between donor and recipient and its impact on the protection against relapses. An adverse reaction of the transplant recipient is GvHD caused by antigenically introduced foreign lymphocytes. The donor T lymphocytes infiltrate the recipient tissues and organs, leading to their destruction. The infiltrates are most often in the skin, the digestive tract and mucous membranes. Reactions can be very severe and the risk of death for the patient is sometimes high. GvHD can be reduced by depleting T cells, but then the risk of transplant rejection increases. The authors showed in their studies that the KIR/HLA mismatch of the donor-recipient after the removal of T cells from the graft material in patients with  (65% versus 51%). The presence of the KIR B/x haplotype with the activating KIR2DS2 receptor was 69% vs. 54% vs. 46% vs. to KIR A/A and KIR B/x, without 2DS2. The presented data show that the mismatch in KIR genes contributed to the reduction of the number of progressions and relapses. Additionally, the presence of the KIR B/x and KIR2DS2 haplotype improved the results on non-relapse mortality. The result of these analyses is the evidence-based system for assessing donor risk factors proposed by the authors, in the form of a point selection algorithm for choosing the best haploidentical donor individually for each patient. This comprehensive analysis clearly demonstrates the importance of genotyping of KIR genes in addition to other clinical parameters, and should be routinely performed as part of the best haploidentical donor selection procedure to optimize survival and reduce the risk of relapse [Haploidentical transplantation was a revolution. Currently, the results of these transplants are continuously improving but they are still not fully satisfactory. There are still no strict criteria for selecting a donor if there are more candidates. In such cases, excluding the donor against whom the patient has DSA antibodies, the donor-mother and preference for the male gender are the most chosen recruitment factors. Furthermore, the issues of developing special desensitization protocols for viral or bacterial immunized patients, so that the level of DSA antibodies are safe for the entire transplant procedure, are also increasingly discussed. In the study by Solomon et al., 208 transplant selections were analyzed. The donor variables analyzed were age, gender, relationship, CMV status, ABO compliance, HLA discrepancy and KIR mismatch. The overall 3-year survival rate was analyzed depending on the data characteristics of the donors. Incompatibilities in the KIR-like receptors resulted in overall  relapse . Current relapse . Recentl relapse .The best option for a patient with end-stage disease is an organ transplantation. Due to the fact that there are few transplants from living donors (kidney), and a shortage of organs from deceased donors, the donor–recipient pair should be selected as carefully as possible so that the transplanted organ survives as long as possible. It is the incompatibility between recipient and donor regarding HLA antigens that leads to the stimulation of the recipient’s immune system and rejection of the transplanted organ. The advances in immunology diagnostic, such as HLA matching, PRA, detecting of alloantibodies, especially DSA and complement binding (ab anti-HLA C1q), cross match -CM-CDC, DSA monitoring, IgM detection and novel immunosuppression strategies have reduced the incidence of rejection and improved survival results in solid organ transplantation patients. Although it does not totally prevent the progressive loss of function of the graft, it allows for a very long functioning of the transplanted organ. Nevertheless, there are still unanswered challenges in this area. The antibody-mediated rejection (ABMR) is the largest reason for a late graft loss. There are three types within the antibody-mediated mechanism: hyperacute, acute and chronic. Hyperacute rejection is mediated by recipient’s preformed DSA donor specific antibodies. It is observed immediately after perfusion of transplanted organs by recipient’s blood. Alloantibodies can bind antigens on the vascular endothelium and cause tissue damage. Prospective complement-dependent cytotoxicity—cross match (CDC-CM) minimizes this type of rejection.Novel reports (2019) diminished the doctrine that microvascular inflammation during rejection (MVI) is caused exclusively by the antibody-mediated pathway . For thiantigen presenting cells (APC) prior to an alloantigen-specific T cell response.Regarding lung transplantation, the major reason of this significant survival reduction is chronic lung allograft dysfunction (CLAD), which develops in about 50% of recipients within 5 years after lung transplantation . The mecIn contrast to lung transplantation, chronic liver transplant rejection occurs in about 3–4% of cases . ReportsThe ‘missing-self’ hypothesis implies that this mechanism of NK activity is of special interest in viral infections as viral antigens are often presented via HLA class I receptors. Moreover, viruses often use decreased expression of HLA receptors as a way to hide from T cytotoxic response. Transplantology is of special interest here as post-transplant immunosuppression results in a high-risk of viral infections. Cytomegalovirus (CMV) infection is the most common severe viral complication after solid organ transplantation (SOT). It remains a major source of morbidity and mortality in solid organ transplant recipients. Currently, only the donor and recipient serological status is used to predict the risk of infection after transplantation, while the cytotoxic potential of immune system is left unassessed. At the same time, NK cells are very much involved in the anti-viral response. NK antiviral activation is modulated by KIRs, so precisely the KIR receptors and HLA genotype could be associated with the risk of CMV disease. Deborska-Materkowska et.al presented the association between the lack of activating KIR2DS2 and lower CMV infection rate after transplantation . The assHabitual miscarriages remain a problem for many couples. It is estimated that live births account for only 20–25% of fertilizations . These oIn order to escape immune surveillance, cancer cells have developed multiple escape mechanisms. One of them is to reduce the expression of MHC molecules on the surface, which hide the cancer cells from the detection by cytolytic T lymphocytes. On the other hand, this fact is used by NK cells. NK cells mechanism of action is based, inter alia, on recognizing and attacking cells with weak or low HLA expression. The decreased expression of MHC by tumor cells results in a greater cytotoxic sensitivity of NK cells. The control of NK cell reactivity is a function of the signals coming from the interaction of NK cell receptors and MHC ligands. Hence, the knowledge of NK and MHC cell receptors seems to be of interest and may constitute a therapeutic point in the treatment of solid neoplasms. KIR receptors are particularly involved in the regulation of NK cells. When the net result of the interaction between KIR and their MHC ligands is negative, NK cells are less sensitive, which is unfavorable in the fight against cancer. If there is an NK activation prevalent, NK cells are activated and eliminate cancer cells. Recently, numerous studies have shown a link between KIR receptors and their ligands, and the protection or susceptibility of solid tumors . For exahttps://clinicaltrials.gov/ct2/show/NCT00552396, accessed on 3 July 2021) [Using the conclusions and the described models of NK cell alloreactivity flowing from numerous centers around the world, numerous concepts of adoptive NK cell therapies are currently being developed. Miller’s team research was one of the first attempts with partial success performed in 2005. The team had shown in previous studies that NK cell infusion post HSCT was safe but failed to ignite the full GvL effect. In the studied group, there was no matching of the inhibitory receptor, which could explain the lack of improvement of the treatment effects . In anotly 2021) . Similarly 2021) . The comly 2021) . The usely 2021) ,113.Adoptive therapy using NK cells appears to be promising. However, the current high costs and organizational effort do not allow for daily use of such sophisticated cellular therapies. In addition, apart from reduced costs, appropriate treatment protocols complementing chemotherapy should also be developed.Despite the PubMed database identifying over 5000 publications on the importance of KIR in the field of medicine since the 1990s and in consideration of the above reports, the area of research on KIR receptors remains insufficiently understood. The results obtained thus far are still inconclusive in many aspects. This is most likely due to the lack of multi-center studies and meta-analyses. Although the awareness on NK cell activity and the KIR/MHC relationship in the clinical management of the patient is rising, this information is still considered auxiliary, not primary. This information can be very useful in selecting the final donor or in order to obtain the best possible patient outcomes and benefits in the therapeutic process. This information can be an auxiliary parameter in the treatment of infertility, or ultimately it can be used in the prediction of antiviral reactions, which are the main cause of post-transplant mortality. The low cost of the testing, in view of the potential benefits, should encourage wider research on KIR receptors. The results will be certain to place this diagnostic procedure next to HLA antigens in the applicable standards.


Although the pathophysiology of coronary slow flow (CSF) has not been fully elucidated, emerging data increasingly support potential role for subclinical diffuse atherosclerosis in the etiology of CSF. We aimed to investigate relationship between atherogenic indices and CSF.130 patients with CSF diagnosed according to Thrombolysis in Myocardial Infarction (TIMI)-frame count (TFC) method and 130 controls who had normal coronary flow (NCF) were included in this retrospective study. Atherogenic indices  were calculated using conventional lipid parameters.p = 0.017) and CRI-II  were independent predictors of CSF. Receiver operating characteristic analysis showed that the optimal cutoff value to predict the occurrence of CSF was 0.66 for AIP  and 3.27 for CRI-II .The logistic regression analyses demonstrated that AIP (OR, 5.463; 95% confidence interval [CI], 1.357–21.991; AIP and CRI-II levels were independent predictors of CSF. Prospective studies in larger cohorts of patients may elucidate the role of atherogenic dyslipidemia in the pathophysiology of CSF. Coronary slow flow (CSF) is a microvascular disorder characterized by the slow entry of radiopaque contrast agent into distal vascular structures in the presence of normal or near-normal epicardial coronary arteries during coronary angiography . Althoug10 (TG/HDL-C)  ≤ 50), moderate to severe valvular heart disease, congenital heart disease, cardiomyopathies , hematological disease, thyroid dysfunction, and/or inflammatory diseases, and if they used lipid-lowering drugs or lacked complete clinical data. Demographic, clinical, and laboratory data of the participants were obtained from the medical records of our hospital.All coronary angiographies were performed either from the femoral or radial access using the standard technique . Iopromide contrast medium  was used in all patients. Images of the coronary arteries were acquired in the right and left oblique planes, as well as the cranial and caudal angles, at 15 frames per second (fps). Two cardiologists who were blinded to the patients’ demographic and clinical features assessed all angiograms to define CSF. The TIMI frame count (TFC) method developed by Gibson et al.  was usedMean reference values of 36 ± 1 TFC for LAD, 22.2 ± 4 TFC for LCX, and 20.4 ± 3 TFC for RCA were reported for the filling of coronary arteries . In the Laboratory findings were collected from the hospital database. Before the angiography, blood samples were collected for complete blood count analyses, following a 12 h overnight fasting. Plasma TC, TG, LDL-C, HDL-C, fasting glucose, creatinine levels were analyzed using the Architect c8000 Chemistry System  commercial kits. LDL-C was calculated via direct LDL-C assays. Then, AIP was determined by the base 10 logarithm of the ratio of the TG level to HDL-C level. The other indices used in this study were calculated as follows: non-HDL-C = (TC-HDL-C), AC = (non-HDL-C/HDL-C), CRI-I = (TC/HDL-C) and CRI-II = (LDL-C/HDL-C).3. Hemoglobin and platelet count were also calculated. Neutrophil to lymphocyte ratio (NLR) was calculated by dividing the neutrophil count to the lymphocyte count, and platelet to lymphocyte ratio (PLR) was calculated as the number platelets divided by the lymphocyte count. Transthoracic echocardiographic evaluation was performed for all patients by using Vivid 5 Pro  brand echocardiography device. LVEF was assessed using Simpson's method [Complete white blood (WBC) counts, including neutrophil and lymphocyte counts, were measured using an automated hematology analyzer CELL-DYN Ruby  and expressed as × 1.000 cells/mms method .The sample size of the study group was determined with 0.80 power and medium effect size using power analysis in R environment . More than 25% of this sample size (n = 260) was included in the study because of the possibility of using nonparametric tests and can be missing values. To reduce the bias when selecting the participants to CSF and NCF groups propensity score with the nearest neighbor method and 1:1 allocation ratio was used. While calculating the score, the gender and smoking status of participants took consideration. However, there was a total of 150 subjects in the NCF group and 175 subjects in the CSF group that fit the criteria. These limited numbers restricted the flexibility to get similar groups very much while selecting subjects into the groups.p < 0.05 was considered statistically significant.All analyses were performed using SPSS, version 23  and R, version 4.0.5  software. Continuous variables were presented as mean ± standard deviation or median (quartile deviation), and categorical variables were presented as numbers and percentages. Kolmogorov–Smirnov test was used to determine whether the continuous variables were distributed normally or not. Independent sample t-test or Mann–Whitney U-test was used to compare continuous variables. Categorical variables were compared within the study group using chi-squared tests. Receiver operating characteristic (ROC) curve analysis was performed to find a cut-off value for AIP and CRI-II according to Youden’s J index. Multiple logistic regression analysis with forward variable selection was used to determine the predictors of CSF. Hosmer–Lemeshow test was used to evaluate model fit. The odds ratio (OR) and 95% confidence interval (CI) were calculated for each independent variable. In all analyses p < 0.001). The WBC, NLR, PLR, neutrophil cell count, platelet count, LDL-C, TG, non-HDL-C, AIP, AC, CRI-I, and CRI-II values were significantly higher in the CSF group than in the control group. Furthermore, the HDL-C level and lymphocyte cell count were significantly lower in the CSF group than in the control group. There were no statistically significant differences between the two groups in terms of other laboratory parameters, TC, and LVEF .In total, 130 patients with CSF and 130 control participants with NCF were included the study. The mean ages of the two groups were 54.05 ± 9.61 years and 54.82 ± 8.78 years, respectively. Both groups showed male predominance . Table p < 0.001). In addition, in 73% of the patients, CSF was observed in the LAD , CRI-II , PLR , current smoking , and male gender  were independent predictors of CSF    Fig. . AccordiThis study was performed to investigate the relationships of CSF with traditional lipid parameters and atherogenic indices  that have been associated with increased cardiovascular risk. Atherogenic indices were higher and HDL-C values were lower in patients with CSF, compared with the control group. AIP, CRI-II, current smoking, and male sex were found to be an independent risk factor for CSF. In addition, LDL-C and non-HDL-C values were significantly higher in the CSF group compared to the control group.In CSF, washout of the contrast agent is prolonged in the absence of any spasm, thrombus, dissection, and any stenosis that causes significant occlusion in epicardial coronary arteries. Although the incidence of CSF in diagnostic coronary angiography is not rare, its pathogenesis has not yet been well elucidated. Various mechanisms have been proposed for its etiology, including subclinical diffuse atherosclerosis. Cin et al.  examinedIncreases in the levels of plasma very-low-density lipoprotein and small dense LDL-C, and reduced clearance of apolipoprotein B-containing particles from plasma have been identified in patients with atherogenic dyslipidemia . CompareAIP is regarded as an indirect indicator of small dense LDL-C . Wang etTo our knowledge, there have been no studies regarding the relationship between CSF and AIP. In the present study, we identified a positive correlation between AIP and TIMI frame count, which is regarded as an indicator of coronary flow reserve. The predictability of TIMI frame count by AIP supports the role of diffuse atherosclerosis in the pathophysiology of CSF. The results of the present study showed that AIP provided a reference for CSF severity. In addition, LDL-C and non-HDL-C, which are defined as the main indicator of atherogenic particles by current guidelines, were higher in the CSF group than in the control group. However, these lipid parameters were not found as predictive variables in the regression analysis.CRI-II (LDL-C/HDL-C ratio) represents the proportion or relationship between the atherogenic and antiatherogenic lipoproteins. CRI-II was a more precise predictor for cardiovascular events than classic lipid parameters  used independently , 35. FujCSF has been reported to be more common in young male smokers. In our study, both smoking and male sex were independent risk factors for CSF. These results are consistent with previous studies. In their cross sectional study, Sanghvi et al.  reportedThere have been multiple investigations of the roles of inflammation and biomarkers that reflect the inflammatory state  in coronary artery disease, as well as their relationships to adverse events –47. AksaThis study had several limitations. First, this was a single-center, retrospective observational study with a small sample size. Because of its retrospective design, inflammatory markers related to atherosclerosis  were not studied, and a more detailed evaluation of the relationship between CSF and inflammation could not be performed. Apolipoprotein B and small dense LDL-C, which reflect the total atherogenic particle load better than LDL-C, were also not measured. Unfortunately, imaging modalities  could not be used, although these would have better demonstrated a potential relationship between CSF and subclinical diffuse atherosclerosis. Endothelial function tests such as pulse wave analysis and flow-mediated dilatation could not be performed. In addition, selection bias may have occurred during selection of the control group due to the retrospective study design, and some individuals with NCF in the control group may have had undetected microvascular dysfunction.The results of this study indicated that AIP and CR II are independent predictors of CSF. Prospective studies in larger cohorts of patients may elucidate the role of atherogenic dyslipidemia in the pathophysiology of CSF. The results may also provide additional support for the importance of lipid-lowering therapy in the management of patients with CSF.